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Proceedings of the 43rd Annual Meeting of the AMERICAN RHEUMATISM ASSOCIATION A Section of the Arthritis Foundation May 30 through June 1 1979 Denver Colorado Abstracts of Papers Presented.

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586
Proceedings of the 43rd Annual Meeting of the
AMERICAN RHEUMATISM ASSOCIATION
A Section of the Arthritis Foundation
May 30 through June 1,1979
Denver, Colorado
Abstracts of Papers Presented
Suppressor T cell dysfunction and anti-suppressor T cell antibody in active early rheumatoid arthritis
N . I. Abdou, E. Pascual, and L. S. Racela; University of Kansas Medical Center, Kansas City, Kansas
Suppressor T cell dysfunction and anti-suppressor T cell
antibody were shown by us in active systemic lupus erythematosus
(SLE) (J Clin Invest 62:789 and in press), and were proposed to play a
key role in the expansion of DNA-binding clones. The status of suppressor T cell in rheumatoid arthritis (RA) is unknown. We evaluated
suppressor T cell in 29 RA patients with various degrees of disease activity and duration, 11 patients with osteoarthritis, and 34 normal
controls. RA patients were receiving gold (8), salicylates (21). nonsteroidal antiinflammatory drugs (8), and prednisone < 15 mg/day
(7). Suppressor T cells were evaluated by their activation in vitro with
20 pg Con A for 24 hours followed by mitomycin-C and cocultured
with autologous or allogenic RA or controls' B cells for 7 days in presence of pokeweed mitogen. From peripheral blood mononuclear cells,
enriched T (>92%) and B (>84%) cells were collected by E rosette
centrifugation method, and monocyte depletion (<2%) was by plastic
adherence. Sera to be tested for anti-suppressor T cell were decomplemented and adsorbed with pooled platelets, AB red cells, and B lymphocytes, and then incubated at various doses; periods, and temperatures with normal T cells prior to Con A activation and testing for
suppressor T cell activity. The latter was evaluated by examining B
cell Ig synthesis and secretion in the coculture by cytoplasmic fluores-
cence and immunofluorescence techniques. Results showed that the 4
RA patients who had active and early disease (ESR>80, rheumatoid
factor > 1:2560, elevated CH50, disease < 12 weeks, 2 patients on <
15 mg prednisone) had significantly lower suppressor T cell activity
when compared to the rest of RA patients or controls; Ig synthesis (P
< 0.02) and secretion (P t0.001). Patients with inactive RA, active
RA with disease > 6 months, osteoarthritis, and normal have comparable suppressor T cell activity (% suppression: Ig synthesis 27 f 1 I
and secretion 5 1 & 2 1 ) and their suppressor T cell suppressed the active early RA cells. Suppression was shown to be mediated by T cells
responsive to Con A and required monocytes in the B cell targets.
Con A activated normal cells pretreated with sera from the 4 active
early RA patients when compared to those treated with other RA sera
or controls' sera had poor suppressor activity (P< 0.001). Suppressor
T cell dysfunction and anti-suppressor T cell antibody could be demonstrated only in early active RA, but not in chronic active or inactive
RA. Anti-suppressor T cell could be responsible for the suppressor T
cell dysfunction and for the initiation, but not the maintenance, of the
autoimmune and/or inflammatory abnormality in RA. The mechanisms or factors responsible for the transient appearance of anti-suppressor T cell at the onset of active RA are unknown.
Antinuclear antibodies abrogate suppressor cell function
Donato Alarcon-Segovia, Alejandro Ruiz Arguelles, and Luis Llorente; Instituto Nacional de la Nutricion, Mexico City, Mexico
Using as a probe a purified, fluorescein-tagged anti-RNP
IgG obtained from the serum of a patient with mixed connective tissue disease, we have been able to show that antinuclear antibodies
(ANA) can enter into T, cells through their Fc receptors and bind to
their nuclei. T, cells have been found to have suppressor cell function
on B cells. We therefore sought to determine if incubation of normal
human mononuclear cells (MNC) in anti-RNP IgG affects their suppressor cell function, and if so, if this is due to T, cell deletion caused
by antibody penetration. For this we used a reverse hemolytic plaque
assay (Lipsky et al: J Immunol 120:902, 1978) where we studied both
spontaneous as well as concanavalin-induced suppressor cell function.
T, cells were separated with T cells with low affinity for sheep
erythrocytes (low affinity-T cells). Cytotoxicity of low affinity-T, cells
Arthritis and Rheumatism, Vol. 22, No. 6 (June 1979)
by anti-RNP antibody penetration was confirmed with "Cr-tagged T
cells in the absence of complement. Results are shown in the Table.
Results with cells incubated in Ig-free medium, with F(ab),
fragments of the anti-RNP IgG, or with albumin-anti-albumin immune complexes were similar to those obtained with normal IgG.
This indicates that the effect of anti-RNP IgG on T, cells is due to
antibody penetration rather than to signal triggering at the cell surface.
Deletion of T, cells caused by penetration of ANA of IgG
class, and the consequent abrogation of suppressor cell function is a
novel mechanism of immunologically mediated damage that can explain why some autoimmune diseases only occur when ANA switch
from IgM to IgG class.
5 87
ARA ABSTRACTS
Suppression, %
Incubation in
Low affinity
Ty cells, %
Anti-RNP IgG
Normal IgG
3.5 f 0.4
19.5 f 0.6
P
<O.o005
Spontaneous
Concanavalin
induced
Cytotoxicity on
low affinity
Ty cells, %
-41.3 f 19.2
+69.3 f 5.3
-58.3 f 18.3
4-67.1 f 1.2
39.8 f 2.9
10.7 f 3.1
<o.o001
<o.o005
<O.o001
Inflammatory arthropathy in leprosy
Jorge V. Alcocer, Rafael Herrera. Carlos Lavalle, Jeslis Gudiiio, and Antonio Fraga; Hospital General Centro Medico La Raza
IMSS, Mexico City, Mexico
Leprosy is a chronic infection which affects mainly the skin,
peripheral nerves, and nasal mucosa. Joint involvement has been reported only as a nonspecific lesion secondary to trauma or infection
imposed upon denervated tissues, or as a rheumatoid arthritis-like
syndrome in association with a reactional state.
We studied the clinical, radiologic, and scanning features of
arthritis of 18 unselected patients with leprosy (12 lepromatous, 2 tuberculoid, I dirnorphous, and 3 indeterminate form).
Fourteen patients had arthritis as a significant feature of their
disease; only in 4 cases was it coincidental with erythema nodosum
leprosum. The remainder had chronic episodic pain, swelling, and impairment of function affecting several large joints (wrists, elbows, and
knees) and small joints (hands). Joint scanning showed in 14 patients
a symmetric pattern of increased activity over the joints, and radiographic studies were abnormal in 16 patients, with erosive and/or cystic changes in 27%.Serum rheumatoid factor was negative, and only 2
patients had positive ANA by immunofluorescence. Ten patients had
polyclonal Igs increased, and none had hypocomplementemia. Synovial biopsy in 4 patients showed edema and lymphocytic infiltration;
acid-fast stain was negative, and the synovial fluid analysis was consistent with an exudate.
Inflammatory arthropathy is common in leprosy, being an
important factor in joint destruction besides the well known neurogenic arthropathy, and may be due to an underlying chronic immune
complex disease.
Significance of elevated Epstein-Barr virus antibodies in serum or synovial fluids from rheumatoid arthritis patients
Margaret A. A Ispaugh, Louisiana State University Medical Center; Gertrude Henle and Werner Henle, Children’s Hospital of
Philadelphia
In this study we have determined the frequency of antibodies
associated with Epstein-Barr virus (EBV) infection in rheumatoid arthritis (RA) and certain other diseases. The particular antibodies studied were directed to the following antigens: RA nuclear antigen
(RANA), early antigens D or R, and EB nuclear antigen (EBNA). Titers were considered elevated if they were: viral capsid antigens
(VCA) 2 80, D or R 2 10, EBNA 2 80. Results of elevated titers are
shown in the Table. Serum levels of antibody to RANA, VCA, or R
did not necessarily agree with the synovial fluid levels. Serial serum
titers were also determined bimonthly over a period of a year on 28
seropositive RA patients. Significant titer changes were noted for antibody to RANA in 15 of 28 patients (54%).but less changes were noted
in VCA titers ( I 1%) or R titers (1 1%).
The data suggest: 1) RA patients may have more active persistent EBV infections than normal individuals; 2) antibodies to EBV
antigens may play some role in general in connective tissue diseases;
3) only antibody to RANA appears to play a major role in seropositive RA.
Antibody to
Source
No. samples
% RANA
% VCA (IgG)
%D
%R
% EBNA
Seropositive RA. serum
synovial fluid
Seronegative RA, serum
synovial fluid
Osteoarthritis, serum
synovial fluid
Systemic lupus erythernatosus, serum
Progressive systemic sclerosis, serum
Burkitt’s lymphoma, serum
Nasopharyngeal carcinoma, serum
Laboratory personnel (LSU), serum
50
12
13
9
16
3
39
10
24
24
16
83
80
8
0
0
33
54
54
46
44
31
66
59
80
92
14
10
0
0
0
0
3
0
0
83
0
38
20
33
22
0
0
0
0
8
0
30
15
10
19
33
6
100
25
I5
66
31
70
79
0
25
30
30
42
6
ARA ABSTRACTS
Correlation of disease activity in rheumatoid arthritis, serial rheumatoid arthritis precipitin levels, antibodies to
Epstein-Barr virus antigens, and their relationships with complement and immune complex levels
M.A. Alspaugh, D. Rosenstock, J.J. Biundo; Louisiana State University Medical Center, New Orleans; W. Henle, Children’s Hospital,
Philadelphia; and R. C. Gupta, University of Colorado Medical Center, Denver
Twenty-seven patients fulfilling the American Rheumatism
Association (ARA) criteria for classic and definite rheumatoid arthritis (RA) were studied serially over a period of 6 months. Keratoconjunctivitis sicca (KCS) was present in 47%. Sera were tested for levels
of Ra precipitin (RAP), rheumatoid factor (RF), and levels of antibodies to the following: Epstein-Barr virus (EBV) antigens, viral capsid antigen (VCA), D, R, and EB nuclear antigens (EBNA). The initial frequency of these antibodies was RAP @I%), RF (74%), VCA
(54%), D (1 I%), R (37%), and EBNA (0).Antibodies to EBV antigens
were only considered if they were significantly elevated above normal
levels. This group of patients was followed on a monthly basis. The
disease activity was assessed and reported as an activity index, the
Westergren sedimentation rate (WSR) was recorded, and these were
compared to levels of antibody, immune complexes measured by Raji
assay, and complement. When analyzing data, patients were studied
individually and divided into subgroups based on the presence or absence of KCS, drug treatment, and the number of antibodies present.
No clear differences were noted between subgroups with and without
KCS or between groups on different drug regimens. However, the
number of antibodies appeared to be important. WSR and activity index were higher in patients with more antibodies. Significant titer
changes (equal to or greater than four-fold) were noted for RAP
(60%), whereas, less changes were noted for R F (30%), VCA (1 I%), R
(18%), and D (2 of 3). Some difficulties were encountered in attempting to correlate to immune complexes, complement, activity index,
and WSR with one antibody since most patients had more than one,
and the activity index and WSR did not necessarily correlate with
each other. In those patients where correlation could be made, immune complexes were correlated with RAP (50%),not with other antibodies; and complement levels tended to be lower with increased immune complexes. However, these two parameters did not necessarily
agree with activity index or WSR, and in others, there was no correlation either.
The data suggest that RA patients may have more active persistent EBV infections than normal individuals and the possibility
that RA precipitin may play some role in the pathogenesis of RA.
Further study will be necessary to better understand the mechanism.
The use of varying penicillamine doses in inducing a remission in resistant rheumatoid arthritis
Ronald J. Anderson, Robert B. Brigharn Hospital, Boston
The purpose of the study was to determine whether the action of penicillamine (PCM) was merely suppressive or capable of inducing a remission in rheumatoid arthritis (RA), and to determine
whether there is a difference in both efficacy and toxicity between
high dose and low dose PCM. The patients studied all met the following criteria: 1) classic or definite RA by American Rheumatism Association (ARA) criteria, 2) active disease for greater than 2 years, 3)
unresponsive or toxic to gold and plaquenil in the past. They were
treated by the following protocol: I ) No placebo controls; 2) 40 patients on high dose (1.5 &day) and 40 patients on low dose (0.75
&day), 3) high dose patients in apparent remission were reduced to
low dose after 6 months, 4) low dose patients who did not respond after 6 months were increased to high dose.
The patients were evaluated by giving a score of + 1 for each
of the criteria met listed below for a remission in RA: I ) No morning
stiffness, 2) all symptoms due to structural lesions, 3) no difference between days on aspirin versus days o f f ,4) no active synovitis on physical exam, 5 ) normal hematocrit (> 39%), 6) normal ESR (C30 mm),
7) improvement in function by 2 1/2 ARA class, 8) increase in function by f30% in grip strengths or 130%in 50 foot walk. A score of - I
was given if new nodules formed.
Patients were evaluated at onset, after 6 months and in late
1978. Those with a score of +7 or +8 were said to be in remission,
those with a score of - 1 to +2 were said to be nonresponders, and
those with scores between +3 and +6 were said to be equivocal.
The current status of the patients at an average of 34 months
(range: 18-68 months) after initiating PCM is shown in the Table.
No nonresponders on low dose remitted when raised to high
dose, and none of those remitting on high dose flared when reduced to
low dose. Seven low dose nonresponders developed toxicity after 6
months upon raising the dose to high dose. No feature before therapy
predicted ultimate response or toxicity.
Penicillamine seems able to induce a remission in certain patients with “resistant” RA. The response and potential toxicity are unpredictable. Toxicity seems to be decreased by doses of 0.75 @day
or less, without altering the efficacy.
Dose
Nonresponders
Equivocal
Remission
Toxic at 6 months
High dose
Low dose
13
0
8
24
4
8
19
4
589
ARA ABSTRACTS
Effect of antibody specificity on the composition and biological properties of soluble IgG-anti IgG immune complexes
William P. Arend, University of Washington School of Medicine, Seattle
The objective of these studies was to examine the effect of
variations in the specificity of anti-IgG antibodies on the structure
and properties of soluble IgG-anti-IgG immune complexes. Rabbit
antibodies (Ab) specific for antigenic determinants on the Fab fragments, Cy2 domain, or Cy3 domain of human IgG were purified by
affinity chromatography. Antigen (Ag) and antibody preparations
which possessed either intact or reduced and alkylated interchain disulfide bonds were utilized.
The results of quantitative precipitin analyses indicated that
the degree of precipitation, when antibodies were combined with human IgG antigen at equivalence, varied between the three preparations in the order Fab specific > Cy2-specific > Cy3-specific. Reduction and alkylation of either the antigen or antibodies, or both, led to
a decrease in the amounts of antibodies precipitated at equivalence.
In spite of the differences in precipitability at equivalence, soluble
complexes prepared in antigen excess possessed comparable amounts
of large-latticed structures (Ag2Ab,) in each antigen-antibody system.
The abilities of the complexes to fix complement and to bind
to macrophage Fc receptors were evaluated by utilizing the purified
antibodies of different specificities. The complexes with intact IgG an-
tigen and reduced and alkylated Fab-specific antibodies were 100times more active in complement fixation in comparison to similar
complexes prepared with Cy2- or Cy3-specific antibodies. There was a
100-fold loss in complement fixing ability, and a 3-fold decrease in
binding to Fc receptors upon reduction of the antigen in the complexes containing reduced and alkylated Fab-specific antibodies. No
such differences in biologic properties were seen with the complexes
prepared with Cy2- or Cy3-specific antibodies. These results indicated
that the IgG antigen in the complexes containing Fab-specific antibodies contributed to the biologic properties of the complexes; the
antibodies specific for Cy2 or Cy3 blocked the IgG antigen in these
activities. Other results suggested that complexes with Fab-specific
antibodies possessed structural characteristics that promoted fixation
of complement to a greater degree than binding to Fc receptors.
It was concluded that the specificity of antibodies ot IgG has
significant effects on the interaction with antigen and on the biologic
properties of soluble IgG-anti-IgG complexes. These findings may
contribute to a further understanding of the role of immune complexes containing IgG rheumatoid factors in human diseases.
Juvenile chronic arthritis: Description of a new HLA-B27subset persisting into adulthood
Frank C. Arnett, Wilma B. Bias, and Mary Betty Stevens, The Johns Hopkins University School of Medicine, Baltimore
Previous studies of HLA-B27 in juvenile-onset chronic arthritis (JCA) have shown an association with pauciarticular disease in
boys which may progress to ankylosing spondylitis. The spectrum of
B27-associated disease, however, especially as it relates to arthritis
persisting into adulthood, has not been fully clarified. Thus, our studies, largely of an adult JCA population, are described. All patients
with childhood-onset (5 16 years), chronic (2 3 months), peripheral
arthritis were selected without regard to rheumatoid factor (RF), antinuclear antibody (ANA), or HLA-B27 positivity. Those with an exclusively axial onset, typical Still’s disease, or psoriasis were excluded.
HLA-B27 was determined in all who were not RF-positive. A total of
50 patients were studied, 35 (70%)were adults and 15 (30%) were children. When groups were analyzed for seropositivity for RF (zk ANA)
Adults
Seronegative (20)
Seropositive (15)
Children
Seronegative ( 1 1)
Seropositive (4)
“Rheumatoid
arthritis”
hands
Acute
iritis
Micrognathia
B27+
B27RF+
ANA+
4
0
0
6
2
2
0
B27+
B27RF+
ANA+
0
0
0
0
0
0
No. (%)
Groups
Females
Polyarticular
or ANA alone versus seronegativity with and without B27, certain
clinical and radiographic features clustered into the seronegative and
B27-positive patients.
These data disclosed a new B27 subset of adults, usually females, with polyarticular onset, seronegative arthritis with “rheumatoid arthritis-like” hands. Cervical apophyseal fusion, especially C2-3,
was prominent and correlated with B27 (P< 0.01, sacroiliitis or ankylosing spondylitis (P< O.OOOl), and acute iritis (Pc0.01), while C1-2
subluxation occurred in those with RF (P < 0.0005). Micrognathia
was common and associated with polyarticular-onset (P< 0.05) and
the cervical fusion lesion (P< 0.01). Therefore, B27 has broader clinical implications than previously thought and should be utilized in the
classification of JCA.
C2-3
fusion
Sacroiliitis
0
I
0
ARA ABSTRACTS
C2 deficiency and a lupus-like illness: Reevaluation of a family
John P. Atkinson, Tommaso Meo, Donald Shrefler, Washington University School of Medicine, St. Louis; Jane Schultz, University of
Michigan Medical Center, Ann Arbor; William Miller, Washington University School of Medicine, St. Louis; C.K. Osterland, McGill
University School of Medicine, Montreal, Canada; and Richard Wahl, Washington University School of Medicine, St. Louis
A large 4-generation kindred with C2 deficiency (C2D) and
rheumatic diseases was reevaluated (see Ann Intern Med 82:323,
1975) and found to have 9 heterozygous and 3 homozygous C2D individuals. The latter group consists of a 27-year-old female proband
with previously severe systemic lupus erythematosus (SLE) that has
been quite responsive to therapy, a 74-year-old female with 2 mild
episodes of discoid LE, and an 82-year-old male with no history of
rheumatic diseases who died of a mycardial infarction while this
study was in progress. The revised and extended HLA typing data indicated that the haplotype HLA-AIO, B18 was in strong linkage disequilibrium with the C2' (deficient gene). Mixed lymphocyte cultures
employing HLA-D homozygous typing cells indicated that the C2D
individuals were Dw2 (mutually nonreactive to each other and to
Dw2 homozygous typing cells). Typing for Factor B of the alternative
complement pathway demonstrated that both common variants were
present in the kindred and that the S (slow) variant was in linkage disequilibrium with HLA-A10, 818, Dw2, C2'. Based on lytic patterns in
gels after isoelectric focusing of serum, three allelic variants for C2
(C2 polymorphism) have been described C2' (common), C2* (uncommon) and C2' (deficient) that occur with frequencies of 96, 3, and
I% respectively. All three polymorphic variants were present in this
family and all 9 members with half-normal C2 levels had only a
single variant. Several individuals in this kindred demonstrated that
the C2 deficient allele segregated at the same locus as the structural
allele. These findings provide strong evidence for a null or silent gene
at this locus.
Fifteen patients with adult onset Still's disease: Life threatening liver failure in two
Daniel G. Baker, H. Ralph Schumacher, and Antonio J. Reginato; Veterans Administration Hospital and University of Pennsylvania,
Philadelphia
We have collected one of the largest series of adult onset
Still's disease (AOSD). We confirm previously described features and
stress other not well recognized systemic manifestations. Fifteen patients ages 16 to 56 (mean 29) with AOSD were studied from 1973 to
1978. The most common criteria for diagnosis were spiking fever (15).
typical evanescent maculopapular rash (14), polyarthritis (15), leukocytosis with neutrophilia (14 patients; mean 23,000), and sore throat
(13) with repeatedly negative bacteriologic and serologic studies including ANA, serum complement, antistreptolysin 0 titers, and HAA.
Other findings were lymphadenopathy 7, pleuropenumonitis 5, metacarpal-carpal arthritis with ankylosis 4, hepatomegaly 3, splenomegaly 2, and pericarditis 2. HLA-B27 was negative in all 7 tested. Synovial fluid in 1 I was inflammatory with a mean of 12,000 WBC and
85% polys. Synovial membrane light and electron microscopy in 7 patients, as in rheumatoid arthritis, showed lymphocytic and plasma cell
infiltrates and proliferation of lining cells. No vital-like particles were
found. Followup from 2 months to 5 years (mean 2 years) revealed no
new diagnostic possibilities.
Other systemic manifestations were hepatic in 5, renal in 3,
central nervous system in 2, pulmonary and gastrointestinal in 1. Two
patients experienced acute hepatic failure with jaundice and marked
SCOT and SGPT elevations. Our first such patient while on high
dose aspirin developed delirium, acute abdominal pain, ileus, interstitial pulmonary infiltrates, and acute respiratory failure requiring
tracheostomy. Urinalysis revealed RBC casts but creatinine clearance
was normal. The second patient with hepatic failure, on high dose aspirin and indomethacin, developed encephalopathy with increased intracranial pressure, elevated prothrombin time, and acute nonoliguric
renal failure requiring dialysis. Both received high dose steroid therapy and experienced complete recovery. Three other patients without
exposure to aspirin or any known hepatic toxins had mild elevations
of alkaline phosphatase, SCOT, and SGPT which were reversible
with treatment that included aspirin. A patient without liver enzyme
elevations was noted to have albuminuria and microscopic hematuria
with a creatinine clearance of 74. Abnormalities disappeared with decreased activity of disease.
This study suggests that AOSD, as in children, may present
with acute life-threatening hepatic failure, whether due only to the
primary disease or in part related to drug therapy.
Late onset systemic lupus erythematosus
Stuart B. Baker, The Johns Hopkins Hospital, Baltimore; Jose R. Rovira, Edward W. Campion, and John S. Mills; Harvard Medical
School and Massachusetts General Hospital, Boston
This study describes the clinical and laboratory features and
the natural history of 3 I patients with late onset (in the sixth decade
or later) systemic lupus erythematosus (SLE). The computerized diagnostic index of the Medical Records Department was searched for all
patients with SLE who were seen as inpatients between the years 1962
and 1977. Drug-induced syndromes, discoid lupus alone, and rheumatoid arthritis with LE cells were excluded. The onset of SLE was
defined both by the date of diagnosis and the date of probable onset,
i.e., the initial manifestation characteristic of the disease without other
explanation. Both onset and diagnosis had to occur after the patient's
fiftieth birthday in order for the patient to be included in this study.
Patients with late onset SLE constitute a distinct subset of the
general lupus population that accounts for approximately 12% of
cases. Advanced age modifies the expression of SLE in terms of clini-
59 1
ARA ABSTRACTS
cal presentation (pleuritis and/or pericarditis are the most common
presenting manifestations) and pattern of organ involvement. Pulmonary abnormalities are more common (33% in our series versus an average of 14.5% in four recent series), whereas lymphadenopathy (10%
versus 3 1.3%), Raynaud's phenomenon (10% versus 30.4%). neuropsychiatric disease (26% versus 46.5%), alopecia (26% versus 45%), and
skin rash (65% versus 82.5%) are less common. Because SLE is not
usually considered to be a disease that affects the elderly, and because
the pattern of SLE in the older age group may differ substantially
from that seen in younger patients, there is often a delay in diagnosis
(median of 10 months, with a delay of over one year in 32% of patients) or an incorrect diagnosis is initially made (55% of patients). In
light of the high incidence of steroid complications in older patients
(40% in our series), and because these patients with SLE have a relatively good prognosis (5-year survival of 92.3%; 9-year survival of
83.1%), therapy should be more conservative in late onset SLE.
Defective monocyte cytotoxicity in rheumatoid arthritis
F. A . Barada, Jr., William M. O'Brien, H. David Kay, David A . Horwitz; University of Virginia School of Medicine, Charlottesville
ADCC were depressed (PC 0.001, P = 0.06 respectively). In another
series of experiments, either 12.5% normal human serum (NHS) or
one of 9 separate RA sera was added to the cells from healthy subjects. RA sera strongly suppressed monocyte natural cytotoxicity
(NHS 14.8 f 8.4% versus RA sera 2.2 f 1.1%. P c 0.001) and also
monocyte ADCC (NHS 11.7 f I% versus RA sera 7.0 f 1.6%, P c
0.OOOl). Only I of 9 RA sera suppressed lymphocyte cytotoxicity. This
monocyte inhibitory factor was heat stable and its effect disappeared
at a 1:5 dilution (2.5%). All RA sera contained IgM rheumatoid factor
(RF), but there was no obvious correlation between RF titer and percent suppression of monocyte cytotoxicity.
These experiments have shown that monocyte, but not lymphocyte, cytotoxicity was depressed in patients with active RA. Sera
from RA patients when added to normal cells reproduced the monocyte defect of RA cells. This monocyte defect may alter immune regulation and consequently may be important in the chronicity of rheumatoid arthritis.
To account for the chronicity of rheumatoid arthritis (RA), it
has been suggested that host defense is impaired, but specific cellular
defects remain poorly defined. In this study the cytotoxic activities of
monocytes and lymphocytes from patients with active rheumatoid arthritis were compared with healthy controls in a 16-hour S'chromium
release assay. Strongly adherent cells, 95% f 5% monocytes, were prepared on plastic microtiter wells and their cytotoxic capacities were
compared with nonadherent lymphocytes. Monocyte preparations
from RA patients and controls contained similar numbers of latex
phagocytic cells and contaminating nonphagocytic cells. These cytotoxic assays were performed in media containing 10%heat-inactivated
fetal calf serum at effector to target cell ratios of 2 0 I. Natural cytotoxicity was measured by using unsensitized K562 target cells, an established myeloid cell line. Antibody dependent cellular cytotoxicity
(ADCC) was determined by using human Chang liver target cells
coated with rabbit IgG.
Lymphocyte cytotoxicity in RA patients was similar to normal controls. However, both monocyte natural cytotoxicity and
Monocyte (Adherent cell)
Sera
Normal (n = 17)
Rheumatoid arthritis (n = 14)
Lymphocyte (Nonadherent cell)
Natural cytotoxicity f SD
ADCC
Natural cytotoxicity
ADCC
29.6 f 13.4%
9.1 z t 5.0%
50.6 f 26.8%
26.3 22.0%
39.1 f 10.5%
38.6 f 16.8%
56.7 f 19.2%
50.7 f 27.2%
*
Renal dialysis in systemic lupus erythematosus: Predictive factors and outcome in 35 patients
John Beary, Michael Lockshin, Hospital for Special Surgery, New York; Raymond Sherman, Janet Mouradian, Jhoong Cheigh,
Cornell University Medical College, New York; and Robert Kimberly, Hospital for Special Surgery, New York
We retrospectively reviewed 35 systemic lupus erythematosus
(SLE) patients who have entered dialysis since 1970. The predialysis
clinical, serologic, and biopsy features of SLE dialysis patients were
compared with an SLE nondialysis group. Experience with long term
dialysis and renal transplantation was also analyzed. Twenty-four of
the 35 patients came from the rheumatic disease unit, and 1 I patients
were referred directly to the dialysis unit. All had SLE by American
Rheumatism Association criteria. One patient also had diabetic nephropathy.
Clinical patterns of onset of renal failure were hyperacute
glomerulonephritis in 16 of 35, gradually progressive glomerulonephritis in 13, and vasculitis in 6 patients. Hypertension, hematuria,
pyuria, hypocomplementemia, and high titer of anti-DNA antibody
were frequent findings in both the dialysis and nondialysis groups and
did not distinguish the groups. Several patients developed renal failure despite good serologic control. Biopsy pattern was relatively nonpredictive. Some patients with mildly abnormal biopsies quickly
progressed to renal failure, while others who had severely abnormal
biopsies did not experience renal failure during the several years of
followup.
Nine of the 35 patients are still on dialysis and 7 were able to
discontinue dialysis; 4 of the 7 patients remain off dialysis. Five have
functioning transplants and 17 died. Most patients who progressed to
dialysis did so within the first two years after diagnosis of SLE. Patients who survived the flare of SLE which precipitated their renal
failure have tolerated dialysis well. The development of endstage
renal disease in SLE is not nearly as predictable as current literature
suggests.
ARA ABSTRACTS
A family study of chronic discoid lupus erythematosus: High serum properdin levels in families with multiple cases
Daniel C. Belin, Robert H. McLean, Bonnie Bordwell, and Naomi Roth$eld; University of Connecticut, Farmington
Multiple family members with chronic discoid lupus erythematosus (CDLE) have rarely been reported. We have previously
demonstrated a high incidence of elevated serum properdin levels in
CDLE patients. We therefore studied families of CDLE patients and
found 3 families with multiple cases. In 2 of the 3 families, there was a
high incidence of elevated properdin in first degree relatives.
No family history of CDLE was obtained from 7 CDLE patients whose family members were not available for study. Twentyfive first degree relatives of 14 CDLE patients had a history and physical examination performed, and properdin levels were measured by
electroimmunoassay in 24 of the 25. C3 and C4 were measured by
radial immunodiffusion and C2 by specific hemolytic assay.
Three families had I first degree relative with CDLE: Family
A-sister/sister; Family B-brother/sister; Family C-father/daughter. Multiple members, including the CDLE patients of Family B, had
heterozygote C2 deficiency. No deficiency of C2, C3, or C4 was found
in members of the other 2 families.
Eight of 21 CDLE patients had elevated properdin levels (>
2 SD > mean) compared to 2 of 42 normal relatives (P< 0.001). In
Families A, B, and C, 4 of the 6 CDLE patients had elevated properdin levels, an incidence significantly higher than in the normal group
(P< 0.001).
Elevated properdin levels in non-CDLE first degree relatives
were found in 4 of 5 in Family A, 2 of 7 in Family B, and not in the
single non-CDLE first degree relatives in Family C. Thus in the 3
families with multiple cases, 6 of 13 non-CDLE first degree relatives
had high properdin levels, an incidence significantly higher than in
the control group (P 0.001).
In the I1 families without multiple cases, only 1 of 9 first degree relatives had elevated properdin, an incidence similar to that of
the controls (P= 0.46).
The findings suggest that families with multiple cases of
CDLE are more common than previously noted. The data suggest
that a higher incidence of elevated properdin levels occurs in non-affected first degree relatives in families with multiple cases than in
non-affected first degree relatives in families in which only I individual has CDLE or than in normal individuals. A possible role for genetic factors in CDLE is suggested by these studies.
-=
Juvenile rheumatoid arthritis and salicylate related liver chemistry abnormalities: Clinical and genetic considerations
Carolyn L. Bell and Peter H. Schur; Robert B. Brigham Hospital, Boston
The incidence of liver function test (LFT) abnormalities was
determined in 187 patients with juvenile rheumatoid arthritis (JRA)
taking salicylates. Thirty-one (17%) were noted to have elevated blood
SCOT and/or SGPT levels on at least one occasion. Twenty-eight patients had only minor LFT elevations, while 3 patients had evidence
of clinical hepatitis. In 23 patients, the LFT abnormalities returned to
normal despite continued salicylate therapy. Salicylate levels did not
correlate with the higher LFT abnormalities. There was no predilection for LFT abnormalities of either sex, age of onset of JRA,
and age of the patients at the time of the LFT abnormalities. Nine patients had a systemic onset JRA, 7 an oligoarticular onset, and 15 a
polyarticular onset (in our total JRA population this type of onset was
present in 19%, 40% and 41% of the patients respectively). HLA typing was done on 131 of the patients, including 25 with LFT abnormalities. The HLA haplotype A2, Bw40 was found in 4 with LFT abnormalities, a frequency significantly higher (Pcorrected for 390 possible
haplotypes = 0.015) than in normal controls. The relative risk for having LFT abnormalities in patients with JRA who have A2, Bw40 was
calculated to be four times higher than in those without this haplotype. These studies suggest that a small portion of JRA patients may
develop transitory LFT abnormalities, and that this susceptibility may
have, at least in part, a genetic basis.
Familial rheumatoid arthritis: Clinical, serologic, and HLA analysis
David A. Bell and Jon Block; University of Western Ontario, London, Ontario, Canada
While recent studies of unrelated rheumatoid arthritis (RA)
patients have revealed a strong association of the disease with HLADRw4, earlier studies disputed a genetic basis for this disorder. The
purpose of the present study was to select families with at least two or
more affected individuals in order to determine if the multiple occurrence of disease in families could be linked to a common haplotype
within each family, revealed by HLA A,B,C, and D locus typing.
Twelve such families have been examined in detail for clinical and
serologic evidence of rheumatoid arthritis, and in each, the proband
had classic seropositive RA.
The following HLA haplotypes were shared among affected
individuals in each family: 1,8 (5 families); 2,s (1 family); 1 I, I5 Cw3
(1 family); 3.35, Cw4 (1 family); 3,49 ( I family); 29,44 Cw5 (1 family);
3,21 ( I family). D locus antigens were identified by employing commercial antisera (Terasaki TE-B 4 Trays) to detect alloantigens on pu-
rified B lymphocytes. The following D locus antigens were identified
in the probands of each family: DRw3 (7 families); DRw4 (2 families); DRw7 (1 family); DRw2 (1 family); DRwl (1 family). HLA-B8
was associated with DRw3 in 6 of 7 probands consistent with the
known linkage disequilibrium between these two antigens. Rheumatoid factor was not always detected among clinically affected members
of some families sharing HLA antigens with the propositus, suggesting that rheumatoid factor may be determined by other genetic or
nongenetic means. Lymphotoxins occasionally present in the serum of
some of the affected patients were not detectable among their normal
consanguineous or nonconsanguineous relatives. ANA present among
most affected individuals was not observed more frequently among
nowaffected consanguineous or nonconsanguineous relatives than
normal controls.
These studies reveal that in some RA families, genetic factors
ARA ABSTRACTS
related to HLA may predispose to the development of disease, but
that additional factors may influence its expression, particularly the
appearance of RF. HLA-B8 and DRw3 were more frequent and
HLA-DRw4 less frequent than expected from reported studies of un-
593
related patients with RA. In contrast with familial systemic lupus erythematosus, serologic abnormalities were infrequently observed
among non-affected consanguineous or nonconsanguineous close
family members.
Generation of anti-DNA antibodies in normal and pre-autoimmune NZB x NZW F, mice
David A . Bell, Larry B. Puncer, and Sharwin K. Singhal; University of Western Ontario, London, Ontario, Canada
The in vivo induction of autoantibody responses to normal
tissue antigens has been difficult to achieve. We have shown that in
vitro the spleen cells of most normal as well as pre-autoimmune
strains of mice spontaneously produce IgM anti-ssDNA antibody
plaque forming cells (PFC). We considered that it might be possible
to induce anti-ssDNA antibodies in vivo by the transfer of such in
vitro activated lymphoid cells to normal or pre-autoimmune mice.
In these experiments, cultured splenic lymphoid cells were
administered to syngeneic normal or young B/W mice. Ten days after
transfer, spleen cells of mice receiving cultured irradiated splenic lymphoid cells harvested on day 3 of culture showed a significant antissDNA PFC response but no increase in anti-SRBC PFC. This response could also be generated with the cell-free supernatant from
such cultures generated in serum-free medium. The response in the
recipient mice was due to antibody produced by recipient B cells
which required the presence of T helper cells to produce significant
levels of anti-ssDNA antibody. The same soluble immunogenic material when added to cultures of young pre-autoimmune B/W mice augmented their spontaneous in vitro anti-ssDNA response. The in vivo
response of the recipient mice was augmented when the donor population was enriched in cultured T cells, but no response was seen with
noncultured T or noncultured whole spleen cells, or when cultured
adherent macrophages were employed. Antithymocyte serum and
complement treatment of the donor cell population abolished the response. It could be shown by a variety of techniques that the immunogenic molecule in the supernatant was DNA and that similar material
was present on the T lymphoid cells used to transfer this response,
since DNase treatment of these cells abolished their ability to generate antibody responses in the recipient mice.
These data indicate that during culture some form of immunogenic DNA probably released from cultured T cells not only seems
capable of enhancing the previously observed spontaneous in vitro response to ssDNA, but also is capable of either augmenting or stirnulating anti-ssDNA antibody responses in vivo in normal or pre-autoimmune mice ordinarily relatively resistant to immunization with
DNA. The mechanism of release of this immunogenic material and its
exact characterization are presently under study.
Exudative arthritis in leprosy
Louis Bermun; University of Texus Mrdicul School, Houston
Destructive joint disease is well known in leprosy. An exudative polysynovitis, however. is rarely described. Nine such patients
have been studied.
Three patients with untreated lepromatous leprosy presented
with polysynovitis of the small joints and early morning stiffness.
Rheumatoid factor was present in all, and antinuclear antibodies were
present in 2 patients. One patient had cryoglobulins and a history of
Raynauds disease. Radiologically, generalized osteoporosis was seen
with occasional juxtaarticular erosions. Six months after treatment
with dapsone and rifampicin, the rheumatoid picture resolved clinically and immune markers decreased.
Four patients with lepromatous leprosy developed an active
polysynovitis precipitated by therapy, but they improved with modification of treatment
Two patients with erythema nodosum leprosum presented
with an exudative polysynovitis that subsided after response to modified therapy which included short term steroids.
Lepromatous leprosy is characterized by generalized cellular
anergy but is associated with an exaggerated immunoglobulin response which may include ANF, rheumatoid factors, and cryoglobulins. However, in tuberculoid leprosy, there is predominantly an exaggerated cellular immune response with tissue and nerve destruction.
“Downgrading and upgrading” refers to changes in the cellular and
humoral immune state with the change of one polar form of leprosy
to another (lepromatous to tuberculous). This may occur during therapy.
It is probable that in lepromatous leprosy, as in erythema
nodosum leprosum, immune complex synovitis produces the exudative synovitis.
Juvenile ankylosing spondylitis: Are adult criteria appropriate?
Bram H. Bernstein, Bernhard H . Singsen, Childrens Hospital and University of Southern California School of Medicine, Los Angeles;
Arthur Lorber, Memorial Hospital Medical Center, Long Beach, and University of California at Irvine; Helen K. Kornreich, Karen K.
King, and Virgil Hanson, Childrens Hospital and USC School of Medicine, Los Angeles
The Rome and New York criteria for ankylosing spondylitis
heavily emphasize x-ray signs of sacroiliitis. Ankylosing spondylitis is
infrequently diagnosed in childhood, although several surveys suggest
that 10-20% of ankylosing spondylitis patients date their symptoms
from the second decade. To ascertain the characteristics and course of
juvenile ankylosing spondylitis (JAS), the records of 25 children (18
males, 7 females) previously diagnosed as having possible JAS were
reviewed. None manifested conjunctivitis, urethritis, skin rash, or underlying bowel disease. All were under age 16 years at onset and had
peripheral joint involvement, predominantly of the lower extremities.
ARA ABSTRACTS
Patients were divided into 3 groups: 1) Definite JAS: x-ray evidence of
sacroiliitis; 2) Probable JAS: low back pain or stiffness and/or a family history of ankylosing spondylitis; 3) Possible JAS: B27-positive peripheral arthritis. All were rheumatoid factor negative, 2 had low titers of ANA, 22 had HLA-B27. Three children had not been tissue
typed but showed sacroiliac disease on x-ray. The table displays mean
onset and followup data in years.
In definite JAS the mean time to develop x-ray change was
4.2 years (range 0.8-1 1.8); 1/9 had radiologic abnormalities when first
x-rayed. Eight children had hip joint involvement (7 definite JAS, I
Age of onset (range)
Length of disease (range)
Length of followup (range)
probable); 3 had shoulder involvement (all probable JAS). Heel pain,
or achilles tenosynovitis was prominent in I I cases (3 definite, 7 probable, 1 possible). Six children had 10 episodes of acute intis (2 definite, 3 probable, 1 possible). Treatment consisted of aspirin and nonsteroidal antiinflammatory drugs. When last seen, 24/25 were in
functional Class I, one child with definite JAS was in Class I1 after 8.7
years. The extended time period required for children to satisfy adult
ankylosing spondylitis criteria suggests a need for revised criteria that
will allow differentiation of JAS from juvenile rheumatoid arthritis,
even in the absence of x-ray change.
Definite JAS (9)
Probable JAS (14)
Possible JAS (2)
Total group (25)
10.2 (3-13)
5.8 (1.4-13.3)
2.6 (0.84.4)
8.8 (2-15)
5.8 (0.3-24.5)
3.0 (0.3-21)
13.0
2.2 (1.62.8)
1.2 (0.1-2.3)
9.6 (2-15)
5.5 (0.3-24.5)
2.7 (0.1-21)
Depressed T cell colons growth in systemic lupus ervthematosus
Maik L. Bernstein, Univeisiyy of Pittsbuigh; Sue A. kobson, Montefiore Hospital, Pittsburgh; and Alan Winkelstein, University of
Pittsburgh
Recently, a technique has been developed for the growth of
human T lymphocytes in vitro. Peripheral blood lymphocytes are
plated on semi-solid agar; in the presence of optimal concentrations of
phytohemagglutinin (PHA), discrete colonies of T cells are observed
at 5-7 days. Each colony contains 10-500 cells and each is believed to
arise from a single stimulated lymphocyte. Studies in 35 normal subjects indicated that 7.5 X lo5 blood mononuclear cells generated a
mean of 6,020 & 2,080 (SD) colonies. These data suggest a plating ef
ficiency of 0.8%. Separation of lymphocytes by sheep red cell rosetting, followed by Ficoll-hypaque centrifugation, demonstrated that
only T cells formed colonies. Colony growth was assessed in patients
with various connective tissue diseases. A high proportion of systemic
lupus erythematosus (SLE) patients (12/28) had subnormal lymphocyte colony growth. The mean colony number for all SLE patients
was 2,326 f 414 (SEM), a value significantly lower than controls (P<
0.005). None of these patients was receiving cytotoxic therapy, and in
vitro growth did not correlate with corticosteroid dosage. Colony formation did not parallel the numbers of circulating T lymphocytes; of
8 patients with decreased T cells, only 2 showed subnormal colony
formation. Furthermore, decreased colony formation did not correspond with clinical or serologic disease manifestations. Sera from SLE
patients were examined for evidence of T cell growth inhibitors by incorporating these sera in the plating media with normal lymphocytes.
Six of 26 SLE sera were inhibitory. Additional studies showed that
lymphocytes from patients with rheumatoid arthritis (12 patients) and
progressive systemic sclerosis (5 patients) produced normal colony
numbers.
These studies indicate that, in the presence of PHA, T cell
colonies are readily produced from peripheral blood lymphocytes.
This technique demonstrated impaired colony growth by using cells
from patients with SLE, but impaired growth did not occur with those
from other connective tissue diseases.
Penicillamine proteinuria: A sequel to gold nephropathy
Lynn M . Billingsley and Mary Betty Stevens; Johns Hopkins University, Baltimore
D-penicillamine (DPA), now recognized as a major agent in
the therapy of rheumatoid arthritis (RA), has generally been reserved
for those patients without response to gold or with drug toxicity interrupting chrysotherapy. In the studies to date, the complication of
DPA-induced proteinuria, variously reported from 2% to 30%. has not
been related to prior gold nephropathy. However, in our experience,
this association is striking.
In our Center since 1976, 25 patients with definite or classic
RA have been treated with DPA. Six (24%) of the DPA-treated patients developed proteinuria. Surprisingly, 5 of these (83%) had a
documented history of gold-induced proteinuria. In contrast, of the 19
patients in whom no proteinuria occurred with DPA, 4 had never received chrysotherapy and only 3 (20%) of the gold-treated subgroup
had had proteinuria (P < 0.01). The emergence of DPA-induced proteinuria could not be explained by the temporal relationship to chrysotherapy, the duration of DPA treatment, or its dosage.
While the common denominator for gold and DPA renal
toxicity is unclear, these data strongly suggest that added caution is
essential when initiating DPA therapy in patients who have had gold
nephropathy .
Prior gold therapy
DPA
proteinuria
Total no. patients
Positive
Negative
6
19
Total patients
Patients with
proteinuria
Interval between
gold + DPA
mean (range) years
6
15
5
3
5.0 (2-8)
3.3 (<1-7)
Duration DPA
therapy
mean (range) months
4.0 (3-8)
9.5 (2-33)
595
ARA ABSTRACTS
Radiographic joint findings in progressive systemic sclerosis and CREST Longitudinal evaluation and clinical
correlations
Kenneth Blocka, Lawrence Bassett, Daniel Furst, and Philip elements; University of California Los Angeles
X-rays in 55 patients with progressive systemic sclerosis
(PSS) (other connective tissue disorders excluded) and 10 patients
with CREST syndrome were analyzed. Followup films were available
in 33 of the PSS patients (mean interval: 28.7 months) and in 7 of the
CREST patients (mean interval: 29.8 months).
In the PSS group, the initial films reconfirmed the high incidence of hand abnormalities including: skin atrophy in 728, calcinosis in 45%, digital tuft resorption in 51%, flexion contractures in
82%. and generalized osteopenia in 40%. We also found radiographic
evidence of an associated inflammatory arthropathy in a significant
proportion of the PSS patients: juxtaarticular demineralization (3.6%).
joint space narrowing (12.7%), marginal erosions (9.l%), or ankylosis
(1.8%). Hitherto undescribed atypical dorsally located erosions occurring singly or in combination with marginal erosions were found in
15% of the patients. The presence of a widespread inflammatory arthropathy was also supported by the finding of: rib erosions (15%).
periositis (12%), and acromioclavicular joint erosions (3%). Rib erosions correlated (r = 0.92)with marginal erosions of the hands or feet.
Foot involvement was similar but less frequent than hand involvement. Progression in frequency and severity of all hand and foot
abnormalities was noted in the PSS group. Unexpected improvement
occurred in some PSS patients with partial reconstitution of previously resorbed digital tufts noted in 9% and resorption of isolated
foci of calcinosis in 6%.
In the CREST group findings were similar but flexion contractures were less (50%) and progression occurred in only one patient. Computer analysis in 22 PSS patients failed to yield any significant correlation between multiple clinical and laboratory parameters
(including rheumatoid factor, ANA, and complement) and the observed radiographic findings.
Our findings suggest: I) A significant incidence of an associated inflammatory arthropathy in PSS, with progression of all radiographic abnormalities over time; CREST patients in contrast showed
little or no progression; 2) dorsal erosions may be a finding unique to
PSS; 3) isolated PSS patients may show reversibility of selected abnormalities such as calcinosis or tuft resorption.
Anti-T cell antibody and loss of suppressor T cell in juvenile rheumatoid arthritis
Yves Borel, Harvard Medical School, Boston; Anthony J. Strelkaukas, Sidney Farber Cancer Institute, Boston; Evangelia
Mantzouranis, Harvard Medical School; Richard T. Callery, and Stuart F. Schlossman, Sidney Farber Cancer Institute
Thirty-nine children between 3 and 19 years of age with juvenile rheumatoid arthritis (JRA) were studied. Seventeen children
with active disease by physical examination had anti-T cell antibody
in their serum as determined by indirect immunoflourescence by using a fluorescent cell sorter. In contrast, 13 JRA patients in remission
without positive physical findings, as well as 10 control sex- and agematched patients without autoimmune disease, did not have anti-T
cell antibody in their serum. Seven children with active disease had
no anti-T cell antibody, whereas 2 children in remission had anti-T
cell antibody. In all patients studied with active JRA (6), the presence
of anti-T cell antibody was always accompanied by a loss of suppressor T cell (JRA + T cell) and enhanced helper cell activity, as measured by B cell immunoglobulin production. In contrast, patients in
remission without anti-T cell antibody possessed these regulatory T
cells in their peripheral blood. The above data suggest that there is a
correlation between disease activity and the presence of anti-T cell
(JRA +). In contrast, anti-T cell antibody does not appear to correlate
with a particular mode of onset in JRA, i.e. pauciarticular, polyarticular, or Still's type. Anti-T cell antibody is not influenced by therapy with salicylates.
Five of the 10 patients with other connective tissue diseases
(2 with dermatomyositis, 2 with rheumatoid arthritis, and 1 with
scleroderma with Raynaud's) have antibody to T cell. It was also
found in 8/10 lupus patients, but this anti-T cell antibody is different
from the JRA patients' since it recognizes a different subset of T cell.
Although anti-T cell antibody does not appear to be JRA
specific, it is proposed that the detection of anti-T cell antibody can be
used as a new diagnostic tool for JRA because of the high incidence
of patients with JRA who spontaneously formed this antibody. Furthermore, the loss of regulatory T cells (i.e. JRA + T cell) may shed
some light on the pathogenesis of the disease.
Implantation of human rheumatoid cells in nude mice
Constance E. Brinckerhoff and Edward D. Harris, Jr.; Dartmouth Medical School, Hanover
The heterogeneous mix of inflammatory and mesenchymal
cells comprising rheumatoid synovium has qualities of a localized malignancy in its ability to invade and/or replace connective tissue in
joints. We used athymic nude mice as recipients of rheumatoid cells
to study the fate of these cells in a milieu that supports growth of abnormal or malignant cells but not of normal ones. We studied gross
morphology, histology, and synthesis of collagenase and prostaglandin E2(PGE2)by rheumatoid cells in this immunologically weak host.
Synovium was dissociated into single cells and 2 X lo7 cells
were inoculated subcutaneously into each mouse. In 8/9 mice, by 3
days a nodule (-1.5 cm) in diameter was seen at the injection site. It
slowly decreased in size and disappeared by 30 days. Histology of tissue removed at 7 days revealed a deposit of coagulated plasma and
massive infiltration of polymorphonuclear leukocytes (PMN) with apparent destruction of some implanted cells. Tissue at 21 days showed
no PMN and organization of original inoculum into a tissue remark-
ARA ABSTRACTS
596
ably like pannus and containing many fibroblasts, occasional multinucleated cells, numerous blood vessels, and diffuse bundles of collagen fibers. The karyotype of the tissue was human.
At 2 1 days, implants were excised and dissociated enzymatically. Cells were cultured and compared with cultures of synovial cells
not passed through mice for morphology and for collagenase and
PGE2 production. Both cultures showed dendritic, multinucleated,
and fibroblast cells. Dendritic cells decreased from 50-75% in cultures
not passed through mice to 10-20% in cultures of passed cells. Cells
passed through mice secreted cumulative collagenase sufficient to degrade more than 1,200 p g collagen fiber/hr/mg cell protein at 37°C
compared to 650 p g for cells cultured in 10% fetal calf serum during
the time of passage in mice. Cumulative PGEl levels in these same
culture media were 1,076 ng/mg cell protein, compared to 113 ng for
cells cultured in vitro.
In 4/4 mice inoculated with 2 X lo7 normal rabbit synovial
or human foreskin fibroblasts, no cells were recovered from the injection site at 21 days.
Rheumatoid cells implanted in nude mice remain at the injection site for at least 21 days. They become organized into a pannuslike structure and retain the ability to make collagenase and PGE2.
Implanting rheumatoid synovium in nude mice may provide a means
of studying factors influencing the proliferative lesion in rheumatoid
arthritis.
Induction of collagenase by phorbol myristate acetate in rabbit synovial fibroblasts
Constance E. Brinckerhofi Rodger M. McMillan, John K Fahey, and Edward D. Harris, Jr.; Dartmouth Medical School, Hanover
Since the first demonstration of mammalian collagenase 12
years ago, much has been learned about the mechanisms of enzyme
action and factors which influence its activity in various cells and tissues in vitro. To more fully understand collagenolysis in rheumatoid
arthritis or other conditions, it is essential to determine the sequence
of intracellular events leading to induction of collagenase synthesis. In
studying this problem, we have used monolayers of rabbit synovial fibroblasts as a model system. Cultures of untreated cells secrete very
little collagenase, but when treated with 0.01 pg/ml phorbol myristate
acetate (PMA), a component of croton oil, large amounts of latent enzyme are produced.
Initial studies examined the changes in intracellular cyclic
AMP levels, prostaglandin E2(PGE2)production, DNA synthesis (assayed by incorporation of 3H thymidine into cells), and collagenase
production after addition of PMA. The following response was found.
Ten minutes after addition of PMA, intracellular cyclic AMP levels
increased 100% to 10 p mol/mg cell protein and returned to control
levels by 20 minutes. Increase of PGE2 in culture medium, reaching
200-500 n u m g cell protein, was maximal within 24 hours, and incorporation of 3H thymidine into DNA decreased to 25% of control lev-
els by 9 hours but rose to exceed that of controls by 24 hours. Negligible amounts of collagenase were released into culture medium
during the first 24-hour exposure to PMA. By 4 2 4 8 hours there was a
sudden and substantial release of collagenase sufficient to degrade
more than 250 p g collagen fibril/hr/mg cell protein at 37°C. Enzyme
production continued for at least 96 hours and was associated with a
20% decrease in cell number and protein content/cell compared to
untreated cultures.
Pharmacologic modulations of these phenomena showed: 1)
concomitant addition of aspirin (50 pg/ml) or indomethacin (3.5 p g /
ml) abolished the PGE2 response but did not affect collagenase release, and 2) cr-amanitin (2 pg/ml), a specific inhibitor of messenger
RNA synthesis, inhibited collagenase production when added at the
same time as PMA.
Our studies imply that induction of collagenase is related to
synthesis of messenger RNA and is not a direct result of PGE2 production. The long lag phase before release of collagenase in PMA
treated cells provides an unusual opportunity to study induction and
synthesis of this enzyme.
Kawasaki disease or mucocutaneous lymph node syndrome: A Massachusetts outbreak
John J. Calabro, Worcester City Hospital, Worcester, Massachusetts; Muhammad Yunus, Peoria School of Medicine, Peoria, Illinois;
and Kenneth Miller, Worcester City Hospital
Kawasaki disease (KD), a disorder of unknown etiology affecting infants and young children, was first reported in 1967 by the
Japanese pediatrician, Kawasaki. By 1978, over 12,000 cases had been
studied by the Japanese Mucocutaneous Lymph Node Syndrome Research Committee. Yet, KD has rarely been reported from other parts
of the world. In the United States, the number of documented cases is
less than 200, representing cases from 24 states, including 45 from Hawaii alone. During 1978, we have observed 7 children with KD; no
cases have been recognized in the central Massachusetts area previously. There were 4 boys and 3 girls, ranging in age from I to 7
years (mean: 2.9 years). From our observation of these 7 cases and review of the literature, we would like to bring attention to the distinctive clinical features of this syndrome, not only because of its rising frequency in the U S . , but also because of its similarity to juvenile
rheumatoid arthritis (JRA), infantile polyarteritis, and the StevensJohnson syndrome.
KD usually begins with fever, rash, conjunctivitis, pharyngitis, cervical lymphadenopathy, indurative edema of the hands
and feet, as well as distinctive oral cavity and extremity lesions that
may last anywhere from I to 3 weeks. These constitute the major clinical signs and symptoms, being observed in over 90% of cases and in
all 7 of our patients. Other clinical features include arthralgia and arthritis, diarrhea, aseptic meningitis, and pancarditis. Sudden death occurs in I-2% of cases, primarily from coronary arteritis that usually
occurs during the convalescent period or in the second to the fourth
month of illness. The febrile pattern has not been characterized previously. In our 7 patients, the fever was quotidian or double quotidian, with one or two peaks daily ranging from 101 (38.5) to 105°F
(40.5OC). thereby mimicking the fever observed in systemic-onset
JRA. Arthritis was present in 6 of our 7 children, including swelling
of knees in 6 and of the hands and feet in 2. Synovial fluid analysis in
I patient was typically inflammatory in nature. Laboratory abnormal-
597
ARA ABSTRACTS
ities include a neutrophilic leukocytosis, anemia, elevated ESR,
thrombocytosis, sterile pyuria, proteinuria, and minimal hepatic function abnormalities. Serum IgE immunoglobulins or complement lev-
els are occasionally elevated but ASO, ANA, and latex titers are uniformly negative. Therapy includes supportive measures and aspirin,
often in doses as high as 150 mg/kg daily.
Demonstration of a monocyte leukotactic defect in ankylosing spondylitis
P. B. Campbell, M. A . Khan, and I. Kushner; Case Western Reserve University, Cleveland
We evaluated peripheral blood monocyte leukotaxis in 22
patients with ankylosing spondylitis (AS). Employing a double filter
in vitro technique, there was no difference between the monocyte leukotaxis responses of normal and AS monocytes to the negative control
solution (TC199). The mean (+ SD) monocyte leukotaxis response of
AS monocytes to zymosan-activated normal human serum, a standard
leukoattractant, was 32.9 & 14.4 monocytes per high-power field, significantly less than the mean response in 60 normal controls, 58.0 f
3.9 (P < 0.001). In 18 of the 22 AS patients, the monocyte leukotaxis
response was depressed at least 2 S D from the normal mean. Mean
monocyte leukotaxis to zymosan-activated serum in 17 B27positive
patients (30.5 f 13.9) did not differ significantly from mean monocyte
leukotaxis in 5 B27negative patients (41.3 & 14.6, P > 0.1). Monocyte
leukotaxis to zymosan-activated serum did not differ between the 12
treated and 10 untreated patients and did not correlate with clinical
activity of AS or serum C-reactive protein levels. Depressed monocyte
leukotaxis to zymosan-activated serum was also found in 2 (26.8, 7.9
monocytes per high-power field) of 5 B27positive individuals without
clinical evidence of AS.
To ascertain whether the leukotactic defect might be due to a
cell-directed inhibitor (CDI) in plasma, normal monocytes were preincubated in normal or AS plasma and the monocyte leukotaxis to
zymosan-activated serum was determined. Mean monocyte leukotaxis
after preincubation in AS plasma from the 18 patients with depressed
leukotaxis was 72.7 f 29.1% of the monocyte leukotaxis of normal
monocytes preincubated in 2% bovine serum albumin. This was significantly less than the mean monocyte leukotaxis after preincubation
in 12 samples of normal plasma ( I 11.2 10.2%, P < 0.01). CDI activity was found in 10 of the 18 AS plasma. Mean CDI activity in plasma
from 8 untreated patients was 43.8 32.6%, significantly greater than
that of plasma from 10 treated patients (14.1 18.5%, P < 0.05). CDI
activity was also detected in plasma from both of the B27positive nonAS controls with depressed monocyte leukotaxis. AS patients and
non-AS controls with normal monocyte leukotaxis did not show
plasma CDI. CDI activity did not correlate with clinical activity of
AS nor with serum C-reactive protein concentration. Physicochemical
characterization showed that CDI activity was heat-stable with MW
> 25,000 daltons.
These data indicate that a substantial proportion of patients
with AS, regardless of HLA phenotype, display defective monocyte
leukotaxis in vitro. This defect may, in some patients, be related to
circulating cell-directed leukotactic inhibitors. Similar changes may
be seen in some B27positive individuals without apparent ankylosing
spondylitis.
*
*
*
Subcutaneous bursae react differently than synovial joints to specific disease stimuli
Juan J. Canoso and Robert A . Yood, Boston University School of Medicine
Morphological similarities between the subcutaneous bursae
(such as the olecranon and prepatellar bursae) and the synovial membrane of the diarthrodial joints have led to the assumption that both
structures react similarly to disease stimuli. This assumption was evaluated in three disorders that may present as acutely inflamed bursitis:
trauma, sepsis, and gout.
Traumatic bursitis (45 cases). As in traumatic arthritis, the
bursal fluid WBC count was low (mean 950/mm3, range 504,950)
and the RBC count was high (median 27,000/mm3, range 100, Hct
25%). However the bursal fluid mucin clot was poor and the mean viscosity relative to water was 3.7 (range 1.3-7.8) compared with a good
mucin clot and a mean viscosity of 29 (range 8.4-83) in traumatic arthritis (Ropes and Bauer, Synoviul Fluid, 1953).
Septic bursitis (24 cases). Bursa1 fluid culture yielded S
uureus in 20, S epidermidis in 1, and streptococcus in 3 patients. Contrary to the expected findings in septic arthritis, blood cultures were
negative, and a cutaneous source of infection was suspected in almost
all patients. In untreated cases, the WBC count was less than 20,000 in
7 of 12 cases of S aureus bursitis but only 1 of 20 cases of S uureus
arthritis (P< 0.002).
Gouty bursitis (11 cases). Intracellular urate crystals were
present in all. The bursal fluid WBC count averaged 2,900 (range
650-6600), compared with 25,500 (range loCr152,400) in 77 cases of
articular gout (P < 0.05). In cases seen within 3 days of onset of attack, the mean WBC count was 3,900 in bursal gout and 39,200 in articular gout (P < 0.05).
These data indicate that subcutaneous bursae and synovial
joints react differently to trauma, bacterial infection, and gout. Light
and electronmicroscopic similarities between bursal and synovial
membranes may conceal important biologic differences between these
structures. The similarities in clinical presentation in these three
forms of bursitis and the blunted inflammatory response of the subcutaneous bursae make detailed bursal fluid analysis and culture essential for proper diagnosis.
Evidence of a primary B lymphocyte abnormality in New Zealand Black mice
Thomas M. Chused, Haralampos M. Moutsopoulos, Susan 0. Sharrow, and Virgil Woods; National Institutes of Health, Bethesda
Most of the current hypotheses on the mechanism of autoimmune disease in NZB mice invoke a loss or malfunction of suppressor
T cells leading to the failure to control forbidden clones of autoreactive B cells. Our current work, however, indicates that NZB mice have
ARA ABSTRACTS
a primary B cell abnormality that leads to spontaneous polyclonal activation of a fraction of B cells. This conclusion is based on the following findings:
1. NZB B cells have normal levels of surface IgM but greatly
decreased levels of surface IgD as shown by flow microfluorometry
(FMF) by using directly fluoresceinated affinity-purified or hybridoma reagents and a fluorescence activated cell sorter (FACS-11). B
cells from mice injected with sheep erythrocytes have normal surface
IgM and reduced surface IgD similar to NZB, suggesting that this is
the phenotype of activated B cells.
2. NZB B cell activation can be easily detected by several
methods. Spontanous production of IgM, which begins at birth and
increases to 40 to 100 times that of control mice by 8 weeks of age,
was measured during a 4-hour in vitro incubation by a sensitive immunoradiometric assay. Cytoplasmic IgM-containing plasmablasts
(detected by FMF after cell fixation) and spontaneous TNP-specific
plaque forming cells are greater in NZB than controls at 1 week of
age, and their frequencies increase in parallel to 10 times that of the
controls by 8 weeks of age. Thus each NZB plasmablast secretes several times more IgM than those in the spleens of normal mice. The
IgM secreting cells were shown by FMF to be large, IgM bright, and
IgD dull.
3. B cell activation is independent of T cells. Adult thymectomized, lethally irradiated NZB mice reconstituted with syngeneic fetal
liver showed the same high levels of IgM secretion and plasmablast
frequency as unmanipulated NZB mice, while similarly treated NZW
controls were normal. Furthermore, homozygous nude NZB mice had
the same level of B cell activation as non-nude NZB, while NIH Swiss
nude mice did not.
4. Peripheral NZB T cells contain cytoplasmic IgM, presumably endocytosed anti-T cell autoantibody, which may perturb their
function. This must be excluded in order to test for a primary T cell
abnormality.
Humoral immunity in native Type I1 collagen-induced arthritis in the rat
Roy B. Clague, Keith Morgan, Mary J. Shaw, and P. J. Lennox Holt; University of Manchester Medical School, Manchester, England
We have confirmed the findings of Trentham et al(l977) that
an arthritis can be induced in rats by the intradermal injection of native pepsin-soluble bovine Type I1 collagen emulsified in complete
(CFA) or incomplete (ICFA) Freund's adjuvant. Arthritis was induced in 76% of rats, but in 45% the arthritis persisted for longer than
6 weeks and was considered to be chronic. No arthritis was induced
by native bovine Type I collagen in CFA or ICFA, denatured Type I1
collagen in ICFA, native bovine Type 11 collagen without adjuvant,
or the adjuvants in buffer alone.
By use of a new solid phase double antibody radioimmunoassay for collagen antibodies in serum, the humoral immunity to collagen has been investigated. Arthritic rats had a higher IgM antibody
response to native Type I1 collagen than nonarthritic rats, which
reached a peak at 15 days, 22.2 f 1.0 (mean f SE) mg/liter versus 8.0
f 1.9 mg/liter; P < 0.002, and then subsided. The IgG antibody response to native Type I1 collagen was also higher in arthritic rats than
in nonarthritic rats, reaching a peak at 21 days (168.8 f 6.0 mg/liter
versus 69.0 & 3 1.5 mg/liter; P < 0.02) and persisting at high levels for
many weeks.
Native bovine Type I collagen in CFA produced high IgG
antibody levels to this antigen (peak levels 133.9 f 24.0 mg/liter), but
unlike native Type I1 collagen, when emulsified in ICFA only very
low levels of IgG antibody were produced to Type I collagen (2.8 f
0.2 mg/liter). High IgG antibody levels to denatured bovine Type I1
collagen (100.2 f 26.7 mg/liter) were produced when this antigen was
emulsified in ICFA.
Our findings suggest that a sequential determinant expressed
on both denatured and native Type I1 collagen possesses the property
of intrinsic adjuvanticity, which is not present in Type I collagen. Native Type I1 collagen, but not denatured Type I1 collagen, induced arthritis, suggesting that the conformational dependent antigenic determinant of the helical region of native Type I1 collagen is essential
for the induction of arthritis. However, the intrinsic adjuvanticity of
the molecule may also be important in enhancing the immune response to the conformational dependent determinant and thus aid the
induction of arthritis.
Humoral immunity to native Type I1 collagen in human arthritis
Roy B. Clague, Mary J. Shaw, and P. J. Lennox Holt; University of Manchester Medical School, Manchester, England
One hypothesis for the chronicity of rheumatoid arthritis
(RA) concerns the role of Type I1 collagen as an autoantigen. Using a
solid-phase radioimmunoassay, we have investigated the incidence of
class-specific antibodies to human Type I and Type I1 collagens in
serum. Thirteen of 34 (38%) patients with definite or classic RA native
Type I1 collagen. Twelve of these patients had elevated levels of both
IgG and IgM antibodies to this antigen, and there was a good correlation between IgG and IgM collagen antibody levels (r = 0.59, f =
4.08, P < 0.001).
Elevated IgG antibody levels to native Type I collagen,
denatured Type I collagen, and denatured Type I1 collagen were
found in 8 (23.5%), 8 (23.5%), and 14 (41%) of patients with RA, respectively. Though there was a correlation between IgG antibody levels to denatured Type I and Type I1 collagens (r = 0.914, f = 12.5, P <
0.001), there was a lack of correlation between 1gG antibody levels to
the corresponding native collagens (r = 0.30, f = 1.75, P > 0.05). This
appears to be due to a proportion of patients with very high antibody
levels to native Type I1 collagen. There were no correlations between
antibody levels to native Type I1 collagen and disease duration or disease activity, as measured by the erythrocyte sedimentation Fate,
rheumatoid factor determined by the Rose-Waaler or radioimmunoassay, and circulating immune complexes ('251-CIq binding activity).
Six of 20 (30%) patients with ankylosing spondylitis had elevated IgG antibody levels to native Type I and Type I collagens, but
IgM antibody levels were all normal. Thirteen of these 20 patients
had peripheral joint involvement, and the 6 patients with elevated
antibody levels were all in this group. All 14 patients with psoriatic
arthritis had normal IgG antibody levels to native Type I and Type I1
collagens.
The patients with RA who had the highest levels of collagen
antibodies had radiologic evidence of severe erosive disease, suggesting that the presence of antibodies to native Tpe I1 collagen is associated with destruction of articular cartilage.
ARA ABSTRACTS
599
Liposome encapsulation of antiinflammatory drugs: Pharmacokinetic and therapeutic studies
L. G. Cleland, M . Shandling, M . Poznansky, J. S.Percy, and T. M. Allen; UniversityofAIberta, Alberta, Canada
Studies were undertaken to explore the feasibility of administering antiinflammatory drugs in lipid vesicles (liposomes). Separate
preparations of vesicles composed of 1) egg phosphatidyl choline
(EggPC) and cholesterol (molar ratio 102) and 2) dipalmitoyl phosphatidyl choline (DPPC) alone were used to encapsulate gold sodium
thiomalate GST labeled with 1 9 8 AGST
~ produced by neutron activation. The preparations were stable for at least several days at room
temperature after removal of free drug. Tissue disposition of gold following intravenous (IV) or intramuscular (IM) injection into rats was
similar for high (5 mg/kg) and low (0.1 mg/kg) doses of gold. When
given IV in EggPC vesicles, gold was found in significantly higher
concentrations in the liver and spleen and in lower concentrations in
the kidney, compared to free gold given IV or IM. Studies of uptake
of gold by inflamed tissue (carrageenan-induced paw swelling)
showed a marked enhancement of uptake of gold given IV in EggPC
vesicles. The kinetics of uptake suggested migration of gold with the
cellular phase of the inflammatory response. Blood clearance studies
with ''C-cholesteryl oleate as a lipophilic liposome marker indicated
close association between gold and the lipid phase of EggPC vesicles
in the blood. Gold given IV in DPPC vesicles dissociated from the
lipid phase in blood and had a blood clearance and tissue distribution
similar to free gold given IV. Cortisol palmitate DPPC vesicles were
made by using cortisol palmitate 10 mg and DPPC 90 mg. Greater
than 99% of the cortisol palmitate was entrapped in the vesicles and
no leakage was detected over two weeks at 4°C. Following intravenous injection, blood levels of 'H cortisol given as palmitate in vesicles were higher than those of free 'H cortisol given by the same
route. Tissue distribution studies showed preferential localization of
'H cortisol given as palmitate in vesicles but not of free 'H cortisol in
inflamed paws. Inhibition of carrageenan-induced paw edema by cortisol palmitate vesicles was equal to that of a ten times larger dose of
free cortisol. An equal dose of free cortisol had no antiinflammatory
effect. A significant thymolytic effect was produced by vesicles at a
dose at which free cortisol had no effect. These studies suggest a role
for liposomes as carriers for drugs in the treatment of rheumatoid arthritis and other inflammatory diseases.
Bovine liver superoxide dismutase crosslinked albumin conjugates: Pharmacokinetics and antiinflammatory properties
L .G. Cleland, K. Wong, and M . J. Poznansky Departments of Medicine and Physiology, University of Alberta, Edmonton. Alberta,
Canada
Conjugates of homologous serum albumin and heterologous
enzymes have been shown to be nonimmunogenic (Remy MH, Poznansky MJ: Lancet 2:68, 1978). In the present study superoxide dismutase (SOD) isolated from bovine liver and rat serum albumin were
crosslinked with glutaraldehyde. The reaction was allowed to proceed
for 4 hours before inhibition of the crosslinking reaction with glycine.
The conjugates were then dialyzed and fractionated on Biogel 0.5M.
Fractions of molecular weight 150.000-300,000 were found to have
more than 50% retention of enzyme activity. The plasma half times
following intravenous injection of 12'1 labeled albumin containing
conjugates of MW 150,000-300,000 were between 6 and 8 hours, with
a trend toward shorter half times for larger conjugates. Plasma clearance estimated by enzyme activity produced similar results, indicating
stability of the complexes in vivo. High molecular weight I2'I labeled
complexes (MW > 500,000) were rapidly cleared from plasma. Complexes of MW 200,000 showed 12% localization by the liver at one
hour, 7.7% at 5 hours, and lesser uptakes in other organs. The anti-
inflammatory properties of the conjugates and free enzyme following
intravenous injection were assessed by inhibition of carrageenan-induced paw swelling. Pooled conjugate fractions of MW 150,000300,000 in a dose of 3,300 units SOD/kg produced 58. I% inhibition of
carrageenan-induced paw swelling (P< 0.0001). At this dose free enzyme produced a 14.5% inhibition (P< 0.05). Studies of uptake of '*'I
labeled albumin SOD conjugates by inflamed paws showed preferential uptake of the conjugates by inflamed paws compared to normal
paws. This effect was seen in animals injected with conjugates up to
24 hours before and up to 3 hours after the carrageenan injection.
These studies demonstrate that SOD albumin conjugates
possess potent antiinflammatory properties after IV injection. The
pharmacokinetic data indicate persistence of the conjugates within the
plasma for practically useful periods. Further investigation of these
conjugates is justified by their potential value as 1) antiinflammatory
agents for clinical use, and 2) an experimental tool in the study of oxygen free radicals in inflammation and its treatment.
Conduction disturbances and arrhythmias in progressive systemic sclerosis: Electrophysiologic and histopathologic
study
Philip Clements, Niger Roberts, Daniel Furst, William Cabeen, and Harold Paulus; University of California School of Medicine, Los
Angeles
Conduction disturbances and arrhythmias may be a major
cause of morbidity and mortality in progressive systemic sclerosis
(PSS); therefore, 4 1 participants in prospective studies of PSS underwent a resting (EKG) and a 24-hour continuous (Holter) electrocardiogram to determine the frequency of arrhythmias and conduction abnormalities. Twenty of these subjects also underwent tight
heart catheterization and intracardiac electrophysiologic study. The
atrioventricular (A-V) conducting systems, obtained enbloc from 5
other PSS subjects, were examined. On EKG, abnormalities of conduction (A-V block, hemiblock, bundle branch block), arrhythmias,
or other abnormalities (abnormal axis, hypertrophy, and/or necrosis)
were found in I5 subjects. Conduction disturbances and/or arrhythmias were noted on Holter in 26 subjects (23 with supraventricular
and 15 with ventricular arrhythmias). Most subjects with abnormal
ARA ABSTRACTS
EKG had abnormal Holters; however, 13 of 26 subjects with normal
EKG had abnormal Holters (see Table). Of 9 subjects with palpitations or syncope, 7 had an abnormal EKG, but all 9 had arrhythmias
on Holter.
Intracardiac electrophysiologic studies were abnormal in 14
subjects; function of sinus node, atria, or A-V node was abnormal in
7,9, and 9 subjects, respectively; however, in only 4 of this group were
conduction and/or impulse formation abnormal on resting EKG. Diminished mass and crosssectional diameter of the A-V node (particularly the proximal node) was noted macroscopically on longitudinal
serial sections of A-V nodes from 5 PSS subjects, even though the microscopic appearance was not strikingly abnormal.
Therefore, we conclude the following: I) arrhythmias and
conduction disturbances not predicted by EKG occurred frequently
in PSS; 2) by intracardiac electrophysiologic and macroscopic studies,
A-V nodal tissue and function are often abnormal in PSS;3) complaints of palpitations or syncope should be carefully evaluated with
Holter studies; 4) this study suggests that arrhythmias may be a more
frequent cause of morbidity and mortality than previously appreciated.
~
___..--_______
Abnormal
Holter
Normal
Holter
Abnormal EKG
Normal EKG
13
13
13
15
26
Total
_____
26
15
41
~____~____
2
Total
Activated B cell subpopulations in NZB mice
Philip L. Cohen, Frances S. Ligler, and Ellen S. Vitetta; University of Texas Health Science Center, Dallas
We have previously demonstrated that, beginning early in
life, NZB splenic B cells bear abnormally large amounts of surface
IgM (sIgM) in relation to surface IgD (sIgD). In order to determine
whether all or only a subpopulation of NZB B cells manifest this abnormality, we have fractionated NZB and BALB/c spleen cells on the
basis of density over discontinuous gradients of bovine serum albumin. Spleen cells from NZB and BALB/c mice were separated into
four discrete layers (A,B,C, and D, in order of increasing density).
Cells from each layer were subjected to surface iodination followed by
lysis, immunoprecipitation, and polyacrylamide gel electrophoresis in
order to quantitate sIgM and slgD. The relative numbers of cells
bearing slgM and slgD were also measured by immunofluorescence
staining followed by analysis on the fluorescence-activated cell sorter.
The number of immunoglobulin secreting cells in each layer was measured by using a reverse hemolytic plaque assay. Our results indicate
that the NZB spleen cells with the highest slgM/sIgD ratio constituted a unique population of low density cells, whereas immunoglobulin secreting cells were evenly distributed among the four layers.
Gradients prepared from BALB/c spleen cells showed a similar even
distribution of immunoglobulin secreting cells but failed to reveal a
corresponding population of low density cells with an increased
sIgM/slgD ratio. These data suggest that, although NZB spleen contains a characteristic subpopulation of low density B cells with a high
ratio of sIgM/slgD, these cells do not constitute the majority of spontaneous immunoglobulin secreting cells. These low density cells may
thus represent a population of activated precursor B cells that have
not yet differentiated into the phase of immunoglobulin secretion.
Our findings lend support to the notion that NZB spleen contains a
substantial population of activated B cells.
Cervical fusions in patients with rheumatoid arthritis
J. Pierce Conaty and Edward S. Mongan; Rancho Los Amigos Hospital, Downey, California
In a 14-year period (1965-78) 37 patients with severe rheumatoid arthritis (RA) (Class 111 and Class IV) had cervical fusions because of clinical and radiographic evidence of cervical instability with
spinal cord compression or vertebral artery symptoms. The average
followup was 3 years (range 0.5-12 years). Results were classified as
good (relief of symptoms, bony fusion, and possibly neurologic improvement), acceptable (subjective improvement with an unchanged
neurologic status), or poor (no relief of symptoms and/or nonuniod).
Four clinical entities requiring different operations and with
different prognoses were noted.
1. Reducible atlantoaxial subluxation on flexion-extension x-rays
occurred in 17 patients. The amount of anterior subluxation ranged
from 4 to 13 mm. Gallie-type fusions, wiring CI to C2, were done
with good results in 13 patients. In 3 patients with nonunion and in I
patient where reducibility lacked 5 mm, the results were poor.
2. Irreducible atlantoaxial subluxation sometimes accompanied by
cephalad odontoid penetration occurred in 9 patients. Fusion from
the occiput to C2 or C3 was done. Five patients had good results, 1
had acceptable and 2 had poor results (1 postoperative death).
3. Subaxial subluxations of 20-5W0 occurred in 7 patients. Three
patients had subluxations at 2 levels; one had 3 level involvement.
Posterior spinal fusions spanning 1-5 levels were done. Three patients
had good results, 2 acceptable ones, and 1 poor results ( I death).
4. Combined atlantoaxial and subaxial subluxation occurred in 4
patients. All had severe RA (Class IV). Two of these patients also had
vertical odontoid penetration of 6 and 10 mm. Fusions from the occiput to below the subaxial level of subluxation were done. Two patients had good results; l patient, who was a severely disabled quadriparetic, did not improve ( I postoperative death).
It is important to determine the exact level and extent of cord
compression because cervical subluxation in RA can occur at the ClC2 level, subaxially, or both.
ARA ABSTRACTS
60 1
Radionuclide imaging in painful prosthetic replacement
Martha Minteer Convery and F. Richard Convery; University of California, San Diego
Pain following joint replacement may be caused by component loosening, infection, or both, and is difficult to assess. The purpose of this study was to evaluate the accuracy of 99m technetium
methylene diphosphonate bone scanning, gallium 67-citrate, and arthrography to establish the diagnosis in thirty painful joint replacements. All had at least one aspiration, a needle biopsy, and operative
intervention. Multiple cultures were obtained during surgery.
Technetium scans were positive in 29 of the 30 joints, with no
difference in uptake between the loose implant and the infected implant. All of the patients were more than 8 months postoperative, and
in 3 neither looseness nor infection could be identified.
Gallium-67 scanning was included in the study since it is a
widely used inflammation and abcess scanning agent. One-third of
the patients had positive gallium scans. However, in only 3 of these 10
joints were the cultures positive, and the remaining 7 were associated
with loose components. The most intensely positive gallium scans correlated best with gross nonseptic looseness. Forty-seven percent of the
arthrograms were inaccurate with 7 false positives and 4 false negatives. The erythrocyte sedimentation rate and hematocrit did not correlate with infection.
The most reliable studies in this series of painful prostheses
were plain film radiography and bacteriologic culture. Seventy-five
percent of infections were diagnosed by sinus tract culture or joint aspiration, and when these cultures are combined with percutaneous
needle biopsy cultures, the accuracy was 97%.
Collagen-induced arthritis: Study of Type I1 collagen for adjuvant activitv
Michael A. Cremer, John M. Stuart, Alex S. Townes, and Andrew H. Kang; University of Tennessee, Memphis
Collagen-induced arthritis occurs in 40-50% of rats injected
with native Type I1 collagen emulsified with incomplete Freund’s adjuvant (IFA) and is characterized by an intense humoral and cellular
response to Type I1 collagen in arthritic animals in the absence of
mycobacterial adjuvants. Clinically and histologically, this disease resembles adjuvant arthritis. These observations led us to investigate
Type I1 collagen for adjuvant activity. Outbred Wistar rats (minimum
of 8 per group) were injected with 10 p g of trinitrophenylated ovalbumin (TNP-OA) or basic protein (BP) emulsified with IFA, IFA
and chick Type I1 collagen (CII), or complete Freund’s adjuvant
(CFA).
Spleen and lymph node TNP plaque forming cells (PFC)
and serum hemagglutinating antibody (HA) titer were measured to
assess humoral immunity. Cell-mediated immunity was determined
by [’HI TdR incorporation by peripheral blood mononuclear cells
cultured with ovalbumin (OA). All studies were performed in parallel
at days 7, 14, and 28.
No significant difference was found in IgM or IgG PFC production following primary TNP-OA immunization with IFA or IFA/
CII; HA titers were similar except at day 28 where IFA/CII titers
were lower (P< 0.05). PFC and HA responses were greater at days 7
and 28 in rats immunized with CFA (P < 0.05-0.025) compared to
IFA and IFA/CII. At days 6 and 14 after boost with TNP-OA, spleen
PFC and HA responses in rats primarily immunized with IFA/CII
were less than IFA although not significant. The boosted CFA group
responded better than the other groups on both dates: PFC (P< 0.050.005) and HA (P< 0.005).
Cell-mediated immunity response to OA was depressed at
each interval in the IFA/CII group, but significantly at day 28 versus
IFA (P< 0.025) and days 7 and 28 versus CFA (Pc 0.01-0.005). Allergic encephalomyelitis was observed in 8/8 BP-CFA injected rats
but in none of the BP-IFA or BP-IFA/CII groups. In addition, mice
injected intraperitoneally with PBS or bovine Type I1 collagen dissolved in PBS 24 hours before intraperitoneal sensitization with sheep
red cells (SRC) showed similar PFC responses to SRC (1910 f 357
versus 1257 f 113 SEM). In conclusion, 1) native Type 11 collagen
lacks adjuvant activity, 2) these results further exclude contamination
of our collagen preparation by bacterial or other adjuvant substances,
and 3) the arthritogenic property of Type 11 collagen in rats is not related to an adjuvant effect.
Muscle histopathology as a progsostic indicator in childhood dermatomyositis
William Crowe, University of Cincinnati College of Medicine; Kevin Bove, Children’s Hospital Research Foundation, Cincinnati;
Evelyn V. Hess, and Joseph E. Levinson, University of Cincinnati College of Medicine. Cincinnati
There are no reliable ways of determining the course and
prognosis of dermatomyositis in children. This Center has prospectively followed 28 cases of childhood dermatomyositis since 1958, and
50% of these have had a complicated course with 3 deaths.
In an attempt to identify those patients with a poor prognosis, the clinical records and available tissue specimens from 19 patients were reviewed. There were 27 muscle biopsy specimens examined by light microscopy.
The patients were classified in the following way: Group Imonocyclic illness of less than 2 years duration (average followup 4.5
years); Group 11-prolonged illness complicated by severe extramuscular involvement but with ultimate remission and independent
function (average followup 1 I .6 years); Group 111-mplicated
ill-
ness with continuing activity and progressive disability (average followup 11.1 years); Group I V d e a t h (average 16 months after onset).
There were no clinical or laboratory features which predicted
the disease course. However, light microscopy of muscle revealed a
nonvasculitic occlusive vascular lesion of muscular arteries that was
predictive of severe disease. The lesion was characterized by marked
endothelial swelling, occlusion by a homogeneous eosinophilic material, and minimal inflammation. This lesion was observed in 4 patients prior to steroid therapy and in 5 following the initiation of steroid therapy, and presence of the lesion correlated well with the
subsequent course.
Clinically significant calcinosis occurred in all 6 of the survi-
602
vors who had the occlusive vascular lesion, but in only 1 case in which
the lesion was not found.
The pathogenesis of this vascular lesion is poorly understood.
It does not seem to be the result of a vasculitis but of a thrombotic
phenomenon secondary to endothelial damage, the cause of which is
obscure. Its presence correlates with the development of subcutaneous
calcification and a poor immediate and long term prognosis.
ARA ABSTRACTS
Group I
Group 11
Group Ill
Group IV
No. with occlusive
vascular lesion
No. without occlusive
vascular lesion
0
3
3
3
7
1
2
0
Total hip arthroplasty in children with juvenile rheumatoid arthritis
William Crowe, Carol Hauselman, Edith Shear, Edward H. Miller, George P. Balz, and Joseph E. Levinson; University of Cincinnati
Medical Center, Cincinnati, Ohio
Experience with total hip replacement surgery is still very
limited in the younger age group of patients. Since 1974, 22 total hip
arthroplasties have been performed on 15 patients with juvenile rheumatoid arthritis (JRA). In all of these, the reason for surgery was severe destructive hip disease with disabling pain and/or limitation of
motion refractory to conventional therapy. The patients' reasons for
desiring the surgery were primarily related to hopes for improved mobility and social acceptance.
Decisions to proceed with surgery were made by a multidisciplinary team after considering the medical, surgical, emotional,
and motivational aspects of each case. There were I5 patients (12 female and 3 male). Five had systemic, 7 polyarticular, and 3 pauciarticular onset JRA. The patients had a mean age of 18.9 years (range
14-26 years) at the time of surgery and had had hip involvement for
an average of 10.2 years (range 1-20 years).
Postoperative followup averaged 30 months (range 5-60
months). Average of flexion deformities has decreased from 28" to 8",
and the average arc of flexion-extension has increased from 50" to
100". Pain was relieved entirely in 21 hips and was improved in one.
Ambulatory status has dramatically improved, as shown in the Table,
though many months of intensive therapy were necessary to achieve
maximum benefit.
Improvement of self image was marked in each patient. The
only significant immediate complication has been considerable abductor weakness occurring in all patients. There has been one late
complication-femoral
component loosening at 6 months postoperatively. Experience to date has been favorable. Functional and
psychologic results are excellent and complications tolerable. The
procedure should be offered to young patients who have the appropriate indications.
Ambulatory status
Wheelchair
Household
Limited community
Unlimited community
Preoperative
At followup
2
2
11
0
2
4
8
1
Severe cutaneous ulceration in childhood dermatomyositis
William Crowe, University of Cincinnati Medical Center; Kevin Bove, Children's Hospital Research Foundation, Joseph E. Levinson,
University of Cincinnati Medical Center
Cutaneous ulceration is not a widely recognized manifestation of childhood dermatomyositis. However, 25% (7 of 28) of the patients followed at this Center since 1962 have had severe cutaneous ulceration as a major manifestation of their disease. In these 7 (5 girls, 2
boys) the mean age of onset of the disease was 8.4 years (range 2.8 to
14 years). The ulcers occurred an average of 8.5 months after onset
and were deep, located most commonly in the axillae, over bony
prominences, on the trunk, and about the perineum. They were often
associated with livido reticularis. None occurred prior to steroid therapy. In 5 patients the ulcers began within 2 months of the start of steroid therapy, and in 2 they began shortly after a marked increased or
exacerbation of the disease. Associated manifestations included severe
gastrointestinal involvement in 6, hypertension while receiving steroids in 5 , and hepatic involvement in 7. Soft tissue calcification occurred in the 5 survivors. Available tissue specimens demonstrated an
occlusive vasculopathy of muscular arteries of the skin (2 cases) or
muscle (4 cases), with minimal inflammatory change and no frank
vasculitis. In two cases this vascular lesion was present prior to steroid
therapy.
The presence of the skin lesions was a poor prognostic sign.
Two patients died in the first year after diagnosis, and 2 developed severe progressive calcinosis and continued to have active disease 3.3
and 6.3 years after onset. Three have residual weakness and calcinosis
but are functionally independent and in clinical remission 6.3, 16.3,
and 20 years after onset.
Cutaneous ulceration was a severe complication of childhood
dermatomyositis in 25% of our cases. It had a temporal relationship to
steroid therapy, correlated with a poor prognosis, and may reflect a
noninflammatory occlusive vasculopathy.
The Occurrence of systemic lupus erythematosus in 14 patients with juvenile rheumatoid arthritis
William E. Crowe, University of Cincinnati College of Medicine; Carol G. Ragsdale, University of Washington School of Medicine,
Seattle; Ross E. Petty, University of Manitoba, Winnepeg, Canada; Donita B. Sullivan, University of Michigan Medical School, Ann
Arbor; Joseph E. Levinson, University of Cincinnati College of Medicine; James T. Cassidy, University of Michigan Medical School,
Ann Arbor
The reported incidence of concomitant, sequential, and
mixed disorders of connective tissue in childhood would suggest that
these phenomena occur much less frequently than in adults. We wish
to report here the experience from 2 large juvenile arthritis programs
where we have had the opportunity to prospectively follow 610 patients with definite juvenile rheumatoid arthritis (JRA) in whom 14
developed systemic lupus erythematosus (SLE) an average of 6 years
(range I to 22 years) after the onset of definite JRA. Eleven were girls,
603
ARA ABSTRACTS
3 were boys. The average age of onset of the JRA was 9.2 years.
Twelve had polyarticular, 2 had pauciarticular, and I had systemic
onset JRA. The joint disease has been erosive in 7 and deforming in
12. At onset, 5 had high spiking fevers and I had a JRA rash. Three
had Raynaud‘s phenomenon. Fluorescent ANA (FANA) was positive
at onset in 5 of 10 patients tested. Rheumatoid factor was positive in 3
of 14. None of the I I patients seen early in the disease had a positive
LE cell preparation.
The subsequent manifestations of SLE have included exacerbation of arthritis (10 of 14), rash consistent with systemic lupus
(6 of 14), persistent Raynaud’s phenomenon (3 of 14), alopecia (2 of
14), pleuropericarditis (7 of 14), leukopenia (6 of 14). seizures (3 of
14). positive FANA (14 of l4), elevated DNA binding (I4 of 14), positive LE cell preparation (13 of 14). positive direct Coombs test (8 of
14), and a positive lupus band test (2 of 3). Six of 10 developed elevated DNA binding 4 months to 4 years prior to the diagnosis of SLE.
Renal biopsy was performed in 8 patients. Mesangial hypercellularity
was found in 2, focal glomerulonephritis in 5 , and diffuse glomerulonephritis in I . Neither azotemia nor nephrotic syndrome have developed, and there have been no deaths to date.
Two percent of patients with JRA in a careful prospective
study have developed a number of the criteria used by the American
Rheumatism Association classification system for SLE. All 14 had
had JRA for a considerable period of time and 1 I were females. Most
were of polyarticular onset. These data suggest that SLE can evolve in
JRA patients and that the development of antibodies to DNA may
identify the population at risk.
A comparison of anti-DNA antibodies from the serum and kidney eluates of NZB
X
NZW F, mice
Howard Dang and Ronald Harbeck; National Jewish Hospital and Research Center, Denver
The qualitative aspects of anti-DNA antibodies from the
serum and those eluted from the kidney of NZB/W mice were evaluated in terms of their class and subclass specificity, avidity, and isoelectric profile.
Utilizing radioimmunoelectrophoresis, the class and subclass
of anti-DNA antibodies from the serum were compared to those
eluted from the kidneys. Of the animals of various ages that were examined, anti-DNA binding was detected in both the IgG and IgM
classes. Furthermore, all subclasses of IgG studied, i.e., IgG,, IgG2,
and IgG2b, were also found to bind DNA. On the other hand, antiDNA antibodies eluted from the kidney were found by immunoelectrophoresis to be only of the IgG, and IgG2, subclasses. Avidity
measurements were made by determining the dissociation of preformed anti-DNA/”’I-DNA complexes by using a 500-fold excess of
unlabeled DNA. These measurements indicated that the avidity of
isolated serum IgG for ‘251-DNAdecreases with age in NZB/W mice,
while the avidity of the isolated serum IgM remains relatively unchanged. No change in avidity of antibodies eluted from the kidney
could be demonstrated when mice of various ages were examined.
Equal amounts of both high and low avidity antibodies to DNA were
found. In addition, the high avidity antibodies eluted from the kidneys demonstrated a longer dissociation rate
than the high avidity antibodies found in the serum of these mice.
Isoelectric focusing was carried out on a discontinuous sucrose gradient by using layers of 50,40, 30, and 20%sucrose containing 5% ampholine solutions. The fractions obtained were assayed for
anti-DNA activity with the Farr technique. The isoelectric profile
confirmed the heterogeneity of the anti-DNA antibodies in the seturn.
Anti-DNA binding activity could be detected between pH 4 to 9. In
contrast, the anti-DNA antibodies eluted from the kidney demonstrated a more restricted pH range (6 to 8) as assessed by isoelectric
focusing.
These results confirm and extend the findings of others and
suggest that the qualitative aspects of the anti-DNA response in murine lupus may be as important as the quantitative aspects of these
antibodies in the development of glomerulonephritis. In addition,
from these results it appears that there exists a specific subpopulation(s) of anti-DNA antibodies that play an important role in
the development of nephritis.
Arrest or reversal of malignant hypertension and scleroderma renal vascular crisis by the angiotensin converting
enzyme inhibitor Captopril
William A . D’Angelo, Jorge A . Lopez-Ovejero, Stuart D. Saal, Jhoong S. Cheigh; State University of New York at Stony Brook; and
John H. Laragh, Cornell Medical Center
Two cases of renal scleroderma crisis were dramatically reversed by the oral administration of the angiotensin converting enzyme inhibitor Captopril (SQ14,225). Both patients had typical
sclerodermatous skin changes, Raynaud’s phenomenon, ischemic fingertip ulcerations, esophageal aperistalsis, severe hypertension, and
renal insufficiency. One patient had a proximal myopathy and
changes compatible with microangiopathic hemolytic anemia. Blood
pressures on admission were 240/140 and 180/118 mmHg. Despite
aggressive therapy with conventional parenteral antihypertensives,
there was progressive clinical deterioration. Blood pressures before
Captopril were 195/110 and 165/120 mmHg and decreased to 125/55
and 145/94 mmHg I hour after oral doses of 10 mg and 75 mg, respectively. Both patients were azotemic, and following Captopril therapy creatinine clearances increased from 40 to 65 ml/minute in one
patient and from 15 to 26 ml/minute in the other. There was rapid
clearing of the microangiopathic findings and marked improvement
in the fingertip ulcerations. Remission has been maintained in both
by using 150 mg 4 times daily. Plasma renin activity (PRA) before
Captopril was very high in both patients (96 and 34 ng/ml/hour) and
during blockade increased further to 86 and 95 ng/ml/hour, respectively. Urinary aldosterone excretion after therapy was suppressed
(4.I and 9.2 88/24 hours, respectively). Nineteen months earlier one
patient was found to be normotensive with normal levels of PRA, urinary kinins, and immunoreactive PGE. Urinary kallikrein levels were
decreased.
These findings confirm the importance of excessive renin and
angiotensin in the pathophysiology of hypertension and renal sclero
derma crisis. In addition to preventing angiotensin 11 formation, Captopril might also cause the local accumulation of the vasodilator bradykinin which could further benefit the renal and peripheral vascular
disease. These findings warrant the further use of converting enzyme
blockade as a new approach to the treatment of renal scleroderma
crisis.
ARA ABSTRACTS
604
Abnormalities of pulmonary vascular dynamics and inflammation in early progressive systemic sclerosis
James A. Davis, Sawtantra K. Chopra, Daniel E. Furst, Philip J. Clements; University of California Los Angeles; Argy'os N.
Theofilopoulos,Scripps Clinic and Research Foundation, La Jolla, California; and Harold E. Paulus, Universityof California Los
Angeles
To characterize the pulmonary lesion in patients with early
progressive systemic sclerosis (PSS) we investigated the hemodynamics of pulmonary blood flow by using s'MKrypton (Kr) perfusion lung imaging, the inflammatory component of the interstitial
pneumonitis by using 67galliumscanning, and measurement of circulating immune complexes (CIC) by using the Raji-cell assay. Eight
patients with PSS of less than 3 years duration were studied. Patients
had increased interstitial marking (7 of 8), decreased vital capacity (6
of 8), and /or decreased diffusing capacity (8 of 8). Kr gas dissolved in
5% dextrose has a half-life of 13 seconds and is a measure of regional
pulmonary blood flow without the risks of technetium macroaggregates. A 300 K posterior image was obtained after injecting Kr into
the patient while in an upright sitting position before and immediately after immersing a hand in ice water for I to 3 minutes (cold challenge).
Baseline perfusion images were normal in all patients and in
4 normal volunteers. After cold challenge, perfusion abnormalities
were noted in 4 of 8 patients but in none of the normal volunteers.
These findings suggested a shunting of blood away from the smaller
peripheral vessels. An increased uptake of gallium was observed in
the lungs of 6 of 8 patients. Five of 8 patients had elevated CIC, but
no consistent correlation between gallium scan and CIC results was
found. Increased uptake on gallium scanning and the presence of CIC
have been shown to correlate with active inflammation on lung biopsy
in idiopathic interstitial pneumonitis (IIP). (Line BR et al: Am Rev
Resp Dis 118: 355, 1978; Dreisin RB et al: N Engl J Med 298:353,
1978). The lack of correlation between gallium scanning and CIC, in
spite of their high incidence of positivity, contrasts with the findings
in IIP. These findings suggest that the pathogenetic processes in early
PSS lung disease and IIP differ, even though the end stage pathologic
findings are similar. The finding of a decreased pulmonary blood flow
in 50% of the patients after cold challenge is similar to the finding of
decreased cortical blood flow in the kidneys of patients with PSS, suggesting a vascular component to the early lesion in both organs.
Effects of a mononuclearcell factor, indomethacin, and prostaglandin E, on protein and collagen synthesis by cultured
adherent rheumatoid synovial cells
Jean-Michel Dapr, Harvard Medical School; Stephen M. Krane, Ruth S. Quinn, and Assa Weinberg,Massachusetts General
Hospital, Boston
Adherent synovial cells (ASC) obtained by proteolytic dispersion of synovium from patients with rheumatoid arthritis produce
high levels of latent collagenase and prostaglandin E2 (PGE2) in primary culture. When incubated in the presence of 8-aminopropionitrile and ascorbic acid, ASC in primary culture and after passage also incorporate precursor proline and lysine into soluble
medium proteins in general, as well as 4-hydroxyproline- and 5-hydroxylysine-containing proteins (collagens). Incorporation of labeled
amino acid into collagens and other proteins is stimulated by indomethacin which blocks PGE2 synthesis. A soluble supernatant factor
produced by cultured human peripheral blood mononuclear cells,
which stimulates collagenase and PGE2 production by ASC, also
stimulates proline incorporation into collagen to a greater extent than
other proteins, an effect which is seen most consistently if PGE, synthesis is blocked by indomethacin. PGE2 at concentrations as low as
10 n u m l overcomes the stimulation produced by the mononuclear
cell factor and indomethacin. Results of a typical experiment are as
follows: Incorporation into (3H] proline (cpm x
per l@ cells):
control. 41; mononuclear cell factor (MCF), 117; indomethacin, 85;
MCF + indomethacin, 175; MCF + indomethacin 100 ng PGE,,
per 1@ cells):
74. Incorporation into [3H]hydroxyproline(cpm X
control, 2.6; MCF, 15.3; indomethacin, 7.0; MCF + indomethacin,
36.8; MCF + indomethacin + 100 ng PGE2. 6.2. Collagen and other
protein synthesis by cultured dermal fibroblasts that produce only low
levels of PGEz are not altered by indomethacin, whereas collagen and
other protein syntheses are stimulated by the mononuclear cell factor.
Thus the mononuclear cell factor may be important in modulation of
collagen synthesis, part of which effect is mediated by prostaglandins
and may influence the extent of fibrosis in inflammatoryjoint disease.
+
Production of collagenase-stimulatingfactor by human monocytes: Modulation by Fc fragments
Jean-Michel Dayer, Harvard Medical School and Massachusetts General Hospital; Justin H. Passwell, Children's Hospital Medical
Center, Boston; and Stephen M.Krane, Massachusetts General Hospital, Boston
Adherent synovial cells (ASC) obtained by proteolytic dispersion of synovium from patients with rheumatoid arthritis produce
high levels of latent collagenase in primary culture. With passage of
ASC, collagenase levels decrease but can be stimulated to more than
100-fold by a soluble supernatant factor (MCF) released in vitro from
cultured normal human blood monocytes (>95% pure). Monocyte
cultures exposed to Fc fragments (50pg/ml) of Ig or concanavalin A
(Con A) (10 to 100 pg/ml) increased production of MCF to an extent
10-fold greater than the control cultures. Although Fc fragments and
Con A stimulate PGEz synthesis and secretion by human monocytes,
the effect on MCF production is not mediated by PGE, since addition
of indomethacin did not alter MCF production. MCF production by
monocytes treated with monomeric IgG, Fab fragments, or lectins
such as phytohemagglutinin or pokeweed mitogen did not differ from
controls. Latex particles (0.04%) or endotoxin (2 &ml) stimulated
MCF production but to a lesser extent (-2 fold) compared to Fc fragments or Con A. The apparent molecular weight of the MCF from the
monocytes is 14.000 based on gel filtration (Ultrogel AcA54). similar to that of MCF from unfractionated mononuclear cells, as previously described. These results suggest that membrane surface receptors on monocytes are involved in the regulation of production of a
factor that stimulates collagenase production by human synovial cells.
Such receptors could be important in mediation of reactions of immunologic factors and substances involved in connective tissue destruction.
-
ARA ABSTRACTS
605
Abnormal regulation of Epstein-Barr virus transformation of rheumatoid lymphoid cells
Joel M . Depper, Peter A. Bardwick, Harry G. Bluestein, Nathan J. Zvaiyer, and J. Edwin Seegmiller; University of California Medical
Center, Son Diego
The observation that rheumatoid lymphoid cells are transformed by Epstein-Barr virus (EBV) into permanent lymphoblast cell
lines more rapidly than normal cells, a process regulated by T lymphocytes, led us to examine the cellular regulation of rheumatoid B
cell proliferation in response to in vitro EBV infection.
Peripheral blood lymphoid cells (PBL) from 20 patients with
rheumatoid arthritis (RA) and 16 normal subjects were separated into
E-rosetting (T cells) and non-T cell populations by density centrifugation. PBL, T, and non-T populations were each cultured at 5 X l@
cells in 0.2 ml RPMI-I640 with 20% fetal calf serum and 0.02 ml of
EBV-containing supernatant from a B95-8 cell culture added as the
source of virus. Cultures were assessed for outgrowth at 2-day intervals by phase microscopy. The time to outgrowth of normal PBL was
18.2 f 0.9 (mean f SE) days compared to 10.6 f 0.4 days for RA
PBL. Removal of T cells from PBL shortened outgrowth to 11.0 f 0.6
days for normal controls and to 8.3 f 0.3 days in RA. Addition of autologous T cells to the normal non-T cell cultures prolonged the time
to outgrowth in proportion to the T/non-T ratio. EBV did not induce
outgrowth in the purified T cell populations obtained from normal
controls, but outgrowth occurred in 18/20 RA T cell populations in a
median time of 13.9 1.1 days. The resulting cell lines were surface
Ig+ and secreted Ig into the culture medium, representing outgrowth
of the 3 4 % B cell retained in the rosetted cell population. In the absence of added EBV, spontaneous outgrowth occurred in 7/20 RA
non-T cell populations, but in only 1/48 experiments with normal
cells. These differences could not be accounted for by previous exposure to EBV as determined by the presence or titer of serum antibody to viral capsid antigen (VCA). Mean log, VCA titers were 6.02
f 0.95, range 0 to 1/640 (normal) and 6.63 f 0.71, range 0 to 1/1280
(RA). There was no significant correlation between outgrowth time
and VCA titer in RA (r = 0.38) or normal (r = 0.46) PBL. These results demonstrate altered T cell mediated regulation of EBV transformation by RA lymphoid cells.
*
Interference of C-reactive protein detection by rheumatoid factor
Richard A. Deyo, Richard M.Pope, and Robert H. Persellin; University of Texas Health Science Center at San Antonio
The serum acute phase protein, C-reative protein (CRP), has
long been employed as a marker of inflammation. Important functions have recently been shown for CRP, such as activation of complement and inhibition of leukocytes and platelets. In addition, it has
been suggested that CRP will differentiate between active systemic
lupus erythematosus (SLE) and bacterial infection. Thus, the detection of CRP is of increasing importance in the study of patients with
rheumatic diseases. The insensitive capillary tube precipitation technique for the detection of CRP has been replaced by more sensitive
methods such as latex agglutination and radioimmunodiffusion. Both
employ specific antibody to quantitate CRP. Since rheumatoid factor
(RF) can interact with IgG antibody, we studied the effect of RF on
CRP detection.
By using a latex agglutination method, CRP was found in 24
of 27 acute rheumatic fever (ARF) sera, only I of which had RF. Of
43 sera from patients with rheumatoid arthritis (RA), 39 had positive
tests for CRP and all patients with high titer sensitized sheep cell agglutination tests were positive for CRP activity. Of 13 SLE sera tested,
1 I had CRP activity, 6 of which were also RF positive. Only 2 of the
SLE patients had active infections, and both had RF and CRP reac-
tivity in their sera. To define the role of RF, sera were treated with either 0.1M 2-mercaptoethanol or were pepsin digested to destroy agglutination by IgM RF. CRP activity in the RF positive sera was
dramatically diminished; all 14 samples tested were significantly reduced, and 8 of 14 became negative following 2-mercaptoethanol reduction, and results were similar after pepsin digestion. On the other
hand, the CRP titer of the majority of ARF sera was unchanged and
in no case did the titer diminish by more than 1 tube dilution following treatment. An isolated monoclonal R F devoid of CRP (0.92 mg/
ml) gave positive CRP reactions in both the latex and radioimmunodiffusion methods. Surprisingly, commercial CRP “positive
controls” were strongly positive for R F by both the RA latex and sensitized sheep cell agglutination methods, and were inactivated by both
2-mercaptoethanol and pepsin. We conclude that RF can interfere
with methods commonly employed to detect CRP which utilize antisera to the acute phase protein, thus limiting their usefulness in any
rheumatic disorder. This interference can be corrected by prior destruction of R F activity in the sera by using either 2-mercaptoethanol
or pepsin.
On the mechanism of the paradoxical effect of salicylate on urate excretion
H. S. Diamond, G. Sterba, K.Ja-yadeven,and A.D. Meisel; Downstate Medical Center, Brooklyn
Low blood levels of salicylate in humans result in marked
renal retention of urate, whereas high blood levels are uricosuric. This
so called paradoxical effect has been explained as representing inhibition of urate secretion at low salicylate blood concentration and inhibition of both urate secretion and reabsorption at high salicylate concentration. This interpretation fails to consider the metabolic
sequence in which ingested salicylate is conjugated and the complex
renal mechanism of salicylate excretion where metabolites account for
over 80% of normal excretion.
Clearance studies were performed on 5 volunteers after 5
days on a constant purine isocaloric diet at varying serum salicylate
concentrations achieved by oral administration of acetyl salicylate.
Excretion of salicylate (S), salicylurate (SU), salicylphenyl glucuronide (SPG), and salicylacyl glucuronide (SAG) was measured by a
fluorometric assay. Urate excretion paralleled excretion of salicylate
and its metabolites, but not serum salicylate levels. At constant serum
salicylate levels, urate clearance increased from 6.8 f 1.4 ml/minute
to 17.7 f 4.5 ml/minute following intravenous administration of so-
ARA ABSTRACTS
dium bicarbonate ( I 1.25 gm over 12-15 minutes followed by 3.75 gm
over I hour). Salicylate excretion increased from 2385 f 2490 pM/
liter to 4929 f 2503 pM/liter (P< 0.05), whereas SU, SAG, and SPG
were unchanged after sodium bicarbonate. Pyrazinamide (PZA)
blocks urate secretion and competes with other inhibitors of urate secretion for excretion. When PZA (3 gm orally) was administered, salicylurate excretion decreased from 5248 & 1459 pM/liter to 3385
*I 190 pM/liter and salicylate decreased from 2707 f 1075 pM/liter
to 1079 f 714 pM/liter, whereas SPG and SAG were changed. Para
amino hippurate (PAH) did not inhibit urate excretion alone, but reduced urate excretion from 8.7k2.1 ml/minute to 4.2 f 1.4 ml/min-
ute when administered during salicylate-induced uricosuria. Salicylate excretion decreased 16.7%, and SAG, SPG, and SU were
unchanged. As salicylate dose was increased from 1.2 gm/day to 4.8
gm/day, urate clearance increased from 2.8 f 0.9 ml/minute to 7.3 f
3.0 ml/minute, and salicylate excretion increased by 3 2 W and SU increased by 163%.
The paradoxical effect of aspirin is accounted for by the combined effects on secretion and reabsorption of salicylate and its metabolites. Salicylurate appears to be the dominant inhibitor of urate
secretion, and salicylate itself appears to be the major inhibitor of
urate reabsorption.
Subpopulations of DNA antibodies in murine lupus
Fanny Ebling, Sandra Freeman, and Bevra Hahn; Washington University Medical School, St. Louis
Female MRL/I and NZB/NZW F, mice and male BXSB
mice develop systemic lupus erythematosus (SLE) with circulating
IgG antibodies to DNA and glomerular deposits of DNA-anti-DNA
immune complexes. Subpopulations of circulating IgG antibodies to
DNA from mice of each strain (bled serially at 2-week intervals) were
studied by isoelectric focusing of urea-treated sera on 5% polyacrylamide gels, which exclude large molecules such as IgM and C3.
Bands of IgG were precipitated with Na2S04. The gels were incubated with calf thymus '"1 DNA, separated into double-stranded
DNA (dsDNA) or dsDNA with single-stranded nicks (dsDNAn) on a
benzyolated naphthyolated DEAE cellulose column, and exposed to
x-ray film.
A single band of anti-DNA activity was found in most normal mouse sera (C57B1/6J and DBA) at pH 7.6-7.7. This "common
band" was present in all lupus sera and was the only band detected in
pre-morbid mice. At the time proteinuria began, many anti-DNA
bands appeared in the lupus sera; the banding patterns were similar in
all 3 strains. Most characteristic (present in 85-100% of mice of each
lupus strain) was a series of 5-10 distinct bands ranging from pH 8.0
to 9.0. Bands between pH 7.2 and 7.4 occurred in 70-80%; bands between 6.2 and 7.0 were seen in a minority. IgG antibodies to DNA
were also detected in glomerular eluates from NZB/NZW F, females
and focused at pH 8.5-8.9. Bands formed with dsDNA and dsDNAn
were very similar. Precipitation of bands with anti-mouse IgG prevented reactivity with DNA.
These data suggest that this technique can be used to identify
subpopulations of circulating or tissue-fixed IgG antibodies to DNA,
that subpopulations of anti-DNA are similar in different mouse
strains, and that anti-DNA with isoelectric points from pH 8.0 to 9.0
may be most pathogenic. This technique has been used successfully in
preliminary studies of sera from patients with SLE.
IgC rheumatoid factors in MRL/I mice
Robert A . Eisenberg, University of North Carolina, Chapel Hill; Lee J. Thor, and Frank J. Dixon, Scripps Clinic and Research
Foundation, La Jolla, California
MRL/ I mice develop fulminant autoimmune disease characterized by massive lymphoproliferation, antinuclear antibodies, high
levels of cryoglobulinemia, and immune complex-type glomerulonephritis, with death occurring around 4 to 5 months of age. Although
in many ways such mice are similar to other autoimmune murine
strains, they show several unique serologic features which make them
of particular interest, including the presence of anti-Sm antibody and
high levels of IgM rheumatoid factor. A further important aspect of
the disease of the MRL/I is the presence of IgG rheumatoid factors.
Such autoantibodies may be demonstrated by a radioimmune assay
using crosslinked mouse IgG Fc fragments bound to plastic surfaces,
pepsin-digested sera, and iodine labeled turkey anti-mouse kappa
chains as a developer. Of greater importance, however, is that the
presence of such rheumatoid factors can be demonstrated by a technique of serum-serum interactions in which immunoprecipitation
lines are formed when two MRL/I sera are placed adjacent to each
other in agarose gel, or in which increased levels of complement are
consumed when two MRL/I sera are mixed together in solution.
Such interactions can be found in the sera of about two-thirds of mice
4 months of age or older. Reacting sera may be divided into two subsets, A and B, such that the great majority of serum-serum interactions is between, and not within, sets. The interacting principle in
sera of both Set A and Set B is an intermediate-sized complex (14 to
16s) containing only mouse IgG. The interactions between two sera
are governed by a rheumatoid factor specificity, as indicated by inhibition with high concentrations of undigested, unaggregated mouse
IgG. Furthermore, the levels of protein involved in such interactions
may reach 1I mg/ml in some cases. Similar reactivities may be seen
in the serum cryoprecipitates. The presence of such rheumatoid factors is especially interesting in MRL/I mice since these animals also
have a systemic vasculitis as part of their disease and demonstrate
chronically swollen hind feet in about 25% of the animals. Histology
of this latter lesion shows acute and chronic inflammation involving
both soft tissues and the joint space, reminiscent of that found in
rheumatoid arthritis. These studies suggest that MRL/I mice may be
a model for providing insight into human rheumatoid arthritis as well
as for the systemic lupus erythematosus.
Type I1 interferon accelerates glomerulonephritis and death in NZB/NZW mice
Edgar G. Engleman, Stanford University School of Medicine; Gerald Sonnenfeld, University of Louisville, Louisville, Kentucky;
George Banting, Thomas Merigan, and Hugh 0. McDevitt, Stanford University Medical Center
NZB/NZW hybrid mice develop a spontaneous autoimmune
disease characterized by the appearance of antinuclear antibodies and
premature death due to immune complex glomerulonephritis. A role
for virus in the pathogenesis of this disorder is suggested by the con-
ARA ABSTRACTS
stant presence, at all ages and in every tissue, of a murine xenotropic
retrovirus. Recently, a new type of interferon has been described, that
is Type 11 interferon (IF2), that has potent antiviral and immunosuppressive properties which are distinct from those of classic Type I
interferon.
In order to assess the effects of IF2 on NZB/NZW disease,
IF2 was obtained from the serum of normal C57BL/6 mice sensitized
with Mybacterium bovis strain Bacille-Calmette-Guerin (BCG) and
challenged with tuberculin; a mock preparation was obtained by innoculation of tuberculin into unsensitized mice. Seven 6-week-old female NZB/NZW mice received daily intraperitoneal injections of
2,000 units for one month followed by 3 injections per week of 350
607
units for 6 months, for a total of 87,000 units per mouse. Seven additional female mice received the mock preparation, and 7 controls were
followed on no treatment. Six of 7 IF2-treated mice died by 9 months
of age, compared to only 2 mock-treated mice and 2 untreated mice
(Pc 0.01). Preliminary data indicate that the premature death of IF2treated mice was due to severe glomerulonephritis. However, there
were no significant differences in mean body weight or hematocrit between IF2-treated, mock-treated, and untreated mice. These data suggest that IF2 accelerates autoimmune disease and death in NZB/
NZW mice. On this basis, IF2 or inducers of IF2 such as BCG may be
contraindicated in patients with disorders associated with immune
complex deposition.
Family studies of the spondylitis of inflammatory bowel disease
Roger W. Enlow, The Good Samaritan Hospital, Baltimore; Wilma B. Bias, and Frank C. Amett, The Johns Hopkins University
School of Medicine, Baltimore
The association of inflammatory bowel disease (IBD) with
ankylosing spondylitis (AS) and sacroiliitis (SI) is well recognized.
HLA-B27, however, is found far less frequently in these patients than
in those with idiopathic AS. Whether a genetic predisposition to inflammatory bowel disease, HLA linked or not, might also confer susceptibility to spondylitis, or whether IBD in situ might, in and of itself, be responsible, is unknown.
Six families containing members having biopsy or radiographically proven IBD, AS/SI, or both were further evaluated with
clinical examination, radiographs of sacroiliac joints in symptomatic
members, and HLA-A,B,C and, in some cases, DR typing (total 25
members).
HLA-B27 occurred in only I family, being present in both
the proband with IBD/SI and his brother with Reiter’s syndrome.
The remaining 5 families contained 5 patients with both AS/SI and
IBD, 4 with AS/SI alone, and 2 with IBD without arthritis. No particular HLA antigens occurred in excess nor did disease segregate by
HLA haplotype. Of special note were the 4 B27-negative spondylitics
who, unlike their relatives, had no clinically apparent IBD. The most
informative pedigree follows is shown in the Figure.
These families strongly suggest a genetic predisposition to inflammatory bowel disease that is not HLA-linked, which also confers
susceptibility to B27-negative inflammatory axial arthritis in the absence of clinically overt IBD.
0 IBD
A2 BS CYL
N:BWi9.cV6
9
.
OAS1SI
t-0
I n2.813
N.B5
B d 6 . B5W 3 8
All BS
DgWS
A3.BW39
X.B8
Variation in characteristics of academic and nonacademic United States rheumatologic practices: 1976-1977
Wallace V. Epstein and Curtis J. Henke; University of California, San Francisco
An accurate statement of the work output of U.S. rheumatologists is one essential component in subspecialty manpower planning.
Nine hundred forty completed questionnaires from 1700 American
Rheumatology Association members in 1977 provide the data for this
report. Of the 837 respondents in active practice, 31% designated
themselves full-time academic, 63% private practice, and 6% nonacademic salaried. Academics spend 15 hours per week attending 23
outpatient and 7 inpatient rheumatic disease (RD) patients, while
nonacademic rheumatologists spend 34 hours in patient care, averaging 55 ambulatory patients and 8 hospitalized (P < 0.0001 for outpatients and hours). Community rheumatologists with academic affiliation saw more R D patients per week (P < 0.02) and spent more
hours (P< 0.03) and a higher share of patient care time seeing RD
patients (Pc 0.000l) than rheumatologists not claiming such affiliation. Participation in group practice did not change the number of R D
patients seen weekly. Highly significant differences exist in the specific RD problems seen in different practice settings and parts of the
country. Academics reported a higher share of their patients with juvenile rheumatoid arthritis (JRA) (PiO.OOOl), systemic lupus erythematosus (SLE) (P < O.OOOl), ankylosing spondylitis (AS) (P =
0.007), and other less prevalent entities, while community rheumatologists reported more osteoarthritis (P < 0.OOOl) bursitis, tendonitis,
and peritendonitis (P < 0.0001); however no differences existed for
RA and gout. Bursitis, tendonitis, and peritendonitis patients are 12%
of the rheumatologists’ patients in the Pacific Census Division and
11% in New England, but only 4% in the East South Central Division
(P = 0.013). Using multivariate analysis, the number of hours per
week spent seeing RD patients by community rheumatologists is
higher for those having academic affiliation (P = 0.003), increases
with the duration of specialty training (P < 0.0005), decreases as the
number of patient care doctors per capita in the state increases (P =
0.003), and is unrelated to the number of rheumatologists per capita
in the state or the solo or group status of the practitioner. Clearly the
quantity of rheumatologic care provided by a particular rheumatologist correlates with defined, measurable practice and community characteristics. Since the numbers and kinds of rheumatic disease problems per capita can be estimated by current prevalence figures, the
present data together with access and referral pattern information
should allow analysis of the present congruence of rheumatologist activities and public requirements.
ARA ABSTRACTS
608
Incidence of malignant disease in patients receiving cytotoxic therapy for rheumatoid arthritis
Stephen J. Farber, Robert P. Sheon, Allan B. Kirsner, and Robert I. Finkel; Medical College of Ohio, Toledo
One hundred twenty-six patients with definite or classic
rheumatoid arthritis (RA) hospitalized between 1965 and 1974 for
cytotoxic therapy were studied for the presence and type of malignant
disease. Each of the 126 cytotoxic patients was age- and sex-matched
to an RA patient hospitalized during the same years but who did not
receive cytotoxic therapy. As expected, the cytotoxic group had more
active, aggressive disease manifested by the greater incidence of nodules and erosions and the higher ESR and rheumatoid factor titer.
Among these patients, 105 received nitrogen mustard and/or cyclophosphamide; 17 received one or both of these in combination with
chlorambucil, thiotepa, methotrexate, or azathioprine; and 4 received
either chlorambucil, thiotepa, or azathioprine alone. Results are
shown in the Table.
From this study we conclude the incidence of malignancy is
not increased by cytotoxic therapy for rheumatoid arthritis.
Malignant disease prior to study
Group
Diagnosis
Malignant disease after entry into study
No. of
patients
Diagnosis
Basal cell carcinoma
Leukemia
Breast carcinoma
Cervical carcinoma
Uterine carcinoma
Basal cell carcinoma
Breast carcinoma
Lung carcinoma
Carcinomatosis
Lymphoma
Cytotoxic therapy? Basal cell carcinoma
Breast carcinoma
Colon carcinoma
Rectal carcinoma
Basal cell carcinoma
Lung carcinoma
Carcinoma tosis
Stomach carcinoma
Control RA*
No. of
patients
* 126 patients; mean age 56.7 years; mean followup 4.7 years.
t
126 patients; mean age 56 years; mean followup 4.9 years.
Monosodium urate monohydrate crystals as spherulites
Justus J. Fiechtner and Peter A. Simkin; University of Washington, Seattle
We have isolated monosodium urate monohydrate, both in
vitro and in vivo, in a crystalline form different from the classic individual “needle-shaped rods. Spherulites (radially arranged crystals
around a common axis that resemble a sphere in three dimensions
and a circle in two dimensions) were precipitated by cooling a 500
mg/dl solution of uric acid in deionized water adjusted to pH 8.9 with
IN NaOH and heated to 60°C. Examination by light microscopy and
scanning electron microscopy revealed 10-50 I.( spherical forms composed of many needle-shaped crystals. These spherulites were
strongly birefringent by polarizing microscopy, and addition of a
quartz red plate produced images reminiscent of yellow and blue
“beachballs” against a red background. The crystals radiating from
the center were consistently yellow when parallel and blue when perpendicular to the gamma axis of the red plate, with an extinction phenomenon present at their interface. The spherulites are thus, by definition, negatively birefringent. Further evaluation by weight of
hydration, sodium content, and x-ray crystallography confirmed the
identity of the spherulites as monosodium urate monohydrate.
We have observed identical spherulites in synovial fluid of
patients with acute gouty arthritis. These synovial fluids usually contained the classic individual needles of extra- and intracellular sodium
urate, but on two occasions a diagnosis of gout was made by visualizing only urate spherulites in the presence of a typical clinical syndrome. Study of previously collected and frozen synovial fluid samples from patients with gout revealed negatively birefringent
spherulites in two of six specimens. These crystals (and those in fresh
synovial fluid) were readily digested by uricase.
It is not surprising that such spherulites would be found in
synovial fluid. First, the spherulite structure is a common one in nature, particularly where long-axis crystals are rapidly precipitated in a
limited space. Second, published photographs of gouty tophi in cartilage, synovium, and kidney bear striking resemblance to these spherulites.
We believe that the spherulites of monosodium urate monohydrate may be important in the pathogenesis of gout. Identification
of these structures in synovial fluid bears the same diagnostic and
therapeutic implications as recognition of the classic needles.
Supported in part by N I H Grant A M 07108.
Skin fibroblast activity in bleomycin induced scleroderma
William R. Finch, Northeastern Ohio College of Medicine, Youngstown; Gerald P. Rodnan, Robert B. Buckingham, University of
Pittsburgh; Alan Winkelstein, end Robert K. Prince, Mon tefiore Hospital; Pittsburgh
Two patients treated with bleomycin developed cutaneous
thickening closely resembling the changes seen in progressive sys-
temic sclerosis (PSS). JR, a 27-year-old man, received a total of 780
units of bleomycin over a 6-month period for testicular carcinoma.
ARA ABSTRACTS
During this time, he developed dermal thickening and induration
over the fingers, hands, forearms, thighs, and legs along with finger
flexion contractures. A 7 mm punch biopsy from involved forearm
skin weighed 65.4 mg (normal 40.1 & 0.9 SEM). Raynaud‘s phenomenon was absent and there were no other clinical or laboratory features
of PSS. These changes have persisted for 18 months after discontinuation of the drug. KS, age 42, received bleomycin as therapy for
lymphoma and noted similar changes over the hands and forearms,
again without any stigmata of PSS. The thickening almost completely
cleared several months after bleomycin was discontinued.
Dermal fibroblast cultures were established from biopsies of
the involved forearm skin of patient JR and from the normal (though
previously involved) forearm skin of patient KS, and collagen accumulation was measured in monolayer cultures. The JR cultures accumulated impressively more collagenase-sensitive protein (CSP) than
normal site-matched control specimens but somewhat less than cul-
609
tures from simultaneously studied PSS patients. In the presence of
supplemental ascorbic acid (50 pg/ml), J R s cells showed a 100% increase in CSP accumulation, a response which is characteristic of PSS
but not of normal skin fibroblast cultures. In addition, J R s fibroblasts
accumulated significantly greater amounts of glycosaminoglycan than
either normal or PSS cultures. In contrast, cultures from patient KS,
whose skin had returned to normal on physical examination, behaved
in a normal fashion.
When bleomycin was added directly to normal cultured skin
fibroblasts, CSP accumulation was significantly increased. A dose of
0.01 pg/ml bleomycin resulted in a 36% increase in CSP accumulation
compared with untreated normal cultures.
We conclude that bleomycin is capable of directly producing
a reversible scleroderma-like cutaneous change in certain patients.
This clinical manifestation is paralleled by changes in skin fibroblast
tissue culture performance that mimic those reported for PSS.
Type I11 collagen in scleroderma skin
Raul Fleischmajer, Hahnemann Medical College, Philadelphia; Stefan Gay, University of Alabama, Birmingham; Jerome S. Perlish,
Hahnemann Medical College; and Jean Pierre Cesarini, Rothschild Foundation, Paris, France
The purpose of this study was to identify and determine the
distribution of Type I11 collagen in scleroderma skin. Skin biopsies
from 3 systemic and 2 localized sclerodermas and 2 normal controls
were divided into papillary dermis, reticular dermis, and fat. All specimens were processed for indirect immunofluorescence electron microscopy (EM) with specific antibodies against Type I11 collagen.
Thin specimens (about 0.5 to 1 mm) were fixed in 1) 1% gluteraldehyde for 20 minutes, 2) placed in Tris buffer pH 7.5, 3) treated with
rabbit anti-human Type 111 antibodies, followed by goat anti-rabbit
IgG antibodies tagged with ferritin. Specimens were then fixed in 3%
gluteraldehyde and processed for regular EM. Collagen types at different skin levels were also estimated by using pepsin digestion followed by SDS-acrylamide gel electrophoresis alone and following re-
duction with mercaptoethanol.
The following observations were made: I ) 100 A fibrils (without periodicity) and about 400 A fibrils (with 640 A periodicity)
proved to be Type I11 collagen. Fibrils of more than 600 A in diameter
did not label and most likely represent Type I collagen; 2) large
amounts of Type I11 collagen was found around capillaries, smooth
muscle cells, and fat, where reticulin is usually present; 3) hybrid collagen bundles consisting of labeled fibrils (Type 111) and unlabeled fibrils (presumably Type I) were also noted. The Type III/Type I collagen ratios were as follows: papillary dermis 0.24; reticular dermis 0.13;
fat 0.8. Fine collagen fibrils (100-400 A) in scleroderma have been
identified as Type 111 collagen. The collagen of reticulin also proved
to be Type 111 collagen.
Serologic study of “dry eye” patients in an ophthalmology clinic
Joseph 2. Forstot, S. Lance Forstot, C.M. Arroyave, Catherine E. Harmon, and Eng M. Tan; University of Colorado Medical Center,
Denver
Recent investigations in Sjogren’s syndrome that make use of
serum autoantibodies have helped in the diagnosis of less typical
cases. Over a 14-month period, we studied 44 consecutive patients referred to a university ophthalmology clinic who complained of “dry
eyes.” Thirty-eight of these patients had keratoconjunctivitis sicw
(KCS) (defined by Schirmer’s test 5 5 mm wetting and positive rose
bengal staining). All patients were screened for the presence of antinuclear antibodies (ANA) and rheumatoid factor (RF). The ANA
screening consisted of a routine ANA plus antibody determinations
for Sm, ribonuclear protein (RNP), rheumatoid arthritis precipitin
(RAP), and Sjogren’s antibodies (SS-A and SS-B).
The ANA positivity (titer ?1:64) of the 44 patients was 56%.
In the 38 KCS patients (61% positive), the ANA positivity was 22/31
women and 1/6 men. Nine of 44 (20%) patients were known to have
a n associated connective tissue disease at the time of examination in
the ophthalmology clinic (6 rheumatoid arthritis, 2 systemic lupus, 1
scleroderma). The remainder 34/44 (77%) did not have a diagnosis of
associated connective tissue disease. Analysis of the ANA specificities
revealed no patients with Sm or RNP antibodies; RAP positivity was
the same (32%) in both the KCS patients and the total study group.
Seven patients, all ANA positive, had SS-B and/or SS-A antibodies.
These 7 patients were characterized by severe xerostomia symptoms
and represented a young patient population (mean age 38, median
age 31). The overall RF positivity was 53%. while it was 60% in the
KCS group.
In summary, serologic abnormalities were present in high incidence in patients complaining of dry eyes who were seen in an ophthalmology clinic. The presence of SS-A and SS-B antibody in 7 patients might suggest an early subclinical form of Sjogren’s-sicca
complex in a young patient population. A second large group of patients with rheumatoid factor may represent early subclinical forms of
rheumatoid arthritis. Studies of this nature might elucidate the early
phases of the natural history of certain connective tissue diseases.
ARA ABSTRACTS
610
Presence of an endogenous sulfhydryl-inhibitorin normal serum: Subnormal levels in rheumatoid arthritis
Donald A . Gerber, State University of New York Downstate Medical Center, Brooklp
The purpose of this study was to look for a metal-containing
endogenous inhibitor of the sulfhydryl (SH) group in normal and
rheumatoid arthritis (RA) serum. The increment in serum SH levels
produced by inactivation of the SH-inhibitor by a chelating agent was
used to estimate inhibitor levels. SH was measured with 5,S-dithiobis
(2-nitrobenzoic acid) (DTNB) and by viscosity changes and aggregation after heat denaturation. The results, as shown in the Table, suggest the presence of an endogenous SH-inhibitor in normal serum and
subnormal levels of this inhibitor in RA serum.
Normalization of the histidine concentration of an RA serum
in vitro substantially normalized its SH-inhibitor level. In vitro studSubjects
SH-assay
Ra
ies indicate that the subnormal serum inhibitor levels in RA are not
due to aspirin, salicylate, low serum SH, hypoalbuminemia, or hyperglobulinemia.
The SH-inhibitor concentration in normal serum appears to
be similar to the pharmacologic serum concentration of gold thiomalate (also an SH-inhibitor). It is hypothesized that the endogenous
SH-inhibitor (perhaps a histidine-dystine-copper complex) may be a
naturally occurring antirheumatic substance with a gold-like mechanism of action. The reported increased disulfide state of the SH
groups of albumin, a2,p, and y-globulin in RA serum could also be
due to subnormal levels of the SH-inhibitor.
Chelator
No.
Inhibitor
SE
EDTA
20
0.66
0.84
(looPW
19
3.11
0.86
EDTA
36
3.16
1.15
(600 M
32
7. I7
1.19
Histidine
41
29.0
3.3
mw
29
40.9
3.2
Histidine
31
--1.13
1.61
(67 m w
25
2.91
1.oo
EDTA
20
(l0PM)
21
DTNB
Control
RA
DTNB
Control
RA
Viscosity
Control
(67
RA
RA
-2.20
% total SH
4.02
Milliabsorbance
% internal gold standard
4.05
% internal NEM standard
4.01
Aggregation, %
2.01
Aggregation
Control
units
(0.05
0.01
Viscosity
Control
P
5.82
2.02
HLA antigens associated with psoriasis and psoriatic arthritis
L. H. Gerber, C. Murray, J.L. Decker, D.L. Mann, National Institutes of Health, Bethesda; S. G. Perlman, W.F. Barth, and T.P.
Nigra, Washington Hospital Center, Washington, DC
Previous studies suggest an association of certain HLA antigens with psoriasis (Ps) and psoriatic arthritis (PsA). We report results
of a comprehensive analysis of HLA antigens and B cell alloantigens
in 56 patients with Ps, 39 with PsA and 89 controls. Ps had >lo%
body surface involved with psoriasis. PsA was defined as having 21
psoriatic plaques, absence of rheumatoid factor, and 3 or more active
joints. HLA typing was performed on anticoagulated peripheral blood
and tested for complete HLA, B, C, and DRw antigens, a total of 51
specificities.
The frequency of multiple antigens differed between Ps or
PsA when compared to controls. The Table shows only those increased frequencies with a corrected probability of <0.008.
The frequency of one or more of the three antigens, B17,
Cw6, DRw7, in Ps was higher than controls (P< 0.0005)but did not
reach significance in comparison to PsA (P> 0.05). The frequency of
one or more of the three antigens, A26, B38, DRw4, in PsA was
higher than in Ps or controls (P> 0.0005). Even upon exclusion of 6
patients with grade III sacroiliitis or New York criteria for spondylitis, the same antigens were associated with PsA. The arthritis of PsA
was more severe in those with 2 or more of the 3 antigens. Five PsA
patients were B27-positive, 2 were spondylitic, and 2 had only syndesmophytes. Three individual antigens A3, B7, DRw2, were decreased below control values in both Ps and PsA (P< 0.01).
These studies support the association of HLA antigens with
Ps and PsA. The finding of marked phenotypic distinctions between
Ps and PsA is unexpected and not easily explained. Haplotype determination is in progress and may provide an explanation.
ARA ABSTRACTS
61 1
Frequency of antigens, %
Groups
A26
B 17
B38
Cw6
DRw4
DRw7
Controls
Ps
PsA
4
2
23t
8
38*
12
4
5
26t
12
50*
26
31
32
64t
24
54.
36
* Differs from controls (P< 0.0005)
t Differs from controls (P< 0.008).
Study of congenitally immunologically mutant New Zealand mice
M. Eric Gershwin and James J. Castles; University of California, Davis
Many of our concepts of autoimmunity in New Zealand
mice, as well as their genetic viral, and lymphocyte subpopulation interactions, depend heavily on the use of surgically manipulated mice.
However, the numerous age-dependent changes, the influence of sex
hormones, and the failure of neonatal thyectomy (even with radiation
and a n t i 4 treated bone marrow reconstitution) and splenectomy to
eliminate the role of the thymus and spleen during gestation have led
to a continued reevaluation of data. Because these models have often
suggested alternative etiologic defects and hence variable therapeutic
programs, our laboratory embarked several years ago to produce, by
repetitive backcrossing and genetic selection, colonies of inbred hereditarily asplenic (Dh/+) and congenitally athymic (nude) New Zealand Black (NZB) mice. These mutant NZB mice were compared with
their intact control littermates with respect to the natural course of
disease and their basic immunobiology. Specifically the appearance of
hemolytic anemia, antinucleic acid, and natural thymocytotoxic antibodies (NTA); age-dependent changes in cell mediated and suppressor cell function; magnitude of polyclonal B cell responses and sera
immunoglobulins; and the incidence of lymphoma were studied. Hereditarily asplenic (Dh/+) NZB mice develop autoimmune disease at
the same rate and frequency as their littermate (+/+) controls. However, they do not develop significant titers of NTA and have normal
suppressor cell function. Congenitally athymic (nude) NZB mice die
very early in life, even under pathogen-free conditions, due to their
compromised state. However, they have an exaggerated appearance of
select autoantibodies and high background responses to a variety of
synthetic haptens.
We believe that these data suggest the existence of both
thymic derived and B cell derived defects in NZB mice. Moreover the
presence of normal antigen nonspecific suppressor function in NZB
Dh/+ mice and its reduction by administration of NTA suggest that
loss of suppressor cells is not a prerequisite for autoimmunity and that
anomalies of this regulating function in NZB mice are secondary to
NTA. Because the combined defects of NZB Dh/+ and nude mice
can be demonstrated at birth, it is proposed that therapeutic trials address the issue of multiple predisposing factors.
Association of sickle hemoglobin with systemic lupus erythematosus in black children and young adults
Donna L. Gibbas, Emory University School of Medicine, Atlanta
Table 1.
ANA+
ANA-
No. patients
AA
AS
ss*
AS or SS*
51
30
41 (80.4%)
5 (9.8%)
2 (6.6%)
5 (9.8%)
1(3.3%)
10 (19.6%)
30 (90.1%)
Sickle cell disease variants (SS*) were noted in 4 of 34 black
patients, ages 6-23, with systemic lupus erythematosus (SLE). In order to determine whether sickle hemoglobin influences the development of collagen vascular disease, hemoglobin electrophoretic patterns were evaluated in 81 children with arthritic diseases. In the U.S.
black population 8% have sickle trait and 0.2% have sickle cell disease. Individuals with positive antinuclear antibodies (ANA) were
compared with ANA negative patients. Results are shown in Table I.
Sickle hemoglobin frequently was significantly increased in ANA
positive individuals (P< 0.005).
ANA patterns also differed as seen in Table 2. Patients with
type AS hemoglobin tended to have speckled ANAs; SS* patterns
were diffuse or nucleolar (Pc 0.01).
Disease patterns varied as well. Of 5 with type AS, 3 had
mixed connective tissue disease (MCTD), and 1 dermatomyositis. The
AS individual with the diffuse ANA is an asymptomatic member of a
MCTD family.
Of 5 sickle cell patients with diffuse or nucleolar ANA, 4 had
3 (9.9%)
systemic lupus erythematosus (SLE) by ARA criteria. One had pauciarticular arthritis with iritis.
Sickle patients with SLE had several similarities: I ) 3 of 4
had f f F (type fetal hemoglobin), fA,+F, or hemoglobin type SC, 2)
milder hemolytic disease-none had received transfusions, 3) prominent splenomegaly, 4) lymphadenopathy, 5) no clinical evidence of
renal disease at diagnosis.
AS and SS* individuals differ in clinical disease and ANA
pattern. SS* may have an increased association with SLE. Sickle hemoglobin may play a role in ANA positive collagen disease and may
well be a significant
factor in clinical manifestations.
Table 2.
Hemoglobin
Diffuse, nucleolar
Speckled
AA
AS
26
15
1
4
0
ss
5
ARA ABSTRACTS
6 12
Subacute cutaneous lupus erythematosus: A form of lupus erythematosus associated with HLA-B8 and DRw3
James N. Gilliam and Peter Stastny; The University of Texas Health Science Center at Dallas
Patients with lupus erythematosus (LE) can be grouped into
subsets having in common certain clinical and serologic features. We
have recently studied a subset of LE characterized by a distinct form
of skin disease which we have called subacute cutaneous LE (SCLE).
Patients with SCLE usually have mild multisystem disease involving
principally the joints and have widespread non-scarring skin lesions.
Skin biopsies show perivascular and periappendigeal lymphoid infiltrates and hydropic degeneration of the basal layer of the epidermis
characteristic of LE. HLA typing was performed in 15 patients with
SCLE, 50 patients with discoid LE (DLE), 20 patients with systemic
LE (SLE), and 68 normal controls, all white and residing in the same
area. The SCLE group stood out because of a marked increase in the
antigens HLA-B8 and DRw3. Thus, the frequency of B8 was 60% in
SCLE patients compared to 29% in controls (P< 0.02); DRw3 was
77% in SCLE compared to 31% in normal individuals (P < 0.005).
The HLA-A1, B8, DRw3 haplotype was found in 62% of the SCLE
patients, while in normal controls it was observed in only 24% (P <
0.02). The strongest association in these patients was with the HLA-D
region, with DRw3 giving a relative risk of 7.5 (P< 0.005). Of considerable interest is the fact that the SCLE group contrasts in this regard
with both the SLE and the DLE patients in whom the HLA-AI, B8,
DRw3 haplotype was not increased compared to the normal controls.
The differences between SCLE and SLE as well as SCLE and DLE
for these antigens were statistically highly significant. The HLA association in this relatively homogenous group of patients was striking.
These findings confirm the clinical impression that SCLE
constitutes a separate entity, and they suggest the existence of a strong
immunogenetic factor. It is of interest that although the HLA association was similar to that of Sjogren's syndrome, clinically no evidence
of the sicca complex was present in these patients. It seems likely that
the HLA-B8, DRw3 antigens known to be associated also with other
forms of autoimmunity signal the presence of an immunologic reaction involving the skin. From these results it was concluded that subacute cutaneous lupus erythematosus is a newly recognized subset of
lupus erythematosus with distinct clinical and laboratory features.
The mechanism of complement activation by monosodium urate crystals
-
Mark H. Ginsberz, Research Institute o f Scripps,
- - La Jolla, California; Patricia Giclas, National Jewish Hospital, Denver; and Neil
Cooper, Researcilnstitute of Scripps
Activation of the complement system may be involved in the
pathogenesis of gout. Monosodium urate crystals (MSU), the probable cause of gouty inflammation, have been shown to activate complement, but the mechanism of activation has not been established.
These studies were undertaken to elucidate the mechanism of complement activation by MSU.
Monosodium urate crystals were synthesized by using uric
acid and glassware that had been heated at 200°C for 2 hours to destroy pyrogens. The uric acid was neutralized with NaOH dissolved in
sterile nonpyrogenic water, and the resulting needle was shaped; negatively birefringent crystals were shown to contain 5 5 0 ng bacterial
lipopolysaccharide per 5 mg crystal in a rabbit pyrogenicity bioassay.
These crystals depleted complement in serum. There were proportionate decreases in C1, C4, and C3 hemolytic activities, suggesting classical pathway activation. To document classical pathway activation,
highly purified C I macromolecule containing radiolabeled CIS sub-
component was employed. Urate crystals bound this C1 and induced
cleavage of the radiolabeled CIS in a pattern typical of C l activation.
MSU binding and activation of C1 required the presence of C l q in an
intact C l macromolecule. Thus these crystals activated C l by first
binding through the C Iq subcomponent; this rigorously establishes
classical pathway activation. Urate crystals avidly bind IgG, and crystal-associated IgG may be responsible for some of the biologic properties of MSU. The purified C l used contained no immunochemically
detectable IgG. In addition, F(ab')2 fragments of anti-Fc antibodies
had no effect on C l activation by MSU; the same F(ab')2 abolished
C 1 activation by IgG aggregates.
We conclude that MSU binds and activates C1; binding and
activation depend on the Clq subcomponent of an intact CI but do
not depend on the presence of IgG. The crystals are thus direct activators of the classical complement pathway.
The mechanism of Hageman Factor activation by monosodium urate crystals
Mark H. Ginsberg, Research Institute of Scripps Clinic, La Jolla, California; B r y n Jaques, University of California San Diego;
Charles G. Cochrane, and John H. Grifin, Research Institute of Scripps Clinic
Activation of Hageman Factor (HF) may lead to pain, vasodilation, and increased vascular permeability. Addition of monosodium urate crystals (MSU) to plasma induces HF-dependent procoagulant activity, suggesting H F activation. The purpose of this
study was to define the molecular events that follow the interaction of
MSU with HF. MSU crystals were synthesized by neutralization of
pyrogen-free uric acid under aseptic conditions. Culture of the crystal
suspension showed no bacterial growth, and the crystals were free of
bacterial lipopolysaccharide (550 ng per 5 mg crystals) in a rabbit
pyrogenicity bioassay. When diluted plasma containing "'I HF was
added to these crystals, proteolytic cleavage of the H F into 52,000 and
28,000 molecular weight (MW) fragments was observed in SDS polyacrylamide gel electrophoresis. This pattern of cleavage is typical of
that associated with HF activation and directly supports the inference
that MSU induced HF activation. Two forms of cleaved HF were observed; the major form was an 80,000 MW molecule containing polypeptide chains of 52,000 and 28,000 MW linked by disulfide bonds
(presumably aHFa). The other form of cleaved HF contained two
free fragments of 52,000 and 28,000 MW (presumably PHFa). The
aHFa remained crystal-bound while the 28,000 MW (which presumably contains the enzyme active site) eluted from the crystals. This indicates that urate crystal-activated-HF may either remain crystal-as-
613
ARA ABSTRACTS
sociated or be released from the crystal. Plasmas genetically deficient
in prekallikrein or high MW kininogen failed to support MSU-induced
HF cleavage. Reconstitution of each deficient plasma with the appropriate purified protein resulted in reconstitution of urate-induced HF
cleavage, indicating that prekallikrein and high MW kininogen are
cofactors in this process. Urate crystals induced cleavage of Iz5I-HF in
synovial fluid from a gouty patient. We conclude that urate crystals
provoke the limited proteolytic cleavage of H F into a crystal-associated and soluble form. This cleavage also occurs in joint fluid and is
enhanced by cofactors such as high MW kininogen and prekallikrein,
indicating a role for these proteins in urate-induced activation of HF.
Thus these proteins may be involved in the process of gouty inflammation.
A multicenter study of survival in systemic lupus erythematosus
Ellen Ginrler and Herbert Diamond; Downstate Medical Center, Brooklyn, and The L u p s Survival Study Group
A multicenter study of factors influencing survival in systemic lupus erythematosus (SLE) was camed out at 9 university-affiliated centers chosen to include a spectrum of treatment philosophy
and geographic, socioeconomic, and racial factors. Data were obtained on 1,103 patients seen for the first time at the participating tenters from January 1965 to January 1976. Overall survival was 90% at 1
year, 77% at 5 years, and 7 I% at 10 years. At individual centers survival ranged from 100-83%, 93-68% and 93-52% at I , 5, and 10 years.
The centers varied widely with regard to race, socioeconomic status,
duration of disease prior to acquisition, and disease severity at acquisition, as assessed by number of American Rheumatism Association
(ARA) criteria fulfilled, degree of azotemia, anemia, and presence of
central nervous system disease. The poorest survival was seen among
patients entering the study with a creatinine >3 mg%: 48%, 295, and
12% at 1,5, and 10 years, and those with a hematocrit ~ 3 0 % 71%,
:
6%
and 2% at I , 5, and 10 years. Survival was also markedly decreased
among patients with more than 6 ARA criteria at acquisition: 84%,
57%, and 37% at 1, 5, and 10 years. Among the 4 centers with the
poorest survival, 77% of the patients fulfilled at least 4 ARA criteria at
acquisition and 14% fulfilled more than 6, whereas 32% of patients at
the 3 centers with the best survival fulfilled 4 ARA criteria; only 2%
fulfilled more than 6. Similarly, presentation with creatininc >3 mg%
was 10 versus 4% at the centers with the poorest and best survival; 36
versus 23% of patients had a hematocrit C30%0 at acquisition, 8 versus
3%had seizures, and 11 versus 5% had evidence of an organic psychosis. Duration of disease prior to acquisition did not account for differences in disease severity, as the mean duration was 30 versus 49
months at the centers with the poorest and best survival. By stepwise
linear regression, the most significant entry variables (P < 0.05) determining survival to 1 year were the creatinine, hematocrit, degree of
urine protein, and the presence of psychosis. By the fifth year, creatinine, hematocrit, and urine protein remained significant variables. In
addition, survival was related to the number of ARA criteria fulfilled,
the age at acquisition, and the duration of SLE prior to acquisition.
After the fifth year, degree of proteinuria at acquisition became the
most important entry variable as a predictor of survival. Thus, differences in disease severity at onset accounted for much of the association of survival with institution, race, and socioeconomic status.
Total knee arthroplasty in classic hemophilia
Victor M. Goldberg and Kingsbury HeQle, Case Western Reserve University School of Medicine
The destructive arthropathy that occurs in classic hemophilia
produces severe joint disorganization and great functional impairments. This report deals with patients with classic hemophilia who
have undergone total knee arthroplasty to demonstrate the problems
and demands of this procedure.
Twelve total knee replacements have been performed in 10
patients who were then followed from 1 to 5 years. All had essentially
no circulating antihemophilia factor (AHF). Their ages ranged from
15 years to 35 years. All of the patients were severely disabled, bein{
either confined to a wheel chair or requiring crutches for ambulation.
The average preoperative knee motion was 55 degrees with flexion
contractures ranging from 15 to 45 degrees. Pain was the greatest limiting factor. The average preoperative quantitative knee score was 25/
100. Type specific cryoprecipitate to replace AHF is utilized and administered preoperatively and for 3 weeks thereafter. An AHF level of
greater than 35% is maintained throughout this period. The ICLH or
Total Condylar prostheses were utilized. One bilateral procedure was
performed under the same anesthetic without problems. Complica-
tions included minor skin problems in 6 knees that eventually healed.
Another patient developed a persistent sinus and an infected deep hematoma with pseudomonas. He required an arthrodesis. There were 3
peroneal nerve palsies and one posterior tibia1 nerve palsy. All of
these recovered full function, except one patient who still had some
residual weakness of ankle dorsiflexion at I8 months postoperatively.
At the time of followup, none of the patients were chairbound, and only 4 patients required some external walking aids. Pain
and function were markedly improved. The average postoperative arc
of motion was 65 degrees with no flexion contracture greater than 10
degrees. The average postoperative knee score was 70. This small series of patients with classic hemophilia demonstrates that total knee
arthroplasty can be a helpful procedure in maintaining ambulation in
this severely handicapped population. The risks and complications in
these patients appeared to be greater than usual. We feel that because
of these problems, this procedure should be undertaken with a complete understanding of the disease and with excellent hematologic expertise available.
6 14
ARA ABSTRACTS
Rheumatoid lymphocytes stimulated with collagen peptides produce a lymphokine which enhances collagenase
secretion from synovial cell cultures
E.E. Golds, H. L p n s , Shriners Hospital, Montreal, Canada; T.D. V. Cooke, Queens University, Kingston, Canada; M. van der Rest,
A.R. Poole, Shriners Hospital
Because of the critical role played by collagenase in the destruction of the rheumatoid joint, it is important to delineate the
mechanisms that stimulate collagenase synthesis and secretion in synovial cells. Previous work from this laboratory demonstrated that
collagenase secretion was increased when peptides derived from Type
I1 collagen were added to culture niedia of rheumatoid synovial explants. An autoimmune reaction was postulated whereby lymphocytes
present in the inflamed tissue respond to collagen peptides by producing a lymphokine, which in turn stimulates collagenase secretion from
synovial cells. In order to characterize the manner in which collagenase secretion from synovial explants was stimulated and to search
more specifically for autoimmunity to collagen, we have cultured isolated synovial cells in the presence of conditioned media derived from
peripheral blood lymphocytes exposed to possible autoantigens such
as collagen peptides. Dayer and Krane have previously shown that
human peripheral blood lymphocytes activated by the polyclonal
mitogen, phytohemagglutinin, produce a lymphokine that stimulates
collagenase secretion from rheumatoid synovial cells. However, it is
not clear that antigenic stimulation of lymphocytes would have the
same effect.
We have exposed peripheral blood lymphocytes obtained
from rheumatoid patients to peptides derived from purified human
Types I and I1 collagen by cyanogen bromide cleavage. The ability of
such conditioned media to stimulate collagenase secretion was assayed on autologous and homologous synovial cell cultures. Our data
show that increased amounts of lymphokine enhancing collagenase
secretion are often released after exposure to either or both collagen
peptides. These results indicate that collagen peptides could act as
auto-antigens in vivo via this pathway. In general, conditioned media
from lymphocytes exposed to Type I collagen peptides had a greater
effect than that from lymphocytes stimulated with Type I1 collagen
peptides. Most, but not all, of the conditioned media from rheumatoid
lymphocytes exposed to collagen peptides of either type produced
more lymphokine stimulating collagenase secretion. The autoimmune
response to collagen peptides appears to be somewhat specific since
production of this lymphokine is not usually accompanied by a strong
blastogenic response nor by production of another lymphokine, leukocyte inhibition factor.
A clustering of Kawasaki disease with new clinical manifestations
Donald P. Goldsmith, Sarah S. Long, Maarten S. Sibinga, Temple University School of Medicine, Philadelphia; Adamadia Deforest,
St. Christopher's Hospital for Children, Philadelphia; and Charles D. Tourtellotte, Temple University School of Medicine
Five years ago Kawasaki disease (KD) was unknown in the
United States. Now, however, K D is recognized as a discrete clinical
entity, possibly related to infantile polyarteritis nodosa (IPN) and
thought to be more prevalent than previously realized. Clustering in
time and/or space of KD has been observed in Japan, but rarely in
the United States. Each year 2 to 3 patients have been admitted to our
primary and tertiary care hospital with this diagnosis. During a 28day period, however, between March and April 1978 we saw 6 hospitalized children with KD which, to us, suggests a small epidemic.
Ages ranged from 2% months to 1 I years and all met the accepted diagnostic criteria. All were male, and none was white (3 American
blacks, I Hispanic black, 1 Indian, and I Korean); this racial pattern
and sex distribution is different from that of our usual hospitalized
population.
All had significant leukocytosis with a predominant neutrophilia, elevated Westergren sedimentation rates, and increased levels
of C-reactive protein. All had significant thrombocytosis, one reaching 1,580,OOO mm3. IgM levels were elevated in 5 patients and IgE
levels increased in 4. Contrary to most previous reports, the total he-
molytic complement (CH50) was markedly decreased in 1 child,
slightly reduced in 2, and normal in I . Serum rheumatoid factor, antinuclear antibody, and hepatitis B surface antigen were negative in all
6 children. Extensive bacteriologic cultures and viral studies were entirely negative. HLA studies did not reveal a common haplotype.
Three previously undescribed findings were observed: 1) One
child developed a benign sixth nerve palsy, further suggesting a possible parainfectious process; 2 ) two patients had initially elevated
CPK levels, one with transient myoglobinuria, and CPK isoenzyme
determinations documented skeletal rather than cardiac muscle involvement; and 3) a 2%-month-old Korean child developed cold cyanotic extremities that progressed to extensive digital gangrene and
the autoamputation of portions of 8 fingers and 4 toes.
Since gangrenous extremities have been previously reported
in 20% of patients with IPN and it is known that the postmortem findings in KD are indistinguishable from those of IPN, this latter complication further supports the concept of a continuum between K D
and IPN.
Role of glass adherent and prostaglandin producing suppressor cells in the depressed mitogen response of patients
with rheumatoid arthritis and systemic lupus erythematosus
James S. Goodwin, Steven Wolinsky, Ronald P. Messner, and Ralph C. Williams, Jr.; University of New Mexico School of Medicine,
A lbuquerque
We investigated the role of suppressor cells in the depressed
cellular immunity of patients with rheumatoid arthritis (RA) and sys-
temic lupus erythematosus (SLE). Twelve patients with active RA
and 6 patients with active SLE were studied and compared to two
ARA ABSTRACTS
control groups: 15 healthy laboratory personnel and 10 patients with
degenerative joint disease (DJD) of similar age distribution to the RA
patients. The RA and DJD patients were on a variety of nonsteroidal
antiinflammatory medications. Two suppressor cell assays employed
were the prostaglandin (PG) producing suppressor cell (J Exp Med
146:1719, 1977) and the glass adherent suppressor cell (J Clin Invest
56:467, 1975), assayed by measuring the effect of addition of indomethacin to mitogen cultures and the effect of removing adherent
cells on the mitogen response, respectively.
Peripheral blood mononuclear cells (PBMC) from the 12 patients with active RA had a significantly depressed response to each of
four concentrations of phytohemagglutinin (PHA), compared to the
young controls or the age-matched DJD controls. Addition of indomethacin ( I pg/ml) to PHA (2 pg/ml) cultures resulted in a 72 f 8%
increase in 3H-thymidine incorporation for the RA patients versus a
21 & 3% increase in the control cultures (mean f SEM, P < 0.001).
615
The mean PHA response of the patients was 60% of control without
indomethacin. This increased to 80% of control with indomethacin.
Thus blockade of prostaglandin synthesis eliminated some but not all
of the depressed PHA response. The SLE patients had a more depressed PHA response (40% of control) and a greater increase (120
2 1%) with indomethacin than the RA patients.
Removal of glass adherent cells resulted in a 50% decrease in
PHA response of both the RA and control PBMC. Thus this is a third
pattern of suppressor cell activity in disease states. In Hodgkin’s disease, either addition of indomethacin or removal of adherent cells restores the response (N Engl J Med 297:963, 1977); in sarcoidosis indomethacin does little, but removal of adherent cells restores the
response (Ann Intern Med, Feb, 1979). With RA, indomethacin improved the response but removal of adherent cells did not change the
response with respect to control.
*
Gold excretion and retention during oral gold (Auranofin) therapy
Norman L. Gottlieb, University of Miami School of Medicine, Miami, Florida
Auranofin (triethylphosphine) is an oral gold compound that
reportedly suppresses synovial inflammation in patients with rheumatoid arthritis (RA). Gastrointestinal absorption has been documented
by progressive increments in serum and urine gold concentrations
during chrysotherapy. However, the excretory pathways and quantity
of gold retained during Auranofin treatment have not been delineated.
Twelve patients with definite or classic RA have been treated
for a mean of 10 months in an ongoing double-blind prospective clinical-metabolic study. One or 3 mg of Auranofin was given orally twice
daily for 8 weeks and then once daily for a minimum of 18 additional
weeks. Clinical course and blood and urine gold concentrations were
monitored at I-to-4 week intervals during outpatient visits. Patients
were hospitalized in a metabolic unit for 3 or more consecutive days
during the first 8 weeks of study for 24-hour daily excreta collection.
Gold levels in blood, urine, and feces were measured by atomic absorption spectroscopy.
No significant hematologic, dermatologic, or renal toxicity
was encountered. Diarrhea and abdominal cramps were observed
during the initiation of treatment, primarily in patients using 6 mg of
compound daily. The clinical response to therapy was variable but
gratifying in some patients.
More than 70% of the administered gold dose was recovered
in the urine and feces of patients who received 6 mg of Auranofin
daily; nearly all was retrieved in patients receiving 2 mg daily. Approximately 95% of the recovered gold was in the feces, and only 5%
was in the urine, regardless of dosage. These findings are in sharp
contrast with those reported previously for intramuscular gold sodium
thiomalate (Myochrysine; GST); 40% of a 50 mg dose was recovered,
70% in the urine and 30% in the feces (Gottlieb et al: Arthritis Rheum
15:582-592, 1972).
Following 20 weeks of conventional chrysotherapy with GST
approximately 300 mg of gold are retained in body tissue. In a similar
time period, less than 75 mg of gold are retained when 6 mg of Auranofin are administered, and lesser quantities when lower doses are
given. Broader clinical experience with Auranofin is required to interpret the clinical significance of these metabolic findings.
Long term therapy of psoriatic and rheumatoid arthritis with oral Methotrexate monitored by serial liver biopsies
James D. C. Gowans, George Arnold, Marshall M. Kaplan, George Wilgram, and Lennig W . Chang; Tufts-New England Medical
Center Hospital, Boston
Psoriatic arthritis is known to be responsive to Methotrexate,
and possibly rheumatoid arthritis responds well, also. Use of this drug
in treatment of nonmalignant disease is controversial because of hepatotoxicity, though the relationship between chronic treatment and
development of cirrhosis of the liver is still unclear. Some studies suggest that the severity of liver disease is related to the cumulative dose
of Methotrexate; others could find little or no relationship.
To gather more information, we performed serial liver
biopsies in 28 patients with chronic diseases before and during Methotrexate treatment. Weekly doses ranged from 5 to 25 mg given orally,
and total accumulated doses 227 to 6698 mg. Diseases treated in-
cluded 14 patients with psoriatic arthritis, 2 with psoriasis, 9 with
rheumatoid arthritis, and I each with pemphigus vulgaris, oral lymphoma, and mycosis fungoides. Needle biopsies were performed at
least twice in all patients both before starting Methotrexate and after
8 months to 7 years of treatment. One patient was biopsied 4 times
and 6 were biopsied three times. Liver biopsies were coded and read
by one of us (MMK) who was provided with no clinical information.
Twenty-two patients showed no significant changes between first and
last biopsies. In 2 patients there was improvement: I had less inflammation and liver cell necrosis after Methotrexate, and in the second
there was less fatty infiltration. Four patients demonstrated an in-
616
crease in portal fibrosis, in 3 the increase was slight, and in I the
change severe enough for us to stop treatment. No patient developed
cirrhosis or overt liver disease. There was no correlation between the
total dose of Methotrexate and either the pathologic changes or liver
function tests. Alcohol was a possible factor in 2 showing fibrosis.
The 23 patients with arthritis were selected because of the se-
ARA ABSTRACTS
verity of disease and failure to respond to usual therapy. They showed
sustained and drug dependent improvement on Methotrexate. It was
concluded that long term Methotrexate could be administered effectively and reasonably safely to such patients, but until further experience, pretreatment and subsequent periodic liver biopsies are advisable.
Evidence for intravascular coagulation in polymyalgia rheumatica and temporal arteritis
R. G. Grau, S.S. Kassan, J.J. Franks, H. Kaplan, and E.M. Tan; University of Colorado Medical Center, Denver
Polymyalgia rheumatica and temporal arteritis are overlapping clinical entities characterized by giant cell arteritis and by
markedly elevated erythrocyte sedimentation rates (ESR). High ESR
is a dominant feature of these diseases, and it may be associated with
abnormalities in the fibrinogen system, although this has not been
clearly demonstrated. Recently, a radioimmunoassay has been developed for the detection of fibrin fragment D-dimer in serum. D-dimer
is a cleavage product of cross-linked fibrin, and increased levels indicate enhanced fibrinolysis, presumably in response to intravascular
fibrin formation. We studied 6 patients with polymyalgia rheumatica
and 4 patients with temporal arteritis. Five patients had received
prednisone therapy for greater than 2 months and had improved with
a mean ESR of 19.4 f 8.0 mm/hr (SE). Five other patients were untreated or treated for less than I week (considered untreated) with a
mean ESR of 75 f 9.4 mm/hr (SE).
Geometric mean serum D-dimer concentrations (with 95%
confidence limits on the means) for untreated patients, treated patients, and for a group of 14 control subjects were 2,171 ng/ml (469 to
4,863 ng/ml), 792 ng/ml(487 to 1,288 ng/ml), and 260 ng/ml(186 to
364 ng/ml). Each of these means differs significantly from the others
(P < 0.05) as determined by analysis of variance. and the StudentNewman-Keuls procedure. ‘251-fibrinogenmetabolic studies revealed
normal fibrinogen distribution and catabolism in 3 patients (2 treated
and 1 untreated). The D-dimer assay appeared to be detecting intravascular fibrin formation and lysis in polymyalgia rheumatica and
temporal arteritis which could not be detected by the fibrinogen kinetic method.
These studies show that patients with polymyalgia rheumatics and temporal arteritis have significantly elevated levels of fibrin
fragment D-dimer. Following adequate treatment with steroids, as determined by clinical response and drop in the ESR, the D-dimer levels
fall but remain higher than normal. The giant cell arteritis characteristic of temporal arteritis and polymyalgia rheumatica may result in a
chronic low-grade intravascular coagulation. The fibrin fragment Ddimer assay serves as a measure of this intravascular coagulation and
may reflect disease activity.
Action of superoxide radical on hyaluronic acid
Robert A . Greenwald, Wai W. Moy, and Ernest M . Moy; Long Island Jewish-Hillside Medical Center, New H y i e Park, NY
The loss of synovial fluid viscosity which accompanies inflammation has been attributed to “depolymerization” of hyaluronate
despite the fact that there is no hyaluronidase in joint tissues or leukocytes (PMNs); furthermore, HA degradation products have never
been found in pathologic joint fluids. Since PMNs generate the highly
reactive oxygen-derived free radical known as superoxide, we have
studied the effect of superoxide radical on the viscous component of
synovial fluid, namely HA.
Superoxide radical can be generated in vitro by the action of
xanthine oxidase (XO) on hypoxanthine (Hx). When HA (850 pg/ml)
was incubated with X O (50 pg/ml) and Hx (0.8 mM), the specific viscosity dropped from 2.88 to 1.87 in 60 minutes and reached I. I1 at 19
hours. A control preparation incubated with XO and uric acid (no superoxide radical produced) lost only 7% of its initial viscosity over the
same time period. The viscosity fall induced by superoxide radical
could be abolished by addition of superoxide dismutase, catalase,
copper-penicillamine complex, mannitol, or histidine, all of which are
scavengers of various toxic oxygen species. Whereas control HA was
excluded from Sepharose CL-ZB, the superoxide radical-treated material eluted at a Kd = 0.6 despite the absence of an increase in reducing
sugars, and in addition 28.7% of the hexuronate became dialyzable.
HA exposed to superoxide radical did not become susceptible to /3glucuronidase activity. Finally, an HA solution was subjected to sequential addition of aliquots of human PMNs that had been stimulated to generate superoxide radical by concanavalin A; a progressive
loss of viscosity over 90 minutes ensued which was not produced by
unstimulated PMNs.
PMNs in inflamed joints are subjected to many stimuli which
lead to superoxide radical production. Since neither PMNs nor other
joint tissues contain hyaluronidase, superoxide radical is the most
likely mediator of the loss of synovial fluid viscosity which accompanies inflammation. Schism of covalent bonds in the HA polymer is
one possible mechanism of such an effect.
Correlations of major histocompatibility (Ir) genes with auto-antibody production
W. Leroy Grifing and C. Garrison Fathman, M a p Clinic, Rochester
In systemic lupus erythematosus (SLE) the major clinical
and laboratory characteristics have been defined, but the underlying
pathogenesis remains unclear. Despite numerous attempts, there have
been no adequate explanations of the suspected genetic and environ-
ARA ABSTRACTS
mental factors which interact to cause disease. Though a n increased
frequency of certain HLA phenotypes has been observed in association with SLE, no relationship to any particular feature of SLE has
been defined. In the murine model of SLE, independent segregation
of glomerulonephritis, hemolytic anemia, and antinuclear antibodies
has been demonstrated. Since major histocompatibility (MHC) linked
immune response (Ir) genes have been demonstrated in experimental
animal models to correlate with antibody production and not with
disease states, we have examined the possible relationship between
MHC linked Ir genes and control of autoantibody production in murine and human SLE. Our results, shown in the Table, have suggested
that an H-2" linked Ir gene controls autoantibody response to native
DNA in (NZB x NZW)F, backcrossed animals at 9 months of age.
These results suggest that there is a qualitative ability to respond to native DNA that is controlled by an H-2d linked Ir gene,
whereas quantitative responses seem to be controlled by other gene@),
(thus explaining the differences in the level of responsiveness in the d/
617
z haplotype backcrossed mice. These differences seem to depend upon
the non-MHC genes of the parental strain used to backcross). Because
of the documented close association of certain HLA phenotypes with
disease, it is possible that Ir genes may be responsible for HLA and
disease association. Rather than disease states, however, this association may rest with autoantibody production which is simply one manifestation of disease. Studies are currently underway to examine possible DRw linkage of defined autoantibody markers in rheumatic
diseases.
Number of Mice
20 (NZB/W)
30 (NZB/W)
28 (NZB/W)
19 (NZB/W)
X
X
X
X
Haplotype
Mean level of DNA antibody
(%/mu
0.0
0.143
1.069
0.3 I 1
NZW = z/z
NZW = z/d
NZB = d/z
NZB = d/d
Isolation of immune complexes from serum by conglutinin precipitated with anticonglutinin
R. C. Gupta, University of Colorado Medical School, Denver; F.C. McDuBe, M a p Clinic, Rochester; C.M.Arroyave, and E.M. Tan,
University of Colorado
In order to study the composition of circulating immune
complexes (ICs) present in some of the arthritic diseases, the isolation
of complexes separate from other serum proteins would be helpful.
To accomplish this objective, we devised a technique using bovine
conglutinin (K), which as a unique property of binding C3d in the
presence of Ca++.In this method, the serum containing high ICs is reacted with K which is precipitated by anti-K. The K-bound ICs are
eluted by O.IM EDTA-saline which chelates Ca++ and releases ICC3d from K. By this technique, we have demonstrated successful elution of purified human C3d and the presence of C3d in the isolated
ICs. Two-fold greater 1Cs were obtained by elution with O.IM EDTA
compared to use of 0. IM N-acetyl D-glucosamine. Using lZSI-C3d,we
found that I mg of K bound 167 ng of C3d. Complexes were isolated
by this method from 7 sera of rheumatoid arthritis (RA) and 5 of systemic lupus erythematosus (SLE) patients having high levels of ICs.
Isolated ICs from sera of HBsAg+ chronic active hepatitis (CAH)
have shown the presence of antigen (HBsAg) in the complexes. The
quantitation of 1Cs in the isolated complexes from these sera and 4
normal sera (NHS) is shown below.
The proportion of the levels of ICs isolated from these sera
have varied. The isolated ICs contained IgG, IgM, C3, and albumin,
but other serum proteins tested, such as glycoproteins, lipoproteins,
and transferrin, were absent. Specific antibodies such as rheumatoid
factors and antinuclear antibodies in varying titers have been found to
be present in the isolated ICs. Thus, the method appears to be a useful
simple technique for isolation of ICs, and our study has revealed some
heterogeneity in the composition of ICs. The implication of the presence of albumin in ICs is presently unknown, but it may be related to
antibodies to albumin found in sera of these patients.
Isolated ICs (pg AHG eq/ml)
No. studied
Mean
Range
RA
SLE
7
186.3
60-488
5
337.6
24656
CAH
2
352.0
184520
NHS
4
68.0
40-80
Suppression of autologous and allogeneic mixed leukocyte cultures by low doses of prednisone
Bevra Hahn, Sharon Burkholder, Susan Loewenstein, Mary Beale, Mary Stacey, and Richard MacDermott; Washington University
School of Medicine, St. Louis
Many lymphocyte functions are reduced by large doses of
prednisone, but few studies of the effect of low doses are available.
Daily doses less than 20 mg are generally considered non-immunosuppressive. Cell-mediated immune responses of patients taking low
doses have been studied by many investigators; lymphocyte dysfunction in those individuals has been attributed to their rheumatic disease. To assess the effects of low dose steroids, peripheral venous
blood was drawn from healthy volunteers before and 2 and 6 hours
after an oral dose of 10 or 15 mg of prednisone. Mononuclear cells
were purified on Ficoll-hypaque, then on a G-10 sephadex column.
Responder cells contained a mean of 53% E-rosetting, 0.4% esterase
positive, and 10.I% surface immunoglobulin positive cells; stimulating cells contained 12% E-rosetting, 62.9% esterase positive, and 29.3%
Ig positive cells. Preliminary studies showed that maximal stimulation
occurred at 6 days in both untreated and steroid-treated cells. Therefore, responder cells (l00,OOO) were cultured for 6 days with 20,000800,000 autologous or allogeneic irradiated stimulating cells and
pooled normal male human serum in flat-bottom microculture wells.
Ten milligrams of prednisone reduced uptake of tritiated
thymidine (mean reduction 65%) in 6 of 9 autologous mixed leukocyte
cultures (MLC) studies by using submaximal stimulation; maximal
stimulation was suppressed more than 50% in 2 of 7 studies. Alloge-
ARA ABSTRACTS
618
neic MLC was reduced in 1 of 4 individuals. Fifteen milligrams of
prednisone greatly reduced autologous MLC responses in 3 of 4 individuals (mean reduction 95%); mean maximal cpm in pre-steroid cultures was 22,241 4,726 SEM compared to 3,753 i~ 3,27 I after prednisone (P c 0.01). Both responder and stimulating populations had
diminished reactivity. Allogeneic MLC was reduced by 15 mg of
prednisone in 4 of 5 experiments, 6 hours after the 15 mg dose. In 5 of
6 individuals, serum drawn 2 hours after a 15 mg dose suppressed al-
*
logeneic MLC of unrelated normal donors by 30-84% (mean 54%);
serum drawn 2 hours after a 10 mg dose suppressed allogeneic MLC
in I of 2 experiments.
In summary, low doses of prednisone are immunosuppressive. Studies attributing suppression of cellular immune function
to disease states such as systemic lupus erythematosus may not be
valid if the subjects are steroid-treated, even with low doses.
Mixed cryoglobulinemia: Association of glomuleronephritis with defective reticuloendothelial system Fc receptor
function
M . I. Hamburger, National Institutes of Health, Bethesda; P.D. Gorevic, State University of New York at Stony Brook; T.J. Lawley,
National Institutes of Health; E.C. Franklin, New York University Medical School; and M .M. Frank, National Institutes of Health
The clinical syndrome of mixed cryoglobulinemia (MC) includes arthralgias, vasculitis, liver disease, hypocomplementemia, and
glomerulonephritis (GN). Immune complexes (IC) including HBsAgHBsAb and rheumatoid factor (RF)-IgG have been described in
cryoprecipitates and may contribute to pathogenesis. We studied in
vivo reticuloendothelial system (RES) Fc receptor function by measuring the clearance from the circulation of IgG sensitized 5 l-Cr labeled autologous erythrocytes (E) in 9 patients. In addition, we assessed serum C l q binding activity (ClqBA; normal <lo%) as a
measure of IC levels, complement component (C) levels, R F titers,
and disease manifestations.
There was a striking correlation between clinical manifestations and Fc specific clearance rates. Three of 3 patients with G N had
Fc receptor dysfunction, as shown by significantly decreased clearance of the IgG sensitized E (mean T 1/2 = 273 f 27 minutes, mean
in normal controls = 89 f 7 minutes, P < 0.001, Student’s t test) while
6 of 6 patients with no signs of renal involvement had hyperactive Fc
receptor activity (mean T 1/2 = 45 f 9 minutes, P < 0.01). In contrast
there were no differences in amount or immunoglobulin content of
cryoprecipitate, R F titer, or C levels (shown by hemolytic CI, C2, C4,
CH50, or antigenic C3 or factor B) in the 2 subgroups. There was also
no correlation between GN and IC levels. ClqBA was high in sera
from all 9 patients (44-87% ClqBA), and remained elevated in 7 of 9
sera after cryoprecipitation for 96 hours. There was no correlation between clearance rates and IC levels, in marked contrast to patients
with systemic lupus erythematosus. Since all of these sera had RF
present, 6 were treated with 2-mercaptoethanol(2-ME) known to dissociate 19s RF into its subunits, and then tested for ClqBA and RF.
2-ME eliminated R F (ranging in titer from 512 and 32,768) in all
cases, but ClqBA remained elevated in 5 of 6 sera, indicating the
presence of IC distinct from 19s RF-IgG complexes. HBsAg was detected in 2 of 3 patients with GN, and HBsAb was present in the
third. HBsAg was not present in the 6 patients with no signs of GN;
one had HBsAb.
Patients with mixed cryoglobulinemia have: I ) reticuloendothelial system Fc receptor dysfunction, and 2) multiple types of
immune complexes. The association of delayed Fc specific clearance
with glomerulonephritis suggests that dysfunctional Fc receptors may
predispose to continued circulation of immune complexes capable of
causing nephritis.
Reticuloendothelial system Fc receptor function in mixed connective tissue disease
M.I. Hamburger, H.M. Moutsopoulos, T.J. Lawley, National Institutes of Health, Bethesda; G.C. Sharp, University of Missouri
Medical School, Columbia; and M .M . Frank, National Institutes of Health
Mixed connective tissue disease (MCTD) shares many clinical and laboratory findings with systemic lupus erythematosus (SLE).
However, complement component (C) levels and anti-DNA titers
tend to be normal, and although immune complexes (IC) are present
in the sera of some patients, the incidence of severe glomerulonephritis (GN) is low. Thus despite similarities to SLE, the course of MCTD
suggests important differences in pathophysiology. We studied in vivo
reticuloendothelial system (RES) Fc receptor function as measured by
the rate of clearance from the circulation of IgG sensitized 5 I-Cr labeled autologous erythrocytes in 9 patients with MCTD. We also assessed serum C l q binding activity (ClqBA, normal <lo%)as a measure of IC levels, C levels, antinuclear antibodies, and clinical
manifestations of patients. The 9 patients had the following signs:
arthralgias (9), Raynaud’s (9), esophageal dysfunction (8), swollen
hands (7), sclerodactyly ( 9 , myositis (3), skin lesions of discoid LE
(3), polyserositis (I), and severe GN (I). Antibodies to ribonucleopro-
tein were detected in high titer (>1/100,OOO) in all patients; in 2 antiSm was also found. Anti-DNA titers (DNA binding, normal <25%)
were elevated in 4 of 9 patients, and IC were present in 5 patients.
One had a low C (C4).
Three of the 9 patients studied had significant RES Fc receptor dysfunction (T 1/2 = 226-352 minutes, upper 95% confidence
limit in normal controls = 62 minutes), and 6 had normal clearance
rates. There was a striking association of markers for SLE with abnormal receptor function. Two of 3 with decreased clearance rates had
anti-Sm and high DNA binding (5460%). IC were detected in all 3
(mean ClqBA = 38%) and one had G N with low C4. All 3 had discoid skin lesions. In marked contrast, the 6 patients with normal
clearance more closely fit the typical picture of MCTD. None had GN
or anti-Sm. Only 2 of 6 had anti-DNA in low titer; only 2 had IC in
low titer. None had discoid skin lesions. Patients with normal clearance also tended to have lower corticosteroid requirements. The find-
ARA ABSTRACTS
619
parameters of SLE with an Fc receptor defect typical of SLE in
MCTD patients with more “lupus-like’’ disease suggests an intimate
interrelation between the clearance defect and clinical manifestations.
ing of normal Fc receptor function in the majority of patients with
MCTD contrasts sharply with SLE, where clearance defects were
found in 90% of patients. The association of clinical and laboratory
Assessment of factors affecting phagocytic function of peripheral blood leukocytes from patients with systemic lupus
erythematosus
David D. Hamm, Giles G. Bole, and Peter G. Meagher, University of Michigan, Ann Arbor
*
13.0, PI 6.7 f 2.2 (normal %P
washed three times in PBS %P 75.8
67.2 f 7.4, PI 6.2 f 0.6). Leukocyte viability was >90% in all incubations (0.2% trypan blue staining). In each SLE patient, marked
depression of phagocytic activity was found when peripheral leukocytes were incubated in autologous plasma. This defect was promptly
corrected when the cells were incubated with pooled normal plasma
or in PBS. Depressed SLE phagocytosis did not correlate with disease
activity (4 patients active, 7 mildly active, 16 inactive), prednisone
dose (2.5-60 mg/day), anti-DNA antibody levels (binding 15-92%,
normal <2O%), serum complement (CH50 83-228~. normal > 1 lop)
activity, or Westergren ESR (15-121 mm/hour).
These data indicated that peripheral leukocyte phagocytosis
is significantly reduced (Pt0.01)in SLE patients and suggest that the
defect responsible is in SLE plasma rather than in the leukocytes
themselves. Suppression of phagocytic activity (PMN and monocyte)
was present in all SLE patients. An autoantibody or inhibitor affecting or blocking cell surface receptors involved in phagocytosis could
explain this phenomenon.
Increased susceptibility to infection has been correlated with
the use of corticosteroid and immunosuppressive therapy in patients
with systemic lupus erythematosus (SLE). Evidence that phagocytic
activity of peripheral leukocytes, especially polymorphonuclear cells
(PMN), is abnormal has also been reported. The current study was
designed to evaluate the effect of systemic disease activity, drug therapy, anti-DNA antibody, and complement levels on SLE blood leukocyte phagocytic function. By using a simple in vitro test system (J
Lab Clin Med 87:98, l976), ingestion of heat-killed nonpathogenic S
albus particles by washed normal or SLE leukocytes in the presence of
autologous plasma, pooled normal plasma, and phosphate buffered
saline (PBS) was assayed microscopically after 60 minutes incubation.
The percent of cells (%P) ingesting particles and the average number
of particles ingested per cell (PI) were determined. Twenty-seven assays on 25 SLE patients (20 female, 5 male) were compared with 10
normal controls. Mean PMN phagocytic activity f SEM for SLE patients was as follows: autologous plasma %P 30.2 f 11.7, PI 2.2 f 1.2
(normal %P 82.3 f 6.9, PI 9.0 f 0.7); pooled normal plasma %P 80.2
f 8.7, PI 7.4 f 2.7 (normal %P 80.5 f 4.8, PI 7.4 & 0.9); and cells
Controlled study of the long term effects of “total hand” injection
Joe G. Hardin, Jr.; University of South Alabama College of Medicine, Mobile
To determine the long term effects of a single triamcinolone
hexacetonide (T) injection of all small hand joints in rheumatoid arthritis (RA), 14 RA subjects with established active synovitis of all
metacarpophalangeal (MCP) and proximal interphalangeal (PIP)
joints (including thumb interphalangeal) and no major deformities
had all LO of these joints of I hand only injected with 10 mg of T suspension in 2% lidocaine, with care taken not to rupture the capsule.
The similarly affected uninjected hand served as a control. Indicated
physical and medical therapy continued except the injected hand was
splinted for several days. Hand radiographs made before injection
and at 6-month intervals thereafter were interpreted blindly at the
completion of the study. Complete functional assessment of both
hands, including PIP and MCP total range of motion and routine grip
strength determinations, and physician quantitative estimation of
magnitude of swelling and tenderness of each joint were done before
injection, and at 3, 6, 12, 18, and 24 months afterwards. Twelve subjects were followed for 2 years and 2 for 1 year only. Data in the
3 months
lnjected hand
Controlhand
P
*I
Table represent percentage changes in mean values from preinjection
means.
Radiographic deterioration was twice as great in injected
hands as in control hands at 1 year and at 2 years (not significant).
Major injected joint subluxations developed in 3 injected hands and
in no control hand. At last evaluation, skin atrophy was present in the
vicinity of 17 injected joints, and x-ray calcifications were noted
around 36. No other complications developed. Improvement observed
in both hands during the study period partially reflects a relatively
complete remission achieved by 3 subjects, but exclusion of their data
does not significantly affect comparative results.
T injection of small hand joints significantly suppresses inflammation for 1 year, but does not significantly improve hand function and may accelerate radiographic deterioration. The procedure
seems not to produce the long term results desired from a chemical
synovectomy.
6 months
18 months
12 months
24 months
1
ROM
GS*
I
ROM
GS
I
ROM
GS
I
ROM
GS
I
ROM
GS
179
133
<O.OI
f20
t9
NST
784
758
NS
167
133
f19
f15
NS
f44
f43
NS
174
153
<0.05
fl5
714
NS
f69
f87
NS
166
f14
TI0
NS
f45
T58
NS
159
147
NS
121
714
NS
fl00
TI25
<0.01
151
NS
NS
= physician quantitative estimation of magnitude of swelling and tenderness of each joint; ROM = total range of motion of PIP and MCP
joints; GS = routine grip strength.
NS = not significant.
t
ARA ABSTRACTS
C3 activation by monosodium urate monohydrate is enhanced by surface IgG
Peter Hasselbacher, Dartmouth-Hitchcock Medical Center, Hanover
Crystals of monosodium urate monohydrate (MSUM) are
capable of activating the complement sequence. There is good evidence that this occurs through the classical pathway. Because MSUM
are also capable of adsorbing immunoglobulin, it is possible that surface IgG is responsible for all or some portion of complement activation. In this study, crystals pretreated with IgG demonstrated enhanced activation of C3 compared to crystals treated with the F(ab’),
fragments of IgG.
Fifty pl of a suspension of MSUM in phosphate buffered saline (PBS) (25 mg/ml) were centrifuged and resuspended for 15 minutes at room temperature in 0.5 ml of Cohn fraction I1 (CF 11) (2 mg/
ml) or the F(ab’), fragment of CF I1 prepared by pepsin digestion and
gel filtration (2 mg/ml). The treated crystals were centrifuged and resuspended in fresh human serum (0.5 ml). The tubes were incubated
for 10-30 minutes at 37OC and complement activation was terminated
by adding EDTA (0.01M). The amount of C3 activation in the supernatant serum was quantitated with crossed immunoelectrophoresis by
measuring the percent of total area occurring under the P I A precipitin
arc.
Pretreatment with CF I1 caused increased conversion of C3
at 15 minutes (46% area) compared to crystals pretreated with F(ab’),
(36%). This difference diminished when incubation at 37°C was continued for 30 minutes (59% versus 53%). Activation by crystals preexposed to F(ab’)* was no different than crystals preexposed to PBS
alone. The CF I1 and F(ab’), preparations alone had no effect on C3
conversion in control tubes without crystals, ruling against an anticomplementary or inhibitory effect of these protein solutions.
These data support the hypothesis that C3 activation by
MSUM crystals is mediated, at least in part, by adsorbed IgG. F(ab’),
fragments, which cannot activate complement, had no effect on complement activation by MSUM. It seems likely that adsorbed IgG is altered on the crystal surface, initiating complement activation through
its Fc fragment.
Crystal induced complement activation: Lack of correlation with IgG or Clq adsorption
Peter Hasselbacher, Dartmouth-Hitchcock Medical Center, Hanover
Crystals of monosodium urate monohydrate (MSUM) are
potent activators of complement when compared with other crystals.
Because IgG is bound to the surface of MSUM, it is possible that
complement activation is mediated by adsorbed immunoglobulin.
These experiments compare the ability of different crystals to bind
IgG and activate complement. Desiccated crystals of MSUM, heated
MSUM (200°C for 2 hours), amorphous sodium urate (amorph
MSU), CPPD, hydroxyapatite (HA), and triamcinolone acetonide
(TA) were added to fresh human serum (150 mg/ml) and incubated at
room temperature for 1 hour. The suspensions were centrifuged at
15,600g for 4 minutes and the supernatant collected. The supernatants
were assayed for remaining IgG, Clq, and other complement proteins
and compared with a control tube containing only serum. The ability
of crystal suspensions to activate C3 in fresh human serum was tested
by measuring the electrophoretic conversion of C3. Crystals were incubated with fresh human serum (2.3 mg/ml) for 60 minutes at 37”C,
promptly centrifuged, and the supernatants diluted in veronal buffer
containing EDTA. Crossed immunoelectrophoresis was used to quantitate conversion of C3.
All crystals studied boun- detectable amounts of IgG an
Clq, but they varied widely in C3 activation. Four different preparations of MSUM varied widely in binding of IgG and Clq but were
equally potent in activating C3. Amorph MSU and heated MSUM
were the most active in binding the two proteins but were much less
active in activating C3 than MSUM. HA and TA bound virtually all
the C l q in the sample but activated little or no C3. In addition, no
correlation of C3 activation could be made with binding of C4, C3,
C5, or properdin factor B. IgG adsorbed by crystals was recovered by
elution with 1M NaC1, and for all crystals with negatively charged
surfaces was restricted to cationic species. IgG adsorbed to positively
charged crystals was restricted to anionic species, confirming the role
of electrostatic adsorption of IgG.
From the results, it is clear that C3 activation by crystals is
not a simple function of quantitative adsorption of IgG or Clq. It remains possible that differences in affinity of binding (with subsequent
conformational change of protein structure) or other physical properties of the crystals are responsible for complement activation.
Monosodium urate monohydrate activates C3 by the classical pathway
Peter Hasselbacher, Dartmouth-Hitchcock Medical Center, Hanover
Monosodium urate monohydrate (MSUM) is a potent activator of complement (C’) when compared with other crystalline material. Previous experiments suggested that this activation is caused by
contaminating endotoxin because heating of the crystals abolished activation. In this study, the pathways of C’ activation by MSUM were
studied by observing the effect of EDTA and EGTA on the formation
of split products of C3 and properdin factor B (FB). EGTA blocks the
classical pathway and allows the alternative pathway to function.
Unheated MSUM was desiccated and incubated (2 mg/ml)
with fresh human serum to which was added EDTA (O.OOSnl), EGTA
(O.OlM), or saline. Suspensions of MSUM or zymosan were incubated
at 37°C for 60 minutes, C’ activation was terminated with EDTA
(O.OlM), and split products of C3 and FB in the supernatants were
quantitated by crossed immunoelectrophoresis. The percent of total
area beneath the precipitin arcs of the split products gives a measure
of activation of that factor. EGTA reduced the formation of the P,A
fragment of C3 in the control tube from 25% to 9% total area, and reduced activation by MSUM from 67% to 8%. EGTA had little effect
on C3 conversion by zymosan, reducing 75% P I C area to 67%. Thus
there was sufficient free Mg++ present to allow alternate pathway ac-
ARA ABSTRACTS
tivation. EDTA completely eliminated both C3 and FB activation.
Formation of the y fragment of FB by zymosan (69% area)
and MSUM (27%) was considerable when compared to control (8%).
EGTA had little effect on FB conversion by zymosan, but reduced activation by MSUM and control to below measurable amounts. Thus
FB activation by MSUM requires activity of the classical pathway
and presumably occurs through the amplification loop.
Heating of MSUM crystals at 200°C for 2 hours caused an
average weight loss of 9.55%and for 18 hours of 10.45%. The theoreti-
62 1
cal water content of MSUM is 8.66%. Heated crystals were therefore
not MSUM, and could be demonstrated to have changes in other biologic properties.
These experiments provide additional proof that complement
activation by MSUM is not initiated through the alternate pathway.
Endotoxin, which initiates C' activation through the alternate pathway, is therefore not responsible for C3 activation by MSUM. Alternative explanations must be found to explain the effects of heating on
C' activation.
Characterization of lymphokh?-mediatedsuppression of chondrocyte glycosaminoglycan synthesis
Jerome H. Herman, C. Scott Mowery, and Marie K Dennis; University of Cincinnati Medical Center
In previous studies evaluating the pathophysiologic significance of lymphokine (LK) in the induction and perpetuation of cartilage degradation, LK derived by T cell mitogen stimulation of Hypaque-Ficoll gradient prepared human peripheral blood mononuclear
cells was shown to suppress chondrocyte glycosaminoglycan (GAG)
synthesis as assessed by 35S04incorporation (Clin Res 25:615, 1977).
In the current studies, biologic and physical properties of the responsible LK have been determined. Although spontaneous lymphocyte
release of the suppressant factor (SF) occurred, its production was significantly enhanced by T cell but not B cell mitogen continuous or
pulse stimulation. There was no significant difference in production
between 24, 72, and 120 hour cultures. Factor production was shown
to be inhibited by puromycin, to be non-monocyte dependent, and of
T-cell origin. Sonicates of mononuclear rich, glass adherent and nonadherent cells lacked activity. The generation of SF was lymphocyte
concentration-dependent, and the intensity of its response varied with
both concentration and the duration of explant exposure. The suppressive effect was totally reversible. Pretreatment of cartilage explants with lysosomal hydrolases and trypsin, but not hyaluronidase,
e n h a n d its response. Aprotinin had no effect on SF activity whereas
EACA and SBI partially inhibited response. Pretreatment of SF with
trypsin destroyed response, but neuraminidase, ribonuclease, and pronase were without effect. Activity was stable at pH 4-10 and at -20"
and -70°C for > 2 months but was destroyed by 100°C exposure and
repetitive freeze/thaw cycling. Based upon its partition coefficient,
maximal biologic activity eluted from Sephadex G-100 at a molecular
weight of 53,000 daltons. SF containing culture supernatants had lymphotoxin (LT) activity as assessed by suppression of L cell fibroblast
protein synthesis. However, SF could be clearly distinguished from
purified LT derived from human adenoidal and tonsillar tissues on
the basis of physical properties and its nonsuppressibility of release
from cells cultured in the presence of agents enhancing intracellular
levels of CAMP. Prostaglandin inhibitors failed to influence SF formation or activity. Thus a lymphokine, distinct from recognized
forms of LT, has been identified that, based upon its suppressibilityof
chondrocyte GAG synthesis, may play a potential role in altered cartilage homeostasis.
Kawasaki syndrome: Rheumatic complaints and analysis of salicylate therapy
Raquel V. Hicks and Marian E, Melish, University of Hawaii, Honolulu
Rheumatic complaints occur frequently in Kawasaki syndrome: 23 of 83 patients (27.7%), with arthralgias occurring in 8(9.6%)
and arthritis in 15(18%). Arthritis was generally a late manifestation
occurring when acute symptoms were subsiding, despite adequate salicylate therapy, with average onset at day 11. Duration of arthritis
ranged from 2-30 days, mean of 6 days. Arthritis was pauciarticular
in 17(73.9%) and polyarticular in 6(26%). Most commonly involved
joints were weight bearing joints: knees in 14, ankles in 1 I, hips in 2.
Involvement of the proximal interphalangeal joints of the fingers/toes
occurred in 4 and of the wrists in 3. Synovial fluid analysis depicted
type I1 inflammatory fluid.
Response to salicylates. Of 69 patients followed prospectively, 25 received no antiinflammatory therapy, while 44 received
ASA 80- 100 mg/kg/day from the time of diagnosis. No statistical significance was found in the total number of febrile days between
treated and untreated patients (untreated 10.08 f 2.79, treated 9.22 f
3.33, P = 0.33).
However, since some patients were treated late in their ill-
ness, 34 patients treated within the first 7 days of disease are analyzed
separately. Fever duration in these was 8.03 f 2.27 versus 10.08 days
untreated, P = 0.01, a statistically significant difference. In the total
group of 44 patients, 27 became afebrile within 48 hours, while 16 remained febrile and ill for up to 10 days on ASA. One patient died on
the ninth febrile day while on vigorous salicylate therapy. Our patients did not exhibit salicylate malabsorption: salicylate levels after
48 hours of therapy at 80-100 mg/kg were satisfactory (20. I mg average) in patients treated at all stages of disease.
Aspirin did not prevent the development of arthritis in 15 nor
the death of one patient. Since Kawasaki syndrome appears to be a
clinical expression of infantile periarteritis nodosa, antiinflammatory
therapy with aspirin may be beneficial, resulting in slight shortening
of fever in some patients, and may have an effect on platelet adhesiveness, in some way preventing coronary thrombosis.
Truly effective therapy for Kawasaki syndrome awaits understanding of its etiology and pathogenesis.
ARA ABSTRACTS
622
HLA-A, B, C, and DRw antigens in dermato- and polymyositis
Thomas J. Hirsch, The Good Samaritan Hospital, Baltimore; Kathryn Pollard, The Johns Hopkins University School of Medicine;
Roger W. Enlow, The Good Samaritan Hospital; Wilma B. Bias, and Frank C. Arnett, Jr., The Johns Hopkins University School of
Medicine
Several studies have suggested associations of certain HLA
antigens with polymyositis (PM) and dermatomyositis (DM), specifically HLA-B8 in childhood DM (Pachman et al) and B8 and B14 in
adult PM/DM (Behan et al and Cummings et al). HLA-DR specificities (B cell alloantigens), especially as they may relate in linkage disequilibrium to the B locus antigens, are unreported.
Thus, a series of 44 unrelated patients (21 whites, 23 blacks)
meeting strict criteria for PM/DM were studied. Thirty-four had pure
adult PM/DM (3 with malignancy), 2 had childhood DM, while 8
other patients had the following diseases with myositis: 5 systemic
lupus erythematosus (SLE), 2 Sjogren's syndrome (SS), and 1 progressive systemic sclerosis. All h a d HLA-A,B, and C antigens determined by a standard microcytotoxicity test, while 39 had DR typing
utilizing a two-color fluorescent cytotoxicity method. Similarly, 42
age-, sex-, and race-matched controls were fully typed. In addition,
the 1977 Oxford Workshop (WS) HLA frequencies were also cornpared to patients and matched controls.
When all 44 patients were considered, there were no HLA as-
sociations, except for a negative correlation with DRw4 (< 0.01).
Analysis by race, however, revealed B8 in 8/15 (53%) white adults
with pure PM/DM versus 28% in matched and 21% in WS controls.
DRw3 occurred in 6/12 (50%) of white adults with PM/DM versus
28% and 24% in controls (5/6 with and 1/6 without B8; DR was not
done in 3/15 adult whites with PM/DM; P = NS).Both SS patients
had B8, DRw3 and both childhood DM patients had B8, while only 1
of 5 SLE patients had B8, DRw3. In black adult patients with PM/
DM, B7 was present in 10/19 (53%) versus 17% and 25% in controls
(P< 0.025), but no DR antigens were found to be in excess. If correction of these P values is made for multiple comparisons, all lose statistical significance.
In view of our data, however, as well as those from other
studies, B8 appears to be associated with PM/DM in white adults.
The possibility of an association with DRw3 in whites and B7 in
blacks with PM/DM requires further study, as does the negative correlation of DRw4, the specificity seen frequently in rheumatoid arthritis, a disease in which myositis is uncommon.
Antibiotic therapy of septic bursitis: A prospective analysis
George Ho, Jr., Eugene Y. Su, and Alan D. Tice; Brown University Program in Medicine, Providence, Rhode Island
Septic bursitis is a common condition that requires accurate
diagnosis and appropriate treatment. Infected olecranon and prepatellar bursae, because of their superficial location, offer a unique
opportunity to study the response of a closed-space infection to therapy. The duration of antibiotic course necessary to sterilize the bursal
fluid was prospectively monitored by serial aspirations and cultures.
In a group of patients, the antibiotic regimen was continued for 5 days
following achievement of fluid sterility.
Fifteen men with septic bursitis had serial bursal fluid cultures (usually daily or every other day, but no greater than 4 days
apart). The time required to eradicate the organism is the sum of
(days with positive culture while on antibiotic) and (days between the
last positive culture and the first negative culture divided by 2). Starting with the day of the first negative culture, 9 patients received 5 additional days of antibiotic treatment to complete the prospective trial.
Staphyfococcus aureus was isolated in 14 cases and Group A
beta hemolytic streptococcus in one case. Eight patients received parenteral antibiotic, and 7 patients received oral drugs. When antibiotic
concentrations were measured, adequate penetration into the bursal
space was achieved with both routes of administration. Symptoms of
bursitis were present from one day to greater than 30 days (median 3
days). The average time required to sterilize the bursal fluid was 4.8
days (range 1.5 to 15.5) in 15 patients. The average duration of total
antibiotic therapy was 11 days (range 7 to 20) in 9 patients prospectively studied. All were cured. Using linear regression analysis, the
time required to achieve bursal fluid sterility correlated with the duration of symptoms prior to initiation of antibiotic with a Pearson correlation coefficient of 0.71 (P< 0.01).
S aureus continues to be the most common cause of septic
bursitis. Bursa1 fluid cultures remain positive longer in those patients
who had symptoms or documented infection of longer duration prior
to definitive antibiotic therapy. All 9 patients followed prospectively
were cured when antibiotic therapy was continued for 5 days following the first negative culture. Prospective studies of similar design can
be done to evaluate the medical management of certain acute bacterial joint infections.
Restriction of the immunoglobulin secreted by rheumatoid synovial tissue to a monoclonal IgG-3
Wayne L. Hoffman, Martin S. Goldberg, and J. Donald Smiley; University of Texas Health Science Center at Dallas
Rheumatoid synovial membranes from 4 patients with classic
rheumatoid arthritis were obtained at the time of joint surgery,
minced, and repeatedly washed. The synovial fragments were cultured for 6 hours in vitro in a specially prepared medium containing
agammaglobulinemic fetal calf serum and ''C-labeled amino acids.
The culture supernatant was centrifuged, dialyzed and fractionated by
DEAE-cellulose chromatography to produce an IgG-rich fraction.
This was then passed through a column of Sepharose 4B to which staphylococcal protein A had been covalently attached, and the bound
and unbound protein fractions were collected. The protein A column,
which has been shown to bind IgG-1, IgG-2, and IgG-4 subclasses
but not the IgG-3 subclass, did not bind 96-98% of the radioactivity
from the DEAE Peak 1. In this unbound fraction, 92-98% of the trichloroacetic acid precipitable radioactivity were also precipitated with
ARA ABSTRACTS
623
anti-IgG. An aliquot of this same fraction was reduced with 2-mercaptoethanol and examined by polyacrylamide gel electrophoresis.
This technique can differentiate the 60,000 molecular weight heavy
chain of the IgG-3 subclass from the 51,000 molecular weight heavy
chains of the IgG-I, IgG-2, and IgG-4 subclasses. The unbound IgG
heavy chain co-migrated with an IgG-3 myeloma marker. Preliminary data based upon mobility differences between kappa and lambda
light chains in this polyacrylamide gel system indicate that the IgG-3
subclass antibodies isolated from the rheumatoid synovial cultures
contain predominately lambda light chains.
The above results suggest that there is a specific monoclonal
response in the rheumatoid synovium to a homogeneous antigen, and
that this antigen was the same in each of the 4 patients whose synovia
were studied.
Supported by USPHS Grants AM-08418 and AM-09989.
The significance of hyperglobulinemia in systemic sclerosis
Peter A , Holt, The Johns Hopkins Hospital, Baltimore; Mary G. Kane, Southwestern Medical School, University of Texas, Dallas;
Frank C. Arnett, and Mary Betty Stevens, The Johns Hopkins University
The clinical significance of hyperglobulinemia (HG) in systemic sclerosis (SS) is not known. During the past 5 years, 87 patients
with SS have been admitted to our Rheumatic Disease Unit, of whom
20(23%) were hyperglobulinemic, defined as serum globulins of 4.0
gm/dl or greater, on one or more occasion. Per protocol, this HG
group was compared to the 67 patients with normal serum globulins
(NG).
Blacks were significantly increased in the HG group (60%
versus 21%; P s 0.0005). Otherwise, there were no demographic differences. The two groups were also comparable with respect to disease
duration, satisfaction of the proposed ARA criteria for SS, organ system involvement, and deaths. Even myositis occurred with equal freFever
HG
NG
PS
9/16(56%)
9/60(15%)
0.005
Anemia
fESR
Latex +
ANA + 2 1/320
anti-nRNP
11/20 (55%)
17/67 (25%)
0.05
20/20 ( 100%)
33/66 (50%)
11/20 (55%)
17/66 (26%)
0.05
15/20 (75%)
19/65 (29%)
0.001
7/20 (35%)
5/56 (9%)
0.025
Arteritis
4/8 (50%)
1/16 (6%)
0.05
quency (25% versus 19%).The significant clinico-laboratory differences are shown in the Table.
Serologically, rheumatoid factor, ANA in high titer, and
antibodies to nuclear ribonucleoprotein (nRNP) characterized the
HG group. Within the entire SS group, myositis was the only clinical
feature that was significantly correlated with antibodies to anti-nRNP
(6/17 or 35% with myositis versus 6/59 or 10%without myositis; P 5
0.05). Of interest, deaths were no less frequent in those with antinRNP (20%)compared to those without (15%).
Thus, in SS, HG occurs more frequently in blacks and seems
to mark a subset of patients more likely to have fever, arteritis, and
impressive seroreactivity.
0.0005
Incidence of gastroduodenal lesions and relation to aspirin type in patients with rheumatoid arthritis on chronic ASA
therapy
Stephen D. Holt, University of Missouri Medical Center, Columbia; Alan D. Morris, James H. Butt, Gerald R. Silvoso, and Kevin J.
Ivey, Harry S Truman Memorial Veterans Hospital, Columbia
There are few prospective endoscopic studies of patients with
rheumatoid arthritis (RA). We studied 57 RA outpatients who were
taking 2 8 aspirin (ASA) daily for 2 3 months, and who had neither
major gastrointestinal symptoms nor history of peptic ulcer disease.
Drug therapy consisted of regular, buffered, and enteric-coated ASA.
Twenty patients took a maximum of one other antiinflammatory
drug. There was no sigmlicant difference in the proportion of patients
on other drugs between regular and enteric-coated ASA (no patient
on buffered ASA was on a second drug). Endoscopic findings were
graded as normal or gastric and/or duodenal erythema, erosions, or
ulcer. Where more than one type of lesion was observed, only the
most serious was tabulated.
Enteric-coated ASA was associated with significantly fewer
gastric erosions-ulcers than were regular or buffered ASA. There were
significantly more erosions and total endoscopic abnormalities in the
stomach compared to duodenum (P< 0.001). There was no significant effect of a second drug.
In summary: I ) the incidence of unsuspected gastroduodenal
lesions in RA patients on chronic ASA therapy is high; 2) gastric lesions are more common than duodenal lesions; 3) enteric-coated ASA
produces less serious lesions than regular or buffered ASA in the
stomach but not in the duodenum; 4) the use of the second drug had
no obvious effect on the endoscopic findings.
Percent of patients showing gastric and duodenal abnormalities at endoscopy
Gastric
Duodenal
Type of aspirin
(no. patients)
Normal
Erythema
Erosion
Ulcer
Normal
Erythema
Erosion
Ulcer
Regular (22)
Enteric-coated (26)
Buffered (9)
Total (57)
9
35
I1
21
32
42
0
32
41
15
78
35
18
8
I1
12
82
65
78
73
14
19
11
16
4
12
I1
9
0
4
0
2
ARA ABSTRACTS
Steroid associated ischemic necrosis of the knee
David S. Hungerford, Thomas M. Zizic, and Russell Moore; The Johns Hopkins University, Baltimore
Eighteen patients with 32 knees with ischemic necrosis of
bone (INB) who have a common denominator of exogenous corticosteroid therapy have been seen since 1975. Thirteen of the 18 patients
had systemic lupus erythematosus (SLE), 3 had renal allografts, and I
each had discoid lupus and viral pneumonia. Fifteen were females
and 3 were males with ages from 1449 years, averaging 27.
All knees were classified according to the method of Arlet
and Ficat: Stage I-normal x-ray, diagnosis by biopsy; Stage IIcharacteristic x-ray showing no bone collapse; Stage III-early bone
collapse; Stage 1V-secondary arthrosis. Fifteen knees in 8 patients,
all symptomatic, were treated by core decompression that consisted of
the removal of an 8-10 mm cylinder of bone from the distal femoral
metaphysis/epiphysis. All patients undergoing core decompression
showed intramedullary hypertension and venous stasis with outflow
obstruction on intramedullary venography. The course of 15 INB sites
is shown in the Table.
Stage of INB per
treatment group
I
I1
111
No. of INB
sites
5
4
6
Of the 17 knees not treated by core decompression, 2 in Stage
I1 were asymptomatic, 2 (in one patient) symptomatic Stage I1 knees
were not done because of patient refusal, 9 in Stage 111 were sufficiently advanced to require eventual total knee arthroplasty, and 4
were treated by total knee arthroplasty.
Steroid related INB of the knees affects primarily young
adults. Core decompression of the knees appears to be as efficacious
as our previously reported results in the hips. In those patients with
significant symptoms and early x-ray changes, it provides immediate
relief of pain and may alter the course of disease in terms of radiologic progression and the requirement of further major reconstructive
surgery. Considering the alternative of total knee replacement in
young adults, this procedure deserves wider application. Longer followup will determine the full effect on the natural history of ischemic
necrosis of bone.
Progression of INB (no. sites)
Duration of followup (mos)
-__
Mean
Range
By symptom
By x-ray
26
27
19
1243
24-40
7-43
0
0
2
0
0
1
0
3
1
~~
-
To prosthesis
Systemic lupus erythematosus in the male: A genetic and endocrine study
Robert D. Inman, The Hospital f o r Special Surgery; Lois Jovanovic, M. Y. Dawood, New York Hospital; and Christopher Longcope,
The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts
Because of the female predominance of systemic lupus erythematosus (SLE) and the immunologic role of sex hormones, 16
male subjects fulfilling the American Rheumatism Association (ARA)
criteria for SLE were studied to assess genetic and hormonal status.
Buccal smears were examined in 13 of the patients and all were negative for X-chromatin. Puberty was delayed in one patient, and hair
distribution was abnormal in the same patient. Libido was decreased
in 3 patients, and impotence was present in 4 patients. Seven patients
had fathered children, none of whom had developed a rheumatic disorder.
Detailed hormonal profiling was restricted to 8 patients receiving no steroid therapy to avoid effects of exogenous corticosteroids on gonadotropin and sex hormone levels. Three patients had
elevated serum estradiol (55, 103, 30 pg/ml; normal 12-23) and elevated serum estrone (160, 155, 115 pg/ml; normal 48-100). One patient had decreased serum testosterone (134 ng/dl; normal 3W1,OOO)
with an elevated serum LH (4.2 ng/ml; normal 1.6-4.0). One patient
had elevation in both serum FSH (17.6 ng/ml; normal 1-5) and serum
LH (10.0 ng/ml). For the group of patients, the mean estradiol/testosterone ratio was 0.010, compared with a mean ratio in normal controls
of 0.003. T o assess androgen metabolism, 2 patients were given infusions of 'H-androstenedione (A) and ''C-testosterone (T). The
metabolic clearance rates for T (670 and 640 I/day/m2) and for A
(1,820 and 1,740 I/day/ni2) were within normal limits The conversion ratios of A to T (0.12 and 0.07) and of T to A (0.07 and 0.05)
were within normal limits.
While chromosomal abnormalities were absent and clinical
indicators of altered androgen status were few, the finding of hyperestrogenemia and hypoandrogenemia in some male lupus patients
supports the hypothesis that female sex hormones may create an immunologic milieu that facilitates autoimmune phenomena.
Cyclophosphamide, azathioprine, or chlorambucil for systemic lupus erythematosus nephritis
M. M. Ivanova, V. A . Nassonova, S. K . Soloviev, A. I . Speransky, V. D. Akhnazarova; Institute for Rheumatism of the Academy of
Medical Sciences of the USSR, Moscow; J. L. Decker, and A . D. Steinberg; National Institutes of Health, Bethesda
Forty patients with lupus nephritis have been entered in a
one year therapeutic trial comparing 4 drug programs. All patients received prednisone up to 0.5 mg/kg/day for control of extrarenal manifestations. The patients were randomized to receive cyclophosphamide, azathioprine, chlorambucil, or placebo in a double-blind
fashion for the first 10 weeks of the study.
The results of the first 37 patients who completed their first
10 weeks have been analyzed. Six indices (serum creatinine, proteinuria, urinary sediment, anti-DNA antibody, complement, and extrarenal manifestations) were used to measure efficacy.
The mean doses (mg/kg/day) administered were: Cyclophosphamide I .9, azathioprine 2.3, chlorambucilO.18; the mean pred-
ARA ABSTRACTS
nisone dose was 22.7 mg/day and did not differ significantly betweei
trial drug groups.
All three cytotoxic drugs were found to be significantly better
than placebo. Cyclophosphamide appeared to be somewhat superior
to azathioprine and chlorambucil. The greatest improvements on trial
drugs were in proteinuria, urine sediment, and serum complement;
cyclophosphamide was associated with the greatest decreases in antibody to DNA. None of the programs produced an improvement in
625
serum creatinine, and changes in extrarenal manifestations were approximately equal in all.
This trial confirms previous short term studies and provides
the first controlled information on chlorambucil. In view of the variable natural history of systemic lupus erythematosus renal disease,
long term information about efficacy is critical. One year followup
data will be available and will be presented.
IgG anti-DNA-rheumatoid factor complexes in sera from autoimmune MRL/1 mice
Shozo Izui, Scripps Clinic and Research Foundation, La Jolla, California; Robert A . Eisenberg, University of North Carolina, Chapel
Hill; and Frank J. Dixon, Scripps Clinic and Research Foundation
MRL/I mice are a newly developed strain that spontaneously develops glomerulonephritis, vasculitis, arthritis, and autoimmune disease characterized by high levels of anti-DNA antibodies
and rheumatoid factor (RF). When sera from 4-5 month old MRL/I
mice were subjected to sucrose density gradient ultracentrifugation,
the majority of sera contained IgG anti-DNA antibodies sedimenting
in the intermediate position between 7 s and 19s markers as well as in
the 7 s position. However, when these sera were centrifuged in dissociating buffer (0.5Macetate buffer, pH 4.0), IgG anti-DNA activity
was located only in the 7s region, and the DNA-binding activity in
the 7 s position from acid gradients was greater than that of neutral
gradients, indicating that anti-DNA antibodies in the intermediate
position are an immune complex form.
The facts that 1) DNase treatment of the sera did not influence the sedimentation rate of anti-DNA antibodies in the inter-
mediate position, 2) the DNA-binding activity of each gradient fraction was essentially identical before and after treatment with DNase,
and 3) none of the gradient fractions contained measurable amounts
of DNA, suggest that the anti-DNA antibodies in the intermediate
position were not complexed with DNA. In contrast, the anti-DNA
activity in the intermediate position was generally associated with
IgG R F activity. In addition, murine IgG-coated immunoabsorbent
columns selectively absorbed anti-DNA antibodies from the intermediate fraction as well as R F activity. Therefore, we conclude that
the intermediate anti-DNA complexes are IgG anti-DNA-RF complexes, but not DNA-anti-DNA complexes. Such complexes were
found only in sera from MRL/I mice, not in those from other autoimmune strains (NZBXNZW) F, and BXSB. Consequently, these
IgG anti-DNA-RF complexes may participate in the glomerular and
other lesions of MRL/I mice.
Arthritidarthralgia and the benign hypermobile joint syndrome
E. Forrest Jessee, Jr. and Duncan S. Owen, Jr., Medical College of Virginia, Richmond
Kirk, Ansell, and Bywaters (1966) examined 21 patients with
benign hypermobility referred for musculoskeletal complaints and
postulated that benign hypermobility may be associated with the development of osteoarthritis. Since then, we and many other clinicians
have had patients referred to us with complaints of arthritis and arthralgia that seemed to be associated with hypermobile joints. For this
reason, we decided to define a group of people with benign hypermobility in a random sample and compare that group with a control
group to determine the significance of arthritis and hypermobility. Six
hundred thirty-seven “healthy” adults at a blood bank were interviewed and examined to determine the incidence and characteristics
of benign hypermobility. Benign hypermobility was defined by the
ability to perform two of the following three maneuvers: 1) passive
hyperextension of the thumb on the volar aspect of the forearm; 2)
passive hyperextension of the fingers parallel with the dorsal aspect of
the forearm; 3) active hyperextension of the elbow beyond 200 degrees. Thirty-one people (4.9%) were found to meet these criteria.
An age- and sex-matched two-for-one control group was
compared to the hypermobile group in reference to arthralgia/arthritis, visits to a physician for arthralgia, easy bruising or bleeding, difficulty with wound healing, thin elastic skin, dislocated or sprained
joints, eye diseases, hernias, history of rheumatic fever, heart murmurs, days missed from work, and relatives with hypermobile joints.
In the hypermobile group, 3 1% complained of arthralgia/arthritis as
compared to 42% of the control group (P< 0.30). The only significant
difference between the two groups for the other above characteristics
was an increased number of relatives with hypermobile joints among
those blood donors with hypermobile joints (48%) as compared with
controls (21%) (P< 0.01). We conclude that the incidence of benign
hypermobility in the healthy adult population is 5%. We find no increased incidence of arthralgias or arthritis in the benign hypermobile
joint syndrome, and we conclude that benign hypermobility has none
of the features of Ehlers-Danlos syndrome except possible hereditary
transmission.
Glycoprotein biosynthesis by scleroderma skin fibroblasts in monolayer culture
Sergio A . Jimenez and Reza I. Bashey; University of Pennsylvania School of Medicine, Philadelphia
Confluent cultures of normal and scleroderma dermal fibroblasts were incubated with radiolabeled fucose, mannose, and glucosamine to study the biosynthesis of glycoproteins by these cells. The
cultures efficiently incorporated radiolabeled carbohydrate precursors
into macromolecules under the conditions employed. Serum supplementation was required for optimal glycoprotein biosynthesis since
ARA ABSTRACTS
626
isotope incorporation was up to 215% greater in cultures incubated in
the presence of 10% fetal calf serum. Gel filtration chromatography of
the biosynthesized macromolecules released into the culture media
was performed employing sodium dodecyl sulfate-agarose columns. It
was found that the normal and scleroderma cultures synthesized one
major glycoprotein species that eluted near the void volume of the
columns. This glycoprotein was isolated and rechromatographed after
reduction of disulfide bonds with P-mercaptoethanol. There was no
apparent change in the chromatographic profiles upon reduction, suggesting that the labeled glycoprotein was probably nor fibronectin.
Marked differences between normal and scleroderma fibroblasts were
noted in the relative distribution of the biosynthesized glycoproteins.
In normal cultures, less than 30% of the incorporated fucose appeared
in the large molecular weight species, whereas in scleroderma cultures
this fraction represented up to 60% of the total radioactivity incorporated. In the absence of serum, normal and scleroderma fibroblasts
synthesized a greater proportion of the large molecular weight glycoprotein relative to other labeled molecules, but in all cases scleroderma cultures synthesized greater amounts of this macromolecule
compared to control cultures. Although the significance of this large
molecular weight glycoprotein is not clear at present, its increased
synthesis by scleroderma fibroblasts may be of importance in the
pathogenesis of the connective tissue alterations present in this disease.
Treatment of knee arthritis with the spherocentric total knee prosthesis
Douglas M. Joseph and C. McCollister Evarts; University of Rochester Medical Center, Rochester, New York
The first 47 patients treated with a spherocentric total knee
replacement were reviewed with a followup of up to 2 years postoperatively. All of the patients’ pre- and postoperative histories, physical exams, and x-rays were reviewed.
Until the advent of the spherocentric knee, the only alternative to arthrodesis in severely deformed arthritic knees was the use of
a metal-on-metal uniaxial hinged prosthesis. The spherocentric knee
prosthesis permits triaxial rotation of the knee joint while maintaining
intrinsic stability. Because of its favorable biomedical features, the
spherocentric knee was selected for use in the patient with a severely
deformed and grossly unstable knee.
The indications for surgery were severe pain with gross deformity and instability of the knee. Also, the procedure was performed in patients whose previous knee arthroplasties had failed.
Each patient received antithrombotic drugs in the form of sodium
warfarin or low molecular weight dextran and antibiotics. All of the
operations were performed by the senior coauthor in an operating
room perfused with ultraviolet light.
Each patient was evaluated in terms of pain, range of motion, stability, angular deformity, and ambulatory status both pre-and
postoperatively. All of the patients improved dramatically postoperatively at only 6 months, and they continued to improve over the
followup period. The complications were exceedingly low as compared to other total knee arthroplasties.
The spherocentric knee prosthesis used in this group of patients provided excellent results when applied to the severely deformed and grossly unstable knee. We believe this prosthesis is the
model of choice for this type of patient.
A controlled study of group counseling in rheumatoid arthritis: Improvement in understanding of disease and patient
self-concept
S. KapIan, F. Kozin; Medical College of Wisconsin, Milwaukee; K. Rytel, Milwaukee Psychiatric Hospital
The value of group counseling and a formal patient education program was studied in subjects with rheumatoid arthritis (RA).
Patient responses were quantitated by use of several psychometric
tests and an objective test of information provided during the educational session.
Thirty-four women were paired according to age, race, marital status, educational level, and socioeconomic position, and were
randomly assigned to an experimental group or control group. Both
groups attended a comprehensive educational session in which the
pathophysiology and therapy of RA were reviewed. Thereafter the experimental group attended 12 weekly meetings led by a patient counselor and psychiatrist. Free discussion was encouraged but specific
questions regarding their disease were not permitted.
Patient responses to several psychometric tests (Zung Depression Scale, Tennessee Self-Concept Scale, Human Service Scale)
and to the patient education test of factual information were measured before and after the educational session and again after 12
weeks. Tests were evaluated for patients’ I ) understanding of their
disease, 2) level of depression, 3) adjustment to role and daily tasks, 4)
interpersonal relationships, and 5) response to therapy.
Both groups demonstrated the expected improvement in the
understanding of their disease immediately after the patient education
session. After 12 weeks, only the experimental group showed further
gains on the patient-education test (Pc 0.05, paired r test). Definite
improvement was noted in self satisfaction (P c 0.01) and family-self
(P c 0.05) in the experimental group, but no significant changes were
seen in the control group. Depression levels or overall disease activity
did not change appreciably in either group.
The formal education session clearly broadened our patients’
understanding of their disease, and these gains were extended by attendance at weekly group counseling sessions. Group counseling significantly improved certain areas of self concept and appeared to help
patients become more realistic in their goals and expectations. These
may be important adjuncts in the total care of patients with RA.
Lymphocyte depletion by continuous flow cell separation in rheumatoid arthritis
Jacob Karsh, Daniel G. Wright,John H. Klqpel, Albert B. Deisseroth, W a p e F l y , and John L. Decker; National Institutes of
Health, Bethesda
The lymphocytic infiltration of the synovium and the beneficia1 effects of lymphocyte depletion by thoracic duct drainage estab-
lish the pivotal role of lymphocytes in rheumatoid inflammation. The
continuous flow cell centrifuge (CFC) permits the removal of selected
ARA ABSTRACTS
circulating cell populations by using peripheral venous access. We
have studied the effect of lymphocyte depletion in 4 patients with
seropositive, erosive RA which has been refractory to conventional
therapy, including gold and/or D-penicillamine. All patients had a
Ritchie-Camp Articular Index (AI) of greater than 40 and were in
Functional Class I11 or IV.
Forearm arteriovenous fistulae were constructed to allow repetitive venous access for 3-hour CFC treatments 3 times a week for a
period of 5 to 6 weeks. In the course of 14 to 18 treatments per patient,
a mean total of 11.1 X 10'" lymphocytes and 2.1 X 1 0 ' O monocytes
was removed. Perfect separation was not technically possible; compared to peripheral blood, the relative removal of mononuclear cells
2x that of platelets, 1% that of plasma, 3% that of granulocytes, and
70X that of hemoglobin. A biweekly blood transfusion was necessary
to prevent anemia. A decrease in circulating lymphocytes (44 f 25%;
mean f SD) was observed but there was no decrease in granulocytes,
627
platelets, or serum proteins. The procedure was free of major complications. In one patient 3 treatments were terminated for chills and low
grade fever attributed to endotoxin in the tubing; her other treatments
were uneventful.
During the first week of therapy, improvement was observed
in morning stiffness and joint swelling in all patients. By the end of
the treatment period, the A1 had fallen by 68 f 6% and swollen joints
by 72 f 1%. Two patients entered an improved Functional Class.
Changes in grip strength, sedimentation rate, and titers of rheumatoid
factor were inconsistent. In followup all patients have maintained improvement of at least 4 weeks. The first patient developed a brisk relapse of RA 18 weeks after termination of CFC treatment.
These observations on an extremely variable disease warrant
controlled studies to establish the role of cell depletion in the long
term management of rheumatoid arthritis.
Risk of pregnancy in mixed connective tissue disease
Ronald L. Kaufman and Rodanthi C. Kitridou; University of Southern California School of Medicine, Los Angeles
Pregnancy in systemic lupus erythematosus (SLE) is associated with increased fetal wastage and maternal morbidity. Mixed connective tissue disease (MCTD) patients share many disease features
with SLE patients. A retrospective review was therefore conducted to
determine if this increased morbidity was also associated with pregnancy in MCTD.
Patients included in the study were: 1) 28 patients with SLE
as per American Rheumatism Association criteria; 2) 32 patients with
MCTD as described by Sharp et al; 3) 5 1 consecutive admissions to
labor and delivery as a control obstetrical population. Fertility rates
(total pregnancies/total pregnant patients) prior to disease onset were
comparable for MCTD, SLE, and controls, being 4.3, 2.9, and 2.8 respectively. After disease onset, significant decreases (P < 0.05) were
noted in MCTD (1.7) and SLE (1.9). Similarly, the parity rates (total
live birthshotal pregnant patients) before disease onset were of equal
magnitude in MCTD (3.3), SLE (2.l),and controls (2.6). After diagnosis these rates decreased significantly (Pc 0.05) to 0.4 (MCTD) and
0.7 (SLE). Interestingly, prior to diagnosis there was a significantly
higher rate (P< 0.05) of total fetal loss in the MCTD (24%) and SLE
(28%) groups as compared to controls (10%). After disease onset, this
increased significantly (P < 0.05) to 76% (MCTD) and 64% (SLE).
The increased fetal wastage was evenly distributed among spontaneous abortions, stillbirths, and therapeutic abortions. There were no
fetal abnormalities noted in either of the patient groups. Forty-seven
percent of MCTD and 31% of SLE pregnancies were associated with
disease-related maternal morbidity. Five MCTD patients had disease
onset during pregnancy while 3 others had an exacerbation of disease
activity. One SLE patient had onset during pregnancy, while 8 patients had increased disease activity.
It is our observation that MCTD shares the same increased
maternal and fetal morbidity with SLE. We suggest that pregnancy in
patients with MCTD be managed with the same caution as pregnancy
in SLE.
Scleroderma: A model to explain collagen accumulation in the deep dermis
Anthony J. Keyser, Sheldon M. Cooper; University of Southern California School of Medicine, Los Angeles; Erkki Ruoslahti, City of
Hope Medical Center, Duarte, California; Francisco P. Quismorio, clnd Marcel E. Nimni, University of Southern California School of
Medicine, Los Angeles
In progressive systemic sclerosis (PSS) there is selective accumulation of collagen in the deep (reticular) dermis of involved skin.
We examined by indirect immunofluorescence the distribution of
fibronectin in sections of normal skin and involved and uninvolved
skin from PSS patients. The reticular area of involved PSS skin was
markedly enriched in fibronectin with a distribution similar to that of
collagen, while subepidermal (papillary) areas were unremarkable. To
further study this depth-related accumulation, fibroblasts from explanted papillary and reticular regions of normal cadaver skin were
exposed for 42 hours to PSS or non-PSS sera in the presence of 'Hproline and ascorbic acid. Radioactive collagen, purified by repeated
salt precipitation after limited pepsin digestion of cell layers and me-
dia, was increased 1% to 3-fold in reticular cultures with PSS sera as
compared to non-PSS sera, while papillary cultures with PSS sera had
30-50% less radioactive collagen than they did with non-PSS sera.
When radioactive hydroxyproline was assayed as a measure of total
collagen biosynthesis, both papillary and reticular cultures made I YZ
times as much with PSS sera as with non-PSS sera. Thus both papillary and reticular cultures are stimulated to increase collagen biosynthesis by PSS serum, but intact collagen molecules accumulate only in
reticular cultures. Reportedly, collagenase activity is confined to the
papillary dermis. In cell cultures, collagenase is present in the form of
an inactive precursor that may be converted to the active enzyme by
pepsin during collagen purification.
ARA ABSTRACTS
628
Our in vitro data are consistent with the following mechanism for collagen accumulation in skin of PSS patients: A circulating
factor stimulates collagen biosynthesis in both reticular and papillary
dermis. Papillary cells have the capacity to degrade collagen, while reticular areas that are lacking collagenase will accumulate collagen.
The result would be a net increase in collagen in the reticular dermis,
the primary cause of which is the blood-borne circulating factor. The
increased fibronectin in reticular dermis may be indicative of overall
metabolic stimulation, and may also play a role in inhibiting the action of collagenase diffusing from the papillary dermis.
Supported in part by Kroc Foundation and Grant #CA 21279
from National Cancer Institute.
Evidence of decreased survival in ankylosing spondylitis by life-table analysis
Muhammad A. Khan, Mohammad K. Khan, and Irving Kushner; Case Western Reserve University, Cleveland
A recent report indicates an excess mortality from a wide
range of causes in patients suffering from ankylosing spondylitis (AS).
The mortality rates were computed from person-years of observation;
such computations represent an average over the entire length of the
study period and do not provide clear information as to whether increased mortality is evenly distributed throughout the period of study
or whether there is a disproportionate excess mortality at some particular stage of the disease.
In order to confirm the finding of increased mortality in AS
by an alternative statistical approach, and to determine when excess
mortality occurred during the course of the disease, we investigated
survival by life-table analysis, using the method of Kaplan and Maier
(J Am Stat Assoc 53:457, 1958). We studied 56 white patients with AS
(7 of them females) who until December 1975 had been followed for a
mean duration of 22. I years since diagnosis. They had initially been
diagnosed by Dr. R. M. Stecher between 1934 and 1960 in the course
of his studies of AS. All patients met the New York criteria, and
HLA-B27 was present in all of the 14 patients who were tested. Mean
age at diagnosis of AS was 35.2 years, and mean duration of symp-
toms before diagnosis was 8.7 years. Aspirin and physical therapy
were the main forms of treatment; only 4 patients received deep x-ray
therapy.
By December 1975, 30 patients had died, 23 were alive, and 3
were lost to followup. The observed and expected survival curves
were obtained expected values derived from life-tables were matched
for race, age, sex, and calendar year with the patients studied. Comparison of the observed and the expected survival curves revealed that
decrease in survival in AS patients becomes detectable after 10 years
and becomes significant (P< 0.05) by 15 years after diagnosis. Causes
of death could be ascertained in 22 (73%) of the 30 patients that had
died; these included cardiovascular causes in 6, cerebrovascular
causes in 5, respiratory diseases in 4, malignancies in 3, and other miscellaneous causes in the remaining 4 patients. These findings support
the recent report of excess mortality in patients with AS and indicate
that a significant decrease in survival begins to be detectable by 15
years after diagnosis. Alternatively, it may be that only patients with
AS severe enough to seek specialized care are at increased risk.
Genetic heterogeneity in primary ankylosing spondylitis
Muhammad A. Khan, Irving Kushner, Case Western Reserve University, Cleveland; and William E. Braun, The Cleveland Clinic
Foundation
Since ankylosing spondylitis (AS) can develop in individuals
who do not possess the HLA-B27 gene, a search for other genetic
bases for this disease has been undertaken. Clinical observations have
suggested that in B27-negative individuals, genetic predisposition to
psoriasis and chronic inflammatory bowel disease (IBD) may similarly predispose to AS even without the development of psoriasis or
IBD. In Holland, van den Berg-Loonen et a1 have suggested that
HLA-Bwl6 is associated with primary AS and with AS in IBD in
B27-negative individuals. Bw16 is known to be associated with psoriasis.
We have studied 160 patients (35 black and 125 white) with
primary AS (unassociated with Reiter’s syndrome, psoriasis, or IBD)
and 545 normal controls (60 black and 485 white), typed for 28 HLA
antigens of A and B loci. Eighteen black patients were found to be
B27-negative; we have previously reported an association between AS
and HLA-B7 in these individuals. Ten white patients were B27-negative and 4 of them were noted to possess HLA-Bwl6 (40%), compared
to 3.09% of 444 B27-negative controls (2with Yates’ correction =
25.87; P < 0.0005; P, < 0.014).
These data indicate an association between B27-negative primary AS and HLA-Bw16 in whites, confirming the findings of
van den Berg-Loonen et al. The results support the clinical suggestion
that in B27-negative individuals, genetic predisposition to psoriasis
and IBD may also predispose to the development of AS, and indicate
that susceptibility to AS in such individuals is associated with HLABw16. Moreover, the finding that B27, B7, and Bw16 are all associated with primary AS suggests that AS May represent a heterogeneous group of phenotypically similar diseases with different genetic predisposing factors.
Computerized axial tomography of central nervous system lupus
Paul J. Killian, Donald J. Schnapf; and Oliver J. Lawless; Walter Reed Army Medical Center
A blind prospective study of 36 patients with systemic lupus
erythematosus (SLE) was performed to assess the role of comput-
erized axial tomography (CAT) scans in the evaluation of central nervous system (CNS) lupus.
ARA ABSTRACTS
These patients (16 with CNS involvement and 20 without)
were scanned on an EM1 5005 CAT scanner with a 160 X 160 matrix.
Scans were performed with and without contrast, at fast and slow
speed. All scans were read independently by two neuroradiologists
whose only knowledge of the patients was their age and the diagnosis
of SLE. The patients were matched for age, sex, and steroid treatment.
Perisulcal atrophy was the predominant CAT finding. Although atrophic changes can be normally seen in patients over age 40,
our accuracy in this age group of CNS lupus (6 patients) was 100%.
Other findings included ventricular dilatation and infarction. Perisulcal atrophy was best seen on scans performed at slow speed. Contrast enhancement was noncontributory. There was 10046 agreement
between the two neuroradiologists.
629
Of 16 patients with CNS disease, I2 (75%) had unequivocal
positive scans; 3 (19%) had unequivocal negative scans; and 1 (6%)
was equivocal. Of 20 patients without CNS disease, 2 (10%) had unequivocal positive scans; and 2 (10%) were equivocal.
CAT scans are a sensitive but nonspecific means of evaluating CNS lupus. The presence of perisulcal atrophy in a patient
with SLE and no other explanation for cortical atrophy is a good indicator of CNS lupus. Since contrast enhancement was not shown to
be beneficial and in view of its potential for precipitating acute renal
failure, these agents should be avoided.
CAT scans offer a safe, reproducible, noninvasive method for
evaluating CNS lupus. It is the only currently available method for
the in vivo demonstration of findings compatible with the reported
pathologic changes in CNS lupus.
Clinical efficacy of high dose intravenous methylprednisolone pulse therapy in systemic lupus erythematosus
Robert Kimberly, Michael Lockshin, Hospital for Special Surgery Raymond Sherman, Cornell University Medical College; J. Steven
McDougal, Robert Inman, and Charles L. Christian; Hospital for Special Surgery
We have studied the clinical efficacy of high dose intravenous methylprednisolone in 28 systemic lupus erythematosus (SLE)
patients, 25 with active renal disease and 3 with nonrenal manifestations, who had either I) marked side effects with previous course(s) of
high dose oral steroids, 2) no response to oral steroids (6 patients), or
3) new onset of disease during adolescent growth (2 patients). Seventeen of 25 renal patients had recent increases in serum creatinine
(ScR);15 of 25 had recent increase in 24 hour urinary protein (Upro);
one patient had fixed but marked renal insufficinecy. Patients received
I gram intravenous methylprednisolone pulse therapy (IV-MP) daily
for 3 doses, except 4 adolescents who received 15 mg/kg/dose. Patients continued their prior medications after IV-MP adjustments in
oral steroids were made within the month following IV-MP in 4 patients. Azathioprine was increased or added in 3 patients during the
entire followup period.
Four patients had a very rapid (<2 weeks), large and sustained (24 months) improvement in renal manifestations; 4 patients
(average SCR5.7 mg/dl at time of IV-MP) progressed to dialysis (no
followup while on dialysis); one patient died with continuingly fulminant disease 8 weeks after IV-MP. Overall 1 I of 23, I I of 20, and 9 of
18 patients had improvement in SCR at 1, 2, and 4 months followup,
respectively; 8 of 18,7 of 18, and 8 of 14 had decreased 24 hour Upro
at 1, 2, and 4 months. Anti-DNA antibodies (Farr, %) fell in all patients and total hemolytic complement increased in all but 2 ( I with
normal pre-IV-MP value; 1 with probable complement deficiency) at
1 month. Serologic improvement was sustained one patient had a
major serologic flare at 4 months. Serial immune complexes (C Iq-BA,
Staph A-binding) fell in most patients but did not appear to be predictive of clinical outcome. No change in clinical status of the 3 patients with nonrenal disease was apparent.
IV-MP in SLE may improve I) renal function and 2) serologic parameters with rapid and large improvement in a subgroup
with nephritis. Renal biopsy has not predicted this subgroup response,
but recent rapid deterioration in function may be a common feature.
IV-MP Was not helpful in our small sampling of nonrenal patients.
Acute corticosteroid-induced depression of renal function in systemic lupus erythematosus
Robert Kimberly, Michael Lockshin, Hospital for Special Surgery Raymond Sherman, Cornell University Medical College; J. Stephen
McDougal, Hospital for Special Surgery Charles Steinman, Mt. Sinai School of Medicine, New York; and Charles L. Christian,
Hospital for Special Surgery
The occurrence of large transient changes in renal function
in several patients with lupus nephritis (LN) who received high dose
intravenous methylprednisolone (IV-MP) prompted a systematic
study of 24 patients receiving 1 gm IV-MP daily for 3 doses. Renal
function was followed serially including daily 24 hour urines in I6 patients. All patients had serum creatinine (ScR) determined l) as a
baseline within 30 hours prior to the first dose of IV-MP, 2) at least
once immediately antecedent to baseline, 3) at least twice during the
3-day treatment period, 4) in 21 of 24 cases at least once in the immediate posttreatment period. All patients ate at least 5 gm salt daily; no
patient was dehydrated at the time of IV-MP. SCRincreased in all patients during IV-MP and in 20 of 24 within 18 hours of the first dose.
The mean increase in SCRat first determination during IV-MP was
0.34 mg/dl (P< 0.001); peak increase was 0.5 1 mg/dl (P< 0.001). This
change did not result from increasing SCRprior to IV-MP since baseline and antecedent values were not statistically different. SCRfell after IV-MP and became indistinguishable from baseline values within
4 days. BUN showed similar increases (mean elevation at first IV-MP
determination: 8.3 mg/dl, P < 0.005; peak elevation: 19.6 mg/dl, P <
0.001) but did not return to baseline within 4 days. Creatinine clearance (CrCl) showed changes concomitant with SCR.Quantitative 24
hour urinary protein (Upro) fell significantly within the first 24 hours
of IV-MP (mean decrease 860 mg/24 hour, P < 0.025) but was indistinguishable from baseline in the first two posttreatment days’ collections. CrCl and Upro were significantly correlated (r = 0.45; P <
0.001). No apparent change in Farr, CH50, immune complexes (ClqBA; Staph A-, binding), or free DNA was seen to suggest an immune
mechanism (Blantz, JCI 61:910, 1978).
ARA ABSTRACTS
630
Transient changes in renal function with IV-MP in SLE appear to represent drug-induced hemodynamic alterations. Immediate
decrements in Upro reflect altered CrCl and must be distinguished
from fundamental change in underlying LN. The kinetics of these
changes, their absence in normal controls (Webel, J Lab Clin Med
80:765, 1972), and the ability of corticosteroids to inhibit phospholipase raise the possibility of prostaglandin synthesis inhibition as the
underlying mechanism, analogous to aspirin.
Phagocytosis of immunoglobulin-containing inclusions from sera of patients with ankylosing spondylitis
T. D. Kinsella, Calgary General Hospital, Calgary, Alberta, Canada
Recent studies have shown that normal polymorphonuclear
leukocytes (PMN) phagocytose immunoglobulin-containing inclusions (ICI) in vitro from sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Accordingly, the
present studies were conducted to ascertain if sera of patients with primary AS would give rise to ICI on exposure to PMN.
To date, sera from 64 AS, 14 SLE, 18 RA, and 48 normal
controls have been tested. Normal PMN were isolated by gravity sedimentation in 0.5% gelatin, washed, and resuspended in Hank‘s solution to give lo6 PMN/O.I ml. Test sera (0.2 ml) were incubated with
lo6 PMN x 90 minutes at 2SoC, washed, and smears prepared in a
cytocentrifuge. Unfixed smears were processed for direct fluorescent
antibody staining to detect combined (polyvalent) or individual, inclusions of IgG, IgM, and IgA, and C3. Positive cells were arbitrarily
considered to contain at least one large and/or three medium-size inclusions; multiple small inclusions were considered negative. Cell and
serum controls in each run included donor PMN stained directly and
after incubation in autologous plasma, and known positive (SLE) and
negative sera; test sera were scored “blind.” Antibody controls included blocking and absorption methods. For each test serum, 200
PMN were counted to determine the percentage of positive cells, according to the formula: % positive PMN in test serum minus % positive PMN in direct smears of cell donor.
To date, controls have shown 2.4 f 1.7 combined ICI (mean
f SD%); AS, 6.2 f 6.4 (P< 0.001); SLE, 22.4 f 20.1 (P< 0.001); and
RA, 10.3
12.4 (P < 0.001). With monospecific antisera, AS-ICI
preferentially express IgG more than IgM; IgA is uncommon. C3 is
present in 20% AS-ICI, relative to combined ICI. The latter were
present (normal mean + 2 SD) in 35 of 64 sporadic AS sera. Serial
testing of 1 I AS for combined ICI has shown significant positivity in
12 of 33 sera.
These results demonstrate that AS sera, sporadically and sequentially, give rise to in vitro ICI. Since studies in SLE and RA indicate that ICI represent immune complexes, it appears that at least
50% of AS sera sporadically contain immune complexes. This finding
may be of pathogenetic significance in patients with AS.
*
A four-year prospective study of gold and antimalarials in rheumatoid arthritis
Allan B. Kirsner, Robert P. Sheon, Robert I. Finkel, and Stephen J. Farber; Medical College of Ohio, Toledo
A prospective, randomized, controlled study of gold and
antimalarials in rheumatoid arthritis (RA) was begun in 1969. Eightyseven patients with classic rheumatoid factor positive RA by ARA
criteria, who had been under a basic home treatment program for
three months or longer, were entered into the study. Patients were
randomized into a gold group, antimalarial group, or control group.
Four-year followup data were available on 80 patients. All patients
received high dose salicylate, physical therapy, and appropriate intraarticular aspiration and injection. Short term usage of phenylbutazone and/or indomethocin was allowed in all three groups. The gold
group received 50 mg of gold salt per week; after lo00 mg the frequency of dosage was decreased. The antimalarial group received 250
mg of chloroquine phosphate per day or 400 mg of hydroxychloroquine per day. Patients allergic or unresponsive to either gold
or antimalarials were switched to the opposite drug. A striking decrease in the development of vasculitis, neuropathy, and rheumatoid
lung disease was noted in the gold and antimalarial groups. The gold
and antimalarial groups did better than the control group in measurements of grip strength, sedimentation rate, protein bound hexoses,
ring size, morning stiffness, and physician’s assessment. Rheumatoid
factor was unchanged. There was little difference between patients
treated with gold or antimalarials.
Developed vasculitis
Developed rheumatoid
lung disease
Required
immunosuppressives
Gold
Chloroquine
Control
2/27
2/22
9/3 1*
0/27
0/22
5/31*
1/27
2/22
9/3 1
* Two patients developed both vasculitis and rheumatoid lung disease.
Stimulation of fibroblast prostaglandin synthesis by supernatants of human mononuclear cells
Joseph H . Korn, University of Connecticut Health Center, Newington; Perry K Halushka, and E. Carwile LeRoy; Medical University
of South Carolina, Charleston
Immune cells may modulate connective tissue metabolism in
a number of rheumatic diseases. We have previously shown that supematants of human peripheral blood mononuclear cell cultures are
capable of suppressing fibroblast (FB) proliferation by stimulation of
endogenous F B prostaglandin E (PGE) synthesis. The origin and na-
ture of this PGE stimulatory-growth suppressive activity were further
investigated.
Proliferation of neonatal foreskin fibroblasts was assessed by
’H-thymidine (3HTdR) incorporation and PGE production by radioimmunoassay. Supernatants of human mononuclear cells cultures
ARA ABSTRACTS
stimulated FB iPGE synthesis to greater than 70 times baseline levels
(81 versus 1.1 ng/104 cells/24 hours). the increased iPGE synthesis
was associated with marked suppression of FB ’HTdR incorporation
(70-90%). Inhibition of FB iPGE synthesis with indomethacin also inhibited the growth suppression; the growth suppression could be restored by addition of exogenous PGE2. The concentration of exogenous PGE required to suppress FB proliferation (50 ng/ml) was
similar to that achieved in mononuclear cells-supernatants treated FB
cultures. Suppression of FB proliferation also occurred with addition
of dibutyryl cyclic AMP (lO-’hf).
Depletion of T lymphocytes from mononuclear cells cultures
had little effect on the generation of FB growth suppressive and PGE
stimulatory activities in the mononuclear cells-supernatants. Conversely, depletion of adherent cells resulted in a marked loss of activity. The PGE stimulatory-growth suppressive activity in mononuclear
63 1
cells-supernatants was nondialyzable and was destroyed by heating
(56°C for I hour) or trypsin exposure. Both the growth suppressive
and iPGE stimulatory activities in mononuclear cells-supernatants
eluted in the same peak on Sephadex G-100 molecular sieve chromatography with an approximate molecular weight of 10-20,OOO daltons.
The ability of mononuclear cells-supernatants to stimulate FB iPGE
synthesis and suppress proliferation could be absorbed by incubation
of supernatants ( I hour at 4OC) with FB but not by similar incubation
with lymphocytes.
Adherent mononuclear cells thus elaborate a factor(s) capable of suppressing FB proliferation by alteration of PGE and cyclic
AMP metabolism. This interaction between immune cells and FB
may play a role in the evolution of certain inflammatory and fibrotic
lesions.
The rheumatic presentation of osteomyelitis
Helen K . Kornreich, Bram H. Berstein. Karen K. King, Bernhard H. Singsen, Fred A . Lee, Hart Isaacs Jr., and Virgil Hanson;
University of Southern California School of Medicine, Los Angeles
This report describes the clinical, laboratory, and pathologic
features of 6 children with osteomyelitis whose prolonged clinical
courses were all characterized by prominent rheumatic symptoms.
There were 3 males and 3 females with age at onset ranging from 2
years to 13 years. Joint pains and limping were present in all; 3 had a
history of preceding trauma. Duration of time from onset of symptoms to diagnosis ranged from 2 months to 4 years. All children were
referred to the rheumatology service with a presumptive diagnosis of
juvenile arthritis (JRA). Three distinct patterns of disease were found
1) Two children developed prominent joint symptoms distant
from the primary site of osteomyelitis. In one, joint pain and swelling
persisted for 2 months in 3 large joints before the diagnosis of osteomyelitis in the left femur was established by bone biopsy. In the other,
constitutional symptoms and joint pains in the lower extremities continued for 1 year until an asymptomatic Brodie’s abscess was detected
in the right humerus. Bone cultures revealed coagulase-positive staphylococcus aureus in both.
2) In the second pattern, sterile synovial fluid cultures in a
child with a 3-month history of ankle pain and swelling led to a diagnosis of monarticular JRA until followup x-rays showed osteomyelitis
of the adjacent distal tibia.
3) In the third pattern, 3 children developed recurrent joint
pain, swelling, and mild constitutional symptoms over a course of 7
months, 2 years, and 4 years. Muhifocal osteolytic lesions were found
radiographically in the metaphyses of long bones in all 3; in addition
I child had clavicular involvement. Cultures from blood and bone
were negative in all. Bone biopsies revealed nonspecific inflammatory
changes consistent with subacute osteomyelitis. One child showed
healing metaphyseal lesions after removal of the diseased clavicle.
The other 2 showed slow improvement over the course of followup.
Subacute or chronic multifocal osteomyelitis becomes a difficult diagnositc problem in children who present with joint symptoms
and often sterile synovial fluid, blood, and bone cultures. The long
lasting or recurrent chronic osteitis suggests an interesting parallel to
the chronic synovitis of JRA.
The treatment of NZB X NZW disease with total lymphoid irradiation
Brian L. Kotzin and Samuel Strober, Stanford University Medical Center
NZB X NZW mice spontaneously exhibit an autoimmune
disease similar to that seen in systemic lupus erythematosus. We have
been able to demonstrate that total lymphoid irradiation (TLI) produces potent immunosuppression and a marked susceptibility to tolerance in normal laboratory animals, as well as marked immunologic
alterations in humans with Hodgkin’s disease. We therefore attempted to measure the effect of TLI on already well expressed NZB
x NZW disease. Littermate female mice, 5-7 months old (mean = 6.0
months) with documented proteinuria were randomly assigned to a
treatment group with 24 animals (total dose = 17 fractions x 200
rads) or a control group with 24 animals. The irradiated animals have
shown significantly less proteinuria at all points of measurements after treatment. After 5 months of evaluation (mean age = 11 months),
none of the treated and 17 of the control animals have advanced to
high grade (>2+) proteinuria. One of the 24 treated animals has died
of nonrenal causes, while 16 of 24 control animals have died with 14
renal deaths (P<O.oooOl). Preliminary studies of anti-DNA antibodies have demonstrated lower titers in treated animals than in control animals. In a further study of very advanced disease, 7-month old
female animals with advanced ( 3 4 + ) proteinuria were treated. All 5
control animals died within 6 weeks while 9 of 12 treated animals remain alive 4 months after irradiation (P< 0.01). many with markedly
suppressed amounts of proteinuria.
We have demonstrated a profound suppression of NZB m
NZW disease by TLI. This and other evidence suggest that lymphoid
irradiation may be unique in its immunosuppressive properties, perhaps suited for the treatment of autoimmune disease in humans.
ARA ABSTRACTS
632
The treatment of rheumatoid arthritis with regional lymphoid irradiation
Brian L. Kotzin, Samuel Strober; Stanford University; Shimon Slavin, Hadassah Hebrew University, Jersualem, Israel; Michael
Gottlieb, Andrei Calin, James Fries, Richard Hoppe, and Henry S. Kaplan; Stanford University; Stanford, California
In the course of investigating the cellular basis of the immunodeficiency in patients with Hodgkin’s disease, we have demonstrated profound long lasting immunologic alterations after radiotherapy. We have subsequently demonstrated that lymphoid
irradiation produces a potent immunosuppression in laboratory animals. We, therefore, designed a prospective study to look at the effects
of regional subdiaphragmatic lymphoid irradiation on patients with
intractable rheumatoid arthritis. Six patients with severe progressive
seropositive erosive disease, who failed conventional therapy (including gold and penicillamine) and who were considered candidates for
cytotoxic drugs, have been entered into the study and evaluated. Radiation was given to an “inverted Y” field that includes paraaortic,
iliac, and inguinal lymph nodes. Patients were assigned by therapists
to high dose irradiation (3600 rads in 200-rad fractions) followed by
low dose placebo or vice versa, while patients and observers were not
aware of the dose of irradiation. Followup has ranged from 4 months
to 2 years after the high dose treatment. Four of the 6 patients re-
sponded to the high dose regimen by a decrease in their disease activity as measured by joint score and duration of morning stiffness.
There were no complete remissions, and all patients required at least
continued nonsteroidal antiinflammatory drugs. The duration of the
response has been long lived (greater than 8 months) but variable. All
6 patients showed expected decreases in the absolute lymphocyte
counts, the percentage of T cells, and the in vivo response to PHA and
increases in the percentage of B cells. Other than transient nausea and
fatigue, the irradiation was well tolerated and there have been no serious complications of irradiation to date.
We have demonstrated that regional subdiaphragmatic irradiation, a regimen chosen for lack of toxicity, can decrease disease activity in intractable rheumatoid arthritis. We are now planning to
study lymphoid irradiation to both sub- and supra-diaphragmatictissues at a reduced total dose in order to obtain greater immunosuppression as suggested by our animal experiments.
Heterogeneity of glomerular immune complexes in patients with systemic lupus erythematosus
Noriyuki Kurata, Ikuo Hara, Shoji Mywaki, and Tadashi Ofiji; O k a y m a University Medical School, Okayma, Japan
The relation of heterogeneity of glomerular immune complexes to immunologic abnormalities in sera from patients with systemic lupus erythematosus(SLE) was investigated. Renal tissues from
141 patients with SLE were tested by immunofluorescent technique.
Immunofluorescent glomerular stainings were classified into 5 distinct
patterns. Briefly, linear pattern (Lin-p) showed no granular immune
complex. Mesangial pattern (Mes-p) showed immune complex localized in mesangium. Subepithelial immune complex was observed in
membranous pattern (Mem-p). Subendothelial, subepithelial, and
mesangial immune complexes were found in granular pattern (Granp). Massive deposition of subendothelial immune complex caused diffuse obstruction of glomerular capillary tufts in lumpy pattern (Lump). All sera for immunologic tests were obtained from acute stage of
the disease. Antibody to double stranded-(ds) DNA was measured by
modified Farr’s assay. Antibodies to acidic nucleoprotein (Sm) and
nuclear ribonucleoprotein (RNP) were determined by counter immunoelectrophoresis. Hemolytic activity of serum complement (CHSO)
was also tested. Elution study was performed on 7 of autopsied kidneys according to the method reported before.
The mean value of dsDNA binding was significantly high in
groups with Gran-p and Lum-p compared with that in other 3 groups
(P< 0.01). Fifteen patients with definite renal damage showed no abnormal binding in spite of multiple tests during acute stage. The incidence of antibody to Sm was between 8 to 42% in 5 groups. Positive
rate of this antibody in group with Lin-p was significantly low compared with that in other 4 groups (P< 0.01).
The incidence of antibody to RNP was from 40 to 73%. The
relation of this antibody to the 5 groups was further studied on 63 patients whose renal biopsies were performed prior to therapy. The incidence of this antibody was low in the group with Lin-p (3096) and
high in the group with Mes-p (91%) compared with that in 63 patients
(62%). Marked decrease of CHSO was found in groups with Mes-,
Gran-, and Lum-p. Groups with Lin- and Mem-p showed slight decrease of CH50. Antibody to dsDNA was found in 3 of 7 eluates,
while antibodies to Sm and RNP were found more frequently. These
results indicate that a variety of antibodies to nuclear antigens in sera
may cause heterogeneity of glomerular immune complexes in this disease; antibodies to Sm and RNP are also of importance in the pathogenesis of lupus nephritis, in addition to the already recognized role
of antibody to dsDNA.
Relationship of exocytosis and ATP metabolism by isolated human peripheral blood neutrophils
C. Kent Kwoh, Donald C. Chow, and John L. Skosey; University of Illinois, Chicago
When isolated human peripheral blood neutrophils are incubated with immune stimuli, endocytosis, exocytosis, and metabolic
alterations are observed. We have previously noted that as these cells
phagocytize opsonized zymosan and release lysosomal enzymes, cell
ATP levels fall. The fall in ATP correlates better with exocytosis than
with endocytosis since it is observed not only when lysosomal enzyme
release occurs in response to phagocytable stimuli, but also when it is
induced by nonphagocytable stimuli such as phorbol myristate ace-
tate and the calcium ionophore A23187. The results reported here
suggest that, although the fall of ATP is associated with exocytosis,
enzyme release does not require ATP utilization.
Isolated neutrophils incubated with opsonized zymosan (1
mg/ml) were stimulated to release lysosomal a-mannosidase (12% of
total enzyme activity in 60 minutes, compared with 4% released from
resting cells). The antibiotic cytochalasin B (CB), 20 pg/ml, had no effect on enzyme release from resting cells but enhanced a-mannosidase
ARA ABSTRACTS
release from zymosan stimulated cells (to 32 f 1%). Endocytosis was
quantified by measuring the uptake of [I4C] inulin. Cells incubated
with zymosan took up 600 f 8 dpm/mg of cell protein (mean f standard error) compared with 266 f 11 dpm/mg taken up by resting
cells. CB inhibited uptake by both resting (144 f 10 dpm/mg) and
phagocytizing (429 f 7 dpm/mg) cells. ATP levels were constant over
the 60-minute incubation period in cells incubated in buffer alone or
with CB. When cells were incubated with zymosan, the ATP level fell
from 1.66 f 0.02 (zero time value) to 1.33 f 0.02 nmoles/106 cells
during the 60-minute incubation. When cells were exposed to both
zymosan and CB, the ATP level fell to only 1.55 f 0.03 nmoles/106
cells. Similarily, when cells were incubated with CB (2 pg/ml) and
633
20% serum which had been activated by prior incubation with zymosan, exocytosis (14% of a-mannosidase released) was associated with a
fall in cell ATP (from 2.13 f 0.10 to 1.23 f 0.05 nmoles/l@ cells). As
the concentration of CB was increased to 20 pg/ml, release of a-mannosidase was further enhanced (to 20%) while the fall in ATP was
abolished.
We conclude that although cell ATP levels fall concomitantly with exocytosis in response to both phagocytable and nonphagocytable stimuli, exocytosis does not require ATP ultilition.
These findings suggest that the fall in cell ATP that coincides with
exocytosis may be blocked by CB and results from, rather than being
required for, enzyme release.
Alterations in estrogen metabolism in systemic lupus erythematosus
Robert G. Lahita, Henry G. Kunkel, and Jack Fishman; Rockefeller University,New York
Estrogen metabolism has been studied in normal individuals
and in various disease states by administration of the tracer, 'H-estradiol, and measurement of the labeled metabolites excreted as the glucuronides in urine. This method was applied to the study of estrogen
metabolism of systemic lupus erythematosus (SLE) patients, and a
number of significant differences from controls were obtained. A
greater excretion of 16a-hydroxyestrone was observed these levels as
percent of total glucuronides excreted were 17 f 3% in the males (controls 6 f 2% from a range of 4-1 1%) and 21 f 5% for females (controls I 1 f 3% from a range of 3-1395). In addition, excreted estriol in
the female patients differed significantly from the female controls; the
patients had a mean estriol level of 21 f 7% (controls 12 f 4% P <
0.001). The SLE subjects also exhibited a compensatory diminished
hydroxylation at C-2 as exemplified by decreased excretion of 2-methoxyestrone. Patients studied with or without steroid therapy showed
these abnormalities. These results indicate an alteration in the direction of estradiol metabolism in SLE patients. This could result in
chronic estrogenic stimulation since 16a-hydroxy labeled metabolities
of estradiol are estrogenic, while the 2-hydroxylated compounds are
not.
In vitro phagocytosis and degradation of DNA/anti-DNA immune complexes
M.C. Lamers, Els R. de Groot, and D. Roos; Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam
The uptake and degradation of 'H-DNA/anti-DNA complexes by neutrophils and monocytes from human blood were studied. The complexes were prepared from 'H-labeled, circular dsDNA
of bacteriophage PM2 with anti-dsDNA-containing sera from patients
with systemic lupus erythematosus (SLE). After phagocytosis, cells
and medium were separated. The cells were treated with DNase to remove noningested complexes before counting of the cell-associated
radioactivity. Thus, only complexes inside the cells were measured.
The medium was analyzed for total and acid-precipitable radioactivity; the difference between these two values was taken as a measure of antigen degradation by the cells. In this way, we found that
both neutrophils and monocytes take up the complexes, but that only
monocytes degrade the antigen. Both processes increased linearly with
the incubation time and cell concentration, were saturable with repect
to complex concentration, and were inhibited by mono-iodoacetic
acid as well as by low temperatures. Large complexes were ingested
and degraded better than small complexes. Complexes made with purified IgG-anti-DNA required 1-2.576 fresh normal serum for processing by the cells. Addition of more than 5% fresh normal serum decreased the complex size and, as a consequence, the uptake and
degradation by the cells. Cobra venom factor-treated serum neither
opsonized nor solubilized the IgG complexes. Complexes made with
purified IgM-anti-DNA were never ingested or degraded. Addition of
purified IgM-anti-DNA to purified IgG-anti-DNA enhanced complex size as well as uptake and degradation.
We conclude that phagocytosis and processing of dsDNA/
anti-DNA complexes require IgG-anti-dsDNA and C3 activation.
The major contribution of IgM-anti-dsDNA seems to increase the
complex size. The results are in accord with the theory that production of only IgG antibodies to dsDNA in SLE leads to formation of
small complexes that cannot be cleared by the phagocytic leukocytes.
Eventually, such complexes may be harmful to the SLE patients.
Serum complement abnormalities in the antinuclear antibody-positive relatives of children with systemic lupus
erythematosus
Thomas J. A. Lehman, Childrens Hospital of Los Angeles; Bernhard Singsen, Helen Kornreich, Bram Bernstein, Karen King, and
Virgil Hanson; University of Southern California, Los Angeles
Hypergammaglobulinemia, false positive tests for syphilis,
and ANA are reported in the asymptomatic relatives of systemic
lupus erythematosus (SLE) patients. They have been interpreted as
evidence of a genetic or combined genetic and environmental etiology
for SLE. Since there is an increased incidence of SLE-like disease in
individuals homozygous for the inability to synthesize early complement components, it was questioned whether partial complement
deficiency might be associated with the serologic abnormalities in the
relatives.
Twenty-one children with SLE and 81 first degree relatives
ARA ABSTRACTS
were evaluated. Twelve of the 8 1 first degree relatives (15%) had ANA
(titers 1 : 10-1 : 80); this group was composed of 5/20 mothers, 3/16 fathers, 3/27 sisters, and 1/18 brothers. The mean values for C3, C4,
and total hemolytic complement (CH50) of the patients, ANA-positive and ANA-negative relatives, and controls (expressed as a percentage of pooled control sera activity) are illustrated in the Table.
The mean serum C4 and CH50 of the relatives with ANA
were significantly depressed. This finding did not occur for C3. The
mean C4 and CH5O of the relatives without ANA were not significantly different from the controls. If decreased C4 resulted from classical pathway activation, a decrease in C3 would also be expected.
Index cases
CH5O
c4
c3
72.0 f 32.0.
77.5 f 46.5
87.4 f 27.7
Isolated C4 decrease could result from a decreased synthetic rate or a
dysfunctional C4 protein. Decreased functional C4 could be associated with the development of ANA through linkage of genes controlling C4 synthesis and immune response. Alternatively, relatives
with decreased C4 may inadequately handle certain viral infections
which in turn stimulate formation of ANA. Whether depressed C4
synthesis is inherited and precedes the development of ANA remains
to be determined. This may provide an explanation of the role of genetic and environmental influences in the etiology of SLE. HLA typing of families and sequential measurement of ANA and complement
component levels are in progress.
ANA positive relatives
NSt
NS
t0.05
77.1 f 11.9
76.7 f 34.2
95.1 f 20.9
<0.005
<0.005
NS
ANA negative relatives
89.8 f 14.1
103.8 f 30.2
99.1 f 18.3
NS
NS
NS
Controls
91.8 f 17.4
94.6 f 27.0
95.4 f 14.0
All values f 1 SD.
t P values for comparison of immediately adjacent columns; NS = not significant.
Reduced T lymphocyte purine nucleoside phosphorylase in systemic lupus erythematosus
Dennis J. Levinson, Deborah B. Chalker, Michael Reese Hospital and Medical Center, Chicago; and William Arnold, Lutheran
General Hospital, Park Ridge, Illinois
Purine nucleoside phosphorylase (PNP) catalyzes the conversion of inosine, guanosine, and xanthosine, as well as their 2 deoxy
derivatives to their respective purine bases. In humans, PNP deficiency is characterized by defective T cell function. In systemic lupus
erythematosus (SLE) the depressed cell mediated immune response is
accompanied by a decrease in the number of identifiable T cells and a
blunted response to certain plant mitogens perhaps dependent upon T
cell dysfunction. The association of the congenital absence of PNP
and defective T cell function, together with cellular immune dysfunction in SLE, prompted a study of PNP activity in 14 patients with
SLE (age 36 years f 10) and 17 healthy controls (age 34 years f 8).
PNP activity was assayed by the rate of conversion of I4C inosine to labeled hypoxanthine in cell free extracts of fresh peripheral
mononuclear cells (PBL) separated by Ficoll-hypaque gradient centrifugation, and enriched T cells isolated on nylon wool columns. The
enriched T cell fraction contained 85% E-rosette forming cells, 5% SIg
bearing cells, and < 1% erythrocytes from patients and controls.
Reduced levels of PNP were detected in T cells from patients
with SLE (mean:1272 f 576 nMoles per mg protein/hour) in comparison with normal control (mean:1959 f 862 nMoles per mg/pro-
tein/hour; P < 0.01). By contrast, values for PNP in PBL were similar
for SLE (meax1843 f 882 nMoles per protein/hour) and normal
controls (mean:1973 f 1042 nMoles per mg protein/hour; P > 0.35).
Reduced PNP activity in T cells correlated with % DNA binding (r =
0.62, P < 0.05), but not with hemoglobin concentration (r = 0.28, P >
0. I), total leukocyte count (r = 0.22, P > 0. I), lymphocyte count (r =
0.22, P > O.l), o r the absolute number of E-rosettes
(r = 0.12, P < 0.01). Steroid therapy in 4 patients had no significant
effect on T cell PNP activity. Adenosine deaminase (ADA) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were assayed
by radiochemical methods in PBL and enriched T cells from SLE and
normal humans as additional controls. No significant differences were
found between groups for either T cell ADA (P> 0.15) and HGPRT
(P> 0.49), or PBL ADA (P> 0.35) and HGPRT (P> 0.47). Furthermore, no differences were found in erythrocyte ADA (P> 0.25) and
PNP (P> 0.4) between groups. In summary, a selective reduction in
PNP has been demonstrated in enriched T lymphocyte from patients
with SLE. The significance of this finding in relation to defective T
cell function remains speculative.
y-Carboxyglutamate excretion: A marker in patients with ectopic calcification disorders
J. B. Lian, Children’s Hospital Medical Center, Boston; L. Pachman, Children’s Memorial Hospital, Chicago; N.E.H.Partridge, Tufrs
New England Medical Center, Boston; C. Gundberg and P.M . Gallop, Children’s Hospital Medical Center, Boston
y-Carboxyglutamic acid (Gla) is synthesized in a vitamin K
dependent posttranslational enzymatic carboxylation reaction of specific glutamic acid residues in a polypeptide. The resultant Gla residues have the specific function of binding calcium. Warfarin type
anticoagulants inhibit the reaction resulting in the loss of calcium
binding. This specialized Ca++ binding amino acid is normally present in prothrombin, factors VII, IX, and X, a serum protein C, in normal kidney, and in a unique protein in bone, osteocalcin. Gla is also
present in pathologically mineralized tissues and in the calcified subcutaneous exudates of patients with scleroderma and dermatomyositis
(DMS). Previous studies have shown that Gla is not metabolically degraded but is excreted almost quantitatively. Since Gla excretion reflects the turnover of all vitamin K dependent calcium binding proteins, urinary Gla was examined in 15 patients with DMS to assess the
turnover of Gla-proteins that might be involved in the ectopic deposition of calcium. In 13 patients, juvenile DMS was diagnosed accord-
ARA ABSTRACTS
ing to the strict criteria of Bohan and Peter and are ENA and ANA
negative. Urinary Gla measurements were performed by two independent methods: I ) direct analysis of a diluted urine sample on a
Beckman 121-M amino acid analyzer programmed for Gla by established methods, and 2) analyses by isotope dilution of Gla isolated
from urine on a Dowex-1 column. Normal excretion levels range from
30-70 nm/mg creatinine. Three patients (1 adult) had normal Gla excretion and no evidence of calcification; 7 juvenile DMS patients with
moderate calcification ranged from 80-95 nm/mg creatinine, and 4
juvenile DMS and I adult, having massive calcium deposition, ex-
635
creted from 110-154 nm/mg creatinine. In addition, only 2 of 6 patients with scleroderma had elevated Gla excretion, and these had
subcutaneous calcium deposition. These data suggest that increased
synthesis of vitamin K dependent Ca++ binding proteins occurs in patients with calcification disorders. The origin of increased urinary Gla
is not known but may arise from turnover of these proteins at the sites
of calcification. Gla excretion measurements may correlate with calcification and be a useful clinical parameter in DMS and scleroderma.
Supported by granis DE 04651, A G 00376, A M 15671, and
A M 21589-OI.
Purification of a nuclear nonhistone protein (Sm antigen) reacting with the sera of patients with systemic lupus
erythematosus
T. S. Lieu and E.M. Tan, University of Colorado Medical Center, Denver
A nuclear acidic protein (Sm antigen) reacting with the sera
of patients with systemic lupus erythematosus has been purified from
rabbit thymus extract to a high degree of homogeneity. The methods
employed included the successive application of fractional ammonium sulfate precipitation (33%to 70%saturation), Sepharose 4B gel
filtration, DEAE-cellulose chromatography (eluted between O.2M and
0.3M NaCl in lOmM borate buffer, pH 7.6), and isoelectric focusing
in pH range 4 to 6. In gel filtration of the isolated protein, the antigenic activity was eluted in the same fraction as human serum albumin with approximate molecular weight of 70,000. In isoelectric focusing the activity was found in pH 5.4 fraction. The purified antigen
reacted with all four prototype anti-Sm sera employed. In addition,
SDS gel electrophoresis of immune precipitates also showed a com-
mon antigen band. The antigen was also isolated from rat liver nuclei
and had similar physicochemical properties. It also showed immunologic identity with that prepared from rabbit thymus extract, as
judged by the double immunodiffusion method. Antigenic activity
was destroyed by treating with 8M urea, 6M guanidine hydrochloride,
IM sodium thiocyanate, 2-mercaptoethanol, and was sensitive to the
modification of lysine residues by maleylation under mild conditions.
These results suggest that conformation of the protein molecule is important for the antigenic activity.
Sm antigen has been isolated in highly purified form. By
biochemical criteria, the antigens isolated from rat liver nuclei and
rabbit thymus are similar, confirming previous immunologic studies.
Circulating immune complexes response to standard steroid therapy in acute systemic lupus erythematosus nephritis
Stephen M. Lindsey, Bernard H. Berne, Kellie L. Snyder, and Oliver J. Lawless; Walter Reed Army Medical Center, Washington,DC
Recent work in systemic lupus erythematosus (SLE) with
new and experimental treatment modalities such as plasmapheresis
and megadose SoluMedrol has shown rapid reductions of circulating
immune complexes (CIC) in acute lupus flares. No data are available
on the earliest CIC changes following standard steroid treatment of
acute SLE. In this study we sought to ascertain the rapidity of CIC reduction in response to standard steroid therapy of acute SLE nephritis. CIC were measured by using the Clq 1-125 PEG (2.5%) method
ofZubler and Lambert in 12 patients with acute SLE nephritis. All serial samples on the same patient were tested on the same run. Serial
studies were done prior to and following treatment every 3-14 days
for up to 2 years, with an average of 9 months followup.
The results show 10/12 had elevated CIC prior to treatment.
Ten of 10 showed a decrease within 14 days with a mean of 8 days.
Five of 10 fell within 2-6 days. A 50% reduction took place within a
mean of 9 days in 10/10. Within a mean of 31 days, 10/10 became
normal; 5 / LO patients developed increased CIC levels following thts
initial normalization. All 5 had a clinical flare within 2 months of ini-
tiation of therapy. In 4 of these 5, CIC showed mild elevations. These
4 had a mild flare and stabilized without a change in steroid dose. In
1 / 5 a marked elevation was associated with a severe clinical flare requiring an increased and divided steroid dose that resulted in a decreased CIC within 1 week and normalization within 30 days. Serial
dsDNA responses paralleled CIC responses in the majority of patients
(7/10). Normalization of dsDNA tended to lag behind CIC. C3 and
C4 levels were slower to respond than either CIC or DNA. Of the 5
patients with subsequent flares, complement levels were still low 2
months into therapy in all, whereas 4/5 without a flare had a normal
C3 and C4 by 2 months of treatment.
These results show a rapid decrease in CIC to standard steroid dose in acute SLE nephritis in as early as 2 days. Early serial determinations may be useful in monitoring therapeutic responses and
predicting early flares. These data suggest that all 3 immune parameters are valuable and that CIC may be the earliest laboratory test to
show changes in response to the treatment of SLE nephritis.
Amino sugar-containing macromolecule distribution and metabolism in articular cartilage from osteoarthritic
human hips
Louis Lippiello, Mary E. Johnson, and Henry J. Mankin; Massachusetts General Hospital, Boston
Numerous studies have supported the thesis of increased synthetic activity by articular chondrocytes in osteoarthritis. More recent
reports have challenged these findings. To further clarify this problem
and also to further define the products of this increased synthesis, sev-
ARA ABSTRACTS
era1 experiments were performed in which the distribution and rates
of synthesis of amino-sugar containing macromolecules in norqal
and osteoarthritic femoral head cartilage were assessed by biochemical analysis and radioisotopic tracer incorporation studies.
Cartilage slices were incubated with 'H-glucosamine in an ex
vivo system and, following harvest, the segments were individually assayed for DNA, glucosamine, and galactosamine. The glucosamine
and galactosamine fractions were also analyzed for incorporated radioactivity. Adjacent tissue was analyzed chemically and histochemia l l y to establish the grade of osteoarthritis.
The biochemical data obtained clearly demonstrate the previously noted significant decline in hexosamine content in osteoarthritic tissue. The decrease is principally due to a diminution in glucosamine concentration and correlates inversely with the severity of
the disease process. Metabolic studies showed a marked increment in
the rates of incorporation of 'H-glucosamine into both the glucosamine and galactosamine fractions of the cartilage. The increased synthesis correlates directly in a nonlinear fashion with the severity of the
disease. The ratio of the rate of incorporation of glucosamine to that
for galactosamine was the same in normal and osteoarthritic samples,
suggesting that the decline in glucosamine concentration is not related
to a qualitative alteration in synthetic activity. The data strongly suggest that the synthetic activities for the chondrocyte remain qualitatively constant in osteoarthritis and that the GAG product is identical, not only as compared with normal but with advancing degrees of
disease. It is concluded that the articular chondrocyte in osteoarthritis
is not phenotypically different from the normal, and that the data can
be translated into a statement in strong support of the theory that the
osteoarthritic chondrocyte is metabolically hyperactive.
Inhibition of human helper T cell function by D-penicillamine and copper sulfate
Peter E. Lipsky and Morris Z @ University of Texas Health Science Center, Dallas
The mechanism of action of D-penicillamine (Pen) in rheumatoid arthritis (RA) remains unclear. The possibility that Pen might
modify the immunologic mechanisms underlying this condition
prompted an investigation of the effect of Pen and mixtures of
Pen+CuS04 on in vitro correlates of immune responsiveness. Specifically, the effect of Pen+CuSO, on the capacity of normal human peripheral blood mononuclear cells (PBM) to generate immunoglobulin
secreting cells (ISC) in response to the T cell dependent polyclonal B
cell activator pokeweed mitogen (PWM) was examined. PBM were
obtained from normal individuals by Hypaque-Ficoll centrifugation
and incubated for 2 hours at 37°C with medium alone, Pen, CuSO,,
or a mixture of Pen+CuSO,. The cells were extensively washed, aliquoted into the wells of microtiter plates, and cultured with or without PWM. After a 7-day incubation, polyclonal B cell activation was
assessed by a reverse hemolytic plaque assay that permitted quantitation of the number of ISC generated. In 9 separate experiments,
PWM stimulated the differentiation of a mean of 7,628 ISC per lo6
PBM. Preincubation of PBM with either Pen (100pg/ml) or CuSO,
(2pg/ml) did not alter subsequent PWM responsiveness. By contrast,
preincubation of PBM with Pen+CuSO, inhibited the capacity to
generate ISC in response to PWM by 93.8 f 3.2%. Inhibition of responsiveness could not be ascribed to cell death, carry-over of the inhibitors, an alternation in the concentration of PWM, or the length of
incubation yielding maximum responses. However, coculture of
Pen+CuSO, preincubated PBM with normal T cells restored PWM
responsiveness, indicating that the mixture of Pen+CuSO, had altered helper T cell function. To examine this possibility further, experiments with purified cell populations were carried out. Purified B
cells cultured alone generated few ISC in response to PWM, while
supplementation with control T cells led to the generation of large
numbers of ISC. In 4 separate experiments addition of Pen+CuSO,
preincubated T cells led to the generation of a mean of only 3% of the
number of ISC found in cultures supported by control T cells. These
data indicate that Pen in the presence of CuS04 can markedly inhibit
helper T cell activity. Such a mechanism may in part explain the therapeutic efficacy of D-penicillamine in RA and especially the capacity
of Pen therapy to decrease rheumatoid factor titers in treated patients.
Supported by N I H AM-09989 and A F Clinical Study Center
Grant.
Comparison of Auranofin versus gold sodium thiomalate on lymphocyte function
Arthur Lorber, William H. Jackson, and Timothy M. Simon; Memorial Hospital Medical Center, Long Beach, California
Auranofin (S-triethylphosphine-gold-2,3,4,6-tetra-o-acetylIthio-B-D-glucopyranoside)(AF) differs significantly from gold sodium thiomalate (GST) in formulation, i.e., aurous gold is stabilized
by dual sulphur and phosphine ligands, hydrophobic rather than hydrophilic characteristics, and lack of ionic charge. These attributes facilitate oral absorption of AF and membrane penetration, resulting in
greater lymphocyte gold content, thereby possibly affecting lymphocyte function. Auranofin therapy was observed to effect primarily T
rather than B lymphocyte function in 16 rheumatoid arthritis (RA)
subjects receiving 6 mg of AF per day for an average of 45 weeks
(range 20-74 weeks) compared with GST treated RA subjects. Lymphocytes from AF treated subjects, separated by using Ficoll-FIy-
paque gradients, manifested prompt and sharp decline in mitogen-induced lymphoproliferative response (LPR); suppressed response to
skin testing with DNCB; blebbing of lymphocyte membranes as
shown by scanning electron microscopy; modest decline in immunoglobulins in 8 of 16 subjects; and no significant change in rheumatoid
factor titer. Suppression of LPR with AF was approximately 60% after the first week and 80% after 20 weeks of therapy, contrasting with
0% and 30% for the respective intervals in GST treated subjects.
DNCB skin testing of AF patients indicated 11 of 14 failed to respond, whereas all GST patients responded. Problems of local or systemic fungal, bacterial, and/or opportunistic infections were not encountered. The effect of AF on B cell effector function, e.g.,
637
ARA ABSTRACTS
suppression of immunoglobulins and rheumatoid factor titer, was significantly less when contrasted with GST therapy of RA subjects as
previously reported. For Auranofin subjects, reductions in IgG, IgM,
and IgA at 20 weeks averaged 3370, 16.5%, and 8.3% respectively,
compared with 4 1 gold sodium thiomalate treated patients averaging
19.3%,38.2% and 31.0%for these parameters. Hence, the therapeutic
actions of these gold compounds, differing widely in formulation and
pharmacologic properties, also seem to differ in their effect on lymphocyte populations.
DNA synthesis and cell proliferation by topographically distinct components of human osteoarthritic cartilage
C. J. Malamud, V. M. Goldberg, and R. W. Moskowitz, Case Western Reserve University, Cleveland, Ohio
Experimental observations of cartilage metabolism in normal
and pathologic states may be compromised by inadequate attention to
variations related to tissue sampling. Metabolic studies comparing
topographically sampled human osteoarthritic (OA) cartilage have
been carried out.
Cartilage specimens were obtained from the femoral head,
femoral condyle or tibia1 plateau of patients undergoing total joint replacement for OA. Cartilage areas designated 1) discolored with no
overt fibrillation, 2) moderate to severe fibrillation or 3) osteophyte
were treated with hyaluronidase and some tissue was fixed for histology. Remaining tissue explants were treated with trypsin and cultured
in Dulbecco-Vogt medium with either 10% fetal bovine serum (FBS),
human serum (HS) or lacking serum. In some experiments, the effect
of omitting trypsin pretreatment or the inclusion of sodium ascorbate
(40 &ml) in the medium was tested. On the day of culture initiation
as well as 3 and 6 days later, explants were incubated in the presence
of 'H-dThd for 20 hours and incorporation was assessed. Results of
these DNA synthesis experiments showed 1) all tissue samples actively synthesized DNA during the first 20 hours of culture; after 3
days, incorporation was substantially increased and was highest in
most cases after 7 days, 2) trypsin pretreatment had little effect on OA
femoral head cartilage, but enhanced DNA synthesis in femoral condylar cartilage; ascorbate had no effect, 3) any serum or the lack of it
had little effect on incorporation on day 1, but FBS consistently stimulated DNA synthesis by day 7 when compared to HS or serum-less
medium and 4) DNA synthesis by osteophyte tissues was similar to
other components during the first 20 hour pulse but significantly
higher by day 7. In study of tissues from 6 different OA femoral
heads, osteophyte cellular outgrowth was marked; no outgrowth from
discolored cartilage was observed. Increased DNA synthesis and
eventual cell proliferation correlated best in osteophytes compared to
discolored or fibrillated material.
These results demonstrate that marked differences exist between OA cartilage components with regard to in vitro DNA synthesis and cellular proliferation.
Supported in part by USPHS General Research Support Grant
RR-05410-17.
Crystal mediated membranolysis in pseudogout
Neil Mandel, Barbara Millstein, Franklin Kozin, and Gretchen Mandel, The Medical College of Wisconsin, Milwaukee, Wisconsin
The membranolytic potential of selected microcrystalline calcium pyrophosphates and phosphates was studied. Triclinic calcium
pyrophosphate dihydrate (t-CPPD), monoclinic CPPD (m-CPPD),
monoclinic calcium pyrophosphate tetrahydrate (m-CPPT), a-calcium pyrophosphate (a-CPP), hydroxyapatite (HA), and brushite
(CaHP) were incubated with "chromium labeled human erythrocytes, and the amount of lysis was quantified.
m-CPPD exhibited the greatest amount of lysis (29.2%hemolysis) compared with t-CPPD (16.7%), m-CPPT (6.4%), a-CPP (9.2%),
HA (8.1%). and CaHP (4.7%). An equal concentration of microcrystalline silicon dioxide (a-quartz) produced 44.3%hemolysis.
To study the effects of surface area, t-CPPD crystals were
ground in an agate mortar and pestle. The normal prismatic morphol-
ogy of these synthetic crystals was markedly altered by grinding, although x-ray powder diffraction patterns were unchanged. Reduction
of particle size (increased the total surface area) essentially eliminated
hemolysis, a finding that was completely unexpected. Similar experiments with silica crystals demonstrated a direct relationship between
surface area and hemolysis. Our results suggest that the natural surface structural organization of t-CPPD crystals is essential in membranolysis.
Naturally occurring microcrystalline t-CPPD and m-CPPD
are more membranolytic than the other phosphate and pyrophosphate
crystals examined. Differences in the atomic structure, which may be
predicted by x-ray diffraction patterns, can be correlated with these
observed differences.
Lymphocyte hyporesponsiveness to PHA in patients with progressive systemic sclerosis: Role of monocytes
Joseph Markenson, Michael Lockshin, Bonnie Rosenfeld, Laszlo Fuzesi, Catharine Joachim. Neil Julie, and Jeff Wallis, The Hospital
for Special Surgery, New York City
In patients with systemic lupus erythematosus (SLE), Hodgkin's disease, and sarcoidosis, abnormal lymphocyte responses of
PHA can be corrected by removal of adherent mononuclear cells.
Further work from our laboratory has demonstrated that in SLE this
lymphocyte hyporesponsiveness is mediated by a soluble product de-
rived from monocytes (Arthritis Rheum 21:600, 1978). The current
study extends these observations to include patients with progressive
systemic sclerosis (PSS), a disease where few immunologic abnormalities have been reported.
Ficoll-Hypaque (FH)-separated peripheral blood mono-
ARA ABSTRACTS
638
nuclear cells from 19 normal controls and 13 patients with PSS were
further fractionated into T cells (T) by passage over an antihuman
F(ab)', column (97% E-rosette, < I % SIg + cells). In some experiments part of the initial FH sample was incubated at 37°C overnight
on glass petri dishes to remove the adherent cell population (FH-M)
(<1% peroxidase positive). Cell number was held constant in all experiments. FH, T, and FH-M cells were assayed with eight concentrations of PHA for response at 3 days by 'H thymidine uptake.
In normals the response of T cells (60,300 f 5900 cpm) was
less than that of FH cells (69,000 f 4400 cpm). In contrast PSS T cells
responded better (42,390 f 6240 cpm) than PSS FH cells (27,270 f
6390 cpm). Removal of monocytes from the FH population also improved the low response of FH PSS cells (+43.6%), but did not im-
prove the response of normal FH-M cells (- 12.3%).Suppression was
not due to media exhaustion.
The data indicate that poor in vitro FH lymphocyte response
to PHA in patients with PSS is mediated by a suppressor cell population among the adherent cells. The elaboration of a soluble product
from PSS adherent cells with similar suppressor properties such as reported in SLE is currently under investigation.
It is likely that all monocyte populations, whether from patients with diseases mentioned or normals, possess both immunopotentiating and immunosuppressive effects. An abnormal balance of
these effects seen in disease may account for the lymphocyte hyporesponsiveness observed.
Measuring health status in arthritis
Robert F. Meenan, Paul M . Gertman, and John H.Mason, Boston University Medical Center, Boston
Evaluation methods in clinical rheumatology focus on biologic variables. Arthritis however, affects all aspects of well-being, so
other components of health status should be assessed. We have completed an initial methodologic trial of a self-administered questionnaire designed to measure physical, social, and mental health status.
The purpose of this study is to develop an effective, efficient, and conceptually comprehensive approach to measuring differences in the
overall health of patients with arthritis. The 73-item questionnaire
consists of sociodemographic items; the Rand health status batteries
for mobility (MOB), physical activity (PA), social activity (SA), anxiety (ANX), depression (DEP), self care (ADL), and general health
perceptions (GHP); and new batteries for pain (PAIN), dexterity
(DEX), and social role (SR). Questionnaires were completed by 104
patients with arthritis of varying duration and severity. An independent physician battery of ARA functional class, joint count, and general disease activity was also obtained (DOC).
There were 3 1 males and 73 females with a mean age of 53
years. Most had either rheumatoid arthritis or osteoarthritis. Scalogram analysis produced good coefficients of reliability and scalability (CR > 0.90; CS > 0.60) for all but two batteries. GHP and
DOC were significantly correlated with all scales (with the exception
of DOC with ANX) and with each other, and selected scales vaned
significantly with age and diagnosis: these findings support the construct validity of the scales. Scales correlated more highly with GHP
than with DOC; with ADL, SA, and PA being most similar and ANX,
DEP, and PAIN most dissimilar. These findings suggest that when
generating global estimates physicians focus on other aspects of
health status than do patients. Multivariate analysis indicated that
PAIN best explained variations in GHP (r2 = 0.55) with DEP, PA,
and DEX also explaining important percentages (? = 0.15, 0.07,
0.02). The scales for PAIN, PA, MOB, SA, ANX, and DEX could be
formed into a global Gutterman scale of physical, mental, and social
well-being. This scale improves on ARA functional class since it has 6
gradations and can be disaggregated to examine specific aspects of
health status.
A spectrum of health status in arthritis which is broadly
based and relevant to patient concerns can be measured by a simple
self-administered questionnaire. This approach should prove useful in
estimating the impact of interventions in this chronic disease.
Cyclic nucleotide levels and mechanism of inhibition of leukocyte function by deaminase inhibition
A . D. Meisel, C. Natarajan, G. Sterba, and H. S. Diamond, Downstate Medical Center, Brooklyn, New York
The association of adenosine deaminase (ADA) deficiency
and immune dysfunction has focused attention on purine metabolism
as a modulator of the immune system. ADA inhibitors inhibit lymphocyte blastogenesis, monocyte, and leukocyte chemotaxis. Although polymorphonuclear cells (PMN) have less ADA activity than
mononuclear cells (MN) (PMN, 0.6 f 0.3 units/h/lO' cells; MN, 19.8
f 5.2 units/h/lO' cells), inhibition of ADA with erythro-9 (2 hydroxyl 3 nonyl) adenine hydrochloride (EHNA) inhibits leukocyte
chemotaxis. Various mechanisms of potential cellular toxicity have
been proposed including accumulation of adenosine, deoxyadenosine,
or cyclic adenosine monophosphate (CAMP), pyrimidine starvation
and inhibition of S adenosyl-methionine (SAM) dependent methylation reactions.
Leukocyte chemotaxis was measured by migration of cells
under agarose, and phagocytic function was assessed by a candidacidal assay. EHNA, 100 pM,decreased chemotaxis from 9.8 f 1.8
mm to 7.8 mm (P< 0.001). lntracellular cAMP increased from 0.48
pic0 moles (pM)/lO' cells in control cells to 0.67 pM/106 cells after
EHNA. Adenosine at I, 5, and 10 mM concentrations had no effect on
chemotaxis in the absence of EHNA, at suboptimal or at inhibiting
concentration of EHNA. EHNA decreased Cundidu phagocytosis by
32% and decreased killing by 12%. Deoxyadenosine (1 mM) decreased
leukocyte chemotaxis from 8.5 f 0.9 mm to 6.1 & 1.4 mm and resulted in an increase in cAMP from 1.27 to 2.48 pM/106 cells.
Deoxyadenosine decreased phagocytosis by 24%. Optimal concentrations of theophyllin (THEO) and EHNA had additive effects on intracellular cAMP (EHNA and THEO, 1.15 pM/106 cells) and produced
additive inhibition of leukocyte chemotaxis (control, 12.2 f 0.2 mm;
EHNA and THEO, 10.0 f 0.2 mm; EHNA and THEO, 10.0 f 0.3
mm). L-homocysteine thiolactone (1 mM) inhibited leukocyte chemotaxis from 8.7 f 0.8 mm to 3.7 f 0.8 mm. Inhibition of chemotaxis
with L-homocysteine occurred in the absence of ADA inhibition and
639
ARA ABSTRACTS
L-homocysteine taxis with L-homocysteine occurred in the absence of
ADA inhibition and L-homocysteine (10 mM, I 0 0 pM,1 mM) did not
potentiate inhibition of leukocyte chemotaxis produced by EHNA (10
phf). L-homocysteine had no effect on CAMP,cGMP or phagocytosis.
The effect of EHNA on phagocytosis is mediated by deoxyadenosine induced increase in CAMP. Inhibition of chemotaxis may
be mediated by both deoxyadenosine and SAM.
Deoxyribonucleoside triphosphate pools in immunodeficiency states
Edwin Mejias, Beverly S. Mitchell, James Cassidy, William N . Kelley, University of Michigan, Ann Arbor
Deficiencies of three enzymes catalyzing sequential reactions
in the purine catabolic pathway have been associated with clinical immunodeficiency states. Immune dysfunction in adenosine deaminase
(ADA) and purine nucleoside phosphorylase (PNP) deficiency is felt
to be mediated, at least in part, by an accumulation of dATP or
dGTP, respectively. Deficiency of lymphocyte ecto-S'-nucleotidase
has been associated with congenital agammaglobulinemia. We have
utilized the DNA polymerase assay to measure deoxyribonucleoside
triphosphate (dNTP) pools in erythrocytes and lymphocytes from patients with several different immunodeficiency diseases. We have observed that 1) normal erythrocyte values (n = 12) were 211
150
pmol/ml packed RBC for dATP and ranged from t 2 0 to 47 pmol/ml
for dGTP, 2) erythrocyte dATP levels were 35,750 and 31,520 pmol/
*
ml in 2 ADA-deficient individuals, 3) dGTP levels were 2,125 and
1,040 pmol/ml in erythrocytes from 2 PNP-deficient patients, and 4)
the remaining erythrocyte dNTP pools were normal in both ADA and
PNP deficient individuals. In contrast, all dNTP pools were normal in
erythrocytes and lymphocytes from 4 patients with ecto-5'-nucleotidase deficiency and from 5 patients with common variable immunodeficiency and normal lymphocyte ecto-5'-nucleotidase levels. We
thus confirm that dATP and dGTP levels are markedly abnormal in
ADA and PNP deficiency respectively and that this may account for
the T-cell dysfunction in these disorders.
The B-cell dysfunction in patients with ecto-5'-nucleotidase
deficiency and in some patients with common variable hypogammaglobulinemia does not appear to be mediated by a similar mechanism.
Release of immune complexes from cellular receptors by complement in systemic lupus erythematosus
Gary Miller, California State College, San Bernadino, David Scarborough, Tujis College of Medicine, Boston, and M. Edward Medof;
The University of Chicago
High levels of circulating immune complexes (ICs) are frequently associated with hypocomplementemia in patients with systemic lupus erythematosus (SLE) during periods of clinical activity.
Previous studies have shown that complement can solubilize experimental immune precipitates and can alter experimental ICs bound to
lymphocyte membrane receptors and induce their release. This property of complement is termed complex release activity (CRA) and involves both classic and alternate pathway components. Complement,
therefore, should theoretically play a role in determining physical and
biologic properties of ICs that are present in SLE, and whether circulating ICs in SLE patients will become bound to cells bearing receptors for ICs.
In this study we allowed ICs from hypocomplementemic
SLE sera to bind to Raji cells and then examined the ability of intact
complement to release the cell-bound 1Cs from receptors. Sera containing the highest levels of 1Cs from each of 10 patients were studied
as follows: Each IC-containing SLE serum was incubated with Raji
cells for 30 minutes at 37'C and the Raji cells then washed. Fresh
normal serum, or alternatively, zymosan-inactivated (ZYM)serum
was then added to the IC-bearing Raji cells. Aliquots of the reaction
mixture were removed after 0, 5, 10, 15, 25, and 40 minutes of incubation at 37°C and immediately cooled to 0°C. 1Cs remaining
bound to Raji cell receptors after progressively increasing times were
quantitated by addition of '251-labeled antiglobulin after removing
serum by washing. In 8 of the 10 cases, at least 40% of ICs that were
initially bound to receptors were released by fresh normal serum. In 4
cases, more than 50% of the ICs were released. Release was complete
after 15 to 25 minutes. There was minimal release by ZYM serum.
In parallel control studies performed under identical conditions, 1Cs prepared in vitro from BSA and guinea pig (GP) anti-BSA
were used in place of the circulating ICs. The experimental 1Cs were
fully released by fresh serum but not by ZYM serum when studied using either Iz5I-anti-GP globulin or 1Z51-labeledICs alone. These results show that intact complement can 1) alter ICs that are present in
hypocomplementemic SLE sera and 2) influence the relationship between these ICs and cellular membrane receptors. The results further
raise the possibility that hypocomplementemia secondarily due to
consumption of complement by ICs contributes to the persistence of
the ICs if CRA plays a role in normal IC metabolism or disposal.
19s IgM hidden rheumatoid factor in juvenile rheumatoid arthritis
Terry L. Moore, Andrew R. Baldassare, Terry D. Weiss, Robert W. Dorner, and Jack Zuckner, St. Louis University School of
Medicine, St. Louis
Fifteen percent of patients with juvenile rheumatoid arthritis
(JRA) have been shown to have 19s IgM rheumatoid factor (RF) by
the standard latex fixation test (LFT) in their serum. However, it has
been shown in preliminary studies that after acid separation of the
IgM-containing fraction from serum, 59% of patients who were RF
negative by the LFT can be shown to have 19s IgM RF by a com-
plement-fixing hemolytic assay (hidden RF). This hidden RF has
been shown to correlate with activity of disease. In this study, we have
extended our findings.
Sera of 76 children with seronegative JRA (109 specimens), 6
with seropositive JRA, 27 with other connective tissue diseases, and
14 normal children were separated by gel filtration of pH 4.05 to ob-
ARA ABSTRACTS
patients with systemic disease. When the disease was inactive, IgM
fractions were positive in only 8 of 22 polyarticular JRA, 1 of 15 pauciarticular JRA, and 2 of 6 systemic. The active polyarticular and
pauciarticular values were significant at P < 0.001.
In conclusion, I ) 67% of sera of patients with JRA who are
seronegative by the LFT contain hidden 19s IgM RF; 2) hidden 19s
IgM R F defined by the hemolytic assay correlates statistically with activity of disease; 3) the assay can be a serologic method of defining
JRA; and 4) the assay may aid in following disease activity.
tain the IgM-containing fraction. The original sera and the IgM fractions were subjected to the complement-dependent hemolytic assay.
Fifty-one of 76 patients with seronegative JRA and 6 of 6
with seropositive JRA had titers > 1:16. None of 14 normal controls
and only 2 of 27 disease controls had titers > 1:16. The mean titer of
seronegative JRA patients was l:109 and seropositive JRA 1:341.
Normals and other diseases showed a mean titer of 1:6. The JRA values were significant at P < 0.001.
Positive results were obtained in 40 of 43 patients with active
polyarticular JRA, 17 of 21 with active pauciarticular JRA, and 5 of 8
Complexity of antinuclear antibodies in scleroderma
Y.Moroi, C. Peebles, M. J. Fritrler, and E. M . Tan, Denver, Colorado and Calgary, Alberta
tract of nuclei and this was identical with antibody to Scl-l antigen
previously described. Of particular interest was the antigen showing
discrete speckled staining. The nuclear antigen was restricted to dividing chromosomes in metaphase cells. In chromosome spreads from
colcemid-arrested cells, staining was limited to centromeres on
chromosomes. All 5 patients with anticentromere antibody had
CREST (calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly,
telangiectasia) without lung, cardiac, or renal involvement and with a
relatively long disease history (mean 19 years) compared to mean 7
years. ANAs are present in high incidence in scleroderma and are
characterized by specificities not seen in other connective tissue diseases.
The use of tissue culture cells instead of organ sections as
substrate in immunofluorescence has shown a very high incidence and
heterogeneity of antinuclear antibodies (ANA) in scleroderma. On a
variety of tissue culture cell lines, including HEp-2 (human laryngeal
carcinoma), Ramos (human B lymphocyte) and Ehrlich mouse ascites
tumor, 21 of 23 (90%) were positive for ANA in indirect immunofluorescence. Several patterns of nuclear staining were observed, including finely granular, nucleolar, discrete speckled, and homogeneous. Some sera showed mixed patterns of these types. Tissue culture
cells were treated with a fixative, paraformaldehyde, lysine, and periodate (PLP), which did not alter the antigenicity of nuclear components and at the same time rendered nuclear antigens stable in
physiologic buffer solutions. The Table shows the incidence and patterns of nuclear staining and susceptibility of nuclear antigens to enzyme digestions.
The finely granular nuclear staining was related to a nuclear
protein antigen, nucleolar staining to RNA and in some instances also
to DNA, discrete speckled only to DNA, and homogeneous staining
to protein antigens which may be chromosome-associated. Only sera
with the homogeneous staining gave a precipitin line with saline ex-
Pattern of staining
%
DNase
RNase
Trypsin/
chymotrypsin
Finely granular
Nucleolar
Discrete speckled
Homogeneous
65
-
40
22
9
-
+-
+
+
+-
*
-
+
-
Clinical and genetic differences of sicca syndrome in the presence and absence of rheumatoid arthritis
Haralampos M. Moutsopoulos, Thomas M.Chused, Dean L. Mann, John L. Decker, N I H , Bethesda
current parotid gland enlargement was present in 18 of 22 patients
with SS and in only 3 of 21 patients with SS + RA. Renal involvement was found in 6 of 22 patients with SS whereas none of the
SS + RA patients had evidence of it. In addition, Raynaud's phenomenon, purpura, lymphadenopathy, and myositis were more common
(P c 0.05) in SS. No significant differences were found in the incidence of splenomegaly, pulmonary involvement, lymphoma, or
Waldenstrom's macroglobulinemia in the 2 groups of patients. We
then investigated the genetic background of the patients and controls
by studying the incidence of the HLA antigens and B-lymphocyte, Ialike antigens. The results are shown in the Table.
The clinical characteristics of these two groups with the genetic differences suggest that SS alone is a distinct pathologic entity.
Sicca syndrome (SS) occurs as an entity alone or in association with other connective tissue disorders, most commonly rheumatoid arthritis (SS + RA). Previous studies have shown significant differences in the incidence of circulating autoantibodies in patients with
SS and those with SS + RA. This study reviews the clinical features
and examines the association of specific HLA antigens in patients
with SS versus SS + RA. The clinical features of all NIH sicca syndrome patients (22 SS and 21 SS + RA), who have been followed for
at least 5 years and do not have a rheumatic disease other than RA,
were analyzed. RA tended to precede the development of SS. No significant differences in the mean age, serum immunoglobulins, C3,
rheumatoid factor titer, or salivary histopathology were found in the 2
groups of patients. The clinical features, however, were different. Re-
Percent positive
Group
Number
HLA-B8
la- I72
la-35
la-7 I5
Normal
184
22
31
59'
9t
34
100'
82't
20
68*
9t
16
68'
9t
ss
SS
+ RA
11
* Differs from normal. t Differs from SS, all P < 0.05.
Ia-350
25
59'
82'
ARA ABSTRACTS
64 1
Histologic and serologic abnormalities of Sjogren’s syndrome in systemic lupus erythematosus
Haralampos M. Moutsopoulos, John H. Klippel, Nicholas Pavilidis, Alfred D. Steinberg, NIH, Bethesda, and Eng Tan, University of
Colorado, Denver
The coexistence of Sjogren’s syndrome (SS) and systemic
lupus erythematosus (SLE) has been well documented. We evaluated
24 patients with SLE for clinical, histologic, and serologic evidence of
ss.
All patients were questioned for clinical evidence of xerostomia or xerophthalmia and were evaluated with a Schirmer’s and slit
lamp examination, parotid scan, and parotid Bow rate, and biopsy of
the minor labial salivary glands. Abnormalities in SLE patients suggestive of SS were identified by questionnaire (54%), parotid flow rate
(13%), parotid scan (58%). and Schirmer’s test (33%). No patient had
keratoconjunctivitis sicca on slit lamp examination. Abnormal histologic findings of minor salivary glands consisting of lymphoid infiltration in clusters, dilatation, or proliferation of ductal epithelium or
glandular atrophy were present in 50% of SLE patients. No correlation was noted between findings of SS and nephritis, myositis, serositis, or neurologic involvement. The sera of all SLE patients as well
as 17 patients with SS unassociated with a connective tissue disease
were examined for rheumatoid factor (RF) and precipitating antibodies to cellular antigens SS-A, SS-B, and RANA. Results are
shown in the Table.
We conclude that approximately one-half of SLE patients
have inflammatory changes of minor salivary glands consistent with
SS. Autoantibodies, particularly R F and anti SS-A, were identified in
one-half the SLE patients with labial histologic abnormalities. These
serologic findings are similar to SS alone with the exception of the absence of anti SS-B in SLE patients.
Incidence of autoantibodies (%)
ss
SLE + biopsy
SLE - biopsy
No.
RF
SS-A
SS-B
RANA
17
12
12
88
42
88
58
0
71
8
0
29
17
25
0
Combined orthodontic/surgical therapy for severe micrognathia in juvenile rheumatoid arthritis
Robert W. T. Myall, Roger A. West, R. William McNeil, Jane G. Schaller, University of Washington, Seattle
Juvenile rheumatoid arthritis (JRA) affecting the temporomandibular joint can alter mandibular growth, dental conformation,
and the motion and function of the jaws. Severe problems can result,
including an increased overbite, crowding of teeth, decreased mouth
opening, and the cosmetic deformity associated with micrognathia.
Although the pain and disability of active temporomandibular arthritis can be controlled or may remit with time, affected patients may be
left in teenage and adult years with permanent facial and dental deformities. We have begun a prospective study to evaluate and treat
patients having the sequelae of severe temporomandibular joint disease secondary to JRA. Six patients with severe micrognathia have
had cosmetic and functional restoration of the jaws through a combined orthodontic-surgical approach. Patients ranged from 10 to 23
years at the start of this therapy. All 6 had severe micrognathia and
orthodontic problems; 3 had restricted mouth opening as well.
Initial evaluations included clinical examination by a team of
orthodontists and oral surgeons, cephalometric head tracings, standard radiographic projections, and articulated dental casts. The observed deformities were analyzed and an attempt made to predict the
outcome of treatment. All patients required pre- and postsurgical orthodontics with tooth movement and utilization of braces to correct
dental deformities. Surgical procedures required to correct the mandibular and facial deformities included sagittal osteotomies of the
mandibular rami to extend the length of the mandible (6 of 6 patients), horizontal osteotomies of the mandible to augment chin size (4
of 6 patients), osteotomies of the maxilla to reduce maxillary height
(Le Fout I procedure; 2 of 6 patients), and alloplastic implantation to
augment chin size (1 of 6 patients).
This approach has been extremely successful in restoring
both adequate jaw and dental function and cosmetic facial appearance in 5 of 6 patients treated; one patient will require reoperation because of inadequate correction of micrognathia.
A human granulocyte factor (CTAP-PMN) stimulates DNA synthesis in cultured synovial fibroblasts
Stephen L. Myers and C. William Castor, University of Michigan, Ann Arbor
Human synovial fibroblasts in culture have been stimulated
to augment hyaluronate synthesis and glucose utilization byconnective fissue activating peptides (CTAPs) extracted from human
spleen, lymphocytes, platelets, and granulocytes. The platelet-derived
mediator CTAP-Ill also stimulated DNA synthesis in synovial fibroblasts, but CTAP-I from lymphocytes and spleen did not. The present
study explored the mitogenic potential of granulocyte extracts
(CTAP-PMN).
Normal granulocytes were prepared using Ficoll-diatrizoate
gradients. Platelet contamination was quantitated by phase micros-
copy and by radioimmunoassay for the platelet-specific protein, pthromboglobulin. Granulocyte CTAP-PMN was extracted with 80%
ETOH, 20% 1.25 N HCl. After acetone precipitation, it was dialyzed
against buffered saline. In double immunodiffusion studies using rabbit antisera to CTAP-I11 no reactivity of the antibody with CTAPPMN was demonstrated. Extracts were assayed for mitogenic activity
by measuring stimulation of ’H-thymidine incorporated into DNA in
synovial cultures.
Crude CTAP-PMN preparations derived from 4 X lo7 cells/
ml stimulated culture ’H-thymidine incorporation to 3.56 f 1.32 (SD)
ARA ABSTRACTS
642
times control levels. Exposure of preparations to 0.1% P-mercaptoethanol and 0.001M dithioerythritol did not affect their mitogenicity. After heating to 100°C for 5 minutes some mitogenic activity remained in crude preparations. CTAP-PMN extracts contained neutral
protease activity which was inhibited by addition of heparin or soy
trypsin inhibitor. These agents alone did not stimulate 3H-thymidine
incorporation and did not inhibit stimulation of DNA synthesis when
added to the mitogen assay along with CTAP-PMN. This suggested
that proteolysis was not required for mitogenic activity.
Crude CTAP-PMN extracts were applied to calibrated Sephadex G-50 columns and elution of mitogenic activity followed the elution volume of lysozyme. By SDS gel electrophoresis the molecular
weight of active fractions was estimated at 11,100 f 1100 daltons.
CTAP-PMN or other granulocyte factors capable of stimulating fibroblast DNA synthesis might play a role in chronic proliferative synovitis or in other settings where exudative inflammation is accompanied by fibroplasia.
Studies on the antigenic determinants in self-association of IgC-rheumatoid factors
Francis A . Nardella, University of Washington,Fred H. Meyer, Louisiana State University,New Orleans, David C. Teller and Mart
Mannik, Universityof Washington,Seattle
In previous studies the intermediate complexes in patients
with rheumatoid arthritis were composed of self-associating IgGrheumatoid factors (IgG-RF) (Pope, Teller, Mannik: J Immunol 115:
365, 1975). The number, location, and nature of antigenic determinants involved in self-association of IgG-RF were examined in the
present study.
F(ab’),, Fab, and Fc fragments were prepared from the isolated complexes from one of these patients with rheumatoid arthritis.
Normal human IgG, Fc, Fc’ (Cy3 domain), rabbit Fc fragments, and
an lgG3 myeloma protein were isolated by standard procedures in
monomeric and immunochemically pure form. High speed equilibrium ultracentrifugation and analysis of data were carried out as previously described.
Normal IgG molecules bound only one IgG-RF Fab fragment, indicating a functional antigenic valance of one for IgG molecules. Normal human and IgG-RF Fc fragments (isolated from the
complexes), however, interacted with two Fab molecules, indicating
an antigenic valence of 2. Thus, normal IgG and IgG-RF molecules
have two antigenic determinants, but at least in normal IgG one of
these sites is sterically hindered by the Fab region. The interactions of
IgG-RF F(ab’), fragments with its own Fc, with normal human Fc
and with rabbit Fc fragments were comparable, yielding association
constants of about I x 10’ liters per mole. Therefore, the IgG-RFs
did not possess unique antigenic determinants to facilitate self-association of these molecules and their preferential dimerization depends
on the formation of two antigen-antibody bonds per dimer.
The IgG-RF F(ab’)2 fragments did not react with purified
normal Fc’ fragments, indicating that the antigenic determinant is located on the Cy2 domain or requires both Cy2 and Cy3 domains of
IgG. The IgG-RF F(ab’), fragments interacted with human IgG3,
suggesting that the antigenic determinant is not synonymous with the
G a antigen for IgM-RF, present on IgG1, IgG2, and IgG4, but not on
IgG3.
The characterization and localization of the antigenic determinants for self-association of IgG-RF will facilitate the discovery
of the reason for development of these unique immune complexes and
their biologic role in rheumatoid arthritis.
Supported by NIH Grants AM 12849, A M 07108, and GM
13401.
Decreased complement solubilization of immune complexes in sera containing high titers of rheumatoid factor
James F. Naylor, Susan A . Ward, Sterling E. Moore, and J. Donald Smiley, University of Texas Health Science Center at Dallas
IgG, IgM-RF, and C3 are deposited in cartilage adjacent to
the joint surface in rheumatoid arthritis (RA). These presumed immune complexes may act as a reservoir for antigen to maintain the
chronic immune response in the rheumatoid synovium. Miller and
Nussenzweig observed that complement components in the sera of
several animal species solubilize immune complexes. For this reason,
the role of rheumatoid factor (RF) in altering this function in patients
with RA was investigated. Assays employed immune complexes of
1251-tetanustoxoidhuman antitetanus prepared in 16-fold (w/w) antibody excess, a ratio of ag:ab readily solubilized by normal serum. The
release of 12’I-label into the supernatant was proportional to the
amount of C3 present. From 1844% solubilization of 10 p g of immune complex occurred in 45 minutes using a volume of whole normal serum containing 60 pg of C3.
Sera from 20 patients (16 RA, I subacute bacterial endocarditis, 3 other) with RA latex titers ranging from 1:80 to 1:25,000
were compared with the sera of 20 other seronegative, non-RA pa-
tients matched for age, race, and sex. The non-RA sera solubilized an
average of 2: times more immune complexes than the RF-containing
sera per milligram of C3 present. There was a significant correlation
between the relative RA latex titer in the serum and the degree of depression of complement solubilization. The 7 RF-positive sera having
a RA latex titer of 1:640 or less produced solubilization in a low normal range whereas 7 other RF-positive sera all with RA latex titers of
more than 1:2,560 showed complete inhibition of complement solubilization. All of the normal sera showed significant complex solubilizing activity. Addition of purified IgM-RF to normal serum also
caused inhibition of this complement function while the same amount
of Waldenstrom’s IgM did not interfere.
These results suggest that RF blocks the fluid phase solubilization of immune complexes by the complement system and may contribute to their continued presence in cartilage, blood vessel walls, and
elsewhere in RA and in other diseases in which R F is present.
Supported by USPHS Grants AM-08418 and AM-09989.
ARA ABSTRACTS
643
Heterogeneity of precipitating antibody systems in polymyositis and dermatomyositis
Masahiko Nishikai and Morris Reichlin, State Universityof New York at Stony Brook, Bufalo
Sera of 26 polymyositis (PM) patients and 22 dermatomyositis (DM) patients were studied for the presence of precipitating antibodies to antigens in calf thymus extracts. Twenty-eight positive reactions were found 18, 8, and 2 sera exhibited 1, 2, and 3 precipitin
lines, respectively. Seventeen of 26 (65%) PM sera and 11 of 22 (50%)
DM sera contained the above reactions.
The 11 PM and 6 DM sera with strong precipitin lines were
studied further and their common specificities established by immunodiffusion. These were classed into three groups of shared specificity
and five reactions which were specific for individual sera. Specificities
were identified by the first two letters of the patients’ names-and in instances where sera exhibited more than one reaction serial numbers
have been used. Of the shared specificities, 8 of the I 1 PM and 1 of
the 6 DM sera were designated Jo-1, 3 of I I PM sera were designated
Jo-2, and 2 of the 6 DM sera were designated Mi-I. The five individually specific sera were designated Va, Ma, Ka, Mi-2, and Ok.These 8
calf thymus antigens were shown to be different from the known soluble tissue antigens nRNP, Sm, Ro, and La. The 4 antigens Jo-1, Jo-
2, Mi-I, and Mi-2 were all shown to be nuclear in origin and have a
molecular weight of 150,000. They were all inactivated by pH 4.0 buffer or heating at 56’ for 30 minutes. The factor in both Jo and Mi sera
reactive with these thymic antigens resided in the DE52 purified IgG
fraction. Clinical specificity was assessed with partially purified Jo-1
(100 fold) and highly purified Mi-1 (1560 fold) antigen. Of 22 patients’ sera with PM associated with other connective tissue diseases
(11 scleroderma [PSS], 4 SLE, 1 PSS-SLE, 2 rheumatoid arthritis
[RA] and 5 with Sjogren’s syndrome) only I patient with PM-PSS had
precipitate
with any of
anti Jo-l in its serum. Purified Mi-1 did not .
.
these sera nor did it react with any of 39 patients’ sera with pure PM.
It precipitated solely with 2 DM sera. Neither of these antigens precipitated with the sera from 22 SLE, 9 RA, and I2 normal sera respectively. Although the majority of PM and DM patients respond to
some thymic antigen, there is considerable heterogeneity. The most
common response was against the Jo-1 antigen and clinical studies
suggest that such reactions might serve as marker antibodies for the
PM syndromes.
Alteration by penicillamine of response of isolated human peripheral blood neutrophils to immune stimuli
Steven Nusinow, John L. Skosey, and Donald C. Chow, Universityof Illinois, Chicago
We previously reported that the nonlethal release of lysosomal enzymes which occurs during phagocytosis of opsonized zymosan (Zym) by human peripheral blood neutrophils in vitro is potentiated by glutathione, cysteine and D-penicillamine (Pcna). Earlier,
we observed that the extent of lysosomal enzyme release was inversely
related to the associated burst of oxidative metabolism. Together,
these findings suggested that an oxidant such as 0,-or Hz02, generated as a byproduct of oxidative metabolism, might interfere with
lysosomal enzyme release, and that the anti-oxidants glutathione, cysteine, and Pcna interfere with this putative action of the oxidants.
However, an alternative explanation exists. The anti-oxidants not
only enhance Zym stimulated lysosomal enzyme release, but also inhibit endocytosis ([‘“C] inulin uptake), a combination of effects similar to those of the antibiotic cytochalasin B (CB). We have previously
suggested that CB, by inhibiting complete formation of the phagocytic vacuole, favors release of lysosomal enzymes extracellularly in
preference to release into the phagolysosome.
We wished to determine if Pcna mimicked other effects of
CB. Serum activated by prior incubation with Zym had no effect on
release of lysosomal enzymes from isolated neutrophils. However,
cells incubated in the presence of CB are conditioned to release lysosomal enzymes in response to Zym activated serum (ZAS). To ascertain if Pcna shared this effect of CB as well as the ability of the antibiotic to inhibit phagocytosis and promote lysosomal enzyme release in
response to particulate stimuli, we tested the ability of Pcna to prime
neutrophils to respond to ZAS. Cells incubated with 10% ZAS as well
as cells incubated with buffer alone released 3% of their total cornplement of lysosomal a-mannosidase in 60 minutes. Pcna, 10 m M ,
conditioned cells to release a-mannosidase (8%) in response to ZAS.
This effect was similar to, although less marked than, that of CB,
which permitted release of 23% of a-mannosidase in response to ZAS.
The effects of Pcna and CB were more than additive; cells incubated
with both agents released 36% of a-mannosidase in response to ZAS.
We conclude that Pcna has effects on isolated neutrophils which are
similar to those of CB. This, as well as a potential anti-oxidant effect
may explain the enhancement of lysosomal enzyme release by neutrophils stimulated in the presence of Pcna.
Studies of lymphoblastoid cell lines derived from rheumatoid arthritis synovial membrane lymphocytes: Cellular and
immunoglobulin characteristics
Stephen A . Paget, Charles Cornell, Robert Inman, and Paul E. Phillips, The Hospitalfor Special Surgery, New York City
Six lymphoblastoid cell lines (LCL) were derived from Epstein-Ban virus (EBV) transformation of rheumatoid arthritis (RA)
synovial membrane lymphocytes (Paget et al: Arthritis Rheum 20582,
1977). The antibodies produced by these cells may be useful to identify a unique antigen inciting RA inflammation. IgG, IgM, and IgM
rheumatoid factor (RF) concentrations in harvested cell culture super-
natant medium were measured sequentially by sensitive radioimmunoassays, as shown in the Table.
Immunoglobulin production rates of each cell line exceeded
those found in four LCL derived from normal human tissues: mean
IgG 1.9 &lo6 cells/day (range 0.8-3.7); mean IgM 1.4 (0.5-2.2).
This suggests an intrinsic immunologic hyperactivity of the RA lines.
ARA ABSTRACTS
pg Ig/106 cells/day
Total Ig harvested (mg)
Culture duration
(days)
IgG
IgM
7.2
3.9
5.8
0.6
4.3
I .7
4.2
0.8
1.5
27.0
5.5
35.0
29
59
37
2
50
5
6
28
19
17
28
19
Mean
18
3.8
12.5
Cell line
1
2
3*
4at
4b
IgM
IgM RF
+
+
16
6
11
10
90
13
338
32
78
+
IgA also produced.
t 4a, 4b from right + left knee of same patient.
Preliminary results indicate that 3 of 6 cell lines are likely to be producing monoclonal Ig as determined by immunochemical methods.
Immunofluorescence studies have demonstrated the presence of: 1)
EBV nuclear antigen in all lines; 2) the Ia antigen (DRw 4 x 7 x 10)
associated with RA in 4 of 6 lines (Gibofsky et al: J Exp Med
148:1728, 1978); 3) unusual surface lg in 3 lines, 2 with IgG, 1 IgA, 4)
rheumatoid arthritis nuclear antigen (RANA) in the one line tested
employing serum known to contain the rheumatoid arthritis precipitin
(RAP). Such cell lines and their Ig products are important tools with
which to study the basic immunologic defects of RA and identify a
unique antigen inciting RA inflammation.
Studies of lymphoblastoid cell lines derived from rheumatoid arthritis synovial membrane lymphocytes: Immunologic
reactivity of secreted immunoglobulin
Stephen A . Paget, Charles Cornell, Robert Inman, and Paul E. Phillips, The Hospital for Special Surgery, New York
Six lymphoblastoid cell lines were derived from the EpsteinBarr virus transformation of rheumatoid arthritis (RA) synovial membrane lymphocytes. These RA lines demonstrate higher immunoglobulin (Ig) secretion rates than those derived from normal tissues; more
than 300 mg of Ig can be harvested from such cultures in less than 20
days. Cell culture harvests (mean total volume 4600 ml) were efficiently concentrated a mean of 70X to final Ig concentrations of 1
mg/ml employing pervaporation, ammonium sulfate precipitation,
G-200 fractionation, and pressure dialysis with Amicon ultrafiltration.
The immunologic reactivity of these final Ig preparations has
been studied in an attempt to identify the antigen(s) inciting RA inflammation. A sensitive passive microhemagglutination assay has
failed to detect antibodies to human types I, 11, I11 collagen (Trent-
ham et al: J Clin Invest 61239, 1978). By use of a highly sensitive complement-dependent adherotoxicity assay, preliminary studies have not
demonstrated Ig reactivity with cultured RA synovial membrane fibroblasts. However by immunofluorescence studies with three ultracentrifuged, fluorescein-conjugated Ig preparations, RA synovial
membranes and cartilage have yielded positive results. A small population of as yet uncharacterized mononuclear cells demonstrated positive fluorescence in cryostat sections of intact RA synovial membranes and in fixed cell suspensions derived from the collagenase
digestion of such tissues. Similar studies involving cryostat sections of
RA cartilage have revealed positive fluorescence localized to cartilage
cells. Thus, some Ig preparations appear to be reactive with cellular
components of RA joints. Such Ig preparations may be useful in the
identification of a unique antigen inciting RA inflammation.
Interruption of articular nerves impairs macromolecular organization of articular cartilage
Marshall Palmoski, Brian O'Connor, and Kenneth Brandt, Indiana University School of Medicine, Indianapolis
Mechanoreceptors are highly specialized nerve endings
within the joint, sensitive to joint position. They initiate reflexes
which control muscle tone around the joint to check excessive movement, protecting the joint from the ever-present microtrauma incurred
in daily activity. Diminished mechanoreceptor function could therefore result in excessive muscle tone which, by increasing the load on
the joint, could lead to cartilage breakdown.
To study that possibility, the posterior and medial articular
nerves (which carry mechanoreceptor input) of the right knees of 2
normal dogs were sectioned (ANS) external to the capsule; 2 other
dogs were sham-operated (SO). Animals were sacrificed 3 and 9
weeks after surgery. In each case contralateral unoperated knees were
used as controls.
Three weeks after ANS water content and "S incorporation
into proteoglycans (PGs) in cartilage from ANS knees were 6% and
22% greater, respectively, than controls; by 9 weeks they were 10%
and 85% greater, respectively, and the cartilage showed loss of Safranin-0 staining and 20% loss of thickness. At 3 weeks 17% of the total
PGs were extracted with a nondissociating solvent (0.4M GuHCl)
from ANS cartilage and 10%from the control. Nine weeks after ANS,
however, 7 times more PGs were extracted from ANS cartilage than
from control (38% versus 5% of total PGs), although PG aggregation
was unaffected (Sepharose 2B chromatography). In all respects, analyses of cartilage from SO knees were the same as controls.
These data indicate that ANS may profoundly disrupt the
macromolecular organization of joint cartilage, possibly by interfering with PG-collagen interaction. Whether this leads to osteoarthritis remains to be determined.
ARA ABSTRACTS
645
Genetic predisposition to gold and D-penicillamine toxicity
G. S. Panayi, A . J. Grifin, P. H. Wooley, T. J. Gibson, Guy’s Hospital, J. R. Batchelor, Hammersmith Hospital, London
The distribution of HLA-DRw antigens was investigated in
91 white patients with classic or definite rheumatoid arthritis (RA), 71
of whom had toxic reactions to gold, D-penicillamine or both drugs.
A confirmation of certain associations in an earlier study was made
(Panayi GS et a 1 Br Med J 21326-1328, 1978), namely, a significant
increase in DRw4 (54% of RA; 34% in controls) a decrease in DRw2
(30% of RA; 12% in controls), an increase in DRw3 in patients with
toxic reactions, and an association with DRw3 and high rheumatoid
factor titer. The distribution of DRw antigens was analyzed according
to toxic manifestations, namely proteinuria, mouth ulceration, rash,
hematologic abnormalities, and eosinophilia. It appears that there is a
genetic predisposition to certain of these. DRw3 occurred in 70% of
patients developing proteinuria (Table 1) and when divided into the
two drugs, 93% of the 15 on gold (P< 0.0005); and 69% of the 13 on
penicillamine, the latter failing to reach significance because of the
high percentage of DRw3 in patients not toxic to penicillamine (43%).
DRw4 was significantly decreased in the proteinuria group when
compared with RA controls (P < 0.01). In the patients developing
mouth ulceration DRw2 was significantly increased when compared
with the control RA patients (12% RA controls, 3 I% toxic RA) (Table
2). The distribution of DRw types in the remaining toxic manifestations did not reach significance but there was a marked trend for the
more severe and sudden hematologic problems to occur in patients
with DRw3. These findings are of interest from the immunologic and
prognostic point of view.
Table 1. DRw antigens in proteinuria.
DRw3
Proteinuria
Present
Absent
TOTAL
Present
Absent
Total
5
24
67
19
18
49
54
37
91
The frequency of DRw3 in 24 patients with proteinuria against
the total 91 RA patients: 2 = 17.92; P < 0.O005.
Table 2. DRw antigens in mouth ulceration*
DRw2
Mouth
ulceration
Present
Absent
Total
Present
Absent
TOTAL
5
6
11
I1
69
80
16
75
91
* The frequency of DRw2 in 16 patients with mouth ulcers against
the total 91 RA patients: 2 + 4.7; P < 0.05.
Arthritis associated with infiammatory bowel disease in children: Relationship between arthritis and activity of the
inflammatory bowel disease
Murray Passo, Kenneth Brandt, and Joseph Fitzgerald, Indiana University School of Medicine, Indianapolis
Few studies exist of arthritis in children with inflammatory
bowel disease. It has been suggested (Lindsley and Schaller, 1974)
that arthritis associated with inflammatory bowel disease in children
may not parallel activity of the inflammatory bowel disease as closely
as in adults. We recently analyzed records of 100 children with inflammatory bowel disease seen between 1966-1978. Age at onset of
idammatory bowel disease ranged from 2-16 years (mean: 10.2
years); 44 had ulcerative colitis (UC) and 56 had Crohn’s disease
(CD). Activity of inflammatory bowel disease in each case was estimated on basis of: number of diarrheal stools, hematochezia, abdominal pain, fever, weight loss, sigmoidoscopy, x-ray, hemoglobin, serum
albumin, and ESR.
Fourteen patients (8 males, 6 females) developed bouts of arthritis (defined as joint pain plus swelling), which lasted 2 days to 3
months. Four had UC; 10 had CD. Ankles, knees, elbows, and hips
were most commonly affected. Sacroiliac joints and spine were not involved.
In 3 patients arthritis preceded bowel symptoms by 2 to 10
months, in 3 they were concurrent and in 8 children arthritis appeared
6 to 56 months after the clinical onset of inflammatory bowel disease.
Four patients had only a single episode of arthritis, 4 had 2 episodes
and 6 had more than 3. Twenty-four episodes of arthritis in I 1 pa-
tients were documented sufficiently to permit detailed analysis of the
relationship of arthritis to inflammatory bowel disease. In these patients clinical bowel activity (diarrhea, abdominal pain and/or hematochezia) was present during 15 attacks of arthritis, whereas 6 attacks
occurred in the absence of gastrointestinal symptoms. During 4 of the
latter attacks, however, extraintestinal features (weight loss, anemia,
hypoalbuminemia) suggestive of active inflammatory bowel disease
were present. In 3 patients in whom arthritis preceded clinical evidence of inflammatory bowel disease, when bowel symptoms developed they were overshadowed by arthritis, and the fact that arthritis
was associated with bowel disease was not appreciated until 2 to 8
months after the onset of bowel symptoms. In 2 of these children
bouts of arthritis were predictive of imminent inflammatory bowel
disease flares, permitting an anticipatory increase in antiinflammatory
bowel disease medication. Most episodes of arthritis occurred at the
time of a “flare-up” of inflammatory bowel disease activity, although
most flare-ups were not accompanied by arthritis.
Our results indicate that in children, as in adults, most cases
of arthritis associated with inflammatory bowel disease occur in the
presence of obvious gastrointestinal symptoms; however, in some
cases gastrointestinal symptoms are absent and only systemic features
(e.g., weight loss, anemia) suggest the correct diagnosis.
ARA ABSTRACTS
Association of a particular B cell alloantigen with susceptibility to rheumatic fever
Manuel Patarroyo, Robert Winchester, Albert0 Veherano, Allan Gibofsky, Fernand Chalem, John B. Zabriskie, and Henry G. Kunkel,
Universidad Nacional de Colombia, Bogota, Colombia, and The Rockefeller University, New York City
The possibility that genetic factors determine susceptibility
to rheumatic fever after infection with Group A streptococci was suggested by epidemiologic and family studies. However, the identification of a definite genetic marker in the patient group including studies
of HLA-A and B allotypes has been unsuccessful.
In the present study the profiles of B cell (Ia-like) alloantigens were studied in 62 patients with documented rheumatic fever
(Jones criteria). Nineteen selected antisera were used including those
with defined DR specificities. One antiserum detected an alloantigen,
designated 883, that was present in 71 and 75% of two patient populations, one in New York City and one in Bogota. The frequency in
the control populations was 17 and 16% respectively (250.14, Yates;
relative risk 12.9). The remainder of the antisera did not discriminate
between the patient and control groups. Study of antigen 883 revealed
no significant correlation with any defined HLA-A, B, or D locus allele, suggesting it was the product of either an Ia locus unrelated to
HLA-D, or a totally distinct B cell locus.
The association of this novel B cell alloantigen with rheumatic fever provides direct support for the concept that an immunogenetic factor is relevant to the development of rheumatic fever afier
streptococcal infection.
Skin reactions complicating D-penicillamine treatment of rheumatoid arthritis
A. Caroline Patterson, Howard B. Stein, Roberta C. Ongley, and AIvena Teufel, Vancouver, British Columbia
D-penicillamine (dP) is a useful agent for treatment of rheumatoid arthritis. It causes a variety of side effects, the most important
being skin rash, thrombocytopenia, and proteinuria. Over 3 years 214
patients have been followed in a penicillamine clinic, receiving dP initially by the “go slow, go low” method of Jaffe and later by the “go
slower, go lower” method. Of the 214 patients 68 (32%) developed a
skin sign or symptom while receiving dP. The types of rashes seen
were urticarial, maculopapular, and eczematous. No patient developed pemphigus. The eczematous rashes were usually itchy, often
macular, and sometimes maculopapular. They were erythematous,
scaly, and irregular in size and shape. Occasionally a single lesion occurred, but more usually there were several patches situated on trunk
or limbs. In most patients there was no relationship between the occurrence of a rash while on gold therapy and a further rash while on
d P therapy. The histology of skin biopsies performed in 14 unselected
patients was uniformly nonspecific and no consistent immunoglobulin
deposition was seen.
Fifteen of 68 (22%) patients continued dP at the same dose,
their skin rash clearing despite continued treatment; 24 of 68 (35%)
continued dP at a reduced dose with or without temporary withdrawal
of the drug; 10 of 68 (15%) discontinued dP permanently because of
the skin rash and 19 of 68 (28%) withdrew for other reasons. Patients
with urticaria had to stop dP but otherwise we could not predict
which rashes would necessitate permanent cessation of the drug.
In conclusion: 1) skin rash occurs in 32% of patients on dP; 2)
skin rash may or may not require permanent cessation of the drug; 3)
a nonspecific, eczematous rash is the most commonly seen.
Mechanism of action of a specific inhibitor of complement (CS)-derived chemotactic activity in serum from patients
with systemic lupus erythematosus
H. Daniel Perez, Ira M. Goldstein, Richard Andron, New York University Medical Center, New York, Robert Webster, David
Chernof, National Jewish Hospital, Denver, and Peter M. Henson, NYU
We have previously reported that sera from some patients
with active systemic lupus erythematosus (SLE) contain a uniquely
specific, reversible inhibitor of complement (C5)-derived chemotactic
activity (CtxA). We now wish to report the isolation and purification
of this inhibitor and its possible mechanism of action. SLE serum,
proven capable of significantly inhibiting C5-derived CtxA in zymosan-treated normal serum (ZTS), was found incapable of inhibiting
the CtxA of the highly purified human anaphylatoxin, C5a (10-20
ng/ml). Similarly, SLE serum was found incapable of inhibiting the
CtxA generated in ZTS in the presence of the carboxypeptidase inhibitor, epsilon aminocaproic acid (EACA). EACA protects C5a from
the action of the anaphylatoxin inactivator present in normal serum
and thereby prevents the conversion of C5a to a peptide (C5a des arg)
which is completely devoid of anaphylatoxin activity. As previously
reported, highly purified human C5a des arg (40- 160 ng/ml) was not
chemotactic unless assayed in the presence of small amounts of normal human serum (NHS), which presumably provides a “helper fac-
tor.” The CtxA of C5a des arg plus NHS was inhibited significantly
by SLE serum, indicating that the complex of C5a des arg-helper factor was the target of the inhibitor. The inhibitor in SLE serum was
isolated by ammonium sulfate fractionation and molecular sieve
chromatography on Sephacryl S-200 and purified by cation exchange
chromatography (CM Sephadex). The inhibitor proved to be a very
cationic protein (p1 = 10) with a molecular weight of 67,000 (SDS
polyacrylamide gel electrophoresis). The helper factor in NHS was
also isolated by molecular sieve chromatography (molecular weight =
22,000) and was determined to be an anionic polypeptide.
These data document that the inhibitor in SLE serum acts
not on C5a but only on the complex of C5a des arg plus a serum
helper factor. This complex can account for most of the CtxA in ZTS.
Furthermore, the data suggest that the cationic inhibitor in SLE
serum does not act directly on C5a des arg but interacts reversibly
with the anionic helper factor.
647
ARA ABSTRACTS
Induction of human antiimmunoglobulinantibody (rheumatoid factor) producing cells by aggregated IgG
Edward J, Pisko, Nancy L. Pruitt, and Robert A. Turner, Wake Forest University, Winston-Salem, North Carolina
The stimulus for the production of rheumatoid factor remains unknown. However, immunoglobulin aggregates or immune
complexes, known to be present in the serum and joint fluid of rheumatoid arthritis patients, have been postulated to be the substances
that induce the abnormal antibody production by leukocytes. This
hypothesis was tested in the present study which demonstrates that
culturing normal, Ficoll-Hypaque isolated, peripheral blood mononuclear leukocytes (PBL) with heat aggregated IgG (HAIgG) for 5 or
6 days induces the production of antiimmunoglobulin antibodies. An
in vitro assay for antiimmunoglobulin production by PBL was developed in which HAIgG was coupled to autologous red cells with
chromic chloride and used in a complement-dependent plaque forming cell (PFC) system. Plaques were enumerated with an inverted microscope on red cell monolayers coupled to flat bottom microculture
plates with poly-L-lysine. Dose response studies showed significant (P
c 0.001) and maximum anti-IgG PFC production when 100 pg
HAIgG/ml media were added to PBL cultures (22.33 f 1.25 PFC/106
PBL). 2-Mercaptoethanol(2-ME) (IO-’M) further increased the num-
ber of anti-IgG PFC when added to HAIgG at the beginning of cultures (28.2 2.21 PFC/106 PBL). This effect of2-ME was not dependent on the dose of HAIgG. 2-ME alone also induced a small, but
significant (P< 0.05) increase in anti-IgG PFC (9.51 f 1.21 PFC/106
PBL) when compared to control PBL cultures with no HAIgG or 2ME added (2.88 f 1.21 PFC/106 PBL). Native (unaggregated) IgG at
the same concentration as HAIgG (100 pg/ml media) failed to induce
anti-IgG PFC. HAlgG also induced anti-sheep red cell PFC (P c
0.01) and anti-ovalbumin PFC (P c 0.025). 2-ME similarly induced
anti-sheep red cell PFC (P c 0.025) and anti-ovalbumin PFC (P <
0.05).
In summary, both HAIgG and 2-ME induce anti-IgG (rheumatoid factor) PFC as part of a polyclonal induction of PFC in which
anti-sheep red cell PFC and anti-ovalbumin PFC are also induced.
The polyclonal activation of mononuclear leukocytes by aggregates of
IgG or immune complexes may be one mechanism whereby antiimmunoglobulin antibodies (rheumatoid factors) are induced in autoimmune disorders such as rheumatoid arthritis.
*
Bladder complications in patients receiving cyclophosphamide for rheumatoid arthritis or systemic lupus
erythematosus
Paul H. Plotz, John H. Klippel, John L. Decker, National Institutes of Health, Bethesda
The bladder complications of 54 patients treated with oral
cyclophosphamide for systemic lupus erythematosus (SLE) (43) or
rheumatoid arthritis (RA) (1 I) were reviewed. During an observation
period of 241 patient years with a mean drug course of 28.4 months
(range 1-91) and total drug dose of 48.1 gm (2-152), we have observed 7 cases of acute hemorrhagic cystitis and 2 cases of transitional
cell carcinoma of the bladder.
Hemorrhagic cystitis developed in 5 SLE and 2 RA patients
after a mean course of 23.6 months (3-47) and total drug dose of 49
gm (6-152). Treatment by discontinuing the drug and increased fluid
intake resulted in resolution of bleeding in all patients. Anaplastic
transitional cell carcinoma was found to be the cause of unexplained
weight loss in one patient with RA. The tumor was identified 5 years
after stopping a therapeutically successful, uncomplicated course of
50 gm of cyclophosphamide. The patient died 4 months after detec-
tion of the tumor with widely metastatic disease involving thoracic
and abdominal viscera and the bony skeleton. A second patient developed gross hematuria due to a noninvasive papillary carcinoma of the
bladder wall 2 years after cyclophosphamide had been stopped because of hemorrhagic cystitis. She had received 152 gm of cyclophosphamide over 47 months for SLE nephritis. Both patients smoked
cigarettes, an additional risk factor for bladder carcinoma.
No bladder complications have been noted in 54 SLE patients treated with intermittent, intravenous cyclophosphamide (12).
azathioprine plus corticosteroids (19), or corticosteroids alone (23).
We consider the frequency of cystitis (7 of 54) and bladder
carcinoma (2 of 54) in SLE and RA patients treated with oral cyclophosphamide to constitute a strong argument for limited use of this
drug in nonmalignant inflammatory rheumatic conditions.
The immunohistochemical localization of proteoglycan subunit and link protein
A. R. Poole, I . Pidoux, A. Reiner, Montreal, Canada, H. Choi, L. Tang, and L. Rosenberg, New York City
Link protein and proteoglycan subunit (PGS) constitute major structural components of hyaline cartilages. They bind to each
other and to hyaluronic acid to form macromolecular aggregates. It is
very important to develop methods with which to specifically localize
these molecules in normal and diseased cartilages. Methods exist for
the demonstration of glycosaminoglycans of PGS using dyes but they
are not always absolutely specific: there are no methods to localize
link protein. Hence monospecific antisera to these molecules have
been prepared to permit the first specific localizations of proteoglycan
core protein and link protein.
PGS and link protein were purified from bovine nasal cartilages, and monospecific antisera were raised in rabbits (to link protein) and sheep (to PGS). These antisera were subsequently demonstrated by exhaustive immunochemistry to be monospecific, by use of
both precipitating and nonprecipitating antibody analysis. Bovine articular cartilage was fixed and treated with chondroitinase ABC to remove chondroitin sulfate. It was stained with antibody F a b subunits
using indirect methods and fluorescein or peroxidase-labeled second
step antibody Fab’. PGS staining was more intense in pericellular
sites throughout the cartilage. Diffuse staining of moderate intensity
ARA ABSTRACTS
was otherwise observed between chondrocytes. In contrast, link protein exhibited similarities and major differences in distribution. Control sections exhibited no extracellular staining. PGS and link were
removed from living articular cartilage by trypsin treatment. During
subsequent organ culture of the cartilage, PGS and link protein were
both secreted from chondrocytes. Although these studies indicate that
chondrocytes can produce both PGS and link protein, the organization of the molecules in the cartilage matrix is not uniform. These differences may be related to the degree of aggregation of PGS and link.
Supported by the Shriners of North America and by grant
AM24198 from the National Institutes of Health.
Antibodies to the immunodominant portion of streptococcal mucopeptide (pentapeptide) in patients with rheumatic
disorders
R.M. Pope, J.E. Rutstein, D.C. Strauss, University of Texas Health Science Center at San Antonio, D. Chang, Palo Alto, California
Infectious agents have long been considered likely causes of
many rheumatic disorders, although confirmation has generally not
been forthcoming. Bacterial cell wall mucopeptide, composed of repeating subunits of N-acetyl muramic acid and N-acetyl glucosamine
with peptide side chains is ubiquitous and has been implicated in juvenile rheumatoid arthritis (JRA), rheumatoid arthritis (RA), and
acute rheumatic fever (ARF). Therefore, the level of humoral immunity to the synthetic analog of the immunodominant pentapeptide
side chain (L-Ala-y-D-Glu-L-Leu-D-Ala-D-Ala)
of group A-variant
streptococcal mucopeptide was examined. The hapten binding capacity of patient and control sera was determined using lZ5Ipentapeptide.
The means for the hapten binding capacities of patients with
ARF(I2), JRA(14), and RA(22) were significantly greater (P< 0.01)
than those of the normal controls (14). Additionally, when compared
to the normals or those with degenerative joint disease (14), the binding capacities for the patients with ARF, JRA, and RA were more frequently (P < 0.01) abnormal (outside the 95% confidence limits of
normal controls). Ninety-two percent of ARF, 70% of JRA, and 68%
of RA patients demonstrated values exceeding the normal range.
There was no correlation between pentapeptide binding and AS0 titers for any patient group. A significant correlation (Pc 0.01) was observed between the IgG concentration and the pentapeptide binding
capacity for the JRA and RA patients but not those with ARF. Previously we demonstrated that patients with ARF and JRA had an increased frequency of elevated concentrations of antibody to isolated
mucopeptide detected by precipitins and radioimmunoassay.
The frequent occurrence of increased pentapeptide binding
and increased levels of antibodies to isolated mucopeptide in ARF
were not unexpected since this disorder is associated with a recent
streptococcal infection. Other investigators have been unable to reproducibly isolate viable organisms from patients with RA or JRA.
The elevated concentration of antibodies to mucopeptide, especially
to the immunodominant pentapeptide, in some patients with JRA and
RA suggests that mucopeptide may be responsible for the self-perpetuating synovitis in these conditions.
IgC rheumatoid factor: Relation to clinical activity in seropositive rheumatoid arthritis and absence in seronegative
arthropathies
Richard M. Pope and Sandra J. McDufy, University of Texas Health Science Center at San Antonio
The relevance of IgG rheumatoid factor (IgG RF) in seropositive rheumatoid arthritis (RA) has been questioned since it has
been frequently detected in a variety of seronegative conditions.
Therefore, sensitive radioimmunoassays for IgG and IgM R F were
employed to examine the prevalence and significance of IgG RF. Six
millimeter plastic beads coated with rabbit IgG were used as substrate
for the RF, and specific 1251anti-Fc and anti-p were employed to
quantify the bound IgG and IgM RF. The mean IgG RF for 28 controls was 2.29 f 0.09 (SEM) ng anti-Fc/bead and for 26 seropositive
RA patients was 14.5 f 3.5. The sera of 54 seronegative patients (23
rheumatoid arthritis, 6 juvenile rheumatoid arthritis, 10 spondyloarthropathy, 4 progressive systemic sclerosis, 2 acute rheumatic fever and 9 systemic lupus erythematosus were also examined. For seronegative RA the mean IgG R F was 2.77 f 0.28. The IgG R F of the
other seronegative groups ranged from 2.41 to 3.22. IgM R F was significantly increased only in the seropositive patients. IgG R F values
for the seropositive RA group were significantly different from the
normal controls and from all seronegative groups. No significant difference existed between any seronegative group and the normal con-
trols. All abnormal IgG R F values in the seronegative groups were
only minimally increased and all but 1 of these patients also had an
elevated IgG concentration. IgG RF correlated (P c 0.01) with IgG
concentration only for the seronegative patients. Additional studies
demonstrated that pooled normal human IgG adsorbed to the solid
phase nonspecifically. These data indicate that nonspecific IgG interactions were responsible for the marginally increased values of the
seronegative groups.
The effect of IgM R F was examined. Reduction and alkylation of RA sera prior to analysis decreased the mean IgG R F by only
34% while the IgM R F values were nearly abolished. IgG RF eluted
after gel filtration principally in the region between 19s IgM and 6.6s
IgG. RA patients with high IgG RF had more severe disease and extraarticular manifestations and the IgG R F corresponded with the
disease activity of the seropositive patients. In summary, elevated levels of IgG R F were detected in the majority of patients with seropositive RA but only rarely in seronegative conditions. Unlike IgM
RF, changes in IgG R F paralleled the clinical course, suggesting IgG
R F may be useful in the assessment of patient progress.
ARA ABSTRACTS
649
Effect of prolonged walking on concrete on the joints of sheep
Eric L. Radin, David Eyre, Jon. L. Kelman, and Alan L. Schiller, Cambridge and Boston Massachusetts
The veterinary literature suggests that animals housed on cement floors are more prone to the development of osteoarthrosis than
animals housed on dirt. We subjected 8 adult sheep to 4 hours per day
of slow steady walking in a concrete floored circular chute. The sheep
were kept moving by a mechanical arm from which was suspended a
skirt fitted with a standard animal electric shocker. Four control sheep
were walked in a similar chute which had a floor of woodchips. The
experimental animals were housed on tarmac; the control animals
were kept pastured. After 9 months the experimental sheep limped.
Serial x-rays revealed some calcification of knee and elbow ligaments
but no evidence of joint narrowing, subchondral sclerosis, or osteophyte formation. Sheep were killed at 12, 18, 24, and 30 months. Mild
to moderate cartilage fibrillation was present in the experimental animals’ knees and elbows. There was also a significant decrease in hex-
osamine content in their weightbearing articular cartilage. Hexosamine levels were unchanged in the non-weightbearing articular
cartilage.
Analysis of the distal femoral subchondral bone by stereological techniques demonstrated a profound change in trabecular architecture. The trajectorial pattern was altered in such a way as to reinforce the tibio-femoral articulation. This area also showed a
significant increase in stiffness as manifest by an increased connectivity of the trabeculae. Thickening of the patellar groove subchondral plate associated with loss of trabecular bone was present in
the patello-femoraljoint.
The results suggest that prolonged repetitive impulsive loading has an effect both on weightbearing articular cartilage and on architecture of its underlying bone.
Interaction of human monocytes with surface-bound immune complexes
Carol G. Ragsdale and William P.Arend, University of Washington,Seattle
Adherent immune complexes are present in the articular and
periarticular tissues of patients with rheumatoid arthritis, and monocytes are abundant in the proliferating joint lesions. The purpose of
these studies was to determine the effect of surface-bound immune
complexes on human monocyte spreading, receptor activity (Fc and
C3 rosettes), and neutral protease secretion. Mononuclear leukocytes
(MNL) were plated onto fibrin substrates, with or without surfacebound immune complexes, and the adherent cells were studied at 3
hours. Neutral protease secretion was determined by quantifying fibrinolysis as MNL settled onto iodinated substrates in the presence or
absence of plasminogen.
Monocytes on a fibrin substrate were uniformly rounded in
appearance and 90% expressed Fc and C3 receptors. These cells
slowly secreted plasrninogen activator; fibrinolysis at 3 hours in the
presence of plasminogen was 2 to 10-fold greater than in its absence.
Monocytes plated onto fibrin with surface-bound immune
complexes were spread with numerous cytoplasmic extensions. Less
than 5% of these cells formed Fc rosettes, but C3 receptor activity was
not reduced. Plasminogen activator was not detected when monocytes
were cultured on immune complexes. Instead, a transient burst of
plasminogen-independentfibrinolytic enzyme secretion was observed.
Assays performed using inhibitors of known specificities indicated
that this activity was due primarily to an elastase-like protease. Preincubation with cycloheximide (10 pg/ml) prevented release of plasminogen activator from cells on plain fibrin but did not reduce the
plasminogen-independent fibrinolysis seen with cells on complexes.
Preincubation of MNL with ethanol (300 mM), octanol (0.5
mM), colchicine (O.1mM) or cytochalasin B (0.02 mM) inhibited
spreading of monocytes on surface-bound complexes. Colchicine and
cytochalasin B reduced by 50% Fc rosetting of monocytes on plain fibrin. No agent prevented the complete loss of Fc receptors seen with
monocytes on complexes or affected the expression of C3 receptors.
Only ethanol and octanol inhibited the immune complex-induced release of plasminogen-independent fibrinolytic enzymes.
These results suggest mechanisms whereby surface-bound
immune complexes affect monocyte receptor functions and release of
neutral proteases, thus influencing the role of these cells in human inflammatory diseases.
An epidemiologic study of households exposed to canine systemic lupus erythematosus
James L. Reinertsen, Bethesda, Richard A. Kaslow, Atlanta, John H. Klippel, Bethesda, Nathan J. Zvager, San Diego, Naomi
Rothfeld, Farmington, Robert M. Lewis, Ithaca, Arthur Hunvitz, New York City, Alfed D. Steinberg and John L. Decker, Bethesda
On the hypothesis that exposure to canine systemic lupus
erythematosus (SLE) is an environmental factor in human SLE development, we studied 83 members (contacts) of 19 households in which
the household dog had a high titer antinuclear antibody detected
within 2 years prior to the study. Fourteen of the dogs had a diagnosis
of SLE. The contacts were compared to 50 members (controls) of 16
households matched for dog age and sex and veterinarian. The following were determined for each contact and control: clinical SLE
symptom score (SxS), dog exposure score (ES), antinuclear (ANA)
and lymphocytotoxic (LCA) antibodies, rheumatoid factor (RF),
serum immunoglobulins (Ig), and antibodies to DNA and RNA
(cyNA). Results are shown in the Table.
No cases of SLE were identified in either contacts or controls. More contacts than controls reported symptoms of arthritis, especially in the older age groups. Three contact households, and no
controls, gave a family history of SLE. These differences in the clinical and family histories may reflect greater concern with rheumatic
disorders in households where such disorders occur in a dog.
Serologically abnormal contacts had a mean ES of 12.2, not
significantly different from the mean ES of 11.9 of serologically nor-
ARA ABSTRACTS
650
Contacts (83)
Controls (50)
1.98
1.44
12.0
11.9
12.0
12.0
ma1 contacts. Titers of ANA and LCA were similar in contacts and
controls. Analyses of subgroups by age and sex did not reveal any
serologic differences between contacts and controls.
9.6
10.0
4.8
2.0
15.7
12.0
0
0
This study did not detect any clinical or serologic effect of
human exposure to canine SLE.
Mechanism of corticosteroid inhibition of prostaglandin synthesis by rheumatoid synovial tissue
Dwight R. Robinson, David Bastian, and Libby Servello, Massachusetts General Hospital, Boston
Inhibition of PGEZ synthesis by glucocorticoids may contribute to the antiinflammatory and other properties of these hormones. In certain tissues and experimental cell lines it has been proposed that the glucocorticoids block prostaglandin (PG) synthesis by
inhibiting the release of arachidonic acid from cell membranes by the
enzyme phospholipase. We have studied the mechanism of action of
corticosteroid inhibition of PG synthesis in rheumatoid synovial tissue explants in culture. Explants of rheumatoid synovial tissue were
maintained in minimal essential medium (Eagle’s) with 10% fetal bovine serum at 37°C. Prostaglandin E2 released into tissue culture media was measured by radioimmunoassay. I-14C-arachidonic acid was
incorporated into tissue phospholipids and triglycerides as demonstrated by thin layer chromatography of tissue homogenates. After incorporation of the labeled arachidonic acid, the tissues were rinsed
and medium was replaced and further incubated for 72 hours. Radioactive lipid products released from the tissue into medium and tissue
homogenates were extracted with ethyl acetate and chloroform/methanol respectively at pH 3, and the products were separated using thin
layer chromatography.
Inhibition of PG synthesis at the level of phospholipase reac-
tion would inhibit the release of arachidonic acid as well as PGs
(phospholipid -+free arachidonic acid +PGE2). In 6 separate experiments, treatment of rheumatoid synovial organ cultures with dexamethasone, I X IO-’M, gave 80-9Wo inhibition of PGEZ synthesis
but no significant difference in free arachidonic acid compared with
untreated tissues. To determine whether conicosteroids affected cyclooxygenase directly, microsomal preparations from rheumatoid synovial tissue were shown to synthesize PGEZ which was inhibited by
indomethacin, but neither hydrocortisone ( I X 10-5M) nor dexamethasone ( I X 10-6M) caused significant inhibition. However, microsomal preparations derived from synovial explants which had been
created with dexamethasone ( I X IO-’M) had reduced PG synthetase
activity by 70-90%.
These data suggest that in rheumatoid synovial tissue, glucocorticoids inhibit PG synthesis by reducing the activity of cyclooxygenase, although not directly interacting with this enzyme.
There is no demonstrable effect of glucocorticoids on the release of
arachidonic acid from membranes, as has been postulated for several
other tissues.
The clinical significance of protein-bound hydroxyproline fractions in sera of patients with adult rheumatoid arthritis
Carmen L. Rosano, Nourollah Parhami, and Charles Hurwitz, VA Medical Center, Albany, New York
Clq levels were measured by 2 methods in sera of normal
subjects and patients with rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), chronic polyarticular gouty arthritis (PAG), ankylosing spondylitis (AS), Paget’s disease of bone (PDB), and lymphoma. The normals consisted of 14 volunteers, mainly hospital staff
(age 20-45 years). The active RA group of 22 patients with classic RA
met ARA criteria (ages 24-58). The SLE group consisted of 7 patients
meeting ARA criteria. The PGA group consisted of 6 patients having
boggy synovitis in several joints, mimicking RA. Their diagnosis of
gout was confirmed by demonstration of urate crystals in synovial
fluid. The 8 AS patients were all HLA-B27 positive. The 7 PDB patients had markedly elevated serum alkaline phosphatase and urinary
hydroxyproline. Of the 4 lymphoma patients studied, 3 had stage 3 to
4 of Hodgkin’s disease and the fourth had stage 3 to 4 non-Hodgkin’s
disease.
Clq levels were measured by the usual radial immunodiffusion (RID) assay and by a procedure based on our finding that
C Iq is the only hydroxyproline-containingprotein in the euglobulin
fraction of serum, and that all Clq in serum precipitates with this
fraction. The C l q content of serum can therefore be estimated by
multiplying the hydroxyproline content of the euglobulin fraction by
23.26, since Clq contains 4.3% hydroxyproline. Clq levels in sera of
patients with RA determined by this method were found to be significantly higher (P<
than levels in sera of normal patients. Levels
of another, acidic hydroxyproline-containing protein appear to decrease in patients with long standing RA (P< 0.05). The increase of
Clq observed in RA was not seen in SLE, PGA, AS, PDB, or lymphoma. The increase of Clq observed in RA is also observed when
measured by radial immunodiffusion. We have reported our C Iq values in terms of the hydroxyproline content of the euglobulin fraction
since our evidence indicates that RID overestimates the Clq content
of sera. Our data also suggest that the level of Clq in sera of RA patients may be an aid in diagnosis of RA since Clq was not elevated in
the other groups. The data also suggest that the level of Clq in RA
may aid in assessing the severity of the disease and response to therapy.
ARA ABSTRACTS
65 1
Gold compounds inhibit mitogen-induced immunoglobulin production in humans
Stuart A. Rosenberg and Peter E. Lipsky, University of Texas Health Science Center at Dallas
The mechanism by which gold therapy slows the progression
of disease activity in rheumatoid arthritis (RA) patients is not understood. However, since chrysotherapy is associated with a significant
decrease in serum Ig and rheumatoid factor levels, it is reasonable to
suggest that the therapeutic effect of gold in RA involves inhibition of
Ig production by involved synovium.
To evaluate this possibility at the cellular level, peripheral
blood mononuclear cells (PBM) were separated from venous blood of
healthy volunteers. PBM were cultured in microtiter wells and stimulated by optimal concentrations of pokeweed mitogen (PWM) or
Staphylococcusaureus protein A (SPA) with or without the addition
of gold compounds. After a 7 day incubation, the cells were harvested,
washed, and antibody production was studied by a reverse hemolytic
plaque assay (RHPA) that identified the total number of IgG, IgM,
and IgA immunoglobulin secreting cells (ISC). In 5 separate experiments gold sodium thiomalate (GST) 10 pg/ml suppressed PWM-induced generation of ISC by a mean of 71 f 8.3% and SPA-induced
ISC by 52.2 f 4.3%. This inhibition was mediated by gold, since cells
cultured with thiomalic acid (TMA) showed no decrease in the number of ISC generated compared to controls, while gold chloride
(GCL) in equimolar concentrations inhibited the generation of ISC
by a greater degree than GST. The results of a typical experiment are
shown in the Table.
The action of gold could not be ascribed to cell death nor to
nonspecific interference with the RHPA. Additional experiments
showed that maximum inhibition of ISC generation occurred only
when GST was added within the first 24 hours of culture. This suggested that gold compounds inhibited an early step in the activation
and differentiation of ISC. These studies demonstrate that concentrations of gold equivalent to those obtained in the serum and tissues of
RA patients undergoing chrysotherapy inhibit the capacity of B cells
to differentiate into Ig secreting cells. Such inhibition may explain the
decrease in RF titers and reduction in joint inflammation observed in
RA patients treated with gold.
Mitogen-induced Ig secreting cells/ 106 cultured PBM
Mitogen
No inhibitor
GST 10 pg/ml
TMA 5 pg/ml
GCL IOpg/ml
PWM
SPA
9,700
6,800
2,050
2,700
10,450
7,050
1,700
600
Quantitation of radionuclide uptake in rheumatoid arthritis
Karen Rosenspire, Monte Blau, Alastair C.Kennedy, and Floyi A . Green, State University of New York at Buffalo
Knees, hands, forearms, and thighs in 20 male patients with
classic rheumatoid arthritis and 20 age and sex matched controls were
imaged with 9 y m Tpyrophosphate.
~
The uptake of 99mTcpyrophosphate in the joints of the hand and knee was quantitated using a
gamma camera interfaced to a computer.
The study was designed to assess the quantitative measurements of joint activity by analyzing the resultant data in a number of
ways. Counts per unit area normalized for body weight and dose of
radiopharmaceutical were found to be the most satisfactory method of
quantitating the differences in disease activity.
Surprisingly, the radionuclide counts per unit area over the
midpoint of the radius and ulna in the patients with rheumatoid arthritis (27.6 f 7.8; mean +. I SD) were found to be significantly higher
than those from the age and sex matched controls (20.9 f 6.8; mean f
I SD P c 0.01). This observation prompted a more detailed investigation of isotope uptake in an animal model of joint inflammation. Inflammation was induced in the right knee joint of New Zealand white
rabbits by injection of the joint with ovalbumin after sensitization of
the animal to this protein. The left knee was injected with sterile saline to serve as control. After administration of the 99mTcpyrophosphate, significantly increased counts were noted over the experimental
knee joint. The animals were then sacrificed and the midsections of
femurs from both sides dissected and counts per gram bone recorded.
There was a 43.6% increased uptake in bone obtained from the inflamed side, tending to confirm the observation made previously in
the patients with rheumatoid arthritis. Dissection of soft tissue, bone,
and cartilage from the rabbits’ knees revealed preliminary data showing that greater than 75% of radioactivity emanated from bone in the
joint whereas soft tissue and synovial fluid accounted for less than
10%.
The results of these studies provide possible evidence of increased radionuclide uptake in bone at a distance from inflamed
joints and may permit quantitative assessment of bone loss known to
occur in this disease. Also, the distribution of counts within the inflamed joints suggests that uptake in bone and not in soft tissue is the
major contributor to the scan image.
Genetic control of murine T cell response to collagen: Definition of the antigenic determinant
Lanny J. Rosenwasser, Rajendra S. Bhatnagar, and John D. Stobo, University of California at San Francisco
Recent interest has focused on the role of collagen as a POtential autoantigen in both experimentally induced animal arthritis
and rheumatoid arthritis in humans. In order to ascertain the immunogenic nature of collagen, we have employed an in vitro murine T
ARA ABSTRACTS
cell antigen recognition assay, utilizing T cells derived from mouse
peritoneal exudates, to gauge the proliferative response of sensitized
cells to denatured beef type I1 collagen. We find that H-2b mice will
respond to collagen (mean ACPM f standard error = 19,587 f 2718
for 5 experiments) while H-2k.a,d.hZ.h4
mice are nonresponders (mean
ACPM f standard error = 149 f 68 for 6 experiments). This pattern
of reactivity suggests that the specific immune response (Ir) gene(s)
that control the response of H-2b mice to beef collagen maps to the
left of the I-B subregion with control localized to either the K or I-A
subregion of the murine H-2 gene complex. Furthermore, sensitized
H-2b beef collagen immune T cells can be cross stimulated, in vitro,
by highly purified native and denatured rat type I1 collagen. These
immune T cells can also be cross stimulated by the synthetic p l y -
peptides (Pro-Gly)n, (Sarc-Pro-Gly)n, and (Pro-Sarc-Gly)n, while the
peptide (-Pro-)n will not cross stimulate. The peptides (Sarc-ProGly)n and (Pro-Sarc-Gly)n, while containing the same amino acids in
a slightly different primary sequence, differ greatly in both secondary
and tertiary polypeptide conformation. These data strongly suggest
that the antigenic determinants which stimulate collagen immune T
cell proliferation are dependent on unique primary amino acid sequence associations between both Gly and Pro residues and are relatively independent of secondary or tertiary conformation.
Experiments that directly assess the ability of macrophages
to present collagen’s antigenic determinants to immune T cells are
now in progress.
Increased production of tissue thromboplastin-like procoagulant activity by leukocytes due to stimulatory effects of
human anti-blood group antibodies
Henry Rothberger, Winston-Salem, North Carolina, James Dunne, Ottawa, Ontario, and Theodore S. Zimmerman, La Jolla,
California
In immunologic inflammatory reactions in which leukocytes
and opsonized particulate antigens are found, tissue damage may be
produced by components of the blood clotting system. Mechanisms
activating coagulation in these reactions are poorly understood. We
find that very small amounts of human anti-blood group antibody
bound to erythrocytes (EA) stimulate leukocyte production of tissue
thromboplastin-like procoagulant activity (TTLA), a potent activator
of the extrinsic clotting pathway. Mononuclear leukocytes 2 X lo6/
ml, isolated from blood on ficoll-hypaque, were incubated with 0.5%
suspensions of EA prepared by in vitro sensitization. After 3-16
hours, these cell mixtures were assayed for TTLA using one-stage
clotting tests. In a typical experiment, EA stimulated a 7-fold increase
of TTLA by 3 hours, a 15-fold increase by 4 hours, and a 20-fold increase by 7 hours as compared to controls with leukocytes and unsensitized erythrocytes. Pathophysiologic human antibodies from 5
patients with post-transfusional immunity to allogenic blood group
antigens were stimulatory in these studies, including human anti-Rh
and anti-A antisera and globulin fractions. In addition, rabbit anti-
serum to ox erythrocytes was stimulatory when ox red cells were present. The monospecific antibodies used were stimulatory only with red
cells bearing appropriate target antigens, e.g., anti-Rh antibody was
stimulatory with Rh+,but not with Ri- red cells. Thus, antigen-antibody complex formation was required for stimulation and artifacts
due to possible contaminants were exluded. Washed EA sensitized
with as little as a I/lOOO dilution of antiserum gave significant stimulation (Sfold), indicating that only very small amounts of antibody
were effective. Rosette depletion studies showed that leukocytes producing TTLA have Fc-R (Fc receptors) and Mrbc-R (monkey red
blood cell receptors). Thus, Fc-R and Mrbc-R enriched leukocyte
preparations produced 45-fold and 90-fold more activity than depleted preparations after addition of EA.
Our results suggest that human mononuclear leukocytes with
Fc-R and Mrbc-R may trigger coagulation pathways in immunologic
inflammatory reactions in which sensitized red cells or other opsonized particulate antigens are present.
Dermal-epidermal junction deposits in nonlesional skin of patients with systemic lupus erythematosus reflects clinical
and serologic disease activity
Naomi F. Rothfield, University of Connecticut, Farmington
Serial biopsies from the deltoid area were obtained in 3 I systemic lupus erythematosus (SLE) patients. The mean interval between biopsies was 22 months. The presence of IgG, IgA, IgM, C3,
C4, and Clq in the dermal-epidermal junction (DEJ) was correlated
with the clinical status and serologic abnormalities at the time of
biopsy.
Clinical activity (signs and symptoms of SLE) was graded
from I + to 3+; no clinical activity was 0. Clinical activity was assessed at the time the biopsy was taken and before the results of laboratory tests or immunofluorescence study of the biopsy were obtained.
In 13 patients there was a decrease in clinical activity from
3+ to 0 or from 2+ to 0. In these patients there was a significant decrease in the number of DEJ proteins (P= O.OOO1, paired f test). In 18
patients there was either no change in clinical activity or a minor
change (3+ to 2+, or I + to 0). In these patients there was no significant change in the number of DEJ proteins (P= 0.02, paired t test). In
individual patients, IgG and IgA were present more often during clinical activity than during quiescence. (P= 0.001, sign test). The presence of more than 4 DEJ proteins correlated with the presence of
serum C3 levels of 4 5 mg% (P= 0.005) and DNA binding of HI%
(P= O.ooOo2), but not with serum C4 of c 2 0 mg% (P = 0.215).
During disease activity 22 biopsies had 4 or more proteins
and 13 had less than 4 proteins in the DEJ. The mean serum C3 was
lower (P= 0.0016). C4 lower (P = 0.004), and DNA-binding higher
(P= 0.01) in the clinically active patients with more than 4 proteins in
the DEJ, than in the clinically active patients with less than 4 proteins
in the DEJ.
Clinical and serologic evidence of disease activity is reflected
ARA ABSTRACTS
653
by the presence of IgG and IgA and in the total number of proteins
present in the DEJ. In patients with clinical evidence of active disease,
serologic abnormalities are significantly worse in patients whose non-
lesional skin contains deposits of 4 or more proteins. Studies of serial
biopsies reflect the serologic and clinical status of the patient.
Serum amyloid protein, P-component, C-reactive protein and erythrocyte sedimentation rate interrelationships in
generalized amyloidosis
Alan Rubinow, Martha Skinner, and Alan S. Cohen, Boston University School of Medicine, Boston
The availability of immunoassays for serum amyloid protein
(SAA) and P-component (SAP), two serum proteins associated with
amyloidosis, prompted a study of the interrelationships between SAA,
SAP, C-reactive protein, and erythrocyte sedimentation rate in a
group of patients with systemic amyloidosis. The sera of 66 patients
with generalized amyloidosis (38 primary, 16 secondary, and 12 heredofamilial neuropathic type) were measured for SAA (radioimmunoassay), SAP (rocket immunoelectrophoresis), CRP (radial immunodiffusion), and ESR (Westergren method). The levels of S A A , SAP,
CRP, and ESR from the groups of amyloid patients and controls are
shown in the Table.
These studies indicate that 1) the SAP remains constant and
in the normal range in all types of amyloidosis; 2) in secondary amyloidosis modest increases in CRP (3.65 mg/100 ml) and ESR (80 mm/
hr) paralleled the strikingly high levels of S A A (mean 8,240 mg/ml
SSA (ng/ml of AA protein)
units AA protein) and the activity of the underlying chronic inflammatory or infectious disorder; 3) in primary amyloidosis the elevated
ESR (64 mm/hr) was accompanied by a mild increase in SAA (971
ng/ml AA protein) and normal CRP levels. These results probably reflect the dysproteinemia associated with primary amyloidosis and are
independent of those changes in acute-phase reactants accompanying
an inflammatory process; 4) all four parameters were in the normal
range in the heredofamilial group of patients.
These results, together with the varied clinical course and
unique biochemical characteristics of the amyloid fibril in the various
forms of amyloidosis, suggest again that the fibril represents the final
common pathway resulting from diverse pathogenetic mechanisms,
including pathologic immunoglobulin light chain formation and/or
degradation, chronic immunologic insult (inflammatory or infectious), and genetically predetermined abnormalities.
SAP (mg/100 ml)
Primary
97 1 (63-3850)
Secondary
8,240 (625-28,500)
Heredofamilial
329(112-1025)
Control
236 (80-500)
Numbers in parentheses indicate range.
6.27
7.60
5.72
7.47
CRP mg/100 ml
1.08 (4.5-8.4)
3.65 ( 0.7-6.8)
C0.5 (C0.5-1.1)
1.0 (C0.5-1.4)
ESR mm/hr
64 (5-137)
80 (34- 135)
16 (6-30)
1CL16
Complement activation in human serum by urate crystals: Evidence for a role for IgC
I. Jon Russell, University of Texas Health Science Center at San Antonio, Christos Papaioannou, Frederic C. McDufie, M a y Clinic,
Rochester, Minnesota
Studies by Naff and Byers, which demonstrated complement
(C) activation in serum by sodium urate crystals (NUC), did not find
a role for immunoglobulins. The demonstration by Kozin and
McCarty of IgG adsorption to NUC prompted us to reexamine this
question. Addition of NUC to normal human serum (NHS) resulted
in nearly complete depletion of hemolytic C. C was not activated by
calcium pyrophosphate, hydroxyapatite, or calcium urate crystals.
NUC were unable to activate C3 when either C2 deficient human
serum, C4 deficient guinea pig serum, or EGTA treated NHS was
used, indicating activation via the classic pathway. Sheep red blood
cells coated with antLC4 formed significantly more rosettes with
NUC which had been preincubated with either untreated NHS (49 f
2.8%) or zymosan treated NHS (SO & 0.7%) than with heat inactivated
NHS (26 & 4.2%) or EDTA treated NHS (36 4.9%). Rosette formation with “untreated NHS coated NUC” was inhibited by purified
C4 (29 f 2.4%). demonstrating specificity. These results further impli-
*
cate classic pathway involvement and suggest that activation of C
components occurs on the crystal surface. The relationship of serum
immunoglobulin to NUC dependent C activation was studied using
hypogammaglobulinemic sera from 8 patients with common variable
immune deficiency (CVID). Only minimal C activation occurred
when NUC were added to 4 of 8 CVID sera, though all had similarly
low levels of IgG and normal C levels. Reconstitution of C reactivity
with NUC occurred in a dose response manner with added IgG (2-8
mg/ml) but not with IgM ( 5 mg/ml) or IgG (5 mg/ml). Washed “IgG
coated” NUC also depleted C from CVID sera. These results demonstrate that urate crystals are capable of activating the classic C pathway by at least 2 mechanisms, one of which is dependent upon crystal
coating by IgG. The ubiquitous distribution of IgG and C plus the
findings of activated C3 and chemotactic factors in gouty synovial
fluids suggest that 1gG dependent C activation may have clinical significance in the early inflammatory mechanism of gout.
Extrusion of inorganic pyrophosphate by mature lapine and canine hyaline and fibrocartilage
Lawrence M. Ryan, Herman S. Cheung, and Daniel J. McCarty, The Medical College of Wisconsin, Milwaukee
Calcium pyrophosphate dihydrate crystals are found most
frequently in fibrocartilaginous tissue and to a lesser extent in hyaline
articular cartilage. Howell et a1 (J Clin Invest 56:1473, 1975) found
that inorganic pyrophosphate (PPi) was extruded into the medium
ARA ABSTRACTS
654
from immature (8 week) rabbit hyaline cartilage and from osteoarthritic articular cartilage, but not from mature (2 year) rabbit or
normal human hyaline cartilage.
Mature lapine and canine hyaline articular cartilage and
fibrocartilage (from menisci) were incubated in organ culture for 4
hours. Hydrolysis of PPi was determined by inclusion of tracer (32P)
PPi in the culture media.
PPi elaboration from both hyaline and fibrocartilage was
closely paralleled in both species by elaboration of uronic acid (r =
0.9;n = 10).
These data show that mature hyaline cartilage releases PPi
into tissue culture medium, although less is released per milligram of
tissue than from immature cartilage. More importantly, meniscal
fibrocartilage, the tissue most involved by calcium pyrophosphate de-
posits, also releases PPi. Since it has been shown that CPPD crystals
can be formed in gels at pH 7.0, we hypothesize that the chondrocytes
in each cartilage showing CPPD crystals released PPi which precipitated in the surrounding gel as its calcium salt.
Tissue
Samples
Lapine hyaline
Lapine meniscus
Canine hyaline
Canine meniscus
Freeze thawed
cartilage
2
2
2
4
3
PPI
Uronic acid
(pmol/mg tissue) (ng/mg tissue)
61
26
40
13
0
330
67
275
85
8
Biologic and methodologic variables of plasma inorganic pyrophosphate quantification
Lawrence M . Ryan,Franklin Kozin, and Daniel J. McCarty, The Medical College of Wisconsin, Milwaukee
Measurement of plasma inorganic pyrophosphate (PPi) is
potentially important in studies of calcium pyrophosphate deposition
disease, osteoarthritis, metabolic bone disease, and renal lithiasis. Interlaboratory disagreement as to plasma PPi concentrations in normal
populations implies unrecognized, uncontrolled variables in the assay
systems, specimen preparation, or metabolic state of the subjects. Six
variables have been identified which must be controlled to allow interpretation of PPi plasma levels: 1) Crystalline tetrasodium pyrophosphate decahydrate (Na4P207.10 H 2 0 ) standard loses 8 waters of
hydration during dessicated storage, potentially elevating standard
curves. 2) Application of a venous tourniquet consistently results in
an elevation = 55%, n = 4) of plasma PPi concentration compared
to that of simultaneous specimens from the opposite nonoccluded
(X
arm. 3) Systemic exercise produces an increase (X = 28%, n = 9, P <
0.01) in plasma PPi, returning to normal after a one-half hour rest. 4)
PPi concentration varies with time of day. 5) Arterial PPi (X = 1.88
pM) is lower than venous PPi (2.56 pM, n = 9, P < 0.01). 6) Plasma
PPi decreases after prolonged fasting. By controlling the standard, the
site, hour, and method of venipuncture and prandial and activity state
of the one normal subject tested on 10 consecutive days, a narrow
range of plasma PPi was noted (2 = 1.26 pM f 0.16 S D coefficient of
variation = 12.7%). (Coefficient of variation of the method = 8’36.0.)
Each of these six variables must be controlled in clinical studies of
plasma PPi levels. Under controlled conditions, variation in plasma
PPi was due mostly to variability in the analytic method.
Synovial fluid and plasma cyclic nucleotide levels in patients with arthritis
Richard I. R y e s and Shailaja Thanki, Albany Medical College, Albany, New York
The cyclic nucleotides, cyclic adenosine 3’,5’-monophosphate
(CAMP) and cyclic guanosine 3’,5’-monophosphate (cGMP), serve as
regulators for several processes which may contribute to inflammation. To assess the role of these substances in the pathogenesis of arthritis, levels of cAMP and cGMP in the synovial fluid (SF) and
plasma (PI) were measured by radioimmunoassay using hydrolysis
with cyclic nucleotide phosphodiesterase to check for specificity.
Forty-four paired specimens of SF and P1 from 41 patients were analyzed. Twenty-six came from patients with rheumatoid arthritis (RA),
8 from patients with osteoarthrosis (OA), and 10 from patients with
miscellaneous inflammatory arthritides (MIA) including 4 with psoriatic arthritis, 4 with pseudogout, 1 with gout, and 1 with Behcet’s
syndrome.
Cyclic nucleotide levels are shown in the Table.
SF cAMP was significantly lower in the group with RA than
in the group with MIA or OA. SF cGMP was significantly higher in
the two inflammatory groups, RA and MIA, than in the group with
OA. There was no significant difference in PI cAMP or PI cGMP levels among the three groups. SF cGMP correlated significantly with P1
cGMP (P < 0.05 for the groups with RA and MIA, suggesting the
possibility of passive diffusion of cGMP from plasma to SF. In contrast there was no significant correlation of SF cAMP with PI cAMP
for these groups; Loglo SF cell count did not correlate significantly
with SF cAMP for any group.
The increase in SF cGMP in the RA and MIA groups relative to the group with OA may be secondary to the inflammatory
process in the former two groups. However, the low SF cAMP found
in patients with RA appears to be related to factors unique to this disease.
Cyclic nucleotide levels*
RA
OA
MIA
SF cAMP
P1 cAMP
SF cGMP
PI cGMP
10.4k0.7t
14.6 f 1.1
14.9 f 1.2
21.3k1.1
21.6 f 1.2
23.9 k 1.4
6.6k0.4
2.5 k 0 . 3 t
6.2 f 1.0
7.1k0.6
4.9 k 0.7
6.8 f 1.0
* Picomoles/ml; mean
t Significantly
P c 0.05.
f standard error.
different from other groups: multiple range tests
ARA ABSTRACTS
655
Defects in T-B communication in systemic lupus erythematosus
Tsuyoshi Sakane, Alfred D. Steinberg, NIH, Bethesda, James L. Reinertsen, Minneapolis, Frank C. Arnett, The Johns Hopkins
University at Good Samaritan Hospital, Baltimore, Ira Green, NIH, Bethesda
Patients with systemic lupus erythematosus (SLE) have a variety of immune defects. The causes of these and their interrelationships remain elusive. Nevertheless, a growing body of evidence indicates that patients with active SLE have an increase in
antibody production and a decrease in T suppressor function. In order
to study in detail the T-B interactions which occur in normal controls
and patients with SLE, we have examined the autologous mixed lymphocyte reaction. This consists of the stimulation of T cells by autologous non-T cells, especially B cells. We have previously found that
patients with active SLE have a marked defect in the autologous
mixed lymphocyte reaction. Those with inactive SLE have a more minor defect limited to the T cell subpopulation with receptors for the
Fc portion of IgG (T gamma cells). In that study we could not be certain if the defect in SLE was in the responsiveness of the T cells or in
the stimulatory capacity of the non-T cells, or both.
In the present study two sets of identical twins discordant for
SLE disease activity were studied in an attempt to answer the questions. In the first set of identical twins (one inactive and one normal)
the T nongamma cells of both subjects responded well to both autologous and syngeneic non-T cells. However, the T gamma cells of the
SLE patients failed to respond to either autologous or syngeneic nonT cells whereas the normal twin responded well to the inactive SLE
non-T cells. This indicated that the defect in inactive SLE was limited
to the responsiveness of the T gamma population.
The second set of twins consisted of a patient with active
SLE and one with inactive disease. The active SLE T cells (T gamma
and T nongamma) all failed to respond to either autologous or syngeneic non-T cells, indicating a defect in responsiveness of all T cells.
Furthermore, the active SLE non-T cells of one twin failed to stimulate her sister’s T nongamma cells (whereas the sister’s T nongamma
cells responded well to autologous non-T cells) indicating an additional defect in stimulatory capacity of active SLE non-T cells. These
studies indicate that the shift from inactive SLE to active SLE is associated with a defect in both B cell and T cell function in T-B interactions and may lead to a better understanding of the cellular basis
for the immune abnormalities of active SLE.
Action of the neutral protease from human articular cartilage on proteoglycan
Asher I. Sapolsky, Kunio Matsuta, David S. Howell, and J.F. Woessner, Jr., University of Miami School of Medicine, Miami, Florida
Since degradation of proteoglycan occurs at the onset of
osteoarthritis, the action on proteoglycan of the neutral proteoglycanase extracted from human patellar cartilage was investigated.
Bovine proteoglycan subunit (PGS) was exhaustively digested by the
2000-fold purified metal-dependent human protease and the products
were chromatographed on Sepharose 2B. A peak of the PGS fragments was obtained with molecular weights ranging from 40,000 to
120,000. The peak was divided into 8 fractions which were characterized for amino acid and sugar content and for molecular weights by
analytic ultracentrifuge. The galactosamine/glucosamine ratio (g) was
inversely related to the fragment size, while the protein/sugar ratio
(p) was directly related. Small fragments (5,8) probably originated
from the chondroitin sulfate attachment region of PGS. Large fragments (1) probably came from the region of chondroitin sulfate and
keratan sulfate overlap. No single chondroitin sulfate chains were
produced. The range of final fragment sizes was smaller than that produced by cathepsins D and G and more like that produced by elastase
and trypsin. However, the specificity of action on PGS by the human
proteoglycanase differed from that of all these proteases, producing
fragments with a smaller average g ratio.
In the digestion of proteoglycan complex (PGC) by the hu-
man proteoglycanase, the viscosity drop lagged behind that of the
same amount of PGS but ultimately reached the same level. With enzyme amounts l,l/5, and 1/10 this lag was 45 minutes, 6 hours, and
140 hours. Complete digestion reduced the average sedimentation coefficient (S) of 44.38 PGC and 25s PGS to 3s. One-twentieth as much
enzyme and 140 hours incubation reduced both components to only
19s. Thus, the large proteoglycan pieces observed in osteoarthritic
joints of animal models, in which only a relatively small amount of
neutral proteoglycanase may occur, could originate by limited action
of the neutral proteoglycanase.
Supported by NIH Grants AM-16940, AM-08662. the Kroc
Foundation, and the W.L. McKnight Arthritis Research Fund.
PGS
Fraction 1
3
5
8
Molecular
weight
-2,000,000
-120,000
90,000
60,000
40,000
g
10.I
6.3
10.2
15.6
22. I
P
1.09
1.74
0.75
0.7 I
0.62
Evidence for a helper cell promoting anti-DNA antibody production in murine lupus
Shigemasa Sawada and Norman Talal, San Francisco, California
MRL/I and MRL/n mice are genetically identical except for
a single lymphoproliferative autosomal recessive gene in MRL/I mice
which is associated with massive lymph node enlargement and a severe autoimmune disease resembling SLE. We have measured the
spontaneous formation of antibodies to dsDNA by female MRL/l
and MRL/n mice both in vivo and in vitro. At 4 months of age,
MRL/l mice had more serum antibodies than MRL/n mice (276 f
50ng DNA bound/ml serum versus 41 f 53ng DNA bound/ml
serum). Furthermore, the major Ig class of anti-dsDNA in MRL/l
mice was IgG, whereas in MRL/n mice it was 1gM. The 1gM:IgG ratio in MRL/I mice was 0.5 (I456 cpm/3240 cpm) compared to 2.3
(1059 cpm/461 cpm) in MRL/n mice.
ARA ABSTRACTS
656
In tissue culture, spleen cells from 4 month old female
MRL/I mice produced more total antibody to dsDNA and more IgG
antibodies than spleen cells from MRL/n mice, as shown in the
Table.
To study whether helper cells are present in MRL/I mice, we
added lymphocytes from MRL/l spleen or lymph node to MRL/n
spleen cells in culture. To enrich for T lymphocytes, the MRL/I cells
were first passed through nylon wool. After the addition of 30%
MRL/I cells, the production of antibodies to dsDNA was increased 46 fold (P< 0.03), but there was no increase in antibodies to poly rA.
There was no increased production of antibodies to dsDNA if lym-
phocytes from MRL/n mice were added instead of MRL/I cells.
These results suggest that the lupus-like disorder of MRL/I mice may
be associated with helper cells promoting fotmation of antibodies to
dsDNA.
ngDNA bound/culture
MRL/n
MRL/I
64 + 9
906+ 117
49* I 1
258 149
*
21 * 2
802 f 120
The Occurrence of congenital heart block in infants of mothers with clinical or serologic evidence of rheumatic
disease
Jane G. Schaller, Carol Wallace, Stanley Stamm, Beverly C. Morgan, Seattle, Washington, Mike Patterson, Vancouver, British
Columbia
A study of mothers of children with known congenital heart
block (CHB) drawn from 3 pediatric cardiology services was undertaken. Mothers and children were examined in 1978-1979 and had
tests for antinuclear antibodies (ANA) and rheumatoid factors (RF).
All children were patients of pediatric cardiologists and had documented complete CHB.
Fourteen children with CHB were available; age at the time
of this study ranged from 5 months to 18 years. Eight were girls, 6
boys. Aside from CHB, all children were well in 1978-1979 with neither clinical nor laboratory abnormalities of rheumatic disease.
Eight of 14 mothers were found to have had clinical stigmata
of rheumatic disease. Six carried a clinical diagnosis of systemic lupus
erythematosus (SLE); 2 had had arthritis of unspecified nature. Rheumatic disease symptoms antedated pregnancy with the CHB child in 3
of 8 mothers, but followed the pregnancy by months to years in 5 of 8.
All 8 mothers with rheumatic disease symptoms were well during the
pregnancy in question; 7 of 8 received no antirheumatic drugs during
that time. Disease has been mild in most of the mothers; all are now
clinically well. Three mothers have had multisystem involvement of
SLE. None has had destructive arthritis. In 1978-1979,6 of 8 mothers
had positive ANA; 2 without positive ANA had positive tests for RF.
An additional mother although without past or present rheumatic
symptoms had a latex agglutination titer of 1:2560 in 1979. Only 5 of
14 mothers of CHB patients had neither clinical nor serologic abnormalities.
In this study a high proportion (9 of 14) of mothers of children with CHB were found to have clinical or serologic abnormalities
consistent with rheumatic disease, particularly SLE. These abnormalities often were first noted months to years after birth of the affected
child. Mechanisms for association of maternal rheumatic disease or
“rheumatic” serologic abnormalities with damage to the developing
conduction system of the fetal heart await definition; the most ready
explanation would be that some transplacentally passed substance is
responsible. Infants born to mothers with known rheumatic diseases,
particularly SLE, should be evaluated for CHB; however, the mildness or absence of symptoms in such mothers until later years may
obscure this association save in retrospect.
A clinical, immunologic, and genetic study of 100 patients with psoriatic arthritis
Peter H. Schur, Gary M. Kammer, and Nicholas A . Soter, Harvard Medical School, Boston, Massachusetts
One hundred patients with both psoriasis and an inflammatory arthritis were studied. These patients have a distribution of synovitis which permits subclassificaiton into three groups: 54 patients had
an asymmetric, oligoarticular arthritis (Group I); 25 had a symmetric
arthritis (Group 11); and 21 had a spondylarthritis with or without peripheral arthritis (Group 111). Individuals with an asymmetric, oligoarticular arthritis (Group I) experience intermittent exacerbation of
synovitis usually responsive to medical therapy; however, about threefourths of patients sustain a mild or moderately progressive disease
course. In contrast, persons with a symmetric arthritis (Group 11) experience a slowly, destructive arthritis in about one-half of cases.
Spondylarthritis (Group 111) may be associated with a peripheral arthritis and has a course similar to that of other spondylarthritides.
Distal interphalangeal joint involvement occurred in 10 patients in
Group I, in 3 patients in Group 11, and in only 1 patient in Group 111.
Arthritis mutilans occurred in 7 patients; it evolved rapidly over
months or more slowly over years to produce a destructive arthritis
and evolved with equal frequency from each group.
There was a significantly decreased prevalence of HLA-B7
( I 1% versus 25% in normals). HLA-B27 was significantly increased in
Groups I1 and I11 (36% and 57% versus 6% n o r m a l s P = 1.2 x
Of those individuals with B27, 8 of 9 in Group I,
and P < 1 X
9 of 9 in Group 11, and 8 of I I in Group 111 also had A2-these are
found together in 1.7% of normals.
Patients with psoriatic arthritis treated with aspirin and hydroxychloroquine experienced a beneficial response in 75% of trials
without exacerbation of psoriasis. Patients treated with gold salts responded in 63% of trials. Nonsteroidal antiinflammatory agents suppressed disease activity in 55% of patients but did not induce remission.
Features that distinguish psoriatic arthritis from similar disorders such as rheumatoid arthritis include the paucity of rheumatoid
factors, radiographic demonstration of predominantly interphalangeal joint involvement of the hands and feet, the presence of
onychodystrophy, a clustering of psoriasis and psoriatic arthritis in
the first degree family members, and the significant association of
HLA-B27.
ARA ABSTRACTS
657
"Strontium reduces autoimmunity in NZB/NZW mice
William E. Seaman, Marcia A . Blackman, John S. Greenspan, and Norman Talal, University of California in San Francisco
We have studied the effects of "Strontium (89Sr) on NZB/
NZW (B/W) mice, which spontaneously develop an autoimmune disease resembling lupus erythematosus. 89Srlocalizes to bone and irradiates the marrow, reducing cells called "marrow-dependent'' (M)
cells. M cells mediate natural killing (NK) and natural resistance to
bone marrow transplantation (NR). Normally, "Sr has little effect on
T or B lymphocytes, but under certain circumstances a reduction of M
cells by R9Sris accompanied by a rise in T suppressor cells (Ts). B/W
mice have high M cell activity (both NK and NR) and an apparent
reduction in T suppressor cells. In order to study the relation between
high M cell activity and autoimmunity, we treated 20 female B/W
mice with "Sr, a total of 270 pCi in divided doses from 1 to 7 months.
NK was followed as a measure of M cell suppression.
In control B/W mice, antibodies to native DNA (antinDNA) rose steadily from 5 months until dealth at 9 months. In 89Srtreated mice, there was no rise in anti-nDNA, even at 9 months. Proteinuria and glomerular immunoglobulin (Ig) were significantly less
in 89Sr-treated mice than in controls. Serum Ig and IgG were equal in
both groups at 2 and at 5 months, but higher in controls at 8 months
(1045 mg Ig/dl versus 613 mg Ig/dl). This Ig difference, however, did
not fully account for differences in anti-nDNA at 8 months, control
mice bound 20.6 f 10.6 ng nDNA/mg Ig, while 89Sr-treated mice
bound only 11.3 f 4.8 ng nDNA/mg Ig (P< 0.01). Despite the reduction in autoimmunity, 89Sr-treated mice died slightly earlier than controls (50%death at 8.7 months versus 9.1 months for controls) because
more than half the 89Sr-treated mice developed sarcomas.
Throughout the study, IgM was 30-8070 higher in 89Srtreated miced versus controls, suggesting that IgM-producing B lymphocytes were sustained or even increased. At 5 months, S9Sr (total
120 pCi) did not reduce splenic plaque-forming cells to sheep erythrocytes nor mitogenic responses to lipopolysaccharide, phytohemagglutinin, or Concanavalin A. Spleen morphology and content of T cells
(by esterase stain) and Ig-positive cells were also normal. These studies suggest that 89Srblocks the accelerated phase of autoimmunity in
female B/W mice, which normally begins at 5 months. We are investigating the possibility that this effect of x9Sr results from either a direct influence on B cell maturation or from a rise in T suppressor cells.
Cross reactivity of antinuclear and antilymphocyte antibodies in systemic lupus erythematosus
Robert P. Searles, Ronald P. Messner, and Arthur D. Bankhurst, University of New Mexico School of Medicine, Albuquerque
Antinuclear antibody (ANA) and antilymphocyte antibody
(ALA) are two antibodies prevalent in systemic lupus erythematosus
(SLE). ANA has been demonstrated in all antibody classes while
ALA has been shown to be a predominantly cold-reactive IgM antibody. This report demonstrates data supporting cross reactivity between the two antibodies. IgM and IgG ANA were selected for investigation using a conventional indirect immunofluorescent (IMF)
technique with mouse liver substrate. IgM ALA was determined by
microcytotoxicity and IMF of suspensions of T-lymphocytes. Sera
were selected from 6 different active SLE patients positive for both
ANA and ALA. Titers for ANA were e 1:256, while cytotoxic or IMF
ALA titers were 5 1:128. Differential absorption of SLE sera was accomplished using fresh preparations of human erythrocytes, human
lymphocytes, chicken erythrocytes, and chicken erythrocyte nuclei.
The results demonstrate that chicken nuclei (intact by phase microscopy) were able to absorb IgM ALA ( 5 5 - 1 W o absorption) while human and chicken erythrocytes had no significant effect. Enzymatic digestion of the chicken nuclei with RNase partially removed their
capacity to absorb ALA. Absorptions with human lymphocytes removed > 80% of ANA activity and 7 1 - 1 W of ALA. Both IgM and
IgG ANA were also absorbed by the chicken nuclei (75-98'70). Human and chicken erythrocytes had no significant effect on the IgG
ANA. In elution experiments, both ALA and ANA were found in
PBS eluates from absorbed nuclei with recovery of ALA being greater
than ANA. These results suggest cross reactivity between nuclear and
lymphocyte surface membrane antigens and support the existence of a
complex interrelated autoantibody system operative in systemic lupus
erythematosus.
Granulocytosis and increased granulocyte adherence in psoriasis and psoriatic arthritis
Julie B. Sedgwick, Paul R. Bergstresser, and Eric R. Hurd, University of Texas Health Science Center at Dallas
Granulocytes are a prominent feature in the epidermal and
papillary dermal infiltrate of psoriasis. Furthermore, granulocytes
from patients with systemic inflammatory disorders have been shown
to be more adherent to endothelial walls in vivo and to nylon fiber in
vitro. To determine whether granulocyte participation in psoriasis
might reflect a more generalized phenomenon, we investigated in
vitro granulocyte adherence (GA) to nylon fiber columns (MacGregor
et a 1 N Engl J Med 291:642, 1974). GA was significantly increased in
28 patients with psoriasis compared to 28 normal controls (33.9% versus 19.2%; P < 0.001). The increase was positively correlated with extent of disease and was greatest in those patients with arthritis
(43.3%). The psoriasis patients also exhibited a significant gran-
ulocytosis compared to the normal controls (5600/mm3 versus 32001
mm3; P < 0.001). GA was not increased in 6 patients treated with lithium carbonate (17.5%) or in 10 control patients with extensive skin inflammation from sunburn or allergic contact dermatitis (16.3%). Both
of those groups exhibited a significant granulocytosis of 6000/mm3 (P
< 0.002) and 5000/mm3 (P < 0.005) compared to normal controls.
Therefore, increased GA is not dependent on the granulocytosis of
psoriasis and is not a general occurrence in cutaneous inflammation.
These data suggest that circulating granulocytes in psoriasis exhibit
altered physiologic function and that the factor@) responsible for
these changes may contribute to the migration and deposition of granulocytes in the cutaneous lesions of psoriasis and psoriatic arthritis.
ARA ABSTRACTS
Increased 0,-generation by serum from psoriasis and psoriatic arthritis patients
Julie Sedgwick, Paul R. Bergstresser, and Eric R. Hurd, University of Texas Health Science Center at Dallas
We have reported increased in vitro adherence of circulating
granulocytes (PMN) from psoriasis and psoriatic arthritis patients.
Both this observation and our finding that these patients have elevated serum C4 levels suggest that psoriasis is not confined to the skin
but has systemic manifestations which include altered PMN function.
To determine the ability of psoriatic serum to stimulate normal PMN
with zymosan treatment, we measured superoxide (02-)generation
by ferricytochrome C reduction (Goldstein IM et al: J Clin Invest
56: 1155, 1975). PMN from normal donors, incubated with serum from
untreated psoriasis or psoriatic arthritis patients, had a significant (P
i
0.01) increase in cytochrome C reduction by 15 minutes when compared to 16 normal sera. This difference increased with time, both at
20 minutes (P< 0.004)and at 25 minutes (P< 0.002). The nmoles of
cytochrome C reduced at the time intervals are shown in the Table.
Complement inactivation of test sera before incubation re-
sulted in a decrease in cytochrome C reduction to < 10 nmoles at 25
minutes. Omission of zymosan from the reaction resulted in a similar
decrease. Under these two conditions, there appeared to be no difference between the groups. These data suggest that patients with psoriasis and psoriatic arthritis have a complement-dependent factor
which leads to increased PMN activation after stimulation with zymosan. This factor may be absent or decreased in normal serum. Alternatively, psoriatic serum may lack a normal inhibitor of PMN stimulation and 02-generation. With systemic treatment, psoriasis patients
revert to the normal state. 02-generation has been reported to cause
autooxidation, and depolymerization of hyaluronic acid and bovine
synovial fluid. Therefore, increased PMN stimulation may play a role
in the pathogenesis of the skin lesions and joint destruction in psoriasis and psoriatic arthritis patients.
Time (minutes)
Normal controls
Psoriasis and psoriatic arthritis
Untreated
Treated
n
5
10
15
20
25
16
3.5
12.4
24.4
34.2
44. I
10
4.5
2.1
17.9
12.9
33.4
24.4
46.9
36.7
56.4
45.2
9
Immune complexes in the cerebrospinal fluid of patients with systemic lupus erythematosus
James R. Seibold, Robert B. Buckingham, and Robert H. Kelly, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Concentrated cerebrospinal fluid (CSF) from 25 systemic
lupus erythematosus (SLE) patients was subjected to high resolution
agarose gel electrophoresis. In 15 of 15 patients with definite central
nervous system (CNS) SLE and 5 of 5 patients with probable CNS
SLE, a distinctive morphologic banding pattern was found in the
gamma globulin region that is considered to represent immune complexes (IC). CSF from 5 control patients with active SLE but without
evidence of CNS involvement had normal electrophoretic patterns. IC
were demonstrable in all CNS SLE presentations including seizure
disorder, motor deficit, organic thought disorder, and multiple sclerosis-like syndromes. Simultaneous analysis of our patients’ sera for
IC and calculation of CSF/sera gamma globulin ratios suggest that
these complexes are generated locally in the CNS and are not present
by virtue of “spill over” through an altered blood brain barrier. Electrophoretic demonstration of CSF IC was the most consistently abnormal laboratory parameter. In the 20 patients with either definite or
probable CNS SLE, I 1 had abnormal EEG and 2 had abnormal brain
scans. CSF abnormalities included 3 with pleocytosis and 10 with ei-
ther elevated IgG or increased proportion of IgG to total protein.
Demonstration of IC by electrophoretic pattern recognition
relies on the altered electrophoretic mobility of antibody when bound
to antigen. Extensive evidence in sera suggests the characteristic
banding pattern is IC. A significant positive correlation with the Raji
cell assay has been found in representative positive and negative sera.
Demonstration of IC by electrophoresis in experimental serum sickness of rabbits indicates sensitivity comparable to that of the Raji cell
assay. Characterization of the rabbit electrophoretic band by immunofixation techniques demonstrates the expected presence of the bovine serum albumin antigen. The pattern is disrupted in acid pH as is
typical of IC.
The demonstration of CSF immune complexes by agarose
electrophoresis appears to be a highly sensitive test for the detection
of CNS SLE. This procedure has the advantages of being rapid, inexpensive, and readily suited to routine clinical laboratory capabilities. These data comprise evidence that immune complexes play a role
in the pathogenesis of CNS SLE.
The sex of siblings of men with connective tissue diseases
Mitchell Selznick and David Kaplan, State University of New York, Brooklyn
Men attending the Arthritis Clinic between July I , 1978 and
October 30, 1978 were interviewed in regard to the number and sex of
their siblings (both full sibs and half-sibs). Twenty-four men with systemic lupus erythematosus (SLE) had 69 brothers and 41 sisters.
Twenty-one men with rheumatoid arthritis (RA) had 99 brothers and
66 sisters. Twenty-five men with other connective tissue diseases
(CTD) lacking gross female preponderance (e.g., Reiter’s, polymyositis, progressive systemic sclerosis, etc.) had 62 brothers and 67
sisters.
The men with SLE, with a sister/total sib ratio of 0.37, thus
ARA ABSTRACTS
had a relative paucity of female siblings (P = 0.03) compared to the
CTD control patients. They also appeared to have an absolute paucity
of sisters, though the difference was not statistically significant compared to the CTD control group ( I .7 versus 2.7 average number of sisters).
Men with RA came from larger families than did the CTD
control patients (Pc 0.05, two-tailed) and also had a relative paucity
of sisters (P = 0.05).
Birth order, age, and sex were similar in all three groups.
Seventy-two women with SLE had the expected ratio of brothers to
sisters (172 to 177).
659
We conclude that maleness is protective against SLE, but
that the protection is probably not hormonal since the men were neither generally prepubescent or aged (85% of both the men and women
were between the ages of 15 and 50, and the mean age for both was 30
years). Rather the data suggest that SLE is a vertically transmitted
disease, and a more severe abnormality, or a larger dose of the causative agent, is required for expression to occur in the male. Fetal wastage of affected female sibs is thus high in sibships in which males are
affected.
Antibody-mediated aplastic anemia and thrombocytopenic purpura in diffuse eosinophilic fasciitis
Lawrence E. Shulman, NIH, Bethesda, Ronald Hogman, Nicholas Dainiak, Yale University School of Medicine, New Haven, John
Nesbitt, The Johns Hopkins University, Baltimore, Harold M. Adelman, Tampa Florida, Oliver J. Lawless and Stephen M. Lindsey,
Washington DC
In 1975 (Trans Assoc Am Physicians 88:70) 4 patients were
described with “diffuse fasciitis with eosinophilia,” a newly recognized disorder resembling scleroderma and characterized by: collagenous hypertrophy and lymphocytic infiltration of fascia separating
subcutaneous fat from muscle, skin bound to underlying structures,
flexion contractures, eosinophilia in blood and bone marrow, plasmacytosis in lesions and bone marrow, high levels of serum IgG; all
preceded by unusual physical exertion, and responding to corticosteroids.
In a 1-year period (1977-1978), beginning with the fourth patient, 5 patients with diffuse fasciitis were found to have serious hematologic disease, aplastic anemia in 3 and thrombocytopenic purpura in
2. In 3 the hematologic disease and fasciitis emerged together; in 2 the
hematologic disease appeared after the fasciitis entered remission.
Two with aplastic anemia died; the third is responding to corticosteroids. Both with thrombocytopenic purpura are responding to therapy.
To determine if the marrow aplasia was immunologically
mediated in 2 of the aplastic anemia patients, in vitro assay systems
for growth of erythroid colonies (burst-forming unit erythroid [BFUEl, colony-forming unit erythroid colonies (CFU-E] and myeloid
macrophage colonies (CFU-C]) were utilized. Addition of both patients’ serum markedly suppressed the number of BFU-E by 98loo%, CFU-E by 90-98%, and CFU-C by go%, generated by control
marrow cells. This inhibitory activity was isolated to a serum IgG
fraction. Bone marrow cells from one aplastic anemia patient generated 30% of the normal number of CFU-E: addition of normal serum
did not influence CFU-E formation, while the patient’s own serum inhibited autologous CFU-E growth by 70%.
Diffuse fasciitis, usually benign and reversible, may be a serious disease, at times associated with life-threatening autoimmune
hematologic disorders. An hypothesis is proposed that the marrow
aplasia, thrombocytopenia, and fasciitis are all immunopathic consequences of an aberrant lymphoplasmacytic proliferation.
Effect of antiinflammatory drugs and metabolic inhibitors on superoxide generation by human neutrophils
Louis Simchowitz, Jagdish Mehta, and Isaias Spilberg, Washington University School of Medicine, St. Louis, Missouri
Current evidence supports the concept of superoxide radicals
(02Jplaying a role in the pathogenesis of tissue injury in rheumatic
diseases. Superoxide dismutase, which enzymatically destroys 02-,
has been shown to ameliorate several types of inflammation in vivo,
including rheumatoid arthritis. To further evaluate the pathways involved in the inflammatory process in the rheumatic diseases, the role
of a chemotactic factor as an inducer of 02-generation by human
neutrophils and the effect of antiinflammatory agents on this neutrophi1 function were assessed. In addition, through the use of metabolic
inhibitors, the role of glycolysis, microtubules, and protein synthesis
on the production of 02-was investigated. This study shows that human peripheral neutrophils generated 02-as measured by fenicytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine4 X 10-8M.
Superoxide generation was inhibited by 2-deoxy-D-glucose (ID50 4 x
10-5M),2-iodoacetate (ID50 5 X 10-5M), and N-ethylmaleimide
(ID50 5 X 10-6M), suggesting a dependence on anaerobic glycolysis
and sulfhydryl groups. Ouabain, microtubule-disrupting agents, inhibitors of respiration, oxidative phosphorylation, and protein and
nucleic acid synthesis were without appreciable effects. Indomethacin
(ID50 1 x 10-4M),ibuprofen (ID50 9 X 10-4M), and phenylbutazone
M ) all caused dose-dependent inhibition of 02-gen(ID50 1 X
eration at doses approximating therapeutic concentrations. Acetylsalicylic acid (125-500 pg/ml) and aurothioglucose ( 10-3-10-6M)
were without appreciable effects. Superoxide generation was inhibited
only by relatively high concentrations of hydrocortisone (ID50
10-3~).
These studies suggest that the production of a potential cytotoxic factor may be subject to pharmacologic manipulation and that
at least some of the antiphlogistic effects of the nonsteroidal antiinflammatory agents could be mediated through effects on O2 - generation.
Studies of Reiter’s syndrome following epidemic shigellosis
David Simon, Richard Kaslow, Centerfor Disease Control, Atlanta, Andrei Calin and Ronald Kaye, Stanford University, California
In order to better define the epidemiology of postdysenteric
Reiter’s syndrome (RS), we conducted followup investigations after 3
outbreaks of dysentery caused by 3 different strains of Shigellu. Several weeks after the episodes of infection, we sent a screening ques-
ARA ABSTRACTS
660
tionnaire to 1,439 individuals who were at risk of developing shigellosis and were accessible by mail; 1,155 people (80%) responded. For
fewer than 10% of these respondents the diagnosis of RS remained
enough of a possibility to require followup by telephone interview
with the subjects or their physicians. HLA-B27 testing was performed
in all instances in which RS was suspected.
We found 3 cases of RS, all B27 positive, among a group of
204 persons who acquired Shigelluflexneri 1 aboard a cruise ship. All
3 were in women, although similar numbers of male and female passengers had dysentery. No RS was discovered among 267 unaffected
passengers. After a second outbreak of Sflexneri 2a in conventioneers,
we evaluated 55 1 people (46 I women and 90 men), 206 of whom were
known to have had diarrhea. Two women with dysentery but no men
were considered to have R S one of the women was HLA-B27 positive. The third outbreak, caused by S sonnei occurred at a summer
camp among 188 people, predominantly children and young adults,
99 of whom were known to have had dysentery. We found no cases of
RS among 133 questionnaire respondents.
Our investigations in defined populations confirm that RS
follows 1-2% of Sflexneri 1 and 2a infections and indicate that
women have at least the same risk as men of developing postdysenteric RS. This is also the first report, to our knowledge, of RS
following documented Shigellaflexneri 1 infection. In the S sonnei investigation, we attempted unsuccessfully to find a counterexample to
the hypothesis that this strain is not arthritogenic.
Gold suppression of human neutrophil function
Anthony J. Sliwinski and Maurice A . Guertin, Georgetown University Medical Center, Washington, D. C.
The mechanisms of action of gold salts remain unclear. The
effects of gold thiomalate (GT) and a new oral gold preparation, Auranofin (AuO), on neutrophil function were measured by quantitative
leukocyte iodination which is a measure of ingestion, degranulation,
and myeloperoxidase activity. Studies were done with and without
carrier iodide. In the absence of carrier iodide, under conditions of
maximal neutrophil stimulation, concentrations of 1, 2, and 5 pg Au/
ml GT suppressed iodination to 91, 80, and 66% while AuO suppressed iodination to 99, 97, and 65% of control values. Under conditions of submaximal stimulation, suppression was enhanced and demonstrable with lower concentrations of both drugs. Decreasing the
amount of serum to decrease protein binding of Au did not change
GT suppression but increased AuO suppression indicating the active
portion of GT is protein-bound while the active portion of AuO is not
protein-bound. To separate effects between suppression of endocytosis and myeloperoxidase activity, the radioactive iodide and drug
were added 15 minutes after neutrophil stimulation with zymosan.
Less suppression of iodination was observed, indicating that the suppression was in part due to inhibition of mechanisms prior to myeloperoxidase activity. Addition of 5 nmoles carrier iodide/5 X 106 neutrophils resulted in increased suppression. GT suppressed iodination
to 86, 76, and 66% at concentrations of 1, 2, and 5 pg Au/ml while
AuO suppressed iodination to 91,73, and 4% of control under conditions of maximal stimulation. This suppression of neutrophil function
may be one of the mechanisms of actions of gold drugs.
The clinical and laboratory features of patients with subacute cutaneous lupus erythematosus: A distinct lupus
erythematosus subset
Richard D. Sontheimer and James N . Gilliam, University of Texas Health Science Center at Dallas
We have observed a clinically distinct type of skin disease in
27 of 299 lupus erythematosus (LE) patients whom we have studied
over a 7-year period. This previously uncharacterized form of cutaneous LE occurs as a nonfixed symmetrical, erythematous eruption in
predominantly papulosquamous or annular patterns over the face,
upper trunk, and extensor surfaces of the upper extremities. It may
persist for weeks, months, or years but resolves without scarring. We
have designated this type of cutaneous LE as subacute cutaneous LE
(SCLE) to emphasize its remitting, nonscamng course which is distinct from the fixed, indolent nature of generalized scarring discoid
LE. Twenty-two of the 27 patients had lesional skin biopsies all of
which showed a lymphohistiocytic infiltration in the dermis and hydropic degeneration of the epidermal basal cell layer, features considered diagnostic of LE. Most of the patients having this type of s k i n
disease were middle-aged white (85%) females (70%). Thirteen of the
22 patients (48%) had four or more ARA criteria for systemic LE. Arthritis/arthralgia was the most frequent extracutaneous manifestation
(74%). Mild forms of central nervous system (CNS) abnormality
(unexplained seizures and thought disorders) occurred in 5 patients
(19%). Renal disease occurred in 3 patients (1 I%) and was severe in
only one. Pleuritis and Raynaud’s phenomenon were uncommon, occurring in only 2 patients (7%) each. Abnormal levels of antinuclear
antibodies were the most frequent laboratory abnormality in this
group (63%). Low levels of double-stranded DNA antibodies were
seen in 10 patients (37%). Only one-fifth of the group had leukopenia
or depressed C3 or CH50 levels. Nine of 15 patients who had lesional
skin biopsies had immunoglobulin depositsat the dermal epidermal
junction (DEJ-Ig) by direct immunofluorescence.This was surprising
since lesional DEI-Ig is observed in 90% or more of scarring discoid
LE patients. Thus, the presence of SCLE identifies a distinct subset of
LE patients who frequently had a mild systemic illness marked by
musculoskeletal symptoms and a low incidence of severe CNS or
renal involvement. These patients generally have an illness intermediate in severity between that of scarring discoid LE and systemic
LE without cutaneous involvement.
ARA ABSTRACTS
66 1
Three courses of juvenile dermatomyositis
Charles Spencer, Helen Kornreich, Bram Bernstein, Karen King, Bernhard Singsen, and Virgil Hanson, University of Southern
California School of Medicine, Los Angeles
Eight children had a unicyclic, 10 a polycyclic, and I4 a continuous course, as shown in the Table; 22/32 are in remission, 9 are
active, 1 died. Thirty of 32 are in functional class I or 11.
The course groups were compared by age of onset, sex, ethNc group, mode of onset, and signs of disease and laboratory findings
at diagnosis. No significant differences were found.
Six of 66 patients died; 5 of 6 died within 1 year of diagnosis:
4 died of complications while receiving steroids (2 gastrointestinal
perforations, 1 sepsis, I pneumonia), 1 died of aspiration pneumonia
while off steroids. The sixth died of pulmonary fibrosis after a 9 year
polycyclic course.
Although the overall prognosis of JDMS in our series is
good, the course is often prolonged and complicated. Three different
courses, with varying duration of disease, may be seen. Onset data do
not predict which pattern a child will follow. This variability in course
suggests the need for individualization of therapy.
Delineation of the course of juvenile dermatomyositis
(JDMS) is essential to development of concepts of pathogenesis and
therapy. Recent reports emphasize a benign, short term course of
JDMS for patients on steroid therapy. Our experience with 66 children (1960-1978) seen at various stages of disease suggested that, although the overall prognosis is good, the course of JDMS for those on
steroids varies greatly. We suggest that there are 3 different courses:
( I ) unicyclic (U): recovery within 2 years of diagnosis without relapse;
(2) polycyclic (P): 1 or more remissions followed by relapse; (3) continuous (C): persistent disease beyond 2 years.
To test this hypothesis, we reviewed all 32 cases (mean followup 7 years, range 2-13 years) that met these criteria: a) availability
of detailed records from this or other facilities when management was
shared b) 2 years minimum followup. Clinical parameters included
muscle remission (rem) (good-normal strength), rash remission (absence), cessation of steroids (> 1 months), and clinical recovery (both
muscle and rash remission).
Time (in months) to:
Muscle remission
Course
U
P (1st rem)
C W14)
Off steroids
Rash remission
Clinical recovery
Mean
Range
Mean
Range
Mean
Range
Mean
Range
8
3-15
6-38
26-90
7
10
3-16
44
12-97
13
41
47
3-27
17-70
9-99
11
42
53
616
17-62
2697
15
51
5-18
The mechanism of action of colchicine in acute urate crystal-induced arthritis
Isaias Spilberg, Brian Mandell, Louis Simchowitz, and David Rosenberg, Washington University School of Medicine, St. Louis,
Missouri
Colchicine is unique among therapeutic agents used in the
treatment of acute arthritis in that its usefulness is generally considered to be limited to crystal induced arthritis and it is not a potent
general antiinflammatory agent. In vitro, it has been shown to diminish polymorphonuclear leukocyte (PMN) chemotaxis and lysosomal
enzyme release. However, it seems unlikely that the major in vivo
pharmacologic action of colchicine is via either of these mechanisms
since neither is unique to crystal induced idlammation. Moreover, a
drug capable of suppressing such PMN functions would be expected
to have more general antiinflammatory effects than colchicine exhibits. The question, therefore, remains as to the existence of a colchicine-sensitive step in the development of crystalline arthritis which is
of secondary importance in other arthritides. Phagocytosis of urate
crystals by human or rabbit neutrophils has been shown to induce the
synthesis and release of a glycoprotein which is chemotactically active
both in vitro and in vivo. It has been proposed that this chemotactic
factor is a prime mediator of the acute gouty attack and colchicine has
been shown to decrease the production and release of this factor in
vitro, suggesting this pharmacologic effect as the mechanism of action
of the drug. If this postulate approximates the events in vivo, colchicine should ameriorate the arthritis induced by crystals but not the
one induced by chemotactic factor. In the studies presented here, colchicine, at nonleukopenic doses (0.05-0.2 mg/kg), is shown to abrogate the acute arthritis induced by monosodium urate crystals in rabbits (10 mg), but to have no effect upon the arthritis induced by the
injection of the purified cell-derived chemotactic factor (10-20 p g ) .
Serum colchicine levels were 5.8 X IO-’M at 30 minutes and 3 x
lO-’M at 90 minutes after intravenous injection. Peripheral blood
PMN obtained from colchicine treated animals migrated normally toward a chemotactic stimulus and its phagocytosis of crystals was not
altered. These data extend previous in vitro studies pointing to the
suppression of chemotactic factor synthesis and secretion as the primary mechanism of action of colchicine in ameliorating acute crystalinduced arthritis. Presumably, colchicine is not effective in many
other inflammatory states because the neutrophil derived chemotactic
factor is not a major mediator and its suppression by the drug can
only modestly abrogate the process.
ARA ABSTRACTS
662
Effects of dibutyl-cyclic AMP on proteoglycan and hyaluronic acid synthesis and macromolecular organization of
articular cartilage
Michael Stack and Kenneth Brandt, Indiana University School of Medicine, Indianapolis
Dibutyl-cyclic AMP (db-CAMP) has been shown to increase
the proportion of total proteoglycan in the medium of cultures of immature joint cartilage. In addition, a shift in collagen synthesis, from
type I1 to type I, occurs when chondrocytes from normal joint cartilage are cultured with db-CAMP. When osteoarthritic (OA) cartilage
is cultured in the absence of db-CAMP, similar changes have been
noted in percent total PGs in the medium and in collagen phenotype.
Since the effect of db-CAMP on macromolecular organization of PGs
in normal adult joint cartilage has not previously been examined, the
present study was carried out.
Cartilage slices were incubated for 24 hours in Ham's F-12
medium with3% or 'H-glucosamine, with or without 2mM db-CAMP.
35S-labeledPG aggregates and disaggregated PGs were prepared from
media, 0.4M and 4M GuHCl extracts of the tissue. Hyaluronic acid
was identified on the basis of 3H-glucosamine eluted from CPC columns by 0.3M NaCl after papain digestion and was confirmed by susceptibility of the 0.3M NaCl fraction to digestion with leech hyaluronidase.
In studies with 9 dogs, 35S incorporation into PGs in the
presence of db-CAMP was 88% of controls. However, the %-PG content of the medium was 60% greater than that of controls and represented 20% of total "S-PGs. In the presence of db-CAMP, HA synthesis was 27% greater than control and the percent of total HA in the
medium was 4 times greater than control. The shift of 35S-PGs into
the medium caused by db-CAMP was not accompanied by an increase
in the proportion of '5S-PGs extracted with 0.4M GuHCl (103% of
control), but the proportion of 35S-PGs in the 4M GuHCl fraction
was only 62% of controls. db-CAMP had no effect on the hydrodynamic size of PG aggregates in any extract and did not affect the
size of disaggregated PGs, which interacted in vitro with HA to form
aggregates, indicating that the HA-binding region of the PG core protein was intact. Thus, these results contrast with the findings in OA, in
which a defect in PG-HA interaction is consistently observed. The enhanced extractability of PGs seen in OA, and produced by db-CAMP,
may be due to destabilization of PG-collagen interaction and may not
be attributable to a defect in PG aggregation, lack of HA, or PG degradation. A similar abnormality may exist in the initial stages of OA.
Felty's syndrome: Humoral and cellular immune aspects
Gordon A. Starkebaum, William P. Arend, Jack W. Singer, Francis A . Nardella, and Sue E. Gavin, University of Washington, Seattle
The objective of these studies was to investigate the mechanisms of neutropenia in Felty's syndrome (FS). The IgG directly
bound to purified polymorphonuclear leukocytes (PMN-bound IgG)
and the serum IgG PMN-binding activity (IgG-PBA) were quantitated using the Fab-anti F(ab')* technique. Additional studies included determination of the serum level of immune complexes (Clq
binding assay) and the ability of peripheral blood mononuclear cells
(MC) from FS patients to inhibit normal marrow PMN colony formation.
Peripheral blood PMN from 8 of 16 FS patients had levels of
cell-bound IgG greater than those seen with 26 normal controls or 17
patients with rheumatoid arthritis alone. Also, the IgG-PBA of sera
from 12 of 20 FS patients was elevated above the normal range compared to 6 of 31 RA sera (?= 7. LO, P < 0.001). There was a high degree of correlation between the direct PMN-bound IgG level and the
serum IgG-PBA for the 16 FS patients on whom both values were obtained (r = 0.93). Twenty FS sera were fractionated by Sephadex G 200. A significant correlation was observed between the serum IgGPBA and the values obtained with both the G-200 excluded pools (r =
0.52) as well as the G-200 IgG pools (r = 0.67).
Sera from 17 of 20 patients with FS had levels of C lq bind-
ing greater than 14% (? = 8.82, P < 0.005). Also, a strong correlation
was observed between the percent Clq binding and the IgG-PBA for
26 sera from these 20 FS patients (r = 0.71). Analytic ultracentrifugation of these sera demonstrated the presence of intermediate complexes in 10 of 19 FS patients; these values were correlated with the
Cl q binding levels (r = 0.54).
Ficoll-hypaque separated MC from 4 of 9 FS patients suppressed normal PMN colony growth by >40%; of these 4 patients, all
had normal levels of PMN-bound IgG and 3 had normal serum IgGPBA. In contrast, 3 of 5 FS patients whose MC failed to suppress normal granulopoiesis had high levels of PMN-bound IgG and serum
IgG-PBA.
The results of these studies document elevated levels of
PMN-bound IgG and of serum IgG-PBA in over half of FS patients;
the serum IgG-PBA may consist of both immune complexes and antiPMN antibodies. These results also suggest the presence of cell-mediated suppression of granulopoiesis in those patients with normal
levels of PMN-bound IgG. Thus, humoral or cellular immune mechanisms of neutropenia may be present in different subsets of patients
with FS.
Lyme arthritis: Immunologic and immunogenetic markers
Allen C.Steere, Allan Gibofsky, John A . Hardin, Robert J. Winchester, and Stephen E. Malawista, Yale University, New Haven, and
the Rockefeller University, New York City
We have recently reported 10 patients with Lyme arthritis
whose joint involvement has lost its usual, intermittent character and
has persisted in knees for at least a year. Although pannus formation
and cartilage erosion were seen in the 5 of these patients who underwent synovectomy, the illness differed from rheumatoid arthritis both
clinically and immunogenetically; at least 7 of the 10 patients had the
ARA ABSTRACTS
B cell alloantigen, DRw2 (frequency in normal controls, 22%; P <
0.005) (Clin Res 27: 338A, 1979).
We have now relaxed the requirement for duration of arthritis and have tested 13 additional patients with neurologic disease and/
or arthritis that persisted for I month to 1 year. Depending on the
grouping (figure), 58-70% of patients had DRw2; each group was significantly different from normal controls. Moreover, most of these patients can be spotted immunologically at onset with erythema chronicum migrans (ECM); at that time, high serum TgM levels, and
cryoglobulins containing IgM (IgM-cryo), predict subsequent involvement; high serum IgG levels, without IgM-cryo predict no later
symptoms (Arthritis Rheum 22: 471483, 1979). If one compares the
12 of 23 current patients seen at onset of ECM, versus 26 patients with
ECM but no later symptoms, the former group had more frequent
IgM-cryo (10/12 versus 5/26, P < 0.0005), higher serum IgM (median: 297 versus 146 mg/ml, P < 0.05), and lower serum IgG (median:
91 1 versus 1060 mg/ml, P < 0.05).
663
Our hypothesis is that both a tick-transmitted infectious
agent and an immunogenetic predisposition on the part of the host are
necessary for the development of at least the more severe aspects of
this immune-mediated inflammatory disorder.
Arthritis
Patients
Arthritis
>1 year
Neurologic
Disease
==
1 month
< 1 year
Total number
No DRw2
O/o DRw2
70%. p c 0 005
vs 22%
5av0,P < o 025
of controls
overall 61%.P < 00025
Salmonella reactive arthritis in British Columbia
H. B. Stein, A . Abdullah, H. S. Robinson, and D.K. Ford, University of British Columbia, Vancouver, British Columbia, Canada
Salmonella reactive arthritis has been reported from Europe
but not from North America. Over a period of I year, 5 cases of salmonella reactive arthritis were identified in British Columbia. Three
patients were adolescents; 4 were males. Stool cultures grew Salmonella tphimurium in 4 cases and Salmonella enteritidis in 1. In 2 cases
tested, serum agglutination titers for Salmonella paratyphi B (“0”)
were mildly elevated to I of 80. The arthritis followed the diarrhea by
7 to 10 days and tended to involve the lower limb joints. Two patients
had low back involvement with I patient developing radiologic sacroiliitis. Joint aspiration in 3 patients showed inflammatory fluid and
negative bacterial studies. The patient with sacroiliitis had Reiter’s
syndrome (conjunctivitis, urethritis, arthritis, and later acute iritis)
and is only the second recorded case of this syndrome after Salmonella typhimurium infection. Patients were ill with marked joint inflammation, constitutional symptoms of fever, fatigue, weight loss,
malaise, and high sedimentation rates. Patients had anemia, mild leukocytosis, and polyclonal hypergammaglobulinimia. HLA-B27 was
present in 4 of 4. Active disease persisted up to 1 year without permanent damage. The sedimentation rate remained elevated for some
time after remission. Patients received antibiotics without effect upon
joint symptoms. Indomethacin was extremely effective and superior to
salicylates and corticosteroids.
D-penicillamine compared to previous chrysotherapy in rheumatoid arthritis
H . B. Stein, R. Offer, C. Atkins, T.A. Brown, Vancouver, Nanaimo and Penticton, British Columbia, Canada
Gold and d-penicillamine (dP) are specific suppressants of
rheumatoid arthritis (RA) with similar actions and reactions. Since
gold is generally used before dP, it would be useful to know if a failure to respond to gold predicts a future failure to respond to dP. Secondly, one would like to know if toxic reactions to gold recur with dP.
To study these questions we reviewed 330 patients with RA treated
with dP in 3 clinics (Vancouver, Penticton, Nanaimo). Ninety-six percent of these patients had received gold in the past and had been on
dP for 2 3 months.
One-hundred eight patients had a poor response to gold.
Fifty-seven (53%) of these patients subsequently benefited from dP
and 28 (26%)did not. Of the remainder, 19 were too early to evaluate,
2 had equivocal responses, and 2 lost the initial benefit of dP. Forty-
four patients lost the initial benefit of gold. Thirty (68%) of these patients subsequently benefited from dP and 7 (16%)did not. Of the remainder, 5 were too early to evaluate, 1 had an equivocal response,
and 1 lost the initial benefit.
Two-hundred two patients had 234 reactions to gold. Onehundred thirty-four of these 202 patients had gold discontinued because of toxicity. Reactions included rash 138, stomatitis 42, proteinuria 18, postinjection arthralgias 15, metallic taste 5, thrombocytopenia 3, leukopenia 3, other blood dyscrasias 3, sensory neuropathy 2,
miscellaneous 5. One dP rash recurred in 49 (36%), stomatitis in 7
(17%), proteinuria in 4 (22%),worse arthralgias 3 (20%),altered taste 2
(40”/.).
Postintestinal bypass arthritis-dermatitis
H. B. Stein, 0.L.A. Schlappner, W . Boyko, R. H. Gourlay, C.E. Reeve, University of British Columbia, Vancouver, British Columbia,
Canada
Various intestinal bypass procedures (IBP) have been devised for the treatment of morbid obesity. Many complications have
ensued including arthritis and dermatitis. Of 350 patients with an IBP
(Salmon technique) performed at St. Paul’s Hospital, 26 (7%) devel-
ARA ABSTRACTS
oped musculoskeletal symptoms considered to be related to the IBP
including 3 who had had milder joint symptoms prior to IBP. Another
5 patients whose IBPs were done elsewhere are included. All 31 patients were females except one. Ages ranged from 22 to 54 years.
Weight loss ranged from 36 to 140 pounds. Joint symptoms developed
1 to 108 months after IBP. Polyarthralgias or polyarthritis tended to
be migratory or episodic although 4 patients had only 1 episode.
Symptoms occurred over a period of I week to 2 years. Tendinitis occurred in 13 patients, myalgias in 5, and morning stiffness in 17. Joint
damage or erosions did not occur. Six patients had sacroiliac sclerosis
on x-ray; 4 of these patients has symptoms in the buttocks. Areas most
commonly involved included knees, ankles, fingers, shoulders, and
wrists. Involvement was usually asymmetric. Neck and back were also
frequently affected. Rashes occurred in 20 patients: pustular vasculitis
(PV) in 8; nodular erythemas in 3; tender red patches in 2; urticaria in
2; painless ecchymoses in 2; folliculitis-vasculitis in 1; rheumatoid
nodule in I; miscellaneous symptoms in 2. PV was characteristic in
appearance and occurred on the extremities just prior to joint flareups. Biopsies of these lesions showed capillaritis and epidermal necrosis; immunofluorescence was positive in 3 of 4. Other features included Raynaud’s phenomenon (lo), paresthesiae (9), and pericarditis
(I). Laboratory features included elevated ESR 22 of 30; cryoprecipitate 9 of 25; low C3 2 of 24; low C4 1 of 24; rheumatoid factor I of 29;
ANF 2 of 28 and normal immunoglobulins in 15 of 15 except for a
mild decrease in IgM in 1 patient. HLA typing in 22 patients showed
no trend, but the 2 B27 positive patients had spondylitis symptoms.
Corticosteroids were effective but nonsteroidal antiinflammatory
drugs and antimicrobials were of variable benefit. A sphincter at the
blind-loop-colon anastomoses to prevent reflux appeared helpful.
Proteoglycan and collagen degradation in rheumatoid arthritis: An organ culture model
James Steinberg, Stuart B. Kincaid, Soichiro Tmkamoto, and CIement B. Sledge, Harvard Medical School, Boston, Massachusetts
Articular cartilage is destroyed in rheumatoid arthritis (RA)
through the action of hydrolytic enzymes which degrade matrix components. The confrontation of synovium and cartilage occurring in
the diseased joint was reproduced in a model culture system. Bovine
nasal cartilage discs cocultured with RA synovial explants released
matrix breakdown products into their media. Proteoglycan (PG)
quantified by Alcian Blue-complexing was shown by molecular sieving and differential enzymic digestion to be PG-chondroitin sulfate
resulting from limited cleavage of the protein core. Collagen degradation products were measured as hydroxyproline (Hyp) in ethanolfractionated media; 80% was present as free amino acid and the remainder in peptides considerably smaller than intact a chains. Cumulative release of PG and Hyp were well correlated and proportional to the quantity of synovium applied. Cortisol inhibited
breakdown in a dose-dependent fashion, generally affecting PG and
collagen in parallel. For PG, the inhibitory response was a function of
the degradative capacity of the synovium. However, in several experiments synovium-stimulated release of PG proved totally resistant to
cortisol at the usually effective dose (10 pg/ml). Yet, a highly significant suppression of collagen breakdown could be readily demonstrated.
These data indicate that degradation of PG and collagen
can be dissociated in vitro and suggest that in the rheumatoid joint
there may be differential responsiveness to a therapeutic agent such as
cortisol. Some cartilage-synovial interactions (e.g. collagen breakdown) may be successfully altered, presumably by suppression of the
synovitis, while other degradative functions (e.g. PG breakdown) proceed unchecked. Application of the organ coculture model can provide important insights into mechanisms of drug action affecting connective tissue turnover and should assist in devising rational methods
of therapy.
Characterization of circulating DNA in systemic lupus erythematosus: Human or nonhuman origin
Charles B. Steinman, Mt. Sinai School of Medicine, New York City
Prominent among proposed pathogenetic mechanisms for
systemic lupus erythematosus (SLE) are those based on the presence
of occult infection, presumably viral. The present study was designed
to characterize circulating DNA in patients with SLE in order to determine whether it is of human origin since several reports have suggested the possibility of its being viral. Previous work from this laboratory and elsewhere has demonstrated that: I ) immunoprecipitable
DNA is released in vitro into serum, thereby rendering ambiguous the
interpretation of previously reported data characterizing DNA from
serum rather than plasma, 2) dsDNA is generally not found in plasma
from normals or even from most patients with SLE, but 3) is frequently found in small amounts in plasma from SLE patients who
have vasculitic or central nervous system (CNS) involvement. Previously reported attempts to characterize such circulating DNA have
led to conflicting conclusions. In the present study a modified urea/
hydroxylapatite method was used to isolate nanogram quantities of
DNA from each of 8 plasma specimens obtained from patients with
SLE and vasculitis or CNS involvement and who demonstrated circu-
lating DNA on multiple occasions. The purified DNA was radiolabeled by nick translation and its nucleotide base composition
compared with that of human DNA by CsCl isopyknic ultracentrifugation. Human DNA that had been added to normal plasma and subjected to the same procedure was shown not to have been altered by
it. DNA differing by at least 5510% in gudnine-cytosine content from
human DNA could be distinguished from the latter by this method.
No evidence of DNA with a base composition differing from that of
human DNA was detected in this manner.
To confirm this further by the more critical criterion of quantitating the human base sequences in plasma-derived DNA, renaturation kinetics were measured in the presence of an excess of either human or control DNA. Reaction conditions allowed renaturation of
unique sequences. The results again supported a human origin of
most of the circulating DNA. Although a minor, nonhuman component cannot yet be ruled out, it is concluded that most or all of the
circulating DNA in these patients is of human and not microbial origin.
665
ARA ABSTRACTS
Recent-onset polyarthritis: A prospective study
Mary Betty Stevens, Lynn Billingsley, The John Hopkins University, Bevra H. Hahn, Washington University, St. Louis, Frank C.
Arnett, and Thomas M. Zizic, The Johns Hopkins University, Baltimore
The course of polyarthritis (PA) of recent onset is often difficult to predict. Sixty-six adults, selected solely on the basis of PA not
exceeding one year in duration but longer than 6 weeks, were entered
into this prospective study of potential factors determining diagnosis
and outcome. Reported here are the first 3 years of observation in the
59 patients (89%)who did not lapse from the study. Based only on the
clinical ARA criteria for rheumatoid arthritis (RA), patients were
stratified as follows: Group 1, with six criteria, Group 11 with five, and
Group 111 with four or less. These groups were comparable with respect to patient demography and disease duration.
Of the 25 patients (42%) initially diagnosed as PA, only 12
could be more specifically categorized during the first 3 years: 7 as RA
after 3-27 months (mean I1.6), and one each with systemic lupus
(SLE), RA-SLE overlap, systemic sclerosis, psoriatic arthritis, and
Behcet’s syndrome. Furthermore, only two additional patients (both
Group I) became latex positive in the interval. The single Group I
patient with PA remained seronegative.
Therefore, “undiagnosed polyarthritis” should be respected
as a necessary diagnosis in a considerable number of patients (22%in
our series thus far) until, perhaps years later, the disease process fully
emerges.
Admission diagnosis
Group
No.F’ts.
RA
PA
Three-year diagnosis.
Other
RA
13
1
15
9(60%)
5(33%)
I ( 7%)
I1
26
I5(58%)
9(35%)
2 ( 7%)
111
18
4(22%)
I 1 (61%)
3(17%)
Total
59
28(48%)
25(42%)
6(10%)
I8
PA
(4)
1
(3)
4
t
4
8
35 (59%)
-
Admission
latex +
patients
Other
1
I2 (8Wo)
(2)
4
21 (81%)
(3)
6
9 (50)
1 I (19%)
42 (7 I%)
13 (22%)
* Diagnostic evolution: Number of patients are shown in parentheses; arrows indicate direction of change.
Cell-mediated immunity to collagen in rheumatoid arthritis
John M. Stuart, Arnold E. Postlethwaite, Andrew H. Kang, and Alexander S. Townes, University of Tennessee, Memphis
These studies were undertaken to determine if cell-mediated
immunity to collagen is present in patients with rheumatoid arthritis
(RA). Peripheral blood lymphocytes (PBL) were obtained from 18 patients with classic or definite RA and from 24 control patients (gout6, ankylosing spondylitisd, osteoarthritisd, n o r m a l d ) . PBL were
cultured in the presence of PHA, Sk-Sd, native types I, 11, and 111 collagen, and a chains from each collagen. After 72 hours of culture, the
supernates were harvested by centrifugation and assayed in modified
Boyden chemotaxis chambers for the presence of lymphocyte derived
chemotactic factor for monocytes (LDCF-M). Production of LDCFM in response to antigens by PBL has been shown to be a sensitive in
vitro correlate of in vivo cell-mediated immunity. Results are stated in
the Table as mean chemotactic index (derived by dividing response of
PBL cultured with antigen by response of PBL cultured with media
alone) f standard deviation.
PHA
RA
Control
P-value
8.3f3.6
8.1k3.7
NS
SkSd
4.5f2.4
5.2f2.7
NS
1
I1
1.5f0.9
1.1f0.6
1.9f1.5
1.OfO.4
(0.05
co.01
PBL from patients with RA produced significantly greater
chemotactic activity in response to all collagen preparations than did
controls. Although a few patients with other kinds of arthritis also responded, the response was in general at a low level. Chemotactic activity produced by RA PBL in response to collagen had a molecular
weight identical to human LDCF-M based on gel filtration. These
data suggest that patients with RA have cell-mediated immunity to
collagen. Our results differ from a recent report of collagen induced
leukocyte inhibitory factor production by RA PBL in that LDCF-M
production was greater in response to (Y chains than to native collagen.
In previous studies of humoral response to collagen by RA sera,
greater reaction to denatured collagen was also observed. Denatured
a chains appear to have antigenic sites important to the development
of both cell-mediated and humoral immunity to collagen in RA.
111
a1 (1)
a2 (1)
a1 (11)
a1 (111)
2.lf1.4
1.1fO.4
co.01
3.9k2.9
1.3f0.8
<O.oOI
3.3k2.2
1.2f0.6
<O.oOl
4.1f2.2
1.4k0.6
t0.00I
4.9f3.1
1.5k1.0
t0.001
The lubrication of articular cartilage by synovial fluid glycoproteins
David A. Swann, Eric L. Radin, and Robert B. Hendren, Boston, Massachusetts
Biochemical and mechanical studies have shown that glycoprotein constituents are responsible for the boundary lubrication of
articular cartilage by bovine synovial fluid in an oscillating whole
joint. Two glycoproteins LGP-I (Swann et al: Biochem J 161:473435,
ARA ABSTRACTS
666
1977) and LGP-I1 (Swann and Mintz: Biochem J 1979) have been isolated from the synovial fluid lubricating fraction and while the role of
LGP-I1 is not known, it has been shown that LGP-I is a required constituent for the boundary lubrication of the cartilage. In order to evaluate the activities of these purified glycoproteins and other synovial
fluid constituents and compare their lubricating abilities with that of
whole synovial fluid under a variety of experimental conditions (concentration, shear, and load), we have developed a cartilage on glass
lubrication testing apparatus. This device employs a rabbit metatarsal
bone fixed into the head of a modified turntable tone arm so that the
cartilage surface is in contact with a rotating glass disc. An annulus of
sample fluid is applied at the radius of contact and the frictional drag
between the surfaces is measured by a force transducer coupled to the
tone arm. Friction measurements can be performed on 1 ml samples
at loads up to 25 kg/cm2 and speeds between 7 and 270 mm/sec. At
each load a characteristic friction versus speed curve is obtained
which allows a detailed comparison of the lubricating properties of
samples with the behavior of whole synovial fluid. The results obtained with this apparatus have confirmed and extended the data obtained with an oscillating whole joint. Hyaluronic acid does not act as
a lubricant and only LGP-I fractions possess lubricating activity. In
addition LGP-I fractions have a lubricating ability equivalent to
whole synovial fluid. These data show that hyaluronic acid does not
play a role in cartilage lubrication e.g., by potentiating the activity of
LGP-I. Lubrication tests on LGP-I fractions before and after modification by proteases and neuraminidase, using the oscillating whole
joint and cartilage on glass system, have shown that the intact molecule is required and indicated that the structure of the oligosaccharide
side chains is important in lubrication.
Supported by N I H Grant A M 19834.
Detection of antilymphocyte antibody in systemic lupus erythematosus with two color method and its heterogenous
specificities against T-cell subsets
Kiyoaki Tanimoto, Kunio Okudaira, Hidenori Nakai, Tetsuo Hayakawa, Takamichi Kashiwado, and Makoto Goto, Tokyo, Japan
Two color method originally described by Van Rood and
others for the typing of HLA-DR was used for the detection of antilymphocyte antibody (ALA) in systemic lupus erythematosus (SLE).
In this method, surface immunoglobulin (Ig) bearing cells were identified with FITC labeled antilg and nuclei of killed cells stained with
ethidium bromide. Therefore, T or B lymphocytes of the target cells
can be identified without separation. ALA were detected in 87.3% of
lupus sera and had preferential reactivity with T-cells. Their major
immunoglobulin class was IgM. These results were consistent with
other published methods.
The subspecificity of ALA was further analyzed with respect
to T-cell subsets. When T-lymphocytes were separated into Fc-recep-
tor bearing (T,)and lacking (T,-) cells, 64.3% of ALA showed preferential reactivity with T, cells and 14.3% with T,- cells. The remainder had undifferential reactivity. When T-lymphocytes were treated
with ALA and rabbit complement and cocultured with B-lymphocytes, ALA with T, dominant specificity gave rise to the enhancement
of in vitro immunoglobulin syntheses, whereas those with T,- dominant resulted in the reduction of them. Similarly when T-lymphocytes
pretreated with ALA were cultured with PHA or Con A, some of
ALA inhibited the response in the dose dependent manner, while others showed biphasic effects on the lymphocyte blastoid formation.
These results indicated that ALA in lupus have the heterogenous specificities against T-cell subsets.
Biochemical and biomechanical properties of osteoarthritic dog cartilage
Jerry Tenenbaum, Roy D. Altman, Wayne Riskin, Loren L. Latta, and David S. Howell, University of Miami, Miami, Florida
Traumatic osteoarthritis (OA) was induced in 20 adult dogs
by stab wound transection of the right hind limb anterior cruciate Iigament in order to assess articular cartilage proteoglycans (PG) and
viscoelasticity.
Size distribution of cartilage PG was assessed in cartilage
samples from abnormal appearing surfaces of distal femoral condyles
and from matched areas of the contralateral knee. Extraction was performed by 4M guanidine HCl and sedimentation properties of the
macromolecules were studied after CsC I differential ultracentrifugation to determine aggregate proteoglycans (PGA) and proteoglycan
subunit (PGS). Viscoelastic properties were tested by calculating unrelaxed (G,) and relaxed (G,) shear moduli from deflection time recordings after a compression stress of 2.35 X IOSN/M2 at abnormal
and contralateral normal knee cartilage sites.
Animals were killed after 2-16 weeks. Right knee lateral condylar cartilage was discolored, soft, and roughened. Light microscopy
confirmed early surface irregularity, chondrocyte proliferation, and
decrease in Safranin 0 staining. The PGA/PG ratio in the OA lesion
decreased with age of the lesion when compared to the control knee (r
= 0.66). This was more dramatic in the 2-9 week lesions, which
showed a decreased proportion of PGA of 14.2%(i33.8 to $9.6) (r =
0.88). In addition the mean sedimentation coefficient for PGA decreased from 60.3s to 52.63 (P= 0.05). There was a tendency for samples from 10-16 week lesions to demonstrate recovery of PGA/PG toward normal, but the recovery of original S values was incomplete.
Increased stiffness of cartilage was demonstrated by an increase in
G,(6.65N/M2 to 10.64N/M2) and an increase in G,(2.12N/M2 to
2.54N/M2). The older lesions did not show a trend toward further increase in stiffness. Retardation-time spectrum was not consistently
changed except for a tendency toward less linearity in abnormal cartilage.
Reduction in proportion of PGA and in size of PGA molecules is interpreted to indicate a disturbance in either breakdown or
synthesis of PG. Spontaneous recovery of these PG parameters in
most late lesions despite continued joint instability indicates the presence of repair, but continued abnormal biomechanical properties are
interpreted to indicate inadequacy of the repair process.
667
ARA ABSTRACTS
Tubular and glomerular proteinuria in patients with rheumatoid arthritis and osteoarthritis
A.L. Thomas, M . Davies, A . Lison, M . Hopkins. A . W. Asscher and G. Nuki, Welsh National School of Medicine, Cardiff, Wales
Studies of renal function and histology suggest that patients
with rheumatoid arthritis (RA) may have evidence of renal tubular or
glomerular damage but the extent to which this is a consequence of
the disease or drug therapy remains controversial.
To investigate this further, sodium dodecyl sulfate (SDS)
acrylamide gel electrophoresis which readily distinguishes glomerular
proteins molecular weight > 60,OO1) from tubular proteins (molecular
weight c 60,000) has been used to screen for proteinuria in osteoarthritis (OA) patients receiving nonsteroidal antiinflammatory drugs
(NSAID) (32 patients) and RA patients receiving NSAID (43 patients), gold (75 patients), and penicillamine (62 patients). There was
no proteinuria by conventional labstix testing. Previous work using
this technique has shown that the urine in end stage kidney failure is
characterized by a consistent four band pattern of low molecular
weight tubular proteins (FBP). Surveys of two representative normal
populations in South Wales showed an FBP in 20% and glomerular
proteins (GP) in 12% of 200 subjects.
Compared to the normal population there was a significantly
increased prevalence of GP in the urines of the OA and RA patients
treated with NSAID; these proteins were detected in 34.4% and 41.8%
respectively (P < 0.001), while the FBP was significantly increased
only in the urines of RA patients receiving NSAID (38.9%, P < 0.05).
Further clinical analysis showed that G P and FBP were not related to
age, sex, or duration of disease.
Although urine protein analysis by SDA polyacrylamide gel
electrophoresis appears to be too sensitive an indicator to be of clinical value in predicting significant proteinuria in rheumatologic practice, the data suggest that the increase of G P in RA and OA patients
may be due to NSAID effects on the glomerulus, while the increase in
FBP in the NSAID treated RA patients is likely to be a consequence
of the disease.
Characteristics of antinuclear antibodies in rheumatoid sera
Carol Tipton, Carol Peebles, Fenneke J o s h , and Eng M . Tan, Universityof Colorado Medical Center, Denver
Although rheumatoid sera are known to contain antinuclear
antibodies (ANAs), the immunologic specificities of the antinuclear
antibodies have not been extensively characterized. By use of classspecific fluorescein-conjugated antisera, it was found that IgM ANA
was positive in 40% (39 of 97) of rheumatoid arthritis (RA) sera, IgG
ANA in 41% (40 of 97), and IgA ANA in 33% (32 of 97). Many sera
contained AN As of more than one immunoglobulin class.
Specificities of the AN As were examined by radioimmunoassay for antibodies to DNA and by immunodiffusion for antibodies to
Sm antigen, nuclear RNP, and the SS-A and SS-B nu-clear antigens.
The incidence was: antibodies to native DNA-3%, to single-stranded
D N A 4 % , to Sm-antigen-1%, to nuclear RNP-I%, to SS-A-5%,
and SS-B-1%. Thus, the ANAs in RA sera are different in specificities from those seen in systemic lupus erythematosus, mixed connective tissue disease, and Sjogren's syndrome. The specificities of
ANAs in RA were further analyzed using an immunofluorescent
method previously described for the detection of antibodies to histones. This consisted of using tissue sections reconstituted with punfied histones as substrates. Twenty-two percent (13 of 59) ANA posi-
tive sera reacted with histones. When specific histone fractions were
used, H2B was the most reactive component and HI, H2A, H3, and
H4 were rarely reactive.
The relationship of rheumatoid factor (RF) to ANA was
studied using RFs isolated on absorbant columns consisting of Sephadex beads coated with aggregated IgG. Ten of 15 isolated RFs
showed ANA activity. Low pH (3.2) was used in Sephadex columns
and sucrose density gradients to dissociate possible complexes of
rheumatoid factor and IgG ANA. It was not possible to dissociate
rheumatoid factor activity from ANA activity, thus suggesting that
both these activities resided in the same immunoglobulin molecule.
This was also suggested by studies showing that ANA activity in some
RA sera could be inhibited by aggregated IgG, whereas ANAs in SLE
sera were not affected.
These studies demonstrate the heterogeneity of ANAs in RA
sera. ANAs of the IgM and IgG classes are equal in prevalence and
22% of the ANAs are reactive with nuclear histones. Two-thirds of
isolated rheumatoid factors possess ANA activity which is not dissociable from rheumatoid factor activity.
lmmunopathogenesis of chronic postrubella vaccination arthritis
Peter D. Utsinger, Temple University, Philadelphia
Two unrelated patients who developed a persistent and
chronic arthritis after rubella vaccination were studied. Both patients
were female. One was 26 years old and had a persistent monarthritis
of the left knee. The other was 28 years and had a pauciarthritis involving both knees and a n ankle. On examination the involved joints
were red, hot, swollen, and tender. Neither patient had a rash, muwus
membrane leison, lymphadenopathy, or hepatosplenomegaly. Because of marked knee synovitis unresponsive to medical management
both patients underwent synovectomy.
T lymphocytes were determined by spontaneous rosetting
with sheep RBC, B lymphocytes by surface immunoglobulin using
F(ab'), antisera, immune complexes (IC) by "'I-Clq and Raji cell assay, C3 and C4 by radial immunodiffusion, CH5O by the method of
Kent and Fife, macrophage migration inhibition (MIF) by the
method of Clausen, and cellular cytotoxicity by a variation of the
chromium release assay of Steele.
Serum lymphocytotoxic, DNA, and antinuclear antibodies
were not found. Serum hemagglutination-inhibition titers to rubella
ARA ABSTRACTS
were 1:32 and 1:16. Serum IC were not found. Serum C3, C4, CH50
were normal. Peripheral blood (PB) T lymphocytes, T-gamma, and B
lymphocytes were normal; PHA and MLC responses were normal.
Synovial fluid contained 83% to 87%T lymphocytes and did not contain IC. PB, synovial fluid lymphocytes, cultured individually with 4
rubella antigens did not produce MIF, nor did they enter a tetraploid
DNA state. Lymphocytes isolated from synovium and cultured with 4
rubella antigens did not produce MIF and did not exhibit cytotoxicity
against rubella infected cells. However, lymphocytes isolated from synovium displayed marked cytotoxicity for autologous synovial lining
cells and also for extensively washed and cultured autologous syno-
vial lining cells, and only minimal cytotoxicity for allogeneic synovial
cells. Such cytotoxicity for synovial lining cells was not exhibited by
autologous PBL. When lymphocytes isolated from synovium were
cultured with autologous synovial lining cells, they developed a tetraploid DNA content and incorporated 3H-TdR when cultured with allogeneic synovial lining cells, significantlyless DNA synthesis was observed. Rubella virus could not be grown from synovial fluid or
synovium and could not be demonstrated by electron microscopy of
synovium.
These data strongly suggest that a synovial lining cell neoantigen produces synovitis in chronic postrubella arthritis.
Systemic immune complex disease after intestinal bypass surgery
Peter D. Utsinger, Temple University, Philadelphia
Although it remains an effective means of accomplishing
weight reduction in morbid obeisity, intestinal bypass surgery has become associated with a growing list of complications. We have had
the opportunity to study 3 patients with postjejunoileostomy who had
a multisystem illness.
The patients were all female. Their ages ranged from 29 to 34
years. The multisystem illness began from 14 to 47 months after surgery. Clinical manifestations included fever, an extensive maculopapular skin rash, lymphadenopathy, renal insufficiency,polyarthritis
including involvement of the wrists, metacarpophalangeal joints,
proximal interphalangeal joints, and knees.
Cryoglobulins consisting of IgG, IgM, C3, and C4 were
found in high titer in the serum of all patients, as were immune complexes determined by the Raji cell technique. E coli antigen was found
in the cryoprotein of 2 patients. Serum CH50, C3, and C4 were normal. Serum IgG was increased in all patients. Peripheral blood T and
B lymphocytes were present in normal percentages and did not produce a tetraploid DNA content when cultured with E coli antigen.
Skin biopsies showed heavy granular deposits of IgG and
IgM at the dermal-epidermal junction in 2 patients. The neutrophilic
dermatitis seen in some bypass patients was not observed. IgG and
IgM could not be eluted by washing skin biopsies with solutions of
enteric bacterial antigen. One renal biopsy showed granular dense deposits along the glomerular capillary walls, consisting of IgG, IgM,
and C3. No staining was found by use of a variety of antibacterial antigen antibodies. In 2 patients control of the polysystem disease was
effected by low dose corticosteroids; in the third, revision of the bypass was necessitated by progressive renal failure and a severe dermatitis.
Postintestinal bypass surgery may cause a systemic immune
complex disease, and now both skin and renal vasculitis have been
documented.
Evidence for circulating cytotoxic autoantibodies to neutrophils in patients with Felty’s syndrome
John A . van Boxel, The Johns Hopkins University, Baltimore, Dennis Torretti, Danville, Pennsylvania, Robert-John Trapani, NIH,
Bethesda
Sera from 8 patients with Felty’s syndrome had a significant
(P< 0.001) incidence of cytotoxic activity for autologous neutrophils
in the presence of rabbit complement, when tested by a dye-exclusion
microcytotoxicity assay. Control groups were 17 patients with rheumatoid arthritis without Felty’s syndrome, 13 multiparous women, 10
patients who had undergone multiple transfusion, and 29 normal subjects. Autologous cytotoxic activity was nondialyzable, heat-stable
(56OC for 30 minutes), complement dependent, and found only in the
IgG fraction by exclusion chromatography. The serum of each patient
with Felty’s syndrome was also cytotoxic for the neutrophils of a
varying number of members of a normal donor panel. The specificity
of this homologous cytotoxic activity was not HLA related. Specific
binding of serum IgG of patients with Felty’s syndrome to autologous
neutrophil membranes was shown by positive indirect immunofluorescent staining with fluorescein-conjugated F (ab’)* fragments of
purified antibodies. After incubation in autologous serum, neutrophils
stained with antibodies to IgG but not IgM. The presence of circulating neutrophil-specific cytotoxic activity was further investigated using affinity chromatography. Purified neutrophils from normal donors
were allowed to adhere to cotton columns. Passage of serum from 3 of
the patients with Felty’s syndrome through these columns markedly
depleted their cytotoxic activity for autologous neutrophils. Protein
eluted from these columns had high titers of cytotoxic activity for autologous neutrophils, but not for autologous lymphocytes tested in
parallel. Autologous cytotoxic activity of the eluted protein of each
patient was in turn removed by, and could be eluted from, anti-IgGSepharose but not control anti-IgA-Sepha-rosebead affinity columns.
These data strongly suggest the presence of circulating cytotoxic IgG autoantibodies to neutrophils in patients with Felty’s syndrome.
ARA ABSTRACTS
6 69
Total wrist arthroplasty: A review of 100 patient cases
Robert G. Volz, University of Arizona Health Science Center, Tucson
In 1973 an artificial wrist prosthesis was developed at the
Arizona Health Science Center which today represents the only such
prosthesis designed and developed in the USA. From 1974 to I978
100 patients underwent wrist replacement by 15 collaborating surgeons. Eighty-three percent of the patients had rheumatoid arthritis.
Eighty-six percent of the patients reported a good to excellent result
with pain relief being the most predictable outcome, followed by a
useful stable arc of motion. Eight percent had a fair result, 6% a poor
result.
Postoperative complications included problems with wound
healing (6 cases), inadequate range of motion postoperatively (6
cases), a single episode of dislocation (4 cases), wound infection (2
cases), motor imbalance, and loosening of the component part (I
case). Patient selection proved to be a critical factor to the outcome.
From an overall point of view, total wrist replacement employing a
cementable metal and polyethylene type of implant appears to be an
acceptable, predictable, and satisfactory solution for patients with far
advanced arthritic change to the carpus.
Resistance to therapy and absence of oncogenesis in mature Palmerston North mice treated with cyclophosphamide
or hydrocortisone sodium succinate
Sara E. Walker and Bertram Schnitzer, University of Michigan, Ann Arbor, Michigan
Palmerston North (PN) mice, a new model of systemic lupus
erythematosus, develop ANA, anti-DNA, LE cells, and glomerulonephritis (J Lab Clin Med 92:932, 1978). To study immunosuppression
and neoplasia in established PN disease, 3 groups of 33-37 mice of
both sexes, 6 months of age, received cyclophosphamide (CY) 8 mg/
kg/day, hydrocortisone sodium succinate (HSS) 10 mg/kg/day, or saline (controls) until death. Longevity, presence of neoplasia, glomerular lesions, and serial anti-DNA (Fan), ANA, and WBC were assessed. In control mice, mean ages at death were 53 weeks f 5 (SEM)
in females and 65 weeks f 5 in males. Renal disease/vasculitis (RD/
vasc) caused death in 27/37 mice. Three control mice developed lymphomas. Mice treated with CY had mean longevity of 57 f 3 weeks;
lifespans were similar in mice of both sexes. In I2 mice with prolonged survival, the CY treatment period was 47 weeks or greater.
Causes of death were RD/vasc (14/37) and lymphoma (1). HSStreated mice lived 61 weeks f 3. RD/vasc was found in 22/33 HSStreated mice, and 4 mice developed neoplasms ( I lymphoma, 1 carcinoma, 2 sarcomas). Tumors in treated mice appeared after 1 2 4 7
weeks of therapy. Other causes of death in all three groups were infection, hemorrhage, pulmonary edema. After 24 weeks of therapy,
mean anti-DNA was 27%f 2 in controls, 24%f 1 in CY-, and 29% f
2 in HSS-treated mice. At this point, ANA were present in 85% of
controls, 77% of CY mice, and 60%of HSS mice. A striking decrease
of total WBC and absolute lymphocyte counts was found in mice
treated with CY or HSS compared to controls. The only significant
improvement was noted in mice treated with CY, whose mean glomerular lesion score was I8 compared to mean scores of 29 in controls
(P < 0.001) and 29 in HSS-treated mice (P< 0.001). PN mice with established autoimmune disease did not benefit from long term therapy
with CY or HSS. Increased oncogenesis was not observed in treated
mice.
These findings contrast with our experience in a small group
of mature B/W mice with active autoimmune disease (17-22 weeks of
age) receiving lifelong therapy with CY, 8 mgkg/day. In these animals, disease was suppressed effectively;neoplasms appeared in 10096
of B/W mice after 38-86 weeks of CY treatment. Successful therapy
in murine models of lupus may depend upon factors inherent in specific strains of animals. Neoplasia does not always complicate immunosuppressive therapy of established autoimmune disease in mice.
Renal histology and subsequent course in childhood systemic lupus erythematosus
Carol Wallace, Gary Striker, Jane G. Schaller, Ralph Wedgwood, Helen M. Emery, University of Washington, Seattle
Initial renal biopsies from 3 1 children with systemic lupus
erythematosus (SLE), median age 13.5 years (5.9-18.8), median disease duration 6 months ( I month-I0 years), were reviewed to identify
features predictive of renal outcome. Sixteen patients had received
steroids for I week or longer prior to initial biopsy. Patients have been
followed for a median duration of 4 years (1 month-10.9 years); 14
had one or more repeat biopsies. Renal specimens were evaluated by
a combination of light, electron, and immunofluorescence microsCOPY.
Initial biopsy revealed: I of 31, no abnormalities; 6 of 31,
mesangial or focal lesions; 3 of 3 1, pure membranous lesions; 2 1 of 3 I,
diffuse involvement. Only 4 of 3 1 patients have developed renal failure; all had initial diffuse lesions. Diffuse lesions later developed in 3
patients who had other lesions on initial biopsy. Subepithelial deposits were present in 15 initial biopsies (only 1 pure membranous); 10
developed massive proteinuria. Deposits in other glomerular locations
were not predictive of outcome.
More than 4 leukocytes per glomerulus were present in 8 of
3 1 initial biopsies, generally in patients studied soon after disease on-
set prior to steroid therapy (6 of 8). Necrosis occurring in more than
30%of glomeruli was found in 10 early biopsies (9 of 10 not receiving
steroids); glomerular obsolescence or synechiae were found in 4 of 4
of these patients on repeat biopsy. Mesangial sclerosis (21 of 31) or
proliferation (23 of 31) were present in initial biopsies regardless of
prior steroid therapy. Glomerular leukocytes, mesangial sclerosis or
proliferation, and glomerular necrosis did not correlate with subsequent renal failure or massive proteinuria.
Interstitial inflammation was present in 12 of 31 initial
biopsies. Four of these 12 children had elevated serum creatinines at
the time of initial biopsy; 3 later had renal failure. Only I of 19 children without interstitial inflammation had an elevated serum creatinine; none of these 19 has had renal failure.
In summary, the majority of children with lupus nephritis
had diffuse renal involvement. Interstitial inflammation and subepithelial deposits were correlated with poor outcome, while deposits
in other locations, glomerular leukocyte infiltration, necrosis, mesangial sclerosis, or proliferation were not.
ARA ABSTRACTS
670
Effects of lymphoplasmapheresis on immunologic status
Daniel Wallace, Peng Fan. Richard Gatti, Dennis Goldjinger, James Klinenberg, and Rodney Bluestone, University of California at
Los Angeles
Lymphocyte function was studied in 4 patients with rheumatoid arthritis who underwent 20 lymphoplasmaphereses over an 11week period. Lymphocytes 3 X lo9 and 40 cc/kg of plasma were removed with each treatment. Two patients were on gold and 2 on penicillamine before and during the treatments. All patients had remission of their disease for an average of 3 months post pheresis. We
attempted to dissect the mechanisms of these remissions by following
certain immune parameters.
Lymphocyte proliferation with PHA and neuriminidase-galactose oxidase (NGAO), T and B cell counts, and monocyte-lymphocyte physical association after NGAO stimulation (J Immunol
119:156, 1977) were measured at pheresis 1, 10 (end of intensive period), and 20. Skin testing with streptokinase-streptodornase,5 inter-
mediate strength tuberculin tests, purified protein derivative, and
mumps was done at pheresis 1 and 20. The results for an average of 4
patients are shown in the Table.
No changes in the skin tests were noted. B and T cell counts
did not change. Increased lymphocyte responsiveness at the end of the
intensive lymphoplasmapheresis was suggested by the greater proliferative association between monocytes and lymphocytes after mitogenic stimulation. Since no changes in the proportions of T and B
cells occurred, it is possible that pheresis removed a soluble inhibitor
or altered the functional status of lymphocyte subpopulations. Nevertheless, enhanced lymphocyte responsiveness was transitory and cannot account for the sustained remission observed after the treatment
course.
Pheresis number (week number)
~
l(I)
10 (4)
275
66
16
58
1599
95
15
56
21
20(11)
~~
NGAO (% 3HTdR incorporation of unstimulated control)
PHA (% 'HTdR of normal donor control)
IgM (B cell %)
E rosette (T cell %)
Monocyte-lymphocyte interaction (%)
12
74
61
16
59
5
Long-term plasmapheresis and lymphoplasmapheresis in the management of rheumatoid arthritis
Daniel Wallace, Dennis Goldfinger, Richard Gatti, Peng Fan, James Klinenberg, and Rodney Bluestone, University of California at
Los Angeles
Lymphocyte depletion and removal of humoral factors by
plasmapheresis have been postulated to be efficacious in treating
rheumatoid arthritis (RA). We investigated the effects of plasmapheresis and lymphoplasmapheresis on 11 patients with RA who were
not responsive to conventional therapy. With a Haemonetics Model
30 Cell Separator, 7 patients were plasmapheresed (removing 40 cc/
kg plasma per treatment), and 4 were lymphoplasmapheresed (removing 40 cc/kg plasma and 3 X lo9 lymphocytes per treatment). Each
patient was pheresed 20 times (three times a week for 3 weeks, twice a
week for 3 weeks, and once a week for 5 weeks), and clinical, serologic
and immunologic parameters were serially measured. Nine of I I patients remitted within 2 weeks of institution of pheresis. The mean
length of post-treatment remission was 3 months. Morning stiffness
diminished markedly, proximal interphalangeal target joint size decreased by an average of 2 mm, and %foot walking time decreased
by 30-70%. During the first 3 weeks, Westergren sedimentation rate
decreased by 80%. RA latex decreased by up to 4 liters; IgG, IgM, and
IgA were lowered by 40-70%. CH50 and C'3 decreased by 1&50%.
Circulating immune complexes, performed by Raji cell and polyethylene glycol methods, grossly paralleled the clinical picture during the
first 3 weeks. Electrolytes, calcium, fibrinogen, liver function tests,
and clotting tests did not change. No consistent change in B and T cell
count and skin tests was noted. The plasmapheresis results were not
significantly different from those of lymphoplasmapheresis. The phereses were well tolerated except for symptoms of dysequilibria. As pheresis was tapered, the serologies, immune complex levels, and protein
levels slowly returned to baseline even though all the patients who
were on gold or penicillamine remained in clinical remission. Possibly
the removal of a plasma factor allows patients who were under-responsive to gold or penicillamine to achieve significant relief from
these agents. Removal of specific plasma components is now being
evaluated in an attempt to further elucidate the mechanisms by which
plasmapheresis is beneficial in treating patients with RA.
Mycoplasma-induced arthritis of rabbits: Results of a oneyear study
Leigh Rice Washburn, Barry C. Cole, and John R. Ward, Salt Lake City, Utah
Preliminary studies on the ability of Mycoplasmo arthritidis
and Mycoplasma pulmonis to induce arthritis in rabbits have now
been extended through a period of 1 year. Rabbits injected intraarticularly with M orthritidis or M pulmonis developed significant
joint swelling peaking at 20 weeks postinoculation. Forty-seven percent of animals still showed a significant increase in joint diameter at
52 weeks. Mycoplasmas were readily isolated from synovial membrane, cartilage, and synovial fluid during the first 2 weeks; however,
ARA ABSTRACTS
after 7 weeks mycoplasmas could no longer be detected by direct culture, by immunofluorescence (IF), or in synovial cell cultures derived
from infected rabbits. Despite the apparent absence of mycoplasmas,
histologic evidence of chronic active inflammation was still present in
all surviving rabbits I year after inoculation. The lesions progressed
from an initial acute suppurative response in the early weeks to a
chronic phase in later stages characterized by villous hypertrophy, hyperplasia of surface synovial cells, follicular accumulations of lymphocytes, and an intense infiltration of the synovial membrane with
lymphocytes, plasma cells, and macrophages. Cartilage erosion and
destruction of bone were characteristic features of M orhitidis-induced arthritis and were first observed at approximately 20 weeks after inoculation. A cell-mediated immune response directed against
67 1
mycoplasma antigens persisted undiminished for 52 weeks. The humoral immune response peaked between 12 and 16 weeks after inoculation and titers were detectable in I 1 of 13 surviving rabbits at 52
weeks. Antibody titers were often higher in joint fluid than in corresponding serum samples; this observation along with the detection of
immunoglobulin (I@-containing cells in the synovial membrane at all
stages suggests local antibody production. Intra- and extracellular
granular deposits containing Ig and complement (C3) were detected
by IF in synovial membranes at all stages, suggesting a role for immune complexes in the perpetuation of chronic inflammation in this
disease. Identification of the antigen participating in these immune
complexes may be a key step toward understanding the mechanism of
chronicity in this and other inflammatory joint diseases.
A randomized blind crossover trial of six nonsteroidal antiinflammatory agents in rheumatoid arthritis and ankylosing
spondylitis
Cody Wasner, R. Guy Kraines, Melvin C. Britton, and James F. Fries, Stanford, California
The most frequent decision facing physicians treating rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients is the
choice of a nonsteroidal antiinflammatory drug. Virtually no studies
compare these drugs to each other, ask if different drugs should be
used for different diseases, or attempt to determine if another drug
should be tried after one fails.
Thirty patients with RA (ARA Criteria) and 24 patients with
AS (New York Criteria 1966) each took Enseals (E), Indocin (I), Motrin (M), Nalfon (NAL), Naproxen (NAP), and Tolectin (T) for consecutive 6-week periods in a randomized crossover design. Patient
preferences, physical evaluations, and laboratory values (20 separate
parameters) were assessed for each drug. Standard packaging was
used, duplicating the situation in clinical practice. Standard PDR
(Physicians’ Desk Reference) doses were employed for each drug.
Each drug was strongly preferred by some patients. Mean
preference rankings for AS (2 = first, 1 = second, 0 = other, - I =
withdrew secondary to side effects or ineffectiveness) were NAP
(0.75), I (0.67), M (0.33), NAL (0.21), T (0.08), E (-0.21), P = 0.03
and for RA were NAP (0.50), M (0.37), NAL (0.37), T (0.30). I (0.07),
E (0.07) P = 0.50. Laboratory values and physical findings, as expected, added little to the patient preference score.
Enseals performed poorly, partly related to the number of
pills/day required and to inconsistent absorption. For AS only 29%
and for RA 44% had therapeutic ASA levels (>I5 mg/100 ml). The
more pills required, the lower the percentage of prescribed pills which
were taken; this compliance finding was strong (P< 0.001). Dosage
required ranged from 2 to 3 pills/day (NAP) to 12 to 20 pills/day (E).
In RA relative compliance had a stronger effect (P= 0.03) than relative drug efficacy (P= 0.51) while in AS drug efficacy had a stronger
effect than compliance (P= 0.03 to P = 0.23). In RA the number of
pills required appeared more important than the specific contents.
AS patients differed from RA patients in drug preference. Indocin performed better in AS than in RA. For AS the preference order was significant (P= 0.03) although in a given individual, a trial of
several drugs might be required to find the most effective. For RA,
preference is more random and the ideal drug choice depends largely
on the degree to which the individual patient is expected to comply
with the number of pills required.
Oblique view radiographs in the diagnosis of sacroiliitis
Cody Wasner, R. Guy Kraines, and Ronald L. K a y , Stanford, and Palo Alto, California
Millions of dollars are spent each year on diagnostic x-rays in
rheumatology. The diagnosis of sacroiliitis depends heavily on radiographs but controversy exists concerning which views should be obtained.
To compare the usefulness of one view (anteroposterior or
posteroanterior of pelvis) versus three views (straight plus left and
right oblique views of sacroiliac joints), 29 patients were randomly selected from a group (at two hospitals) in whom the diagnosis of sacroiliitis was suspected clinically. The only requirement was that a
straight view of the pelvis with both obliques taken concurrently had
been obtained. Four physicians with experience in reading sacroiliac
(SI) joints, two radiologists, and two rheumatologists, graded each S1
joint according to the New York Criteria for ankylosing spondylitis
(1966). No clinical information was furnished. Films were presented
in random order at two separate readings 5 to 7 days apart with either
one or three views available on the first reading and the alternate situation present on the second reading. The distribution of abnormalities
ranged from 0 (normal) to 4 (complete fusion).
There was remarkable consistency between readings for the
same physician with 128 of 232 joints (55%) having identical grading
and 208 of 232 (89%) differing only 1 grade or less. No statistically significant benefit (P> 0.30 sign test) was seen with addition of oblique
views. Distribution of gradings were grade 0 - 1 13, grade 1-89, grade
2-55, grade 3-136, grade 4-71, Only 5 patients (8 SI joints) had a
clinically significant change in grade (from normal to abnormal or abnormal to normal). In 3 of the 5 patients this change was judged as
misleading (as determined by blind evaluation of the clinical record
and HLA-B27 status before the study) and in 2 patients it was helpful. Thus, in most cases oblique films didn’t change the grading and
when they did were misleading as often as helpful.
The increased cost, increased radiation (especially in the
young population who are at risk for sacroiliitis), and technical difficulty suggest that oblique views are not routinely indicated for the diagnosis of sacroiliitis.
672
ARA ABSTRACTS
Monosodium urate crystals in asymptomatic metatarsophalangeal joints in gout
A braham Weinberger, University of Pennsylvania, Philadelphia, Carlos A. Agudelo, Wake Forest University, North Carolina, H.
Ralph Schumacher, University of Pennsylvania, Philadelphia, Javier Molina, Medellin, Colombia, and Robert Turner, Wake Forest
University, North Carolina
The diagnosis of gouty arthritis in the interim between attacks is often based on complex clinical criteria and serum uric acid
(UA) levels. We have studied whether aspiration of asymptomatic
first metatarsophalangeal (MTP) joints for the demonstration of
monosodium urate crystals (MSUC) might be helpful in interim diagnosis. When crystals were easily identified from currently asymptomatic joints, we extended the study to first metatarsophalangeal
(MTP) joints never clinically involved in gouty patients and in hyperuricemics.
Ten asymptomatic gout patients with a history of podagra, 9
gouty patients with no previously clinically affected MTP joints, 10
asymptomatic hyperuricemics (uric acid > 8 mg/dl), and 3 controls
with normal serum uric acid were studied. Of the 19 gouty patients
the diagnosis had been confirmed previously in 15 by demonstration
of MSUC during attacks in knee joints. None had visible tophaceous
deposits. Serum uric acid was high in only 10 of 19 gouty patients at
the time of MTP aspiration. X-rays of the aspirated joints in 18 patients disclosed a cyst-like erosion in only one. Under aseptic technique with topical anesthesia, the first MTP joint was entered with a
22 gauge needle I cm lateral to the extensor hallux longus. One to two
drops of material usually including identifiable viscous synovial fluid
were obtained in all. Aspirates were examined as wet preparations using a compensated polarizing light microscope. Aspiration was well
tolerated in all patients.
In 10 of 10 aspirated joints of gouty patients with asymptomatic but previously involved joints, extracellular short rods or long
and short needle shaped crystals typical of MSUC were seen (mean =
2/high power field [HPF]; range = 2-4/HPF). MSUC could also be
detected in 6 fluids of 9 from the asymptomatic not previously clinically involved joints (mean = 5/HPF, range = I-I3/HPF). In the
normouricemic controls and idiopathic hyperuricemic subjects no
crystals have been found.
As noted in isolated cases by Garrod and others, articular
crystal deposits can occur without detectable inflammation and in
joints reportedly never clinically involved. Aspiration of even asymptomatic first MTP joints may allow crystal identification and definite
diagnosis, eliminating the need for complex clinical criteria in patients with quiescent disease. Reasons for the absence of a phlogistic
response at some time despite the presence of crystals remain to be
determined.
Antibodies to the Sm antigen in systemic lupus erythematosus: Clinical significance
Arthur Weinstein and Margaret Sullivan, University of Connecticut, Farmington
The presence of antibodies to the Sm antigen (anti-Sm) has
been described in a minority of systemic lupus erythematosus (SLE)
patients, but has been shown to be a highly specific marker for the
disease. The correlation of the presence of this antibody with specific
clinical features of SLE is controversial. Serial measurements of antiSm with changes in disease activity in individual patients have not
been described.
Antibodies to ribonucleoprotein (anti-RNP) and Sm were
measured by counterimmunoelectrophoresis in I 17 consecutive SLE
patients, 50 patients with rheumatoid arthritis, 30 patients with nonSLE connective tissue diseases, and 50 normal controls. Serial antiSm titers were measured in 12 of the anti-Sm positive patients selected because they had been seen by us during periods of disease activity and during remission.
Anti-Sm was not found in controls or patients with rheumatoid arthritis, but was present in 2 of the 30 (6.6%)patients with other
connective tissue diseases and in 24 of the I17 (20.5%) SLE patients.
Only 2 SLE patients had anti-Sm in the absence of anti-RNP. There
was no difference between SLE patients with or without anti-Sm in
the incidence of seizures and/or psychosis (P= 0.684), urinary casts
(P= 0.772), profuse proteinuria (P= 0.965), or any other manifestation of the ARA Preliminary Criteria for the Classification of SLE.
There was no difference in the number of manifestations of the ARA
Criteria between those patients with and without anti-Sm (P= 0.23).
Five of the 12 patients in whom serial samples were studied had a decrease in anti-Sm by 2 2 tube dilutions during periods of remission. In
the remaining 7 patients, titers remained constant independent of the
clinical status of the disease.
The data confirm that anti-Sm is a highly specific marker for
SLE and suggest that the presence of anti-Sm does not correlate with
any specific clinical manifestation of SLE. In some patients anti-Sm
titers did correlate with clinical disease activity and declined significantly or disappeared during clinical remission. In this respect, antiSm is similar to other antinuclear antibodies in SLE.
Regulation of phosphate and calcium metabolism by vitamin D metabolites: Studies in a patient with oncogenic
osteomalacia
Mark Wener, Loren Cohen, Robert S. Bar, M. Paul Strottmann, University of Iowa Hospital, Iowa City, and Hector DeLuca,
University of Wisconsin, Madison
We have studied a patient with diffuse bone pain and myopathy who had hypophosphatemia secondary to a mesenchymal tumor
of the hand (oncogenic osteomalacia). Serum phosphate was 1.9 f 0.3
mg/dl and bone biopsy confirmed osteomalacia. Serum calcium and
parathyroid hormone (PTH) were normal and fractional excretion of
phosphate was high (21 k 3%). Measurements of vitamin D metabolites revealed normal serum levels of 25-(OH)D2 and 25-(OH)D3;
however 1,25-(OH)2D3was undetectable and 24,25-(OH),D3 was elevated at 8.6 ng/ml (normal 0.25-2.5 ng/ml). When PTH was suppressed by a calcium infusion (peak serum calcium 12.3 mg/dl), frac-
673
ARA ABSTRACTS
tional phosphate excretion fell to 1% and serum phosphate increased
to normal levels. Treatment with 1,25-(OH)2D3(1 &day x 3 weeks)
reversed the biochemical abnormalities. Following resection of the
12.5 gm atypical chondroma, phosphate homeostasis normalized and
clinical improvement was rapid.
Therefore the oncogenic osteomalacia was associated with a
reversible alteration in vitamin D metabolism, best explained by inhibition of renal I-hydroxylase activity by a tumor product. Correction
of phosphaturia by 1,25-(OH)2D3indicates that this hormone was important in the regulation of phosphate reabsorption. Since suppression of PTH by a calcium infusion was also effective in eliminating
phosphaturia, it is likely that vitamin D metabolites directly modulated PTH-sensitive phosphate transport in the kidney. Finally, the
presence of normocalcemia in the face of absent 1,25-(OH)2D3 and
normal levels of PTH raises the possibility that either 24,25-(OH),D3
or a hiterto unmeasured vitamin D metabolite can maintain serum
calcium when I,25-(OH)*D3 is not present.
Thus, oncogenic osteomalacia was associated with low serum
levels of 1,25-(OH)*D3 and elevated 24,25-(OH)2D3 levels, which resulted in inappropriate phosphaturia. We currently are assaying tumor extracts for specific modulators of I-hydroxylase activity.
A controlled experiment to evaluate the use of a time oriented summary medical record in a rheumatic disease clinic
Quinn E. Whiting-O’Keefe, Donald W. Simborg, and Wallace V. Epstein, Universityof California,San Francisco
A central problem in the organization of services for persons
with chronic rheumatic diseases is the volume of clinical, laboratory,
and therapeutic response information that must accompany each patient yet be accessible in variable time and space locations. Systemic
lupus erythernatosus (SLE) is prototypic of a chronic disease in which
past laboratory data and clinical information form the basis for therapeutic decisions over extended periods. Several automated systems
designed to replace or augment the traditional medical record are
being advocated but no controlled experiments have been reported
demonstrating the effectiveness of these systems as conveyance instruments for clinical information.
A randomized single blind experiment was done in order to
determine whether a flowsheet (FS) type of summary medical record
could validly serve as a means to communicate clinical information in
the absence of the traditional medical record. The study was done in a
clinic specializing in the care of patients with SLE in which data flowsheets with entries limited to a single column per encounter have been
maintained for 15 years. In the 68 study encounters (Group S), rheumatologists were given flowsheets and an option to receive the stan-
dard medical record. In the 27 control encounters (Group C), both
flowsheets and standard medical record were provided. In 41% of
Group S encounters, physicians requested the full medical record.
Prior to randomization, pre-encounter chart review identified an average of 11.5 clinical, laboratory, and therapeutic informational elements potentially requiring physician recognition during the encounter. As determined by blind postencounter chart review, the rate of
documented followup of the pre-encounter identified information elements was 0.75 for Group S and 0.74 for Group C. There is a 98.7%
(Student’s r-test) certainty that the true followup rate for Group C is
no more than 0.10 greater than the true followup rate for Group S .
Using chart review done immediately after the encounter, physicians
in Group S were unable to detect overlooked clinical information
with greater frequency than physicians of Group C.
The outpatient care of persons with complex chronic diseases
such as SLE apparently can be undertaken with a simple, potentially
automatable data flowsheet without regular access to standard medical record and without deterioration in the communication of essential clinical information.
In vitro synthesis of prostaglandins: Differences between peritoneal and pulmonary alveolar macrophages
Fredrick Wigley, Joseph Chang and David Newcombe, The Johns Hopkins University,Baltimore
Peritoneal (PM) and pulmonary alveolar (PAM) macrophages obtained from outbred Swiss mice by saline lavage and cultured under identical conditions, including cell densities, incorporate
60% and 70%. respectively, of the total arachidonic acid-14C (ARAI4C) added to cultures. Greater than 90% of the ARA-I4C was incorporated into cell phospholipids, especially phosphotidylcholine and
phosphotidylethanolamine.Since both types of macrophages incorporate comparable quantities ARA-I4C, the in vitro synthesis and release of prostaglandins by AM and PAM were compared. Bradykinin
and thrombin, agents that cause the synthesis and release of ARA and
prostaglandins from platelets and fibroblasts, were not active when
added to PM and PAM cultures. PM or PAM in monolayer culture
(1.5 x 106 cells/plate) phagocytosed zymosan (Z) particles at a constant dose (50 pg/ml) and the release of radiolabeled prostaglandin
E2 (PGE2) was measured over time. Both macrophage types showed
greater than 90% viability after 5 hours; however, PM released significantly more PGEZ into the media as a function of time at a constant
dose of Z than AM. Nontoxic doses of Z ( 5 , 10.25, and 50 &ml) presented to identical cell densities of PAM or PM in the presence or absence of 10% fetal calf serum (FCS) showed an increasing release of
PGE2 as a function of Z dose. PM released significantly greater quantities of PGEZ at each dose of Z than PAM plated at identical densities (P < 0.001). Under these conditions, no significant differences
were found between the quantities of PGEZ released in the presence
or absence of FCS. Immune complexes also resulted in significantly
more PGEZ release by PM than by PAM (P < 0.001). These data
show that macrophages from the same species yet from different sites,
incubated and stimulated in an identical manner, synthesize and release significantly different quantities of PGE2.
6 74
ARA ABSTRACTS
Antineuronal antibodies in the cerebrospinal fluid of patients with systemic lupus erythematosus
Gary W. Williams, Harry G. Bluestein, University of California Medical Center, and Alfred D. Steinberg, NIH, Bethesda
Antibodies reactive with neuronal cells are present in the
serum of patients with systemic lupus erythematosus (SLE) and have
been implicated in the pathogenesis of central nervous system (CNS)
involvement in this disease. Previous studies have demonstrated increased levels of lymphocytotoxic activity in the cerebrospinal fluid
(CSF) of SLE patients with CNS disease, but direct evidence of CSF
antineuronal antibody has been lacking.
We have examined CSF from 26 SLE patients with and without CNS involvement and 25 non-SLE patients with a variety of neurologic disorders for antibody to the human neuroblastoma cell line
SK-N-SH. IgG antibody was detected in a direct '*'I-Staph-A assay.
IgM antibody was detected in an indirect assay using rabbit anti-human IgM plus "'I-Staph-A. Binding was expressed as the average
counts per minute (cpm) of duplicate determinations. In the direct assay, measuring IgG, SLE patients bound 627 f 178 cpm (mean f SE)
compared to 90 24 cpm in the non-SLE neurologic disease controls.
When the SLE patients were grouped according to presence or absence of CNS disease, the 16 patients with CNS involvement bound
*
905 +. 265 cpm compared to 182 f 66 cpm in the SLE patients without CNS involvement.
IgM antibody, measured by the indirect assay, was increased
in SLE patients 391 +. 82 cpm versus non-SLE neurologic disease
controls 24 f 9 cpm but was not significantly higher in the subgroup
of SLE patients with CNS disease compared to those without CNS involvement. There was a weak positive correlation between the CSF
IgG antineuronal activity and total CSF IgG (r = 0.4, P 0.05).
DNA bindings were determined in the CSF of 19 patients with SLE.
They were elevated (> 5% binding) in 4 patients, all of whom had
CNS disease.
These results demonstrate that both IgG and IgM antineuronal antibodies are present and elevated in the CSF of patients
with SLE compared to non-SLE patients with various neurologic diseases. IgG antineuronal antibody levels are higher in the subgroup of
SLE patients with CNS involvement. These findings support a role
for antineuronal antibodies in the pathogenesis of CNS disease in
SLE.
-=
Serologic studies in late onset systemic lupus erythematosus
H. Alexander Wilson, John B. Winfield, University of North Carolina at Chapel Hill, Maurice E. Hamilton, State University of New
York at Brooklyn, Daniel A. Spyker, Carolyn M. Bnmner, and John S. Davis, ZV, University of Virginia at Charlottesville
Systemic lupus erythematosus (SLE) in the elderly is recognized as a milder disease than that in younger patients. In order to
characterize serologic differences in relation to age, we have examined
122 patients including I5 with disease onset after age 55. Age of diagnosis was defined by the first disease manifestation attributable to
SLE. Total hemolytic complement (CH50), anti-DNA antibodies (determined by CIE), and cryoglobulins were assayed routinely. Selected
sera were assayed for rheumatoid factor (!atex fixation) and for reactivity with rabbit thymus extract (ENA) uy agarose gel diffusion. Data
analysis was performed by stratification and linear regression methods.
Common disease manifestations in older patients were arthritis, muscle pain or stiffness, alopecia, and Raynaud's phenomenon. In those patients evaluated prior to steroid therapy, CH50 levels
were significantly higher with increasing age (P = 0.005). This association also pertained in followup observation of the entire group (P =
0.02). Anti-DNA antibodies followed a similar pattern, but with
weaker correlation. The incidence of cryoglobulins was not age related. Persistent latex fixation titers > I : 160 were observed only in pa-
tients over 30 years. This group generally followed a benign course
characterized by episodes of polyarthritis. Anti-RNP antibody was
not increased in elderly patients nor in the subset whose major manifestations were muscle pain and/or Raynaud's phenomenon. The incidence and severity of renal involvement declined with age. Onset of
proteinuria was less associated in elderly patients with an antecedent
fall in complement (P= 0.01). Cryoglobulins, although present in 9 of
15 elderly patients, were correlative with disease activity in only one,
and were either sporadic (4 of 9) or persistent (4 of 9 ) in the remainder. In younger patients cryoglobulins tended to vary inversely with
complement levels and to correlate positively with disease activity,
suggesting a shift in the composition of immune complexes with advancing age.
Hence, late onset SLE is serologically as well as clinically
characterized by an absence of abnormalities seen in younger patients. These observations are consistent with either differences in genetic predisposition or less florid autoimmune responses in patients
with late onset disease.
Association of IgG anti-brain antibodies with central nervous system dysfunction in systemic lupus erythematosus
H. Alexander Wilson, John B, Winzeld, University of North Carolina School of Medicine, Chapel Hill, Robert G. Lahita, The
Rockefeller University, New York City, and David Kofler, Hahnemann Medical College, Philadelphia
In order to define further the basis for brain injury in systemic lupus erythematosus (SLE), sera from 20 patients with active
central nervous system (CNS) dysfunction were examined for antibodies reactive with neuronal membrane determinants by use of indirect immunofluorescence techniques. Substrates in these assays
were viable cells from neuroblastoma cell lines SK-N-SH and IMR32. Detection of antibody and determination of immunoglobulin class
were performed using rhodamine-conjugated F(ab'), fragments of
rabbit antibody monospecific for human IgG or IgM. Warm-reactive
IgG antibodies were demonstrable in 82% ( 9 of 1 I ) of patients with
clinical evidence of seizures or other types of diffuse CNS disease, but
were absent in non-CNS SLE sera or when focal neurologic deficit or
psychosis was the primary CNS manifestation. Serial observations in
6 patients established a temporal relationship between onset of active
ARA ABSTRACTS
CNS dysfunction and the appearance in serum of IgG anti-brain antibodies. Presence of such IgG antibodies was not specific for SLE-related brain disease, however, because similar antibodies were found in
sera of 3 of 10 non-SLE patients hospitalized with nonimmunologically mediated CNS injury. Cold-reactive antibodies of the IgM
class were equally prevalent in patients with or without CNS disease
and appeared to be more directly correlated with extra-CNS systemic
illness. Absorption experiments with lymphocytes, brain homogenate,
and various other tissues suggested a predominant brain-specificity
for IgG antibodies and partial lymphocyte cross-reactivity for IgM
675
antibodies. Although the presence of IgM anti-neuroblastoma antibody correlated positively with cold-reactive antilymphocyte antibody activity, a specific relationship between lymphocytotoxic activity
and brain dysfunction was not observed.
The special association of IgG anti-brain antibodies with diffuse CNS dysfunction in SLE may prove useful diagnostically. Additional study will be required to clarify whether such antibodies play a
role in pathogenesis or are merely an epiphenomenon after brain injury mediated by an as yet undefined mechansim.
Molecular mechanism(s) of deoxyribonucleosidetoxicity in T-lymphoblasts
James M. Wilson, Beverly S. Mitchell, William N. Kelley, University of Michigan, Ann Arbor
Deoxyadenosine and deoxyguanosine are toxic to human
lymphoid cells in culture and have been implicated in the pathogenesis of the immunodeficiency states associated with adenosine deaminase (ADA) and purine nucleoside phosphorylase deficiency respectively. We have compared the effects of these deoxyribonucleosides
on the relative incorporation of several labeled nucleosides into DNA
and into nucleotide pools of T-lymphoblasts to further elucidate the
mechanism@) of their toxicity. We have shown: I) Both deoxyadenosine plus EHNA, an inhibitor of ADA, and deoxyguanosine progressively inhibit incorporation of 'H-uridine into DNA but do not affect
'H-uridine incorporation into RNA; inhibition of DNA synthesis correlates with an accumulation of dATP or dGTP respectively and a depletion of the dCTP pool. 2) Deoxyguanosine (10 p M ) as well as hy-
m,
a known inhibitor of ribonucleotide reductase,
droxyurea (100
inhibits the incorporation of 'H-uridine but not of I4C-thymidine into
DNA; deoxyadenosine plus EHNA does not show this differential inhibition in the incorporation of labeled uridine. 3 ) Deoxyguanosine as
well as hydroxyurea markedly increases the incorporation of 'H-cytidine into the ribonucleotide pool while decreasing the incorporation
of 3H-cytidine into the deoxyribonucleotide pool; deoxyadenosine
plus EHNA produces much smaller changes in pool sizes.
From these data we conclude that inhibition of DNA synthesis by deoxyguanosine is due to dGTP mediated inhibition of ribonucleotide reductase. Deoxyadenosine mediated inhibition of DNA
synthesis, however, must be explained by mechanisms other than, or
perhaps in addition to, inhibition of ribonucleotide reductase.
Characterizationof a distinct nuclear acidic protein antigen-MA-and
erythematosus patients with MA antibodies
clinical findings in systemic lupus
Donna M. Winn, J. Frederick Wove, and Gordon C. Sharp, University of Missouri School of Medicine, Columbia
This study characterizes a distinct nuclear acidic protein antiCirculating MA antigen was detected by immunodiffusion prior to
gen (NAPA), called MA, and describes the clinical features of 11 patients who have antibody to the MA antigen. Enzyme and physiochemical treatment of calf thymus nuclear extract, the antigen source,
revealed that MA differed from other NAPAs in enzyme sensitivity,
thermal lability, and pH sensitivity. Immunodiffusion using reagent
antisera also showed MA to be distinct from n-RNP, Sm, PM-1,
SCL-1, SS-A, SS-B, and RAP antigens. Electrophoretic and chromatographic techniques showed the serum factor precipitating with
MA to be a gamma globulin. MA antibodies have been detected in 11
of 57 SLE (ARA criteria) patients screened, and in none of 900 sera
from patients with other rheumatic diseases. All patients had a positive antinuclear antibody test with a distinct pattern of large speckles
localized at the periphery of the nucleus.
SLE patients with MA antibodies usually had manifestations
of severe disease including recalcitrant skin rashes, marked lymphadenopathy, and anemia with positive direct Coombs test. Comparison
with other SLE patients who were negative for MA antibodies and
positive for Sm antibodies or DNA antibodies are shown in the Table.
acute nephritis in 3 patients.
Thus the MA antigen is a NAPA distinct from any previously described. MA antibodies appears to identify SLE patients
with very severe disease. The alternating presence of MA antigen and
antibodies prior to clinical flares suggests a biologic role for this system in an immune complex nephritis.
% MA
Condition
Renal disease
Splenomegaly
Neurologic disease
Hypertension
Raynaud's phenomenon
(n = 1 I )
% Sm
(n = 9 6 )
% DNA
(n = 5 6 )
82
41 P t O . O I *
64
23 P t 0 . 0 0 5
14 P c 0 . 0 2 5
17 P c O . 0 5
57 P t 0 . 0 0 5
57
17 P c 0 . 0 0 5 t
37
28
35 P c 0 . 0 2 5
45
45
0
* Comparison of MA and Sm patients.
t Comparison of MA and DNA patients.
Objective derivation of clinical discriminators for mixed connective tissue disease
J. Frederick Wove, Seiichi Takasugi, Lawrence Kingsland, Donald A . B. Lindberg, Donna M. Winn, and Gordon C. Sharp, University
of Missouri, Columbia
As part of our long range objective to establish diagnostic
criteria for mixed connective tissue disease (MCTD) by using objective methods, we investigated whether objective statistical analytic
techniques could document a separation of MCTD from systemic
lupus erythematosus (SLE) by taking into account carefully defined
clinical and commonly available laboratory findings and not relying
676
on specialized antibody studies. A modified ARA database on 570 patients with connective tissue diseases was computerized for analysis.
Clinical diagnosis and antibody specificity were also entered: 308
cases were considered to be MCTD and 262 cases fulfilled A M criteria for SLE.
Stepwise discriminant analysis of 17 patient record attributes
(i.e. clinical and laboratory observations) revealed the rank order in
which these attributes were useful in discriminating cases and yielded
an estimate of the relative importance of each attribute. The separation of MCTD and SLE cases was s i g d c a n t (P< 0.01) and 80% of
this distinction was attributed to only 4 of the 17 attributes: negative
LE cell test, myositis, Raynaud’s phenomenon, and absence of renal
disease. Additional distinguishing attributes in descending order of
importance include arthritis, absence of anemia, esophageal hypomotility, absence of alopecia, and swollen hands. By means of sequential cluster analysis and x square statistics, the presence of Raynaud‘s
ARA ABSTRACTS
phenomenon was the best single attribute distinguisher for MCTD as
opposed to SLE (P < 0.01). Of the patients positive for Raynaud’s
phenomenon, the concurrent use of myositis yielded the best separation of MCTD from SLE. Of the patients negative for Raynaud‘s phenomenon, the absence of renal disease set off MCTD from SLE (P<
0.0 I).
From these objective analyses we conclude that MCTD can
usually be distinguished from SLE without recourse to special antibody studies. Raynaud’s phenomenon and myositis in the absence of
renal involvement appear to be the most discriminating combination
of clinical factors for the separation of MCTD from SLE. Thus, the
use of specialized computer technology with large numbers of patients
with unusual diseases permits identification of combinations or clusters of clinical attributes which are helpful in diagnosis of subsets of
connective tissue diseases.
Biochemical basis for differential deoxyadenosine toxicity to T- and B-lymphoblasts: A role for 5’-nucleotidase
Robert L. Wortmann,Beverly S. Mitchell, N. Lawrence Edwards, and Irving H. Fox, Universityof Michigan, Ann Arbor
Deoxyadenosine and an adenosine deaminase inhibitor
(EHNA) are toxic to T-lymphoblasts, but not B-lymphoblasts. dATP
accumulates in high concentrations only in the T-cell. This in vitro
model of adenosine deaminase deficiencywas studied in MOLT4 (Tcell) lymphoblasts, MGL-8 (B-cell) lymphoblasts, and cultured human fibroblasts to elucidate the biochemical basis for the selective
toxicity of deoxyadenosine to T-lymphoblasts. By use of 30 to 300 p M
deoxyadenosine and 5 p M EHNA, T-lymphoblasts phosphorylate
deoxyadenosine to deoxyadenosine nucleotides at a 20- to 45-fold
greater rate than B-lymphoblasts or fibroblasts. During the synthesis
of dATP, dADP accumulates only in the T-lymphoblasts.
B-lymphoblastshave 7-fold or 44-fold greater 5’-nucleotidase
activity using dAMP or AMP as substrate, respectively. Fibroblasts
also have high 5’-nucleotidase activity. There is no difference in
deoxyadenosine kinase or dAMP kinase activity. The apparent Km
values of deoxyadenosine for deoxyadenosine kmase or dAMP for
dAMP kmase and 5’-nucleotidase are similar in T- and B-lymphoblasts.
The data suggest that 1) B-lymphoblasts and fibroblasts do
not accumulate large amounts of dATP because of increased dephosphorylation of dAMP by the greater 5’-nucleotidaseactivity and 2)
T-lymphoblasts accumulate dATP because of a relative inability to
dephosphorylate dAMP by low 5’-nucleotidase activity. Low activity
of 5’-nucleotidase may have a central role in sensitizing cells to
deoxyadenosine toxicity by allowing dATP to accumulate from the
phosphorylation of deoxyadenosine.
Disability in rheumatoid arthritis: Interaction of medical, occupational, and social determinants
Edward Yelin, Universityof California at San Francisco, Robert Meenan, Boston University School of Medicine, Michael Nevitt and
Wallace Epstein, Universityof California at San Francisco
Because rheumatoid arthritis (RA) is a frequent cause of disability, rheumatologists are often called upon to predict the effect of
RA on their patients’ work capabilities. This study of 245 patients
with RA from 25 rheumatologic practices in the San Francisco and
Boston areas analyzes the interaction of medical and occupational
factors in disability.
The patients were an average of 53 years old, 213 were female, and they had had RA for an average of 10 years. Patients’ selfassessment placed 70% in class 1 or 2 and the rest in 3 or 4; rheumatologists’ assessment placed 45% in stage 1 or 2 and the rest in 3 or 4.
Fifty-nine percent of those employed at the time of disease onset were
no longer working; only 29% worked 40 or more hours who formerly
did so. The data indicate that workplace factors combined with stage
of RA improve upon stage alone in predicting disability. Thus, within
the stage 3 or 4 group: 2.7 times as many professionals/rnanagers continue working at some kind of job as do service workers (P< 0.05);
1.8 times as many more self-employed individuals continue working
as do those employed by someone else (P < 0.02); and 2.2 times as
many individuals who control their own pace of work continue working as do those whose supervisor controls the pace of their work (P<
0.05). Within the stage 3 or 4 group: seven times as many professionals/managers maintain a 40+ hour workweek as do service workers
(P< 0.01). When stage alone is compared to workplace factors alone,
workplace factors emerge as the better predictor. One and a half times
as many of those in stage 1 or 2 continued some kind of work compared to those in stage 3 or 4 (P< 0.01). Almost two times as many of
those self-employed continued working as did those employed by
someone else (P< 0.005) and 2.7 times as many workers controlling
the pace of their own work continued working than did those whose
superior controlled the pace of their work (P< 0.0003). One and a
half times as many of those in stage 1 or 2 maintain a workweek of
40+ hours compared to those in stage 3 or 4 (NS).However, 2.1 times
as many self-employed individuals maintain a 40+ hour workweek
than those working for someone else (P < 0.02), as do 2.1 times as
many workers controlling the pace of their own work than those
whose superior controlled it (P< 0.02).
This study demonstrates that specific workplace characteristics as well as stage of disease are important factors in determining
the disability of patients with RA.
677
ARA ABSTRACTS
Soft tissue uptake of bone-seeking radionuclide in amyloidosis
Robert A . Yood, Martha Skinner, Alan S. Cohen and Victor W. Lee, Boston University School of Medicine
Although soft tissue uptake of the “bone-seeking’’ radionuclides is unusual, a few patients with amyloidosis have had bone
scans documenting soft-tissue radionuclide uptake. We initiated a
prospective study to evaluate the diagnostic potential of bone scanning in amyloidosis when a retrospective review of our patients disclosed 3 abnormal scans in individuals with primary amyloidosis. The
first patient had amyloid cardiomyopathy and increased cardiac uptake on scan. The second had hepatic amyloidosis and extensive hepatic uptake. The third had amyloidosis involving the soft tissues
around her shoulders (“shoulder p a d sign) and knees and had periarticular uptake in these areas. Eleven consecutive subjects were then
evaluated prospectively; 7 had primary, 2 secondary, and 2 heredofamilial amyloidosis. Bone scans with 15 mCi methylene diphosphonate (MDP) were normal in 2 and abnormal in 9. Soft tissue uptake was found in 3 patients, all of whom had hepatic uptake of MDP.
These 3 included I of 8 with normal liver function tests and 2 of 3
with abnormal liver function tests and biopsy-proven hepatic amyloidosis. None of the 3 patients with cardiac amyloidosis or 9 with gastrointestinal amyloidosis had uptake in those organ systems. Five pa-
tients had articular abnormalities due to underlying inflammatory
arthritis (2 patients) or unrelated osteoarthritis (3 patients). Two patients had unexplained increased uptake of MDP in the femur.
The uptake of radionuclide was also measured in vitro by
adding approximately 0.5 pCi of MDP to 5 mg extracts of purified
primary and secondary amyloid fibrils as well as normal and amyloidladen tissues. The fibrils or tissues were allowed to react with the
MDP under nitrogen and washed with buffer. Although MDP clearly
bound to primary and secondary amyloid fibrils, binding also occurred with normal and amyloid-laden tissue extracts.
These studies demonstrate a spectrum of soft tissue uptake of
“bone-seeking” radionuclide in 6 patients with amyloidosis including
3 of 11 consecutive patients. Although hepatic uptake of tracer occurred primarily in patients with hepatic amyloidosis, our current
data indicate that the bone scan cannot be used to evaluate the extent
of amyloidosis. The diagnosis of amyloidosis should be considered,
however, in patients in whom a bone scan shows activity in noncalcified areas.
The relation of HLA-B5 to the development of immune complexes in acute rheumatic fever
Sadayoshi Yoshinoya and Richard M. Pope, Universityof Texas Health Science Center at San Antonio, Texas
Evidence exists suggesting that certain manifestations of
acute rheumatic fever (ARF) may be due to immune complexes and
that the immune response to streptococcalantigens is genetically controlled. Therefore, 5 radioimmunoassays were employed to detect immune complexes in ARF. The assays included: The Clq binding assay (Clq-BA), the C l q solid phase assay (Clq-SP), Clq inhibition
assay (C 1q-INH), the monoclonal rheumatoid factor inhibition assay
(mRF-INH), and the monoclonal rheumatoid factor solid phase. assay (mRF-SP). The ARF sera examined included 46 samples from
patients in the acute phase and 27 from those in the convalescent
phase. Patients with systemic lupus erythematosus (SLE) (25), rheumatoid arthritis (RA) (21) and normal individuals (19) served as positive and negative controls. A significant difference existed between the
control and ARF sera by the Clq-BA (Pc 0.01), the Clq-SP (P c
0.001), and the mRF-INH (P< 0.05). Immune complexes were detected in the acute phase sera sedimenting between 6.6s and 19s after
preparative ultracentrifugation. All ARF patients had acute polyarthritis, and no other disease manifestation was associated with the
presence of immune complexes. The values with the convalescent
ARF sera were significantly diminished from the acute phase by the
Clq-BA (P< 0.001) and the Clq-SP (P< 0.01), although occasional
abnormalities persisted. The synovial fluids of 6 ARF patients were
examined and did not contain detectable immune complexes. HLA
antigens of the A and B locus were determined for 35 of the 46 ARF
patients. Those positive for HLA-B5 (10) demonstrated a more exaggerated immune response. The mean values for 4 of the 5 radioimmunoassays were higher in the B5 positive compared to the B5 negative
patients. Also, 70% of B5 positive patients had 3 abnormal assays
compared to only 12%of B5 negative individuals (25). This difference
was significant, P < 0.002.
In summary, circulating immune complex-like materials
were detected in the majority of sera from patients with ARF. Although values decreased in convalesence, some abnormalities persisted. Since HLA-B5 has been previously associated with a heightened in vitro immune response to streptococcal antigens, our findings
suggest a genetic basis for the development of immune complexes in
ARF.
Ticrynafen in patients with hyperuricemia and/or gout
Jack Zuckner, R. Eugene Arthur, Andrew Baldassare, Terry Weiss, St. Louis University School of Medicine, St, Louis
Ticrynafen [2, 3-dichloro-4-(2-thienylcarbonyl)-phenoxy
acetic acid] is a new diuretic, antihypertensive compound. It has the
unique quality of also being uricosuric, thus lowering serum uric acid
levels.
In a 24-week double-blind study, ticrynafen was compared
with probenecid in 24 patients with hyperuricemia, 21 of whom had a
history of gout. Data from 6 other patients are not included because
of insufficient information. After a placebo period, probenecid in
doses of 250, 500, or lo00 mg a day was compared with ticrynafen in
doses of 125, 250, or 500 mg daily. Nine individuals were on probenecid and 15 on ticrynafen. The double-blind study was followed by a
washout period, then I5 of the patients entered an extended open label study utilizing only ticrynafen in a similar dosage schedule, thus
enabling a crossover comparison of drugs in some individuals.
678
There was a dose related reduction of serum uric acid in all
patients. The mean decrement in serum uric acid levels on probenecid
therapy was l8%, 31%, and 38% with dosages of 250, 500, and lo00
mg/day, respectively. Decrements in serum uric acid with ticrynafen
therapy were 31%and 5 I%, respectively, with dosages of 125 and 250
mg daily. The 500 mg daily dose of ticrynafen was administered to
only 2 patients. Thus, the 125 mg dose of ticrynafen was about
equally as potent as the 0.5 gm daily dose of probenecid in its hypouricemic effect, and the 250 mg daily dose of ticrynafen was more
effective than 1 gm of probenecid.
ARA ABSTRACTS
Ticrynafen was generally well tolerated. Six patients had
acute attacks of gout while on the drug, while 5 individuals on placebo and one on probenecid had similar episodes. Mild transient elevations of liver enzymes were noted in 5 patients on ticrynafen, in 2
patients on probenecid, and in 3 others on placebo. In patients on ticrynafen a slight increase in blood sugar occurred in 5 individuals and
transient low potassium values in 3.
Ticrynafen is an effective and relatively nontoxic hypouricemic agent which should prove beneficial for both gouty and nongouty patients requiring diuretic and antihypertensive medication.
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