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Proceeding of the 36th Annual Scientific Session of teh American Rheumatism Association. June 89 1972

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AMERICAN RHEUMATISM ASSOCIATION
A Section of the Arthritis Foundation
1212 Avenue of the Americas, New York, New York 10036
President: John L. Decker, MD, National Institute of Arthritis and Metabolic Diseases, Bethesda. Maryland 20014. Vice President and President-Elect: Thomas E. Weiss. MD, Ochsner Clinic, New Orleans,
Louisiana 70121. Second Vice-president: David S. Howell, MD, University of Miami, Miami, Florida 33152.
Secretary-Treasurer: Evelyn V. Hess, Md. University of Cincinnati, Cincinnati, Ohio 45229. Executive
Secretary: Miss Lynn Bonfiglio, 1212 Avenue 6f the Americas, New York. New York 10036.
Proceedings of the 36th Annual Scientific Session of the
American Rheumatism Association
June 8-9,1972
Abstracts of Papers Presented
(Affiliations given for senior authors only)
Low Molecular IgM in Rheumatoid Synovial Fluid
ANNABELLE ANDERSON-IMBERT,
The Hospital for Special Surgery, v. HARISDANGKUL,MARIA DE BRACCO and CHARLES L.
CHRISTIAN, New York
A subspecies of IgM with a molecular weight less than
900,000 has been identified in a variety of pathologic sera.
Antigens reactive with low molecular weight IgM have included: nuclear constituents, cow’s milk casein, tetanus
toxoid and erythrocyte isoantigens.
Stage and Mannik (confirmed by Theofilopoulos et nl)
noted a relationship between serum levels of low molecular
weight IgM and severe systemic RA-especially
manifestations of vasculitis. The present report concerns studies
of RA synovial fluids in which correlations were sought between levels of low molecular weight IgM, complement
(CH,, and four components), rheumatoid factor (RF),
complexes precipitable by Clq and R F and clinical patterns. In addition, some of the physicochemical properties
of low molecular weight IgM were determined.
The concentration of low molecular weight IgM was estimated by radial immunodiffusion (RID) analysis of fractions from zone centrifugation. The RID characteristics of
19s IgM, low molecular weight IgM and reduced-alkylated
subunits of 19s IgM were established-at a concentration
of 20 mg%, ring diameters were 67.79 and 96 mm, respectively. These three materials were separable by Sephadex
426
G200 chromatography and zone centrifugation, indicating
molecular distinctions between low molecular weight IgM
and reduced-alkylated subunits. The sedimentation behavior of low molecular weight IgM was not modified by an
acid (pH 3.2) environment, and purified material’ had
neither properties of R F nor of complexes precipitable by
Clq or RF.
Of 25 synovial fluids examined, the majority of specimens
from subjects with RA possessed detectable low molecular
weight IgM (up to 150 mg%). The patients with the highest
synovial fluid concentrations invariably had (in synovial
fluids) very low CH,, levels ( < 5 units/g% protein), high
titers of R F and complexes reactive with RF; and the clinical character of articular disease was severe. Pleural fluid
from 1 such patient contained 140 mg% low molecular
weight IgM and large quantities of complexes. There was
no significant correlation between levels of low molecular
weight IgM and 19s IgM in synovial fluids, and the concentration of low molecular weight IgM in synovial fluids
always exceeded that of serum.
Present data do not exclude the possibility of low molecular weight IgM deriving from catabolic processes. Or-
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
gan cultures are being examined for evidence of production
in vitro.
Although there is no direct evidence implicating low ma-
lecular weight IgM in pathogenesis, the associations noted
suggest that it is one more manifestation of immunologic
disorder in RA synovia.
Adult Onset Juvenile Rheumatoid Arrhririr
ROBERT G. APTEKAR,
National Institutes of Health, JOSEPH s. BUJAK, JOHN
Maryland
Fifteen patients have been evaluated because of fever, iritis or arthritis with onset in young adulthood. Five had
unexplained episodes of prolonged fever in childhood. After
thorough study and extended followup, all are regarded as
having ,juvenile rheumatoid arthritis (JRA). All are negative for rheumatoid factor and none has bowel disease,
sacroiliitis, psoriasis or systemic lupus erythematosus. Several clinical patterns were observed.
Two patients (1 male, 1 female) presented with polyarthritis accompanied by fever, mesenteric adenitis and leukocytosis. Erosions of the knees and wrists were found.
Ten patients, all males, had a similar syndrome, except
that arthritis was not prominent. This group had a marked
propensity for maculopapular rash (7), lymphadenopathy
(7), pleuritis and/or pericarditis (7), splenomegaly ( 6 ) ,selflimited abdominal pain (3), sore throat (7), myalgias (10)
and arthralgias (10). Fever was hectic with a temperature
elevation greater than 105' F (9). Laboratory parameters
included: elevated ESR (mean: 86 mm/hr), leukocytosis
(mean WBC: 23,5OO/cu mm), anemia (mean hemoglobin:
9.6 g%) and hepatic abnormalities were present in 3.
L. DECKER
and SHELDON M. WOLFF, Bethesda,
Forty-one biopsies of skin rash. nodules, lymph nodes.
bone marrow and liver were taken. hIany showed chronic
nonspecific inflammation. Four had arthritis. hut only
1 had erosions after 2 years of disease. O n e had antecedent unilateral panophthalmitis requiring enucleation.
One female patient, presenting with micrognathia, had
bilateral iritis which developed at age 30. Fifteen years later
she had unilateral gonarthritis and radiologic cervical spine
fusion.
Two females had large ,joint, pauciarticular disease.
Progressive bilateral hip involvement incapacitated 1 patient. The other, with a history of childhood fever and lymphadenopathy, presented at age 43 with micrognathia and
erosive disease of the wrists, knees and ankles.
Seven of the 15 patients had undiagnosed manifestations
of JRA in childhood; the disease was first recognized in
adulthood. The remainder, with identical adult features,
had no antecedent manifestations. It is concluded that the
specific disease, .JRA, can have its onset in adult life. Such
patients constitute a portion of adults usually labeled
seronegatvie rheumatoid arthritis.
Comparison of the Biologic Properties of Soluble Immune Complexes
and Aggregaredr 0-Globulin
WILLIAM P. AREND,
University of Washington School of Medicine, and MART MANNIK, Seattle, Washington
Previous studies have concluded that soluble immune
complexes and homologous aggregated $2 have equivalent
biologic properties on a weight basis. We compared soluble
complexes and aggregated $2 of comparable size as to their
abilities to adhere to fi receptors on macrophages and fix
complement. HSA-antiHSA complexes were prepared in
antigen excess using radiolabeled specific rabbit antibodies.
Test protein
Complexes possessing more than two antibody molecules in
their lattice work, but still less than 20s in size, adhered to
stimulated rabbit alveolar macrophages in vitro, in the absence of complement. Small aggregates of rabbit $2, possessing the same sedimentation coefficients as complexes
that adhere to the cells, and large heat aggregates of 6,
were prepared. The relative strength of binding to macro-
50% Inhibition
of adherence ( p g )
50% Complement
fixation (sg)
5.0
42
95
725
710
0.92
180
250
0.70
415
Immune complexes with intact antibodies
Immune complexes with red. and alkaline antibodies
Minimally aggregated rabbit y G ( ~ 2 0 s )
Heavily aggregated rabbit rG (20 t o 40s)
Monomeric rabbit y G
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
427
ABSTRACTS
phages was indicated by determining the micrograms that
inhibited adherence of a standard amount of labeled complexes by 50%. The efficiency of complement fixation was
indicated by the micrograms fixing 50% of 1.5 U of complement.
Soluble immune complexes adhered more strongly to
macrophages and fixed complement more efficiently than
did aggregated 6of comparable size. Large aggregates of
6 and the complexes possessed comparable complement
fixing properties on a weight basis. However, these 20 to
40s aggregates were much less effective inhibitors of the
adherence of complexes than the <2OS aggregates. These
findings suggested that mechanism of adherence to macrophages and complement fixation are not determined
solely by the number of $3 molecules in the soluble complexes. Two possible mechanisms of increased activities of
the complexes are: a) a specific arrangement of
molecules in the lattice work leading to potentiated biologic
properties; or b) a change in antibody conformation upon
reaction with antigen leading to increased biologic activity
of individual antibodies.
HL-A Antigens in Systemic Lupus Erythematosus (SLE)
FRANK
c. ARNETT, The Johns Hopkins Hospital, WILMA B.
Susceptibility to murine leukemia virus and specific immune responsiveness are both linked to the major histocompatibility (H2) locus in inbred mice. Recently, certain
human diseases have been associated with the major histocompatibility (HL-A) antigens in man. Grumet et a1 (1971)
reported increased frequencies of HL-A Type 8 and HL-A
Type W15 (LND) in 40 patients with systemic lupus erythematosus (SLE). T o explore this further, HL-A antigens,
using a standard lymphocytotoxicity test, were determined
for 60 unrelated patients with SLE and 60 normal controls
matched for age, race and sex. The frequencies of antigens 8
and W15 were similar to those in controls. Of the 26 HL-A
antigens studied, only Type 13 was increased; 15% in the
patients with SLE, as compared to 3% in the controls
( x 2 = 3.63, P = 0.06). However, since 26 antigens were
tested, data for Type 13 may not be statistically significant.
The data were then analyzed to determine whether there
was any segregation of specific HL-A antigens according to
BIAS and LAWRENCE E. SHULMAN, Baltimore, Maryland
age, race, nephritis, central nervous system (CNS) involvement, hypocomplementemia and antinuclear antibody
(including specific staining patterns). Six (15%)of 40 black
patients had HL-A Type 13, as compared to none in black
controls (xz = 4.50, P = 0.03); the frequency of Type 13 in
the black population is estimated at 0.4%.Five (33%)of the
15 patients with CNS disease had Type 13, as compared to
4 (9%) of the 45 patients without CNS involvement
( x 2 = 3.53, P = 0.06). No other correlations between specific antigens and disease attributes were found.
Therefore, whether a given HL-A specificity is associated
with the diagnosis of SLE is uncertain; clearly, more data
are needed. Variations in the HL-A findings among different series of patients with SLE may relate to clinical and
racial differences between patients included in these series.
The findings presented raise the possibility that certain
HL-A antigens may, in some way, mediate the expression
of specific features of SLE.
Antibodies to Reovirur RNA in Systemic Lupus Erythamatosus (SLE)
Detected b y a Cellulose Ester Filter Radioimmunwssay
MOISES R. ATTIAS,
Veterans Administration Hospital, ROBERT A. SYLVESTER and NORMAN TALAL, San Francisco, California
Antibodies to DNA and to synthetic double-stranded (ds)
RNA occur in SLE and in NZB/NZW F, mice with a lu3H-reovirus ds RNA
No. positive
No. tested %
SLE
NZWNZW F1 mice
Sjogren's syndrome
Rheumatoid arthritis and
Sjogren's syndrome
Rheumatoid arthritis
Normal
428
pus syndrome. We now report the presence of antibodies to
viral RNA ('H-reovirus ds RNA) in 70% of patients with
14C-KB KNA
No. positive
No.tested %
'4c-Poly I . Poly
No. positive
No. tested %
35/50
22/29
9/46
70
76
19
38/50
15/29
0145
76
52
0
37/50
14/29
4/45
74
48
9
4/23
3/21
4/88
17
14
2/24
1/21
1/44
8
5
2
6/24
1/21
3/45
25
5
7
4
c
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
SLE and 76% of NZR/NZW F, female mice at 9 months of
age. These antibodies can be detected by ammonium sulfate
precipitation and by a superior radioimmunoassay method
in which radioactive immune complexes are collected and
counted on cellulose ester filters. This latter assay is highly
sensitive, easy to perform, gives results comparable to ammonium sulfate precipitation and can be used with a wide
variety of radioactive antigens.
T h e Table presents results with the filter assay using )Hreovirus ds RNA, I@-KB cell native DNA and ICpolyinosinic.polycytidylicacid.
In a competitive inhibition assay, antibodies to 3H-
reovirus ds RNA showed greatest specificity for viral RNA
and less specificity for r l 4 or DNA. These antibodies were
found in three pairs of identical twins even though only I
member in each twinship had clinical SLE. Both the concentration and specificity of these antibodies remain remarkably constant in individual patients throughout many
years of disease.
T h e presence of antibodies to viral RNA in SLE supports
the possibility of immunization to viral antigens. T h e rellulose ester filter radioimmunoassay is a simple, reliable and
sensitive method for measuring antinucleic acid antibodies
in this diasease.
Cardiac Involvement in Rheumatoid Arthritis-An Echocardiograph Study
P . A . BACON, University
of California at Los Angeles, and
Pericarditis in rheumatoid arthritis (RA) is a recognized
entity that may easily escape notice. Careful clinical investigation will show it exists in 10% of patients with chronic
RA, but the incidence in postmortem series is higher averaging 30%. Endocardial and myocardial involvement with
granulamata ‘similar to rheumatoid nodules have been
described only at postmortem. The development of echocardiographv has made it possible to examine both the pericardium and mitral valve by a harmless noninvasive technic. We have used echocardiography to investigate a series
of 22 patients with chronic nodular RA, and 22 age and sex
matched osteoarthritic controls.
Pericardial effusion has been found in 11 (50%) of the
nodular and 4 (15%) of the nonnodular patients. It was
D.C. GIBSON, London,
England
found at all stages of the disease. An effusion was not detected in any of the controls. T h e incidence of pericardial
involvement in severe RA is found to be higher by this
technic than the usual clinical estimate, and approximates
the postmortem incidence.
A slow rate of initial mitral valve movement was seen
more frequently in the nodular than nonnodular patients
with RA. T h e mean rate of movement in both groups was
less than that of the control group. A very slow rate of mitral valve opening, comparable to that seen in rheumatic
mitral stenosis, was found in 3 nodular patients but in none
of the others. It is suggested that this may represent granulomatous infiltration of the valve, while the more general
slowing may be related to myocardial disease.
Characterization of an Acute Rheumatoid Pericarditis Fluid
GENE v. BALL,
University of Alabama in Birmingham, RALPH E. SCHROHENLOHER and
RAYMOND B. HESTER, Birmingham, Alabama
T h e rare occurrence of cardiac tamponade in a man with
acute pericarditis and rheumatoid arthritis provided an
opportunity for studying fluid obtained by perieardiocentesis. These studies were undertaken to determine if the
pathogenesis of rheumatoid pericarditis were similar fundamentally to the pathogenesis of rheumatoid synovitis. Conventional examinations on pericardial fluid gave results
expected for rheumatoid effusions: the fluid was sterile on
culture for bacteria, fungi and mycobacteria, and contained
88,000 predominantly neutrophilic white blood cells. T h e
total protein was 4.5 g. Protein electrophoresis and immunoelectrophoresis patterns were unremarkable. Rheumatoid factor, shown to be of IgM class, was present in both
serum and fluid in a titer of 1 :40,960 as measured by latex
fixation; antinuclear antibody was not present; sugar was
14 mg%. T h e third component of complement, measured as
fllC/fllA by radial immunodiffusion, was 140 mg%; the
corresponding value for serum was 155 mg%. Total hemolytic complement activity was too low to read. while a
normal value was obtained for serum. T h e pericardial fluid
was not anticomplementary.
Acid phosphatase, a representative lysosomal enzyme,
was present in a concentration 160 times greater in pericardial fluid than in the patient’s serum. Complexes containing I g C were detected using monoclonal rheumatoid
factors in an agar diffusion plate system. Their precipitation was not altered by heat treatment or reduction and
alkylation of the fluid. T h e complexes were precipitated by
0.62 and 0.81M sodium sulfate, but were soluble at lower
salt concentrations. Density gradient ultracentrifugal
studies of the sodium sulfate fractions demonstrated sedimentation of the complexes at approximately 18s. Similar
complexes were not detected in the serum. These pericardial fluid findings, considered in the context of reported
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
429
ABSTRACTS
findings in rheumatoid synovial fluid, support the concept
that inflammation in rheumatoid arthritis is a unitary
phenomenon in which immune complexes, complement and
lysosomal enzymes are of major importance.
Gout Associated with Increased PRPP Synthetase Activity
MICHAEL A. BECKER,
University of California, LAURENCE J. MEYER, ALEXANDER w
Jolla, California
5-Phosphoribosyl-1-pyrophosphate(PRPP) is synthesized from ribose-5-phosphate and ATP in a phosphatedependent reaction catalyzed by the enzyme PRPP synthetase. Recent studies have indicated that the concentration
of PRPP is an important factor in determining the rate of de
nova purine synthesis. We have identified a patient in a
family with gout in whom increased intracellular PRPP
concentration and purine overproduction are associated
with increased activity of PRPP synthetase of erythrocytes.
Clinically, this patient's disease has been characterized by
recurrent uric acid stones with relatively mild arthritic
manifestations. Daily urinary excretion of uric acid is 1400
to 1600 mg/day (normal: <600 mg) on a purine-free diet.
Incorporation of glycine-I-'C into urinary uric acid is 3 to
4 times normal. Fibroblast and erythrocyte PRPP concentrations are approximately twice normal, and the patient's
erythrocytes synthesize 60% more PRPP than control erythrocytes. PRPP synthetase acitivity in the patient's dialyzed
red cell hemolysate is normal at phosphate concentrations
. WOOD and J . EDWIN SEECMILLER,
La
below 2.0 mM but is elevated up to twice that of the control
range at higher phosphate concentrations. The patient's
erythrocyte inorganic phosphate concentration is within the
normal range. The increased rate of purine synthesis, demonstrable in the patient's fibroblasts (by measuring the
accumulation of formylglycinamide ribotide in the presence
of azaserine) is relatively resistant to inhibition by adenine,
hypoxanthine or 6-mercaptopurine ribonucleoside.
These findings are consistent with the hypothesis that
this patient's overproduction of purines results from an increased generation of PRPP by a more active PRPP synthetase. The abnormal pattern of phosphate activation of
PRPP synthetase demonstrated here is clearly different
from that described recently by Sperling et al, in a gouty
family (personal communication). Thus, a range of abnormalities in PRPP synthetase activation appears to exist
in association with overproduction of uric acid, and defines
another class of human metabolic errors affecting regulation of purine synthesis.
Biologic Properties of Isolated IgG and IgM Anti-7-Globulins
("Rheumatoid Facton")
NICOLAS E. BIANCO,
Robert B. Brigham Hospital, and
High levels of y€ and yM anti-y globulins have been
associated with low complement levels in rheumatoid synovial fluids. y M rheumatoid factors (RF) are known to fix
complement. This study compares the latex agglutinating
and complement-fixing abilities of 6 and y M RF. Sera
from normal individuals and patients with osteoarthritis
(OA), and juvenile and adult rheumatoid arthritis (JRA,
RA) were heat-inactivated and adsorbed onto a Sepharoseaggregated y-globulin immunoadsorbent. Supernatants
from latex positive sera became latex negative. Protein was
eluted from the immunoadsorbent with 0. 1 M acetate
buffer, p H 4. 1. Eluates contained only IgM and/or IgG, as
determined by immunoelectrophoresis and radial immunodiffusion using monospecific and polyspecific antisera. IgG
and IgM were separated by Sephadex 13-200chromatography with p H 4. 1 buffer and neutralized. Isolated I$
430
PETER H. SCHUR,
Boston, Massachusetts
RF gave a postitive latex test (<1:80) only at 4 ° C while
IgM R F gave a positive latex test ( 5 1:1280) at both 37 and
4°C. Both IgG and IgM preparations gave precipitin
curves with heat-aggregated, 2-mercaptoethanol-treated yglobulin. Two IgM preparations fixed complement (139%)
at equivalence, and three IgM preparations fixed complement (550%) at four times antibody excess. IgG preparations from patients with OA, RA and .JRA fixed up to 66%
complement at equivalence and up to 43% complement at
eight times antibody excess. On a molar basis, IgM was
about ten times more efficient than I@ in fixing complement. One IgG preparation from a normal gave a positive
latex test and precipitin curve but did not fix complement.
The complement-fixing ability of $ R F may account, in
part, for the low synovial fluid complement levels seen in
seronegative RA, as well as contribute significantly to the
complement fixation seen in seropositive RA.
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
The Chomotuctic Effect of Cathopsin-D
J.T. BOYLE, Cornell
University Medical College, F.E. TABACHNICK and J.L. GRANDA, New York, New York
Cathepsin-D, in addition to its proteolytic effect, may
play other roles in inflammatory processes. This study deals
with the chemotactic role of cathepsin-D in an in vitro system.
Highly purified cathepsin-D, isolated from human
rheumatoid synovium, was prepared by a series of chromatographic separations on G-100, C-75, G-200 Sephadex
and DEAE cellulose. Final purity was obtained by preparative polyacrylamide gel electrophoresis. Polymorphonuclear (PMN) leukocytes were obtained from the peritoneal cavity of rabbits. Chemotaxis was measured using
modified Boyden chambers, and quantification was obtained by averaging fragment counts at standard depths into
650 mp Millipore filter.
In all experiments, at all stages of purification, cathepsin-
D was found to attract P M N leukocytes in significant excess
of controls. In general, the higher the concentration of cathepsin-D, the greater the chemotactic effect. This effect
was essentially the same throughout the p H range of 6.0 to
7.4. Gold thiomalate, in concentrations that inhibit 90% of
the enzyme activity, was found to inhibit totally this chemotactic effect.
These experiments show that cathepsin-D is chemotactic, suggesting that, in addition to its proteolytic effect, it
may play a role in maintaining chronic inflammation by
attracting P M N leukocytes to the rheumatoid joint. The
fact that gold inhibits both enzyme activity and chemotactic
effect, may help to explain its therapeutic role in the treatment of rheumatoid arthritis.
Transient and Sustained Polyarthritis A d i a r e d with Typo IV
H yporlipoproteinemia
R.B. BUCKINGHAM,
University of Michigan Medical School, GILES G. BOLE and DAVID R. BASSETT, Ann Arbor, Michigan
Previous reports have suggested that unique forms of articular disease may be found in patients with Type I1 or
Type IV hyperlipoproteinemia. One preliminary report
included 10 Type IV hyperlipoproteinemic patients who
had arthralgias primarily involving large joints. Inflammatory signs were uncommon.
During a 2-year period we have identified 12 patients (8
males, 4 females, aged 37 to 64) with undiagnosed articular
disease in the patient population attending the lipid clinic at
this institution. The patients had Type IV hyperlipoproteinemia. Objective .joint findings involved both large and
small ,joints (PIP or MCP). In 8 of 12 cases the disease was
bilateral, but tended to be assymetric and oligoarticular.
Synovitis was mildly inflammatory and persistent (3 weeks
to many months), rather than episodic in nature. Articular
pain was moderate, disability minimal and constitutional
symptoms, including morning stiffness, infrequent or ab-
sent. Serial clinical, laboratory and radiographic assessment
failed to establish these cases as due to any of the currently
recognized forms of rheumatic disease. Joint radiographs
demonstrated,juxtarticularosteoporosis in 9 patients; 2 had
unusual bone cysts, but no articular erosions were found.
Latex fixation tests were positive in 6 of 12 patients (1 : 160
to 1:1280 titer). In 2, brief extraction of serum specimens
with cold diethyl ether converted the tests to negative. Synovioanalysis in 2 patients revealed mononuclear cell predominance with total cell counts of 1400 and 9050, respectively. Lipid stains demonstrated no lipid droplets in
synovial fluid. Synovial biopsies showed minimal to moderate subsynovial mononuclear infiltrates. These cases differ
from those previously described and appear to constitute a
clinically homogenous group. The concurrence of articular
disease and Type IV hyperlipoproteinemia suggests a potential pathogenic relationship between the two phenomena.
Local and GeneralizedSchwartzmann Reaction Induced in Rae with
6-Sulfanikamidoindazole Arthritis
FRANK BURCH,
University of Utah College of Medicine, MORGAN MILLER and JOHN
The present study was undertaken to investigate the role
of fibrinogen-fibrin factors in the pathogenesis of arthritis
i n d u c e d i n r a t s by t h e o r a l a d m i n i s t r a t i o n of 6sulfanilamidoindazole (6-SAI).
Male Sprague-Dawley rats, 500 g, were given 6-SAI
suspended in methylcellulose by gavage in a dose of 250
R. WARD,
Salt Lake City, Utah
mg/kg/day for 7 days. The animals were examined daily
for evidence of swelling in joints. E coli lipopolysaccharide,
400 to 6.25 %, was injected intravenously 24 hours after
the seventh feeding. Blood fibrinogen levels were determined on the seventh day of feeding and serially after the
intravenous injection of endotoxin. White blood cell and
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
431
ABSTRACTS
platelet counts were also obtained. Kidneys, spleen, lung,
adrenals, liver and hind paws were fixed in 10% formalin
from autopsied animals, and tissues were stained with
H&E, Trichrome and Lendrum’s stain for histologic evaluation.
Rats given only endotoxin were clinically unaffected.
Rats given seven feedings of 6-SAI developed arthritis of the
hind paws, and when given as little as 6.25 pg of endotoxin,
demonstrated marked hemorrhage and sw+ling into the
hind paws; death occurred in 90% of the animals by 12
hours. Fibrinogen levels increased from a normal of 350 to
1200 mg% after 7 days of 6-SAI feedings. Heparin prevented 6-SAI arthritis as well as ,joint hemorrhage and
death in rats prepared with 6-SAI after the injection of en-
dotoxin, fibrinogen decreased to 90 mg%, the white blood
cell count fell from 13,000 to 2400/cu mm and platelets decreased from 1.2 x l o bto 2.5 x lo5.
Histology showed: a) edema, hemorrhage and vascular
disruption in the joints, b) fibrin thrombi in glomerular
capillaries, and c) congestion of the liver and spleen, and
fibrin depositions in the liver sinusoids, portal veins, red
pulp of the spleen and septa1 capillaries of the lung.
In summary, 6-SAI prepares the rat for the generalized
and local Schwartzmann reaction. Heparin blocks the clini c a l m a n i f e s t a t i o n s of 6 - S A I a r t h r i t i s a n d t h e
Schwartzmann phenomenon in endotoxin-challenged animals. This suggests a role for fibrinogen-fibrin products in
the pathogenesis of 6-SAI arthritis.
Successful Results with an Aggressive Approach to Systemic lupus Erythematosus (SLF)
ERSKINE CAPERTON,
Brooke Army Hospital, San Antonio, Texas, GERALD MULLIN, FRED R. DICK and
ALFRED MICHAEL, Minneapolis, Minnesota
The result of an aggressive therapeutic approach to SLE
is reviewed. All patients had definite SLE, and were followed a minimum of 1 year or until death. The 126 patients
were treated during the last 6 years (mean followup: 4
years).
Patients with life-threatening disease (renal 64, hematologic 52, CNS 13, lung 24 and heart 11%) were treated
aggressively; the aim being to achieve a quick and complete
remission. Prednisone was administered to noncritically ill
patients for 4 to 8 weeks; patients showing a good response
were maintained on steroids alone. Critically ill patients
and all patients with life-threatening SLE who did not respond satisfactorily to steroids alone were treated with a
combination of prednisone and azathioprine (2 mg/kg,
WBC > 4000). Ideally, alternate-day steroids were employed after an initial 2- to 4-week course of daily steroids.
Once remission was achieved and the patient was stabilized, drugs were very slowly withdrawn. Hypertension
was treated early and effectively.
Patients were frequently evaluated initially, with
exacerbations, and while changing therapy; followup was
extended from 1 to 3 months for healthy patients. Renal
function testing, serum complement and anti-DNA antibody titer were done at each visit. Evidence of disease
exacerbation by clinical or chemical parameter led to careful evaluation and reinstitution of therapy as outlined
above.
Sixteen patients died, 9 of non-SLE-related causes or
without receiving therapy. Seventy-four patients had renal
disease. Of 55 renal biopsies, 24 patients (mean followup:
3.7 years) had diffuse proliferative nephritis (DPN), while
13 patients (mean followup: 4.5 years) had membranous or
focal proliferative nephritis. Nine patients, 5 with DPN,
had multiple renal biopsies; 6 showed morphologic improvement, and 3 remained stable. Of the 37 patients with
biopsy-proven nephritis, 5 died, 3 of therapeutic complications; the 2 who died of renal disease had terminal renal
failure from DPN when fint seen.
A very favorable prognosis is possible with aggressive,
early treatment and close followup of SLE, despite lifethreatening complications including active renal disease.
Renal Histology and Clinical Course of Systemic lupus Eryrhematosus (SlE)
DON E. CHEATUM,
The University of Texas Southwestern Medical School, Dallas, ERIC R.
MORRIS ZIFF, Dallas, Texas and Denver, Colorado
Renal biopsies were carried out in 30 patients with SLE
who were subsequently followed over an average interval of
9.4 years. The patients were classified into two groups on
the basis of light microscopy: 19 patients had definite renal
abnormalities and 11 had either normal or equivocal
changes. Of the 19 patients in the renal group, 8 died from
renal and 2 from nonrenal causes; a mortality rate of 53%.
432
HURD, STANLEY
w. STRUNK and
Of the 11 nonrenal patients (excluding 1 patient lost to followup), 7 died (mortality rate: 70%). all from nonrenal
causes. All deaths in the renal group occurred in the 13 patients with focal or diffuse proliferative lupus nephritis, after an average of 4.6 years. In contrast, the other 6 renal
patients with either membranous glomerulonephritis or
mesangial glomerulitis are still alive after an average of 9.4
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
years.
toloe;ic abnormalities on renal biopsy; neurologic involvement appeared to be the chief cause in the group with
minimal or negative findings. The greater frequency of neurologic and psychiatric complications in the nonrenal group
may have been a result of the lower steroid dosage given.
Although the mortality rate was much higher in the renal
patients during the first 5 years (37%), after 8 years the
nonrenal mortality rate exceeded that in the other group.
indicatine that the absence of renal disease does not implv a
better prognosis in patients surviving beyond an 8-year
period.
I
In the nonrenal group, major neurologic involvement
and/or psychosis were eventually noted in 100’70, as opposed to 47% in the renal group (P< 0.003). These complications were the probable cause of death in 5 of 7 instances. Patients in the nonrenal group received an average
of 15.9 mg/day of prednisone, while renal patients averaged
24.6 mg/day during the last 12 months of followup.
.
In the patients studied, proliferative glomerulonephritis
was the chief cause of death in the group with definite his-
I
Immunofluorrcent Studies on Skin Biopsies From Patients with Systemic lupus
Erythematosus and Other Antinuclear Antibody Positive Diseases
JOHN J . CONDEMI, University
of Rochester School of Medicine and Dentistry, J A Y GROSSMAN and M A R Y LOU CALLERAME,
Rochester, New York
T h e deposition of gammaglobulin along the epidermal
basement membrane (EBM) has been reported to be a useful diagnostic test for systemic lupus erythematosus (SLE).
In order to determine the significance of this finding, punch
biopsies from normal appearing skin were obtained from 54
antinuclear antibody (ANA) positive patients and studied
by the immunofluorescent technic for the presence of IgG,
IgA and IgM along the EBM.
The deposition of gammaglobulin along the EBM was
found in the majority of patients with clinically active SLE
~~
(73%), but not in any patients with clinically inactive SLE
or other ANA positive patients, except in 1 with inactive
procainamide-induced lupus. This patient expired from
severe arteriosclerotic heart disease and sepsis shortly after
the biopsy was performed. Serial biopsies i n 1 patient with
SLE gave a positive result only when her disease was clinically active. We feel that basement membrane fluorescence
of normal skin is fairly specific for clinically active SLE, hut
a negative test does not rule out SLE. This test may be of
help in differentiating clinically active SLE from procainamide-induced lupus.
~
Biopsies showing positive
fluorescence a t EBM
No. of skin
biopsies
Diaenosis
SLE (clinically active)
SLE (clinically inactive)
Possible SLE
Procainamide-induced lupus (clinically active)
Procainamide-induced lupus (clinically inactive)
Rheumatoid arthritis
Scleroderma
Dermatomyositis
Felty‘s syndrome
Chronic active hepatitis
No.
YO
15
5
11
9
1
0
1
0
0
0
0
0
73
0
11
6
8
6
1
2
1
1
0
0
12
0
0
0
0
0
The Immunofluorescent Identification of Immunoglobulins and Complement in
Rheumatoid Articular Collagenous Tissue
T. DEREK COOKE,
McMaster University, Hamilton, Ontario, ERIC R . HURD, JOHN BIENENSTOCK,
ZIFF, Hamilton, Ontario and Dallas, Texas
In the synovitis induced by the intraarticular injection of
antigen in rabbits previously immunized with antigen and
HUGO E. JASIN and MORRIS
Freund’s adjuvant, prolonged synovial membrane synthesis
of immunoglobulin occurs. In this model. immunoglobulin
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
433
ABSTRACTS
and C 3 have been found in association with persisting antigen in surface layers of articular cartilage, menisci and intraarticular ligaments. T o determine whether there is similar sequestration of immunoglobulin and complement in
rheumatoid articular tissues, in the present experiments,
articular cartilage and other collagenous ,joint tissues of patients with RA and other arthritides have been examined by
the immunofluorescent technic. Biopsy specimens of cartilage and menisci were washed, sectioned and stained by the
direct immunofluorescent method for IgG, IgM, IgA, PIC,
fibrinogen and albumin, using monospecific antisera, with
appropriate blocking controls. Dense granular deposits of
the following were consistently found in identical locations
in the surface layers of RA joint cartilage: IgG and PIC: (14
of 15), IgM (13 of 15) and IgA (7 of 7). In some specimens
from patients with osteoarthritis (2 of 7) and miscellaneous
arthritides (3 of 6), weak, patchy immunofluorescence for
IgG and PIC was seen, Specimens from post-traumatic and
meniscectomy ,joints ( 8 ) , and joints with neurogenic foot
deformities (7) all showed negative staining.
These data suggest that immune complexes localize in
the surface layers of RA cartilagenous tissues. The similarities in localization of immunoglobulin and glC in both
RA and antigen-induced arthritis suggest: a) a similar pathogenic mechanism for both and b) that the chronicity of
RA synovitis may be a consequence of persistent stimulation
by antigen sequestered in articular collagenous tissue in the
form of immune complexes.
A Controlled Trial of High and Low Doses of Cyclophosphamide in 82 Patients
with Rheumatoid Arthritis
THE COOPERATING CLINICS COMMITTEE OF THE AMERICAN RHEUMATISM ASSOCIATION, Charlottesville, Virginia
This thirty-two week controlled, double-blind trial was
designed to determine if .lower doses of cyclophosphamide
(<75mg/day) would accomplish the same therapeutic result
as shown with higher doses (<150mg/day) with fewer adverse effects. This abstract is based on 54 of these 82 patients who had completed the trial as of February 29. All
patients have severe, active disease which was resistant to
conventional therapy. None had received recent gold or
previous immunosuppressive therapy; corticosteroid and
analgetic were permitted only if the doses were kept absolutely constant during the trial. The high-dose group
(average dose: 1.78 mg/kg/day) fared as well as a similar
group in a previous trial, and showed substantial im~
provement in five of six objective measures. The low-dose
group (average dose: 0.74 mg/kg/day) fared only slightly
better than the control group of a previous trial.
Degree of improvement in the high-dose group correlated
poorly with the dose in mg/kg/day, but did correlate better
with the fall in the absolute lymphocyte count. Minimal
side effects were frequent in both groups. Hair loss occurred
in Over two-thirds of the patients in both groups. Eight patients were withdrawn before completing the trial: 4 for
reasons of convenience, 1 because of an unexplained convulsion and 1 because of an episode of pneumonia. Two
low-dose patients died with infections which were probably
related to therapy.
~~~
Previous trial dose
This trial dose
Previous trial
Median improvement
<
Morning stiffness (hr)
Grip strength (mmHg)
Painful joints (No.)
Swollen joints (No.)
50-Foot walk (sec)
150
<150
1
16.35
11
7.5
2.5
1
21.5
10
8.0
1.4
5 75
Controls*
.5
0
-6.7
-1.5
-1 .o
-0.7
4.4
5
3
1
* = deterioration
Evidence for a Distal Postsecretory Reabwrptive Site for Uric Acid
HERBERT S. DIAMOND,
State University
Of
New York Downstate Medical Center, JAMES
Brooklyn, New York
We have reported increased urate excretion with increased urine flow. Pyrazinamide (PZA) blocked 83% of
434
S. PAOLINO
and DAVID KAPLAK,
this increase. This suggested distal reabsorption of uric
acid. In order to test the three-component hypothesis for
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
renal handling of uric acid in man, the effects on uric arid
excretion of various drug combinations were observed in 56
studies in 15 subjects.
RESULTS: a) A 3 g dose of PZA reduced urate excretion in
subjects taking probenecid (PB) by 71% (PB: 448 pg/min;
PB + PZA. 129 pg/min). Pyrazinamide did not significantly affect urate excretion in subjects on high-dose aspirin
(ASA). It was roncluded that the uricosuric effects of PB
and ASA are exerted at different sites. b) PB had no uricosuric effect in subjects taking P Z A or low-dose ASA
(Control: 697 pg/min; PZA + PB: 139 pg/min; Control:
573 pg/min; low ASA + PB: 562 pg/min). It was concluded that PZA and ASA probably inhibit at a common
site. c) Low-dose ASA did not alter PZA-induced urate re-
tention. Low-dose ASA decreased excretion in subjects on
PB (724 to 548 pg/min). Conclusions were consistent with
a and b. d) O ne 0.9 g dose of ASA reversed the urate retention induced by PZA (PZA: 74 pglmin; PZA + high
ASA: 275 pg/min), and, unlike PB, was uricosuric in 2 of 5
subjects. It was concluded that ASA and PB inhibit reabsorption at different sites. e) T h e decrement in uric acid
excretion produced by PZA or low-dose ASA in subjects
taking PB exceeded control excretion in 2 subjects. It was
concluded that some urate reabsorption occurs distal to the
secretory site.
These findings are not in accord with predictions based
on the three-component hypothesis, and are more consistent
with the existence of a second, postsecretory distal reabsorptive site for uric acid.
Insulin Resistance, Growth Hormone Hypersecretion and Abnormal Glucose Tolerance:
Intrinsic Defects in Gout
HERBERT
s. DIAMOND, State University of New York Downstate Medical Center, ELAINE B.
FELDMAN and ANNE
c. CARTER,
Brooklyn, New York
Specific abnormalities of carbohydrate metabolism have
been associated with secondary gout, and impaired glucose
tolerance has been thought to be common in primary gout.
Plasma glucose, insulin, free fatty acids, glycerol and
growth hormone responses to lOOg of ylucose were
measured in 21 subjects with primary gout (mean age: 48),
normal renal function and normal fasting plasma glucose
(92=t2.7 mg%). In these gouty subjects, insulin resistance,
growth hormone hypersecretion and abnormal glucose
tolerance appeared to be secondary to hyperuricemia, and
could not he related to its underlying cause.
Mean glucose tolerance was decreased; hyperglycemia
was present in 12 subjects. Insulin secretion was increased,
expressed as absolute values, percent increment above basal
and relative to plasma glucose. This differs from findings in
presumed genetic diabetes where insulin secretion relative
to basal insulin or glucose is decreased. Neither the glucose
intolerance nor the hyperinsulinism was explained by age,
weight or adiposity. T h e incidence of glucose intolerance
and hyperinsulinism was similar in 7 overproducers of uric
acid and in 6 subjects without evidence of overproduction
but with diminished tubular secretion of urate. This suggests that these abnormalities of carbohydrate metabolism
are secondary to hyperuricemia per se. There was a significant correlation ( r = .53, P < .05) between serum uric arid
and plasma glucose 1 hour after glucose. Levels of free fatty
acids after glucose were increased in subjects with gout.
Subjects with an abnormal glucose tolerance showed hypersecretion of growth hormone after glucose, sugaesting
impaired hypothalamic inhibition of growth hormone secretion. T h e elevated levels of free fatty acids and impaired
hypothalamic response of growth hormone to glucose may
lead to the hypersecretion of growth hormone, thus inducing insulin resistance in response to hyperuricemia.
Arthritis with Hepatitis: Clinical Features, Course and Laboratory Studies
JOSEPH DUFFY,
Baylor College of Medicine, JOHN
T. SHARP, MARTIN D. LIDSKY and F. BLAINE HOLLINGER,
Fourteen patients with polyarthritis and hepatitis have
been observed; 13 displayed hepatitis B antigen or antibody. T w o distinctive rheumatic disease patterns were apparent: Group7-10 patients with an acute, disabling but
transient polyarthritis; Group 2-4 patients with a lifethreatening disease consistent with a diffuse vasculitis.
,Jaundice appeared in 6 of 10 patients in Group 1 and 1 of
the 4 patients in Group 2. Skin involvement was observed in
3 Group 1 patients and 1 Group 2 patient; manifestations
were protean and included urticaria, maculopapular-pe-
Houston, Texas
techial rash and purpura. Duration of synovitis was variable. Those in the first group were markedly improved or
asymptomatic after treatment with large doses of aspirin
was started. All patients in Group 2 had cardiac, renal or
bowel involvement in addition to synovitis and hepatitis.
One Group 2 patient went into clinical remission while receiving prednisone. T h e remaining 3 patients in Group 2
treated with both prednisone and cyclophosphamide manifested persistent signs of multisystem involvement 8 to 12
months after the onset of disease.
Arthritis and Rheumatism,Vol. 15. No. 4 (July-August 1972)
435
ABSTRACTS
Rheumatoid factor titers I 6 4 0 were detected in 5 of 10
Group 1 patients; titers of 640 to 20,480 were present in 3
of 4 Group 2 patients. Antinuclear antibody in a titer 2 64
was present in 1 patient, from G r o u p 1. Serum C3 was depressed in 2 of 10 individuals with transient polyarthritis
and 3 of 4 individuals with multisystem disease.
Synovial fluid white blood cell counts varied hut were
commonly somewhat higher in Group 2. Synovial fluid C3
values, after correction for protein content, were normal in
9 of 10 fluids. Hepatitis B antigen was present in synovial
fluid from 5 of 10 patients. Hepatitis B antibody was detected in synovial fluid from 1 of 10 patients.
Infection with hepatitis B virus appears to be associated
with at least two patterns of rheumatic disease.
Role o f Thymic and Bursol lymphocyte Subclasses in Chronic Allergic Synovitis
D.C. DUMONDE,
T h e Mathilda and Terence Kennedy Institute of Rheumatology, London,
London, England. and L.N. PAYNE, Huntingdon, England
Studies of experimental allergic monoarthritis (Dumonde. Glynn: 1962) indirate that both cellular and humoral immune mechanisms are involved in the pathogenesis of
these laboratory models of the rheumatoid joint (Glynn:
1965; Dumonde: 1971). T h e present experiments investigated the ability of thymic and bursal lymphocyte systems to
support antigen-induced chronic synovitis in sensitized
chickens. Adult chickens were sensitized to bovine yglobulin (BGG) by intramuscular injection of BGG emulsified in a mycobacterial adjuvant; 3 weeks later, a suspension of BGG coated onto silica particles was injected into
the ankle joints. A chronic proliferative synovitis developed
with widespread synovial infiltration by lymphocytes, macrophages and plasma cells, and the gradual formation of
two types of ectopic lymphoid foci: a) large lymphoid follicles with mature germinal centers; and b) large aggregates
of macrophages and lymphocytes. Neonatal thymectomy
markedly suppressed synovial mononuclear cell infiltration
and both types of lymphoid foci. Agammaglobulinanemic,
C.M. OATES and R . N . MAINI,
neonatally bursectomized birds supported a chronic allergic
synovitis with intense lymphocyte-macrophage infiltration.
hut with an absence of germinal center follicles and plasma
cells. Cell-mediated (thymus-dependent) mechanisms alone
were therefore capable of supporting a chronic allergic
synovitis; hut both thymic (T cell) and bursal (B cell) systems were necessary for full development of the rheumatoidlike histology. Likewise, in vitro studies showed that the
peripheral lymphocytes of bursectomized chickens were
able to generate mediators of delayed hypersensitivity
(lymphokines), hut that both T and B cells were needed for
maximum lymphokine activity. I t is suggested that an early
event in the development of the ectopic synovial lymphoid
foci involves the production of T-cell lymphokines which
then recruit other lymphoid cells (eg, B cells and macrophages) into activity in the local (synovial) environment. O n
this hasis, local persistence of antigen might provide the
continuing stimulus to generation of further T- and B-cell
activation products which would facilitate the histogenesis
of the synovial lymphoid fori.
Failure of Human lymphocytes and Fibroblasts to fncorporate Allopurinol
MICHAEL H.ELLMAN,
University of Chicago, LEIF B. SORENSEN and SERGIO G.JACOBELIJ, Chicago, Illinois
T h e possibility of allopurinol being incorporated into
nucleic acid was studied by incubation of 6-1Y:-allopurinol
with transformed human lymphocytes and human fibroblasts. Allopurinol incorporation was compared with its
analog hypoxanthine. Nucleoside and nucleotide formation
from the incubation of allopurinol with human erythrocyte
lysate was estimated and compared with hypoxanthine.
6-~%-allopurinol was synthesized with a specific acitivity
of 0.67 mCi/mmoles. Lymphocytes were incubated with
either 'C-allopurinol or I%-hypoxanthine (0.67 mCi/
mmoles) with and without PHA, using 4 x lo6 lymphocytes per tube or a microtechnir, using 0.1 ml of whole
blood per tube. Normal skin fibroblasts from a second
transfer were incubated for 5 days with either IT-
Whole blood cultures
Isolated lymphocyte cultures
(counts/min/O.l m l whole blood) (counts/rnin/4 x lo6 lymphocytes)
Purine added
14C-aIlo pu rino I (0.8 pmo Ies)
14C-hypoxanthine(0.8 pmoles)
436
PHA-
PHA+
PHA-
PHA+
Fibroblast
cultures
(counts/rnin/
1 x 10~cells)
1.2
1.3
0.2
39.8
2.0
40.4
2.4
191.5
2.0
24.700.0
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
allopurinol or '4C-hypoxanthine. Radioactivity in the acid
insoluble protein fraction of the harvested lymphocytes and
trypsinized fibroblasts was determined. Representative incorporation results on triplicate samples are summarized in
the table.
Allopurinol and hypoxanthine can be converted to their
nucleoside and nucleotide, although at markedly disparate
rates. Nucleoside formation from IT-hypoxanthine and
14C-allopurinol incubated with erythrocyte lysate was approximately 96 and 0.3 mrmoles/mg of protein/min and
nucleotide formation by the phosphoribosyltransferase
reaction was approximately 1.47 and 0.07 mpmoles/mq of
protein/min, respectively.
Elion et al, in 1966, could not detect nucleic acid incorporation of '4C-allopurinol in mouse liver after intraperitoneal injection with l*C-allopurinol approximately onetenth the specific activity we employed. Our present studies
failed to detect incorporation of "C-allopurinol into nucleic acids of rapidly dividing human lymphocytes and fibroblasts.
Favorable Outcome in Diffuse Proliferative Glomerulonephritis of Systemic
Lupus Erythematosus (SfE)
WALLACE EPSTEIN,
University of California, and
Numerous retrospective studies of the course of biopsyproven, diffuse proliferative glomerulonephritis have established a death rate of approximately 70% by 5 years after
the onset of renal disease. T h e present experience concerns
a total of 67 patients with SLE, of whom 32 were found to
have diffuse proliferative glomerulonephritis by percutaneous renal biopsy. T h e mean duration of multisystem
disease for this latter group was 52.3 months. A total of 7
deaths have occurred in this group for a 21.8% mortality.
Four of the 7 deaths were related to renal insufficiency; and
of these, 2 presented for treatment in advanced azotemia. Of
the surviving members of this group, only 1 has progressed
to advanced renal insufficiency while under close observation, and, in this case, drug treatment was based predominately on clinical rather than laboratory observations.
T h e administration of prednisone. chlorambucil or the
H E N R Y CRAUSZ, San
Francisco, California
two drugs in combination during periods between overt
clinical disease activity has been based on a group of laboratory as well as clinical determinations repeated on an
average of every 10 days. In all 32 patients with diffuse
proliferative glomerulonephritis the urine concentration of
free L-chain protein was over 80 &ml (normal: 3 to 5
pg/ml) at the time of active disease. T h e urine L-chain
concentration returns to levels below 20 pq'ml after almost
all other expressions of disease activity. In no instance has
the renal lesion progressed when the urine concentration of
L-chain remains below 20 pg/ml. In several instances, rising levels of urine L-chain protein anticipate clinical evidence of exacerbation of disease. T h e favorable outcome of
these cases is thought attributable to the ability of a selected
group of laboratory tests, repeated frequently during clinically asymptomatic periods. to guide therapy.
Production of Fibrils In Vitro by Action of Normal Human Kidney Enzymes
on 1-Chain Proteins
WALLACE v. EPSTEIN, University
of California and
Studies, by a variety of technics, have established that
human amyloid fibrils consist of fragments of immunoglobulin L-chain protein of either the I( or the A antigenic
type. Based on molecular weight and amino acid sequence
analysis, the variable half of the L-chain was found to enter
into amyloid fiber formation. Glenner el a1 (Science
174:712. 1971) have recently reported that the action of
pepsin on type A, but not I(, Bence ,Jones proteins results in
fibrils that show both physical and chemical properties of
naturally occurrinq amyloid fibrils.
Much evidence suggests that intracellular enzymes of the
proximal tubule cells are responsible for the normal catabolism of L-chain proteins. We have extracted lysosomal
enzymes from normal human kidney homogenates capable
MARGARET TAN,
San Francisco, California
of degrading both type I( and A Bence ~Jonesproteins to
small peptides. In the proteolysis of many, but not all,
Bence ,Jones proteins of both antigenic types, an insoluble
polymer forms after 1 to 24 hours, which is composed of
fragments as well as intact Bence ,Jones protein. Electron
microscopic examination of precipitates, formed in this
manner from two type I( and A Bence ~Jonesproteins, reveals
fibrils approximately 9 0 to 100 A in width and having the
appearance of naturally occurring amyloid fibrils as well as
those produced in vitro by Glenner et a1 using pepsin. Appropriate staining characteristics, using Congo red and
crystal violet, suggest that these are amyloid-like fibrils.
These studies indicate that both the enzyme and the Lchain substrate for amyloid fiber formation may be found in
the human kidney.
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
437
ABSTRACTS
Criteria-Testing in Rheumatic Disease: The Preliminary OLE Criteria
JAMES F. FRIES,
Stanford University School of Medicine, and
When new clinical criteria are proposed, several important questions arise: a) How representative is the patient
population from which the criteria were derived? b) How
well do the criteria perform in other populations? c) How
much does the effectiveness of the criteria depend upon the
point in the course of the patient at which they are applied?
d) What is the effect of interdependence between individual
criteria? and e) Which diseases might exhibit false-positives
and how frequently? This study evaluates the prelirnina:-y
criteria / o r classification of SLE in the context of these
questions, and emphasizes the utility of computer-stored
and computer-sirnulaled patient populations in testing
proposed criteria.
A computer databank containing detailed clinical information on 112 patients with SLE and 227 patients with
other rheumatic and connective tissue diseases was utilized.
Testing was performed using cumulative data for each patient, data available at the time of the first visit and data of
individual subsequent visits taken alone. T h e clinical characteristics of the ARA patient population, the databank
population and two other large series were compared. In-
ROBERT
c . SIECEL, Stanford, California
terdependence of individual criteria was assessed statistically. Frequency of false-positive results was derived from
the databank, and simulated populations of patients with
rare diseases were created to estimate false-positives in these
groups.
T h e ARA test population was found to he unusually
classic. T h e proposed criteria were met in only 73% of the
databank SLE population, and this proportion dropped
even further when shorter observation periods were
utilized. Some pairs of individual criteria were clearly interrelated, resulting in approximately 14% reduction in efficiency. False-positive results were found in 19% of patients
with rheumatoid arthritis. 35% with systemic sclerosis and
significant numbers of patients with Wegener's granulomatosis. discoid LE, lupoid hepatitis and polyarthritis.
Good discrimination from sarcoid, acute rheumatic fever.
gonococcal arthritis, spondylitis and gout was achieved by
the criteria. Reasons for the imperfect performance of these
criteria will be discussed and suggestions for revision advanced. Modern technics of information-handling may he
used to anticipate performance of criteria prior to their
publication.
Drug Synergy in Therapy of NZB/NZW Mice
hiicHAEL
c . CELFAND, Walter Reed Army Institute of Research, Washington,
JAMES H. KNEPSHIELD,
DC, ALFRED D.
STEINBERG and
Washington, DC and Bethesda. Maryland
This study compares different immunosuppressive regimens in the treatment of the lupus-like nephritis of
NZB/W mice. Groups of 5-month-old female NZB/W
mice were given azathioprine, cyclophosphamide and
methylprednisolone in all one-. two- and three-drug regimens, each drug in the relatively low dose of 1.5 mg/kg/
day. Treatment for 3 months with one or two drugs resulted
in modest suppression of NZB/W disease. Mice receiving
all three drugs had significantly less proteinuria, lower
titers of anti-DNA antibody and less severe histologically
evident renal involvement than mice treated with one or two
drugs. Survival at 1 year was 10% for untreated controls,
44% one-drug- treated, 37% two-drug treated and 86%
three-drug treated mice. T h e survival for the three-drug
regimen was significantly longer than any other group
(P< 0.01). The three-drug regimen was synergistic, since
mice treated with each drug at three times the dose had
significantly more proteinuria after 3 months of treatment
and lowered 1 year survival (33%). T h e beneficial effects of
triple-drug- therapy were attained without increased toxicity.
This study represents the first rontrolled evaluation of
single versus combination therapy in a model of autoimmune disease. Based on these results, a controlled evaluation of triple-drug therapy in human SLE appears warranted.
Assay of Antibodies to Double-Stranded DNA by Millipore Filter
BARRY GINSBERG, Albert
Einstein College of Medicine, and
We have developed a rapid, simple and economic quantitative assay for antibodies to double-stranded DNA based
on the observation that double-stranded DNA passes
through cellulose nitrate filters, whereas complexes of DNA
438
I I A R O L D KEISER,
Bronx, New York
with anti-DNA antibodies are retained.
Sera were heated at 56" C for 1 hour to lower nonspecific
binding of DNA. Preheated serum (0.025ml) was incubated
with 1 pg (5000 counts/mm) of radioactively-labeled.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August1972)
ABSTRACTS
double-stranded D N A in 0.1 ml of tris buffer p H 7.5 at
37" C for 15 minutes. T h e solution was then diluted with 3
ml of buffer and passed through a Millipore filter (type
HAWP). T h e filter was washed with 16 ml of buffer, dried
and its radioactivity measured. Tritium-labeled preparations of lambda bacteriophage, E cofi and Hela cell doublestranded DNA, sonicated to a molecular weight of approximately 1 million, have all been used as antigens in this
assay.
The amount of radioactivity retained by the filter was
proportional to the amount of serum added over a wide
range, allowing the detection of low levels of anti-DNA activity as well as quantitation of very high antibody levels.
Retention of radioactivity in the filter was inhibited by the
addition of nonradioactive DNA, but not KNA, oligo- or
monodeoxyribonucleotides. Most of the serum DNA-
binding activity appeared in the 7 s IgG fraction after gel
filtration on Sephadex (3-200.
T h e results obtained with this assay for anti-DA antibodies were comparable to those reported with the hemaqglutination and ammonium sulfate precipitation (Farr)
technics. Normals and 55 patients with SLE in clinical
remission showed less than 2.5 pg DNA bound/ml serum.
Twenty-nine of 36 patients with clinically active SLE had
levels greater than 2.5 Gg DNA bound/ml serum. T h e level
of binding found with this assay correlated well with the
percent binding activity determined by the Farr technic on
21 sera tested by both methods.
T h e millipore filter assay for antibody to double-stranded
DNA is far more rapid and simpler to perform than previously described technics, and should allow wider use of this
valuable determination in monitoring disease activity in
patients with SLE.
Suppression of Neutrophil Chemotaxis and Random Migration by a Factor Derived from
Human Leukocytes
EDWARD J.
COETZL,T h e Robert B. Brigham Hospital, and K. F R A N K AUSTEN, Boston, Massachusetts
Human peripheral blood leukocytes exposed to endotoxin for 1 to 2 hours at 37" C, washed and then incubated in
saline for 1 hour at 37" C, release a soluble inhibitor of both
the in vitro chemotactic response of other human neutrophils and their random migration. Maximal release of this
neutrophil immobilizing factor (NIF) was achieved with 0.1
pg or greater of endotoxin per 2 x 10' leukocytes. Human
leukocytes incubated in a balanced salt solution at a p H of
6.0 or lower release a similar N I F which also inhibits both
directed and random migration of neutrophils. The release
of N I F at p H 5.0 was maximal after 1 hour of incubation of
leukocytes at 37" C. T h e neutrophil immobilizing factor
from either source was effective regardless of the specific
chemotactic stimulus and when added to either side of the
chemotactic chamber, but was more inhibitory when pres-
ent on the cell side. Further evidence that N I F acts directly
on the neutrophils is the failure to reverse chemotactic inhibition by washing test neutrophils after their preincubation
with N I F for 5 minutes or longer. T h e random migration of
Ficoll-purified, human neutrophils from slass capillary
tubes was suppressed by NIF, which was equally effective
whether present in the migration fluid or preincubated with
the neutrophils. Partial purification of N I F from either
source by gel filtration on G,,,Sephadex has provided an
apparent molecular weight of about 5000. This partially
purified N I F was stable at 56" C for 2 hours and was inactivated by chymotrypsin. T h e N I F released from normal
leukocytes by endotoxin treatment or inrubatinn in acid
media may play a role in the in vivo regulation of inflammation by holding neutrophils in the exudate.
Decreased Delayed Hypersensitivity Responses to Tuberculin (PPD) and
Trichophyton in Systemic Lupus Erythematosus (SLE)
BEVRA H.
HAHN,Washington University School of Medicine, and c. KIRK OSTERLAND,
St. Louis, Missouri
Although many studies have indicated that humoral
immune responses to a variety of antigens are increased in
SLE, there is little data pointing to alterations in cellmediated immunity. Delayed hypersensitivity skin responses to P P D and trichophyton were measured in 33 patients with SLE (22 untreated) and 39 normal controls.
Positive responses (>,5 mm induration at 48 hours) to 10
Todd units of P P D occurred in 2 of 32 patients with SLE
(mean induration: 2.16 1.78 mm), and 13 of 32 normals
(mean induration: 9.22 + 2.82 mm). These differences
were all statistically significant. Mean induration to a 1 :30
dilution of trichophyton at 48 hours was 8.80 f 1.5 1 mm in
patients with SLE and 15.47 + 1.81 mm in normals; this
was also significant. Differences in the skin responses remained significant when data from patients with SLE receiving steroid or cytotoxic therapy were eliminated. In contrast, peripheral lymphocytes from 1.5 of the patients with
SLE (13 untreated) and 15 age-, sex- and race-matched
normal controls responded similarly when cultured in vitro
with PPD. Lymphocyte transformation was measured by
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
439
ABSTRACTS
uptake of tritiated thymidine. Ratios of counts/min in cultures containing PPD to counts/min in unstimulated cultures were
1.48 f 0.27 in patients with SLE and
1.70 f 0.41 in normals; these were not statistically different.
Thus, cellular immunity to these two antigens seemed hy-
poactive in patients with SLE when measured by one
parameter (delayed hypersensitivity skin tests) and normal
when measured by another parameter (in vitro lymphocyte
transformation). These data are compatible with the hypothesis that selective defects of cellular immunity may occur in SLE.
Superiority of Combined Azathioprine-Prednisolone Therapy Over Single Drug Therapy
in Suppression of NZB/NZW Nephritis
BEVRA H . HAHN,
Washington University School of Medicine, and C.
Since NZB/NZW mice develop an immune nephritis
similar to the nephritis of SLE, a study was designed to test
the relative efficacy of three immunosuppressive drug regimens in these mice. Ten-week-old mice, divided into age.
sex and litter-matched groups of 20, received daily oral
doses of azathioprine (5 mg/kg), prednisolone (5 mg/kg),
azathioprine-prednisolone(5 mg/kg of each), or no therapy
for 72 weeks. As we reported previously. after 36 weeks
deaths from nephritis were significantly and similarly reduced in prednisolone and azathioprine-prednisolone
groups. However, by 54 weeks, azathioprine-prednisolone
proved superior, with death rates from nephritis being 80%
in controls, 71% in azathioprine, 50% in prednisolone and
7% in azathioprine-prednisolone groups. Similarly, mean
survival times (wk) were 35.1 (controls), 39.5 (azathioprine), 44.3 (prednisolone) a n d 54.5 (azathioprineprednisolone). Statistically, death rates and survival times
were significantly better in the prednisolone group com-
K I R K OSTERLAND,
St. Louis, Missouri
pared to controls, and significantly better in the azathioprine-prednisolone group compared to the prednisolone
group. The ability of a drug regimen to suppress nephritis correlated with its ability to suppress antibodies to native
DNA. measured by a modified Farr technic. At 48 weeks,
mean percents of ’%-DNA (0.09 pg) bound by mouse sera
(10 pliter) were 20.0 in controls, 14.3 in azathioprine, 8.0 in
prednisolone and 4.9 in azathioprine-prednisolone.None of
the regimens significantly reduced glomerular immune deposits, ANA, Coomb’s antibody or proteinuria. Long-term
results indicated that, although no regimen abolished the
autoimmune disease, prednisolone was better than azathioprine and azathioprine-prednisolone was better than
azathioprine or prednisolone alone in preventins deaths
from nephritis. Autopsies showed impressive histologic hepatic damage in mice receiving prednisolone, alone or
combined with azathioprine. No malignancies were found
in any group.
Fffect of Hydrocortisone and Gold on Degradation of labeled Cartilage by Rheumatoid
Synovium in Tissue Culture
R I C I I A R D H. H A W K I N S , Robert
B. Rrigham Hospital, Boston, RONALD E. ROSENTHAL and CLEMENT H. SLEDGE, Boston,
Massachusetts
Rheumatoid synovium is rich in lysosomal enzymes, such
as acid phosphatase, &glucuronidase and cathepsin D. A
prevalent theory of pathogenesis of rheumatoid arthritis
holds that release of these enzymes initiates articular cartilage breakdown. The modifying effect of hydrocortisone and
gold on this process was studied using an in vitro tissue culture technic. Rheumatoid synovium taken at the time of
synovectomy and/or arthroplasty was incubated in tissue
culture with chick embryo cartilage of known radioactivity.
T h e cartilage had been prepared by injecting 7-day-old
chick embryos with radiosulfate and harvesting the rudiments 4 days later. T h e dissected rudiments were freezedried and ground to a fine powder. Five hundred microgram portions of known activity (about 4000 counts/min)
were incubated in tissue culture with synovium and Weymouth’s 752/1 Medium for 24 hours. T h e medium was
440
then passed through a millipore filter to remove p a r t i d a t e
cartilage and prepared for counting in a Packard liquid
scintillation counter.
Specimens were incubated with radioartive cartilage
powder alone, and also with either hydrocortisone or gold
added to the medium. The hypothesis was advanced that
these drugs would retard enzymatic degradation of cartilage
powder by the rheumatoid synovium and, hence, lessen the
amount of radiosulfate released into the medium. T h e mean
counts/min of media from 14 rheumatoid synovia was
1195.2; whereas, the mean counts/min when hydrocortisone was added was 656.9. T h e addition of gold salt similarly lowered the mean to 698.9. It was concluded that the
drugs acted to inhibit breakdown of t h e proteinpolysaccharide cartilage molecule with the attached sulfate
radicals, presumably by decreasing enzyme release.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August1972)
ABSTRACTS
An Assessment of the Value of the New York andlRome Criteria for
Rheumatoid Arthritis (RA) in Population Studies
JEAN-CLAUDE HENRARD,
Southwestern Field Studies Section, NIAMD,
Phoenix, Arizona
A prevalence study of RA was made on 1748 persons,
aged 15 years and over, constituting 87% of the adult Pima
Indian population living on the Gila River Reservation.
T h e overall prevalence rate of probable and definite active RA did not differ significantly when graded by Rome
criteria (5.2%) or by New York criteria (5.9%). Using the
Rome criteria, a slightly higher prevalence of RA was found
among females (6.0 us 4.3% in males; sex ratio: 0.7),
whereas, using the New York criteria a higher prevalence
was observed in males (6.3 us 5.270 in females; sex ratio:
0.2). The difference in ratios was significant (P<0.05).
Less than 30% of persons positive by one set of criteria
were so by the other; 20% of those having RA by Rome
criteria had none of the New York criteria, and 60% of
those having RA by New York criteria had less than two
Rome criteria. T h e differences were due in part to a higher
PETER H . BENNETT and THOMAS A. BURCH,
frequency of limitation of motion in elbows and M C P joints
in males resulting in many who were positive by the second
New York criterion, and partly to a higher frequency of soft
tissue swelling of knees and ankles in females resulting in
many who scored up to three points for joint swelling in the
Rome clinical criteria.
In order to correct some of the apparent weaknesses of
the New York criteria while still preserving the weight attributable to the pattern of joint involvement, which is an
important feature of RA, the second New York criterion
was applied after excluding limitation of joint motion as one
of its components. T h e new overall prevalence rate obtained
was 3.0%, with a predominance among the females (3.8 us
2.0% in males), results approximating more closely to those
of the other population surveys. The modified criterion appeared to identify subjects in whom the experienced clinical
rheumatologist would more often agree with the diagnosis.
Correction of Hurler Fibroblasts by Enzyme Replacement
J. KENNETH HERD,
State University of New York at Buffalo, Buffalo, New York
T h e intrarellular accumulation of acid mucopolysaccharides (AMPS) in fibroblasts cultured from patients with
Hurler’s disease appears to result from an inability to degrade these macromolecules. Hyaluronidase, one of the
primary enzymes responsible for this action, has been reported deficient in the livers of some patients. Herein, we
report the absence of hyaluronidase in cultured Hurler
fibroblasts, its presence in normal fibroblasts and the biochemical correction of Hurler fibroblasts by replacement of
hyaluronidase to detectable levels in the Hurler cells.
A staining method for the detection of as little as 0.04
units of hyaluronidase on electrophoresis membranes consists of digestion of an overlay membrane saturated with
hyaluronic acid followed by staining with alcian blue.
Hyaluronidase activity of the purified enzymes for dermatan sulfate was measured by viscosity reduction and release
of N-acetyl hexosamine. Corrective effect on Hurler fibroblasts was determined by loss of metachromatic staining,
augmented loss of S3504-AMPSand decreased cell content
of AMPS isolated by classical methods.
Electrophorectically identifiable hyaluronidase was absent from Hurler cells and present in normal cells. A 2pg
hyaluronidase/ml medium caused the loss of 96% of the
metachromasia in 24 hours. Aryl sulfatase or beta galactosidase (2 rcg/ml) caused the loss of only 14 or lo%, respectively, of the metachromasia. A 2 rcg hyaluronidase/ml
medium increased the loss of SW,-AMPS from Hurler
cells by 500% in 24 hours, but had no effect on normal cells.
A 2 pg hyaluronidase/ml medium lowered the AMPS content of Hurler cells to less than that of normal cells in 10
days. T h e presence of hyaluronidase was demonstrated in
extracts of Hurler cells following treatment with hyaluronidase. Different samples of hyaluronidase possessed corrective activity in proportion to their ability to degrade dermatan sulfate. Hyaluronidase appears to have corrective
activity which is additive to that of normal serum. These
results suggest a new therapy for Hurler’s syndrome.
Immunopathologic Significance of Cartilage Degradation Products in
Rheumatoid Joint Imflammation
JEROME H . HERMAN,
University of Cincinnati Medical Center, DAVID w. WILTSE and MARIE DENNIS, Cincinnati, Ohio
A ma.jor consequence of intraarticular rheumatoid .joint
inflammation is degradation and destruction of articular
cartilage, presumably principally mediated by granulation
tissue pannus as well as proteolytic enzyme release from
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
441
ABSTRACTS
polymorphonuclear ( P M N ) cells and synoviocytes. A recent
study has demonstrated the capacity of degradation products of cartilage to activate kinins. There have been few
immunologic studies performed on cartilage per se and little attempt to define the immunologic significance of degradation components as regards causation and/or perpetuation of rheumatoid joint inflammation.
In the current study, human cartilage antigens employed
included chondromucoprotein (CMP) and its PP-L and
PP-H derivatives, intact chondrocytes grown in monolayer
culture, their homogenates and products of chondrocytes
synthesis. Antibody to these antigens was not detected in
rheumatoid serum or synovial fluid by immunodiffusion,
hemagglutination or immunofluorescence technics. Using.
anti-CMP specific antibody however, distinct cartilage antigenic constituents could be identified in synovial fluid
specimens and occasionally in the phagolysosomes of
infiltrating P M N cells.
T h e peripheral blood lymphocyte response of patients
with rheumatoid arthritis to cartilage antigens was studied
to define a potential delayed hypersensitivity mechanism.
This was assessed by blast transformation employing 'Hthymidine incorporation as in index, liberation of macrophage aggregation factor and the release of cytotoxin as determined by de novo synthesis of protein by a strain of
mouse L-cells incorporating 'C-leucine. By these parameters, 13 of 29 patients with rheumatoid arthritis having
sero-positivity and radiographic evidence of joint destruction, demonstrated a positive response to one or more antigens. Studies performed in 18 normal controls were negative. T h e response observed in patients with degenerative
joint disease, gout. SLE, Reiter's disease, scleroderma and
infectious arthritis will be contrasted. This study suggests
that cartilage degradation products may cause or facilitate
perpetuation of rheumatoid joint inflammation by: a ) their
phagocytosis causing release of lysosomal enzymes and b)
cellular immune mechanisms.
Hypocompkmentemia in Rheumatoid Arthritis (RA)
GENE G . HUNDER,
Mayo Clinic and Mayo Foundation, and FREDERIC c .
Although patients with RA usually have normal or elevated serum levels of whole complement (CH!,), we have
observed 16 patients with classic RA who had low CH,,
levels. In addition to severe joint involvement, high rheumatoid factor (RF) titers and elevated IgM, extraarticular
manifestations were particularly prominent. Rheumatoid
nodules were found in 15 patients, skin ulcers in 7,
splenomegaly in 5 (leukopenia in 4 of these), keratoconjunctivitis sicca in 2 and pericarditis and pulmonary fibrosis
in 1 each. An unusually high incidence of recurrent serious
bacterial infections occurred during the period of observation (septic joints in 4 patients and other infections in 2)
Serum Clq, C4. B,c/B,A, C I I n h proteins were quantitated by radial immunodiffusion, and functional CI, C4.
C2, C3, C5 by use of red cell intermediates and purified
components.
Lowest CH,, values were found d u r i n g s u b a c u t e
exacerbations of the patients' joint disease or development
MCDUFFIE,
Rochester, Minnesota
of extraarticular complications. Complement component
studies revealed low values for C 4 (all of 14 tested) and C2
(6 of 12 tested). Other components were mostly normal.
both by hemolytic assay and protein measurement. Complement-fixing activity could be demonstrated in several
hypocomplementemic sera. Highest levels of R F and IgM
(both 19s and 7s) were found when CH,, was lowest.
Fourteen sera produced precipitin bands against heat-agqregated IgC.
T h e low levels of CH,, and presence of complementfixing material in these sera suggest utilization of complement by immune complexes. T h e pattern of decreased C 4
and C2 with more normal amounts of other components
implies complement activation via C1. T h e concurrence of
hypocomplementemia with exacerbations and complications of RA suggests that complement-fixing immune complexes may contribute to these events. In addition, the
frequent infections may mean that these immunologic
abnormalities lead to impaired host resistence to infection.
Parameters of Improvement in Patients with Rheumatoid Arthritis Treated
with Cyclophosphamide
E R I C R. HURD,
T h e University of Texas Southwestern Medical School, and M O R R I S Z I F F , Dallas, Texas
Cyclophosphamide has been shown to be effective in
treatment of rheumatoid arthritis (RA). Nine patients with
severe RA have been treated with cyclophosphamide and
various parameters studied to determine which might be
helpful in predicting improvement. Eight of the 9 patients
442
had at least a 50% decrease in joint score after an average of
8.6 weeks after start of therapy. T h e average cyclophosphamide dosage at the time of improvement was 121 mg/
day (range: 75 to 200). Seven patients were receiving
prednisolone at an average dose of 4.9 mg/day. At this time
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August1972)
ABSTRACTS
there was a 10% decrease in IgG and IgA, an 18%decrease
in total y-globulin and no fall in IgM. Total polymorphonuclear cells fell 21% and total mononuclear cells 25%.
Large lymphocytes showed no significant change but small
lymphocytes decreased 31 % and monocytes 58%. There was
no significant change in C3, SSCA titer, hematocrit, ESR or
albumin.
In 7 of the 9 patients the small lymphocytes were depleted to 0 after an average of 31 weeks (range: 7 to 60)
with concomitant increase in large lymphocytes. Though
monocytes were decreased at this time, they were never depleted. In 3 patients with systemic lupus erythematosus
similarly treated, all had total depletion of the small lymphocytes after an average of 30 weeks (range: 14 to 46) with
concomitant increase in large lymphocytes.
Following initiation of cyclophosphamide a transient in-
crease in small lymphocytes was frequently observed. A rebound was also noted with cessation of cyclophosphamide
early in the treatment course. When cyclophosphamide was
discontinued after the small lymphocytes had been depleted,
the rebound was not observed.
T h e data demonstrate that at the time of significant improvement of RA on cyclophosphamide there is: a) a striking decrease in monocytes; b) a considerable decrease in
small lymphocytes and c) no significant change in yglobulin levels. Total depletion of small lymphocytes with
concomitant rise in large lymphocytes occurred with longterm treatment. T h e findings suggest that improvement on
cyclophosphamide is related to: a) an antiinflammatory
effect via a decrease in circulating monocytes and b) an
immunosuppressive effect resulting from a fall in circulating small lymphocytes.
Deposition of Urate Crystals in Gout
WARREN A. KATZ,
Albert Einstein Medical Center, and
T h e purpose of this study is to define the mechanism of
urate deposition antecedent to the crystal-induced inflammation in gout. Excess urate was agitated with human
synovial fluid and homogenates of bovine nasal cartilage,
liver, kidney, lung and heart muscle. A threefold augmentation of urate solubility ocrurred in the synovia and cartilage when compared to the control buffer. T h e solubility
was minimally increased in the other tissues. PPL, a protein
polysacrharide and major connective tissue component, was
primarily responsible for this effect. PPL’s isolated from
both human synovial fluid and bovine nasal cartilage were
similar in composition and electrophoretic migration. There
PRISCILLA LICOT,
Philadelphia, Pennsylvania
was no appreciable difference in their ability to hold urates
in solution. Chondroitin sulphate alone, or a variety of proteins did not augment urate solubility. When trypsin,
hyaluronidase, lysosomal enzymes or inflammatory synovial fluids were incubated with the P P L and urate solution,
crystals of the latter appeared as the P P L molecule was digested.
These results would indicate that the in vitro uratesolubilizing effect of intact P P L of synovial fluid and the
development of urate rrystals after the addition of leukocytic
lysosomal enzymes is akin to the in vivo urate deposition
phenomenon proposed to occur in patients with gout.
lmmunofluorescent Study of Kidneys in lupus Nephritis: Correlations and Implications
C.D. KENYON,
University of Cincinnati Medical Center, J .
T h e finding of DNA antibodies with lowered serum
romplement (SlC) is considered a poor prognostir sign in
systemic lupus nephritis. Fourteen patients with SLE
nephritis have been followed with serial clinical and immunologic studies. All the patients had renal biopsies, and
detailed imrnunofluorescent studies were performed. Immunoqlobulins G, A and M and three breakdown components of C3 (PIC) were looked for in the biopsies. T h e clinical state of the patient, the severity of the renal disease, the
type of nuclear antibody and the serum j3lC have been correlated with the immunofluorescent data and all of these
related to the prognosis in each patient.
Two of the 14 patients graded as having severe renal
disease, as judged by clinical and histologic changes, died
from lupus nephritis. Neither had DNA antibodies by immunofluorescence; 1 had a moderate depression of BlC, the
other a normal value. Four patients had disease of moderate
severity; 2 had DNA antibodies; all had depressed j3lC. Of
MCADAMS
and E.V. HESS, Cincinnati, Ohio
8 patients with mild disease, 3 had DNA antibodies and 3
had lowered j3lC levels.
T h e immunofluorescent studies of the kidneys showed
that all three immunoglobulins and all three C3 components were present. IgG was detected in 4 of 8 patients in
the mild group, 4 of 4 in the moderate group and in both of
the patients who died. T h e most prominent of the C3
breakdown components was the D antigen of C3b, which
was found in all 14 kidneys. T h e staining patterns were
granular, linear and mesangial in type but did not correlate
with disease severity. T h e degree of deposition by immunofluorescence, particularly IgG and the D antigen, did
correlate with disease severity.
It would appear that the immunofluorescent method for
the detection of DNA antibody and the BlC alone for serum
complement level do not provide sufficient information to
predict the outcome of lupus nephritis, nor do they reflect
the actual immunofluorescent findings in the kidney.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
443
ABSTRACTS
Enhanced Lymphocyte Transformation in Plasma Depleted of Complement ( C )
b y Aggregated7 -Globulin
T.D.
KINSELLA,
Queen's University, Kingston. Ontario, Canada
Studies in this laboratory indicate that, in some subjects,
in vitro lymphocyte transformation is modulated in the
presence of plasma depleted of C by heating or immune
precipitates. Since denatured y-globulin can also deplete
plasma C, the effect of this reagent in'this experimental
system was investigated.
Cohn fraction I1 (FII) of human plasma was sterilized by
filtration, aggregated (aggFII) by agitation or heating and
deaggregated (deaggFII) by ultracentrifugation. Appropriate cultures received 200 pg of each preparation. Liquid
scintillation counting of the uptake of tritiated thymidine by
lo6buffy coat lymphocytes assessed transformation. Results
were calculated by dividing radioactivity in experimental
tubes (FII added) by that in control tubes containing only
fresh autologous plasma. A ratio of 2.0 or more was arbitrarily ronsidered significant. Hemolytic complement, C 4
and C3 were measured in each plasma.
T o date, 9 patients with rheumatoid arthritis (RA), 6
with ankylosing spondylitis (AS) and 5 normals have been
studied. With addition to aggF11 significant transformation
occurred with 5 patients with RA (mean: 3.5, range: 2.1 to
4.7), 6 patients with AS (mean: 5.4, range: 2.2 to 9.5) and
none of 5 normals (mean: 1.5. range: 0.9 to 1.6). With addition of dcdggFI1, no enhanced transformation of RA, AS
or normal lymphocytes occurred. FII-induced transformation occurred only after C depletion, but not all C-depleted
plasmas supported enhanced transformation.
These studies demonstrate that lymphocytes from some
subjects with RA and AS undergo enhanced transformation
in autologous plasma depleted of C by aggFII. Although the
response is small relative to some mitogens, a similar in vivo
mechanism could contribute to the lymphoid hyperplasia of
patients with AS and RA.
Comparative Immunosuppressive Effects of Azathioprine and Cyclophosphamide in
the Treatment of Rheumatoid Arthritis
JOSHUA LEVY,
University of California at Los Angeles, MICHAEL w. WHITEHOUSE,
E U G E N E v.
PEARSON, Los Angeles, California
Azathioprine and cyclophosphamide have been reported
efficacious in the treatment of rheumatoid arthritis (RA).
T o study their mechanisms of action we have evaluated the
effect of these drugs on humoral and cellular immunity in
24 patients with RA.
Eighteen patients on azathioprine and 6 on cyclophosphamide were evaluated with turnover studies of radioiodinated gammaglobulins to assess the synthetic rates of IgM
and IgG before and after 6 months of therapy and 6 months
of placebo. In addition, serial studies of cellular immunity
as measured by the incorporation of 'H-thymidine into
DNA of PHA-stimulated lymphocytes in 3-day culture
were performed in 6 patients on azathioprine, 4 on cyclophosphamide and 4 on placebo.
Gammaglobulin turnover studies revealed azathioprine
BARNETT
and
CARL M
therapy to decrease IgM synthesis 4 1 % (P<O.001)and IgG
synthesis 16% (P<0.01). Cyclophosphamide therapy decreased IgM synthesis 61% (P<O.OOI)and I ~ & synthesis
51% (P<0.001).T h e synthetic rates of IgM and IgG after
placebo showed no essential change. Lymphocyte reactivity
to mitogen as measured by the ratio of 'H-thymidine incorporation by PHA-stimulated to that by nonstimulated
cells decreased 66% (P<0.02) in cyclophosphamide-treated
patients by the fifteenth week of therapy. T h e ratio in patients treated with azathioprine did not differ from that of
placebo controls.
Both azathioprine and cyclophosphamide, when given to
patients with RA, demonstrated a common effect of suppressing immunoglobulin synthesis. Only cyclophosphamide, however. appeared to suppress the cellular response of rirculating lymphocytes to mitogen.
T-cell Specificity of Lymphocytotoxins from Patients with
Systemic Lupus Erythematosus (SLE)
RICHARD B. LIES,
University of New Mexico School of Medicine, RONALD P.
Albuquerque, New Mexico
If the lymphocytotoxic antibodies which occur with particularly high frequency in patients with systemic lupus
erythematosus (SLE) are related in some way to the basic
444
MESSNER
and
RALPH
c. WILLIAMS,
JR,
disease process, it would be of considerable interest to ascertain whether such lymphocytotoxins showed any degree
of specificity for the T- or B-cell system. T o examine this
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
question as directly as possible, the lymphocytotoxicity of
sera from 23 patients with SLE was compared in three sets
of lymphocytes-peripheral
blood lymphocytes from 18
normal control subjects, peripheral blood lymphocytes from
8 patients with chronic lymphocytic leukemia (CLL) and
thymic cells of 3 human fetuses. T h e C L L cells (presumably
B cells) showed a variable degree of immunoglobulin
markers (2 to loo%), while the thymic cells (presumably T
cells) showed no markers of this type.
T h e microcytotoxicity test, as described by Terasaki, was
used for testing. All tests weredone at 15" C with 3.5 hours
incubation time. There was no predilection of the SLE sera
to react with control lymphocytes from any 1 donor, nor
was there an obvious correlation with HL-A type of the test
lymphocytes. T h e results of parallel testing of the three
lymphocyte types showed a marked decreased reactivity
with CLL cells as compared to controls (1 1 % positive reac-
tions of all tests performed with C L L cells us 45% reactivity
for control cells--P<.0005), and marked increased reactivity with thymic cells (86% reactivity us the 45% for control cells--P<.0005). One of 10 normal sera was positive
when tested with these thymic cells. T o further document
specificity and rule out the absorption positive, cytotoxicitv
negative phenomena with C L L cells, absorptions of four of
the S L E sera with thymic and C L L cells were carried out.
In general, cytotoxicity was considerably reduced after absorption with thymic cells, while it was not reduced after
absorption with C L L cells.
These data suggest T-cell specificity of lymphocytoxic
antibodies in SLE, and appear analogous to the coldreactive thymocytotoxic antibody in N Z B mice described by
Shirai and Mellors. It is possible that T-cell specific lymphocytotoxins in SLE sera function as built-in immunostats, and may be related to disturbances of cellular immunity previously recorded in these patients.
Significance of Persistent Serologic Abnormalities in Systemic Lupus Erythematosus (SLE)
ROBERT
w. LICHTFOOT, JR, Hospital for Special Surgery, New York, New York
It is not clear whether the persistence or reappearance of
serologic abnormalities in asymptomatic patients on therapy for SLE implies incipient clinical exacerbation or not.
The present study has attempted a retrospective correlation
of clinical course with changes in serum total complement
(CH,,) and anti-IF-DNA binding. Eleven of 27 available
records have been reviewed to date. Average duration of
disease was 38 months and the average follow on the above
tests 23 months, a period encompassing 28 definite and 20
suspected exacerbations. A test was considered predictwe if
found prior to a recognized exacerbation, conjrrnalory if
the abnormality was found after exacerbation and invalid if
neither of the above were true. CH,, was predictive on 13 of
48 occasions, confirmatory of 19 of 48 and invalid on only
16 of 48. Anti-'E-DNA binding levels were predictive in 14
of 32 instances, confirmatory in 13 of 32 and invalid in 5 of
32. Significance of persisting seropathology in patients
bein? treated was assessed by noting the interval until the
next subsequent exacerbation and its severity as reflected in
days of hospitalization. Duration of remission averaged 167
days on those occasions when CH,, normalized and 158
days in those with persisting CH,, depression. Duration of
the ensuing hospitalization was 13.8 days in those with
normalized CH,, and 27.5 days in those with persistent
hypocomplementemia. Duration of remission averaged 268
days in those with normalized anti-IC-DNA binding levels, only 82 days in those remaining abnormal in this respect, while days of subsequent hospitalization averaged 3.8
and 34.4, respectively, for these groups. T h e data indicate
that abnormal anti-'C-DNA binding levels may be a valuable indicator for predicting exacerbations of SLE, and
that persisting elevations of anti-"-DNA
binding tend to
presage more immediate and more significant exacerbations. T h e implications of these data on management will
be discussed. (Supported by the New York Chapter of T h e
Arthritis Foundation).
N-Acetyl Cysteine for Heavy Metal Chelation and Treatment of Gold Toxicity
A R T H U R LORBER,
University of Southern California School of Medicine, and RICHARD HOLLCRAFT,
Los Angeles, California
Therapy of gold toxicity is currently limited to a few
chelating drugs,-ie, BAL and D-penicillamine which are
hazardous. Thus, there is need for safer and more effective
treatment of toxicity encountered with chrysotherapy.
Clinical studies were conducted to evaluate the chelating
potential of N-acetyl cysteine, which can be administered
orally or intravenously to human subjects in relatively large
quantities (3 to 6 g/day). Gold levels in serum and urine
were analyzed by polarographic technics. Urinary gold excretion was monitored in 20 subjects following a 50 mg injection of gold sodium thiomalate. During the control period, this was noted to decrease 32% (mean value) from the
sixth to seventh day. In contrast, consistent and significant
increase in gold excretion (mean: 38%, P=.Ol) was observed for the same interval following intravenous N-acetyl
cysteine administration (3 g). T w o patients receiving gold
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
445
ABSTRACTS
were referred to us for therapy of bone marrow depression.
N-acetyl cysteine was administered intravenously for 5
consecutive days. Total reversal of profound granulocytopenis and thrombocytopenia was achieved
1 week,
In vitro studies utilizing ,9aAu demonstrated N-acetyl cysteine to be an effective chelator for heavy metals bound to
serum proteins. T h e chelating coefficient for N-acetyl cysteine based on molar displacement of Ht from its S H
group by heavy metal was approximately equal to that of
o-penicillamine. Previous work conducted in animals
yielded high concentration of N-acetyl cysteine, including
its S H groups in bone marrow, liver and kidney within 5
minutes of intravenous administration. It is postulated that
the high concentration of S H achieved in the tissues with Nacetyl cysteine treatment may compete for heavy metal or
restore essential S H groups independent of chelating
function. ~h~ use of N-acetyl cysteine may thus have
therapeutic application for conditions that deplete tissue
SH-eg, heavy metals, alkylating agents and radiation.
Effect of High-Dose Systemic Corticosteroidson the Metabolism of Articular
Cartilage of Rabbits
HENRY J. M A N K I N , Massachusetts
General Hospital, Boston, WILLIAMJAFFE and
New York City
Prior studies have demonstrated a profound interference
in the matrix anabolic activities of articular cartilage following interarticular injection of steroid hormones. A study
was designed to investigate the effect of systemically administered corticosteroids on the same tissue.
Adult New Zealand white rabbits were treated for 9
weeks with cortisone, (approximately 3.5 mg/kg). At intervals, the animals were killed and articular cartilage from
the distal femur harvested and assayed for DNA and hexosamine content and rates of incorporation of labeled
cytidine, glycine and sulfate. T h e results were compared
against identical studies on cartilage from untreated controls, and also from a special group of starued controls,
whose weight loss was monitored to approximate that of the
group treated with cortisone.
Analysis of the data showed that cortisone treatment
produced no change in DNA concentration, a gradual de-
ANTRA Z A R I N S , Boston,
Massachusetts and
crease in levels of hexosamine to approximately 50% of
normally fed controls and marked diminution of incorporation rates of labeled glycine and sulfate. Less change was
noted in the rate of incorporation of cytidine-’H. T h e
starved controls showed a similar decline in hexosamine
content and metabolic activities, but this was less marked,
except for glycine-’H incorporation which showed approximately the same degree of inhibition as in the animals
treated with cortisone.
O n the basis of these data, it is possible to state that highdose cortisone therapy and, to a lesser extent, starvation
produces ronsiderable interference with the matrix anabolic
activity of articular cartilage of rabbits, and that this results
in a significant reduction in the concentration of acid mucopolysaccharides. T h e data allow speculation regarding
the mechanism of action of corticosteroids on articular cartilage and its relationship to the negative nutritional balance of starvation.
Hypocomplernentemia, Cutaneous Vasculitisand Arthritis: Possible New Immune
Complex S yndrome
F.C. MCDUFFIE,
Mayo Clinic and Mayo Foundation, W.M. SAMS, J.E. MALDONADO,
Rochester, Minnesota
We have encountered 4 patients with rerurrent attacks of
a skin eruption resembling erythema multiform, arthritis
and abdominal distress. T w o patients have developed mild
membranoproliferative glomerulonephritis over the course
of followup. Symptoms have been present for 5 to 13 years
in these patients. Low levels of serum total complement ( < I
to 22 units/ml us normal of 40 to 80 units/ml) have been
present during attacks with occasional return to normal between attacks. Both early (C1 and C4) and late (C3 and
C3-C’))reacting components of complement have been decreased. Trace to small amounts of cryoglobulins containing
I& and ocrasionally IgM have been present in these sera.
Sera from 2 patients have produced lines in immunodiffusion against a monoclonal rheumatoid factor capable of
446
D.L. CONN, P.H. ANDREINI
and
E. SAMAYOA,
precipitating immune complexes.
A skin biopsy in 1 patient showed necrotizing vasculitis
with deposition of IgM in vessel walls. Biopsies of 2 others
showed nonspecific dermal inflammation without vasculitis
or deposition of immunoglobulin. One renal biopsy was
studied by immunofluorescence, and showed granular deposits of immunoglobulin and complement. All patients had
negative tests for rheumatoid factor, L E factor, hepatitis
associated antigen (HAA) and dermal basement membrane
staining for immunoglobulin. These negative findings, together with the every small amounts of cryoglobulin present,
suggest that these patients do not have SLE or mixed cryoglobulinemia. They appear to have an immune complex
disease resulting from interaction of an undefined antigen
with antibody and complement.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
Survival Factors in Males with Scleroderma: A National Sample of 358 Veterans
THOMAS A.
MEDSGER, JR,University of Pittsburgh School of Medicine, H A R R Y ROBINSON and ALFONSE T. MASI, Pittsburgh,
Pennsylvania and Memphis, Tennessee
Patterns of survival in scleroderma have not previously
been evaluated on a national scale. This study utilizing
hospitalized Veterans Administration (VA) patients was
designed to accomplish this purpose for males. All male
veterans discharged with a first VA hospital diagnosis of
scleroderma during the interval 1963 through 1968 were
included for study if they satisfied defined clinical criteria
for diagnosis. Followup through 1970 was successfully accomplished in 96% of these 358 patients accepted for study.
Using the life-table method, cumulative survival rates were
calculated from entry to study until death or last followup.
Demographic and clinical variables found at entry to study
were analyzed in terms of survival effects.
In the total patient series, survivorship declined most in
the first year, decreasing to 70% cumulative survival rate,
and continued to decline to 40% after 5 years. Patients under age 50 at entry survived significantly better than those
50 and older, even after adjusting for the better expected
survival of the younger group (P<O.01). Surprisingly, these
significant survivorship effects of age occurred only in the
first 2 years of followup. No significant differences in sur-
vivorship were found by race or geographic location of
residence.
Regarding clinical manifestations described at entry, all
17 patients with kidney involvement died within 10
months. After excluding these patients, the 41 with heart
involvement had the next worst prognosis, followed by the
112 patients with lung but not heart involvement. All of
these subgroups differed significantly from each other in
survival (P<O.OI). In comparison, patients with none of
these organs involved at entry had the best survival, with a
60% cumulative survival rate after 5 years. The prognosis
in this latter group was not affected by the degree of cutaneous involvement or the presence or absence of sclerodermatous involvement of the gastrointestinal or musculoskeletal systems. In this same group of patients, factors
associated with significantly reduced survival were: older
age (50+ years), elevated sedimentation rate (Wintrobe
uncorrected 32+ mm/hr, anemia (hematocrit < 35%) and
proteinuria (1+ or greater). There was no evidence that
any of the common therapies, including rorticosteroids,
influenced survivorship.
An Evaluation of Hand Function in Rheumatoid Patients Undergoing Wrist Fusion
LEWIS H.
MILLENDER, Robert B. Brigham Hospital, and
T h e purpose of this study is to evaluate the overall hand
function of rheumatoid arthritic patients who have undergone wrist fusion. W e have reviewed 70 wrist fusions in 60
patients (10 bilateral) done by us during the past 4 years.
Forty-seven of these patients have been followed for more
than 2 years, and each has been personally evaluated by one
of us in a long-term, follow-up examination. Although alleviation of pain was the most significant benefit from the
patients' standpoint, we were especially impressed with the
absence of functional loss from the arthrodesis. Patients
uniformly demonstrated substitution patterns using the elbow and shoulder to compensate for lack of wrist flexion
and extension. Patients with bilaterial wrist fusions in a
neutral position were able to carry out most all activities,
including personal hygiene. A few patients had difficulty in
wiping things up from the floor or window, and some had to
adjust their handwriting. They all felt that the increased
EDWARD A. WALEBUFF,
Boston, Massachusetts
function resulting from alleviation of pain and increased
strength overcame any minimal impairment from loss of
flexion and extension. Even patients who were fused only
for pain and had good preoperative motion and stability felt
their hand function was improved with a painless fused
wrist.
Based on the results of this study, we have broadened o u r
indications for wrist fusion. Initially, fusion was done for
gross instability, deformity or destruction. With more experience, we have learned that a painful wrist, even though
it has normal motion and stability, can significantly weaken
and impair hand function. We now feel that early wrist fusion is indicated in patients showing persistent wrist joint
pain who derive no relief from splints or occasional steroid
injections, and whose x-rays show loss of joint cartilage and
early carpal destruction.
Effects of Salk ylates on an Experimentally Induced Model of Osteoarthritis
ROLA\D
w. %IosimwiTz.( h e Western Reserve University. wiRT DAvIs, WILLIAM A. JONES,
MALONE,
MICHAEL MAYOR
and
RRVCE
Cleveland, Ohio
Salirylates reportedly aid cartilage healing after scarifiration. This study investiqates their effect on an animal
model which more closely simulates human osteoarthritis.
Partial meniscectomy i n rabbits is followed by osteophyte
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
447
formation. Cartilaqe degeneration also occurs. inrluding
ulceration, fibrillation. fissurinq, cyst formation and decreased matrix prnteinpolysaccharide. Chondrocyte proliferation represents attempts at repair. I n 24 partial meniscectomy animals. tibia1 osteophytes were seen in 96%,
femoral osteophytes in 32% and cartilage degeneration in
60% at 12 weeks. ’Twenty-five partial meniscectomy animals treated with sodium salicylate for 2 weeks prior to and
during the period of study revealed similar results with tibial osteophytes in 92’70, femoral osteophytes in 56% and
cartilage degeneration in 68%. Size of ulcers was similar in
both groups. T h e average for each animal of four blood
salicylate levels taken at surgery, 4. 8 and 12 weeks, varied
from 3 to 26 mg% (mean average: 14 mg%). Blood levels
and pathology did not correlate.
Studies in vitro, utilizing viscometric technics, confirmed
presence of a hydrolytic enzyme in both human articular
and rabbit ear cartilage, which degrades proteinpolysaccharide (PPS). T h e mean decrease in viscosity of PPS from
7 human samples by this enzyme varied from 6 to 16%. T h e
mean decrease in viscosity of 3 rabbit ear cartilage samples
of PPS varied from 5 to 10%. Sodium salicylate, 3.5 mg%,
did not significantly affect these results.
Sodium salicylate did not prevent or ameliorate experimentally induced osteoarthritis, nor did it affect in vitro
degradation of proteinpolysaccharide.
Septic Arthritis in Sickle Cell Stares: Pathophysiology of Impaired Response to
Infection and Implications for Management
DONALD
w. PALMER, University of Chicago,
MICHAEL H . ELLMAN
Two patients with septic arthritis and sickle cell disorders
responded poorly to therapy. Although prompt responses
are reported in nonsicklers, E coli and S enteritidis persisted
in .joints of sicklemic patients for 53 and 42 days in spite of
parenteral antibiotics in extraordinary doses. Synovial fluid
gases were measured in one case before, during and after
septic arthritis. The 0, saturation of articular capillary
blood in equilibrium with the joint fluid was calculated, and
the percentage of sickled cells and resultant increase i n viscosity were predicted for local capillary blood. T h e ratio of
articular blood flow to articular oxygen ronsumption was
determined by the Fick principle. Time curves of synovial
fluid antibacterial activity were obtained against the isolated organism, and its antibiotic sensitivity was studied in
regular and hypertonir media.
and
SERGIO C. JACOBELLI, Chicago,
Illinois
Extreme metabolic abnormalities were found-eg, pH
6.8, PO, 29 mm Hg, PCO, 92. lactate 6.98 mM/liter. Loral capillary sickling ranged up to 10070, and increase in
blood viscosity up to 4.7-fold. T h e ratio of blood flow to O2
consumption ranged from .I0 to 5.0. S enferifzdis was recovered from synovial fluid containing ampicillin bactericidal to a titer of 16 for the same isolate in regular media.
T h e recovered Salmonella reverted to an L-form on hypertonic medium, and its antibiotic sensitivity was altered.
Optimal management therefore includes: a) reversal of
local sickling by correction of anemia or exchange transfusion and b) parenteral antibiotics according to sensitivities
on regular and hypertonic media, in increased doses or
combinations directed at both unaltered bacteria and
spheroplasts.
Arteriographic Evaluation of lntraarterial Reserpine in R a ynaud’s Phenomenon
JAMES PAOLINO, State
University of New York Downstate Medical Center, EZRA SHARON,
Brooklyn, New York
MAHESH KINKHABWALA and DAVID KAPLAN,
The response to intraarterial reserpine treatment in
Raynaud’s phenomenon was evaluated clinically and by
percutaneous transfemoral brachial angiography before,
immediately following and 5 weeks after the intraarterial
injection of 1 mg of reserpine. Prereserpine angiograms in 7
patients with Raynaud’s phenomenon showed the typical
changes of marked spasm of large arteries, diffuse tapering
and abrupt occlusions in the digital arteries and diminished
venous return from the fingertips. In all patients an angiogram was performed 15 minutes after a 1 mg reserpine injection into the brachial artery. Four patients responded
with increased filling of digital arteries. These 4 patients
showed healing of skin ulcers and amelioration of
Raynaud’s phenomenon. Repeat angiogram 4 weeks after
448
injection demonstrated persistent effect of reserpine treatment. Clinical improvement has been maintained for more
than 12 weeks. Three subjects not demonstrating arteriographic response to reserpine failed to show clinical improvement. Increased arterial filling following reserpine
was also demonstrable in the hand of a patient 5 weeks after
complete cervical sympathectomy. T h e immediate angiographically demonstrable response to intraarterial reserpine is therefore predictive of the clinical response. These
observations suggest that dilitation of large arteries contributes to the therapeutir effect of intraarterial reserpine.
Arteriograms clearly illustrating these changes will be
shown.
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
Electron Microscopy of Giant-Cell Arteritis: Unique Changes in Internal Elastic lamina
FRANK PARKER,
University of Washington,
L.A. HEALEY
Temporal artery biopsies from 26 patients (age 25 to 82)
were examined by electron microscopy. The 18 nonarteritic
vessels revealed a progressive intimal thickening with increasing age, due to accumulation of smooth muscle cells,
also loss of segments of internal elastic lamina and occasional atheromatous plaques.
T h e eight arteritic vessels showed three distinct changes
in addition to those of aging: a) Histiocytes, epithelioid and
giant cells infiltrated between smooth muscle cells of intima
and media on either side of internal elastic Jamina. T h e
granulomatous cells contained many intracytoplasmic lysosomes and surface microvillous processes. b) Internal
elastir lamina entrapped within t h e granulomatous
and
KENNETH R . WILSKE,
Seattle, Washington
infiltrate was fragmented and degenerating with unusual
density and granularity of elastin matrix. Although in close
proximity, no internal elastic lamina fragments were found
within the giant cells. c) Smooth muscle cells contained increased intracellular organelles. This is considered a nonspecific reaction and seen in other pathologic vascular reactions. No virus-like inclusions were present.
While serum immunoglobulins and complement are
normal in giant-cell arteritis, the electron microscopic
changes are considered consistent with cell-mediated, immune response. Papajiannis (Arch Pathol89:434, 1970), by
injecting heterologous elastic tissue intradermally in rats,
has evoked a giant-cell response similar to the reartion of
giant-cell arteritis.
Epstein-Barr (€6)Virus Antibody and lgG levels in Systemic lupus Erythematosus (SLE)
PAUL E. PHILLIPS, T h e
Hospital for Special Surgery, and
Although numerous virus antibodies are increased in
SLE, Evans et a1 found a somewhat greater increase of EB
virus antibody than of other virus antibodies and suggested
this might have pathogenetic significance. We previously
found a direct correlation between measles antibody and
gammaglobulin levels and suggested that the proximate
cause of elevated measles antibody in SLE is hypergammaglobulinemia. We have now studied antibody and IgG
levels in the same population of patients with S L E who had
siqnifirantly increased levels of measles and parainfluenza
Type I antibodies. Coded sera were obtained from 57 patients with SLE and 57 age- and sex-matched blond donor
controls. Epstein-Barr virus antibody was measured by indirect immunofluorescenre on the Jijoye cell line in the
laboratory which reported the increased levels of EB virus
antibody in sarroidosis. IgG levels were measured in duplirate by radial immunodiffusion.
Mean E B virus antibody titer (Log,) in the group with
SLE was 7.18 (=t2.02 SD), which was not significantly
(P> .05) greater than the mean titer of the controls, 6.62
( *2.04). ,Mean IgG level in the group with SLE was
1793.6 (+ 772.1) mg%. A highly significant direct correlation was found between EB antibody and IgG levels:
YASHAR HIRSHAUT,
New York, New York
r=0.51, P<.OOl. A significant direct correlation was also
found between EB virus and measles antibody levels in the
51 patients with SLE with both determinations: r = 0.36.
P < .02. Epstein-Barr virus antibody did not correlate
with age or with parainfluenza Type I antibody. EpsteinBarr virus antibody and IgG levels were measured in 2
patients with SLE sequentially over 2- (8 sera) and 4-year
(11 sera) periods. There were marked variations in both
parameters with maximum changes of 8- and 32-fold in
EB virus antibody, and of eight- and fourfold in I&, respectively. Fair qualitative correspondence was found between changes in the 2 parameters. Excellent correspondence was found between changes in measles antibody and
IgG in these patients. That EB virus is often a chronic
or recurrent infection may explain the difference. T h e
slight elevation of EB virus antibody in SLE rasts doubt
on a special role for EB virus in the pathogenesis of SLE.
The direct correlation between EB virus antibody and
IgG levels supports the hypothesis that IgG level may be
a determinant of other virus antibody levels besides E B
virus and measles, and that the elevation of IgG in S L E
is the proximate cause of the increases in virus antibodies
found in this disease.
Immunoglobulin Production b y Synovial Organ Cultures
PACL E. PHILLIPS,
T h e Hospital for Special Surgery, STEFANO BOMBARDIER], CHARLES L. CHRISTIAN, RITA H A R C R A V E
and PETER c . BULLOGH, New York, New York
Intraarticular immune complex formarion may be an
important pathogenetic mechanism in rheumatoid arthritis
(RA). Microbial agents may be involved, but efforts at isolation have yielded inconsistent results. Determination of
the specificity(s) of antibody produced by RA synovia might
indicate the nature of the antigen(s) involved. Here we describe the prolonged production of immunoglobulin by RA
synovial cultures and a suspension culture method for producing milligrams or greater amounts of IgG.
Surgical synovial specimens were rinsed with BSS,
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August1972)
449
ABSTRACTS
minced, rinsed again and placed in Falcon organ culture
dishes. Enough medium 199 with 20% fetal calf serum was
added to wet the tissue. The cultures were incubated at
37" C in a humidified atmosphere of air and 5% CO,. Medium was harvested daily for 3 weeks and then weekly for
up to 9 months; after low speed centrifugation, it was frozen
at--7O0 C until immunoglobulin assay. Presence of growth
and cell type were determined microscopically in vitro and
by histopathology. IgG and IgM concentration was determined by a previously described radioimmunoassay. The
sample was incubated with polyaminostyrene-coniugated
anti-lgG (or IgM) antibody, then 1251-labelled IgC (or
IgM) added, the mixture incubated and the radioactivity
remaining in the supernate measured. Standard curves
using known amounts of IgG (or IgM) were constructed for
each experiment.
Thirty-two synovia grew visibly; 16 producing IgC.
Eighteen synovia did not grow; 1 produced IgG. Of 23
growing RA synovia, 11 produced IgC in maximum
amounts of 3 to 21 pg/ml medium/day for periods of 2 to
35 weeks. Of 4 growing juvenile RA (JRA) synoviai 3 produced 2.5 to 5.5 pg IgG/ml/day for 2 to 8 weeks. Of 5
growing non-RA synovia, 2 produced 3.5 to 6 pg IgG/ml/
day for 2 to 4 weeks (1 was a proliferative synovitis from a
hemophiliac). IgM production was measured in 23 cultures, but was found only in IgG producers: 5 RA and l
JRA, 0.3 to 1.5 g/ml/day for 2 to 1 3 weeks. Maximal
IgG production was by a RA synovium containing lymphoid follicles. Medium from a 100 ml Maitiand-type
suspension culture of minced RA synovium was harvested
at 3-day intervals for 7 weeks, cumulatively producing > 3
mg IgG and 250 pg IgM. Rheumatoid arthritis and JRA
synovia produce IgG and IgM in vitro for prolonged periods; large volume suspension cultures may produce enough
immunoglobulin to allow characterization of its antigenic
specificities.
Nucleolus-Specific Anfibdies in Autoimmune Diseases
JACOB L. PINNAS, Scripps Clinic and
Research Foundation, J.
Sera from 26 patients with autoimmune diseases which
showed antibodies to nucleoli by immunofluorescence on
mouse kidney sections formed the basis of this study to
characterize a soluble precipitating nuclear antigen. Ten of
26 patients showed only nucleolar staining at all dilutions
of sera, and the remainder showed mixed nucleolar with
speckled or diffuse staining patterns. In the group with
mixed staining patterns, nucleolar staining became more
evident at higher dilutions of sera. Nucleolar staining was
eliminated when the tissue sections were pretreated with
ribonuclease, but was unaffected by deoxyribonuclease. In
order to analyze the nucleolar antigens further, calf thymus
or rabbit liver nuclei were sonically disrupted in isotonic
sucrose followed by differential centrifugation of the sonicate over 0.88M sucrose to obtain a nucleolar pellet. Nucleoli isolated in this manner and examined by immunofluorescence retained the antigens reactive with the above
sera. Solubilization of nucleolar antigens was obtained by
deoxyribonuclease digestion in tris-HCI buffer p H 7.5
followed by treatment with deoxycholate in the presence of
D. NORTHWAY
and ENG M. TAN, La Jolla, California
polyethylene sulfonate, a ribonuclease inhibitor. This nucleolar extract reacted in immunodiffusion with the above
sera to give two nonidentical precipitin lines. One of these,
described previously, is a nuclear ribonucleoprotein which
present evidence suggests is also in the nucleolus. Sera precipitating with this antigen showed mixed patterns of nucleolar staining associated with speckled nuclear staining.
Sera with pure nucleolar staining reacted with nucleolar
extract to give another precipitin system. The latter reactive
nucleolar antigen was not DNA, deoxyribonucleoprotein or
the nuclear Sm antigen, but precipitating activity was
eliminated by spleen phosphodiesterase digestion. The nucleolar antigen sedimented in the 4 to 6s region by sucrose
gradient ultracentrifugation, and its ultraviolet absorbancy
showed a 260/280 nm ratio of 1.8. Present evidence suggests that the antigen contains RNA, which is unique to the
nucleolus. Sera which contained antibodies to this nucleolar
antigen were obtained from patients with scleroderma,
Sjogren's syndrome and undiagnosed illnesses manifesting
Raynaud's phenomenon.
Heterogeneity of the Proteoglycanr Dispersed in the Extracellular Fluid of C b t k g e
JULIO c. PITA,
University of Miami School of Medicine, FRANCISCO J. MULLER, JUAN E. MADRUGA and DAVID s. HOWELL,
Miami, Florida
The physiologic importance of cartilage matrix sulfated
proteoglycans and their role as regulators of endochondral
calcification has been studied in the microscopic samples,
(about 20 to 50 ml) of extracellular fluid aspirated in vivo
from epiphyseal cartilage. A consistent concentration of 2.0
450
to 2.5 mg/ml of hexuronate has been demonstrated in the
fluid aspirated, in vivo, by micropuncture (Cn) from the
tibia1 epiphyseal cartilage of rats. The chemical composition
of the cetylpyridinium precipitate (ie, protein 8 to lo%,
hexuronate 18 to 22%, hexosamine 20 to 23% and sulfates
Arthritis and Rheumatism, Vol. 15,No. 4 (July-August 1972)
ABSTRACTS
7.0 to 7.5%) was congruent with a concentration of 9.5 to
(0.0 mg/ml of sulfated proteoglycans. Only about 30% of
this amount sedimented with added amorphous calcium
phosphate. This fraction, which is responsible for the inhibitory capacity exhibited by the Cn to prevent mineral
accretion in vitro had a weight average sedimentation coefficient of about 120s. T h e aggregated structure of this fraction has been demonstrated by disaggregation in 4 M
guanidinium HCI. An ultramicro, analytical, ultracentrifugational technic was developed based in the transport method. By adaptation of the theory and equations reported by
Wattanabee et af, (Biochem Riophys Acta 15:38, 1954). it
has been possible to characterize the polydisperse distribution of the proteoglycans present in the aforementioned
(Cn). T h e large aggregates included polydispersed components in a range from 6 0 to 140s. Proteoglycans exhibiting
lower values had a range from 5 to 25s approximately; no
intermediate components were found. T h e scaling down of
the purification technic, using CsCl gradient, has permitted
the preparation of the proteoglycan subunit present in the
Cn. T h e sedimentation coefficient distribution has been established on the proteoglycan subunit (PGS) and a synthetic aggregate prepared from combing this Cn PGS with a
protein component in the supernatant. Similar, but preliminary, data have been obtained on bovine articular cartilage
samples run through CsCl gradients. These proteoglycan
aggregates, in addition to inhibitory effects on mineral
growth, are believed to contribute importantly to the
rheologic properties of articular cartilage, due partly to
their enormous water domains. These data provide the first
firm evidence for the physiologic presence of the proteoglycan subunit and aggregate proteoglycans in native interstitial cartilage fluids.
Chlorpromazine-Induced Antinuclear Antibodies ( AN A )
F. P. QUISMORIO, University
of Southern California School of Medicine. D.
Los Angeles, California
Drugs such as procainamide and hydralazine induce a
lupus-like syndrome and ANA. One of us recently observed
a lupus-like syndrome following chlorpromazine. This case
and a previous report (Acta Med Scand 187:67, 1970) interested us in further study of the problem.
Sixty-three patients, mostly schizophrenics, were
studied. Groups 1 and 2 were receiving rhloropromazine at
the time studied; Group 1 400 mg/day for at least 7 weeks,
and Group 2 50 to 300 mg/day for varying periods. In
Group 3, 5 patients received no chloropromazine for at
least 3 months, and 21 received none for at least 10 months.
Antideoxyribonucleoprotein (anti-NP) and anti-DNA was
tested with indirect immunoflourescence using human leukocytes and rat liver. Rheumatoid factor (latex fixation test)
was detected in only 1 serum. Antinuclear antibodies are
tabulated as number of positives in each group:
Anti-DNA reacted with denatured. not to native DNA.
F. EIJARNASON, E. L. DUBOIS
and GEORGE J .
FRIOC,
~~~~~~~
No. of Median
AntiGroup patients age LEcell NP
1
2
3
20
40
7
17
47
2
2 6 4 0 0
8
3
0
AntiDNA
Total
ANA
4
0
0
10
4
2
No patients had clinical evidence of lupus or leukopenia. A
positive ANA did not correlate with total cumulative chloropromazine dose, nor did a history of photosensitivity or
skin rash. T h e data confirms and expands earlier observations on chloropromazine-induced ANA, and suggests relationship to continued daily dose rather than cumulative
total dose. Specificity of antibody is similar to procainamide-induced ANA. Prospective studies and examination
of mechanism(s) of induction are underway.
Long- Term Followup in Juvenile Rheumatoid Arthritis: Factors Influencing Distribution of
Joint Involvement, Prognosis and Serologic Abnormalities
MARK RCDERMAN,
University Of COIlneCtiCUt School of Medicine, EDWARD SCCLL,
ROTHFIELD, Hartford, Connecticut
One hundred cases of juvenile rheumatoid arthritis (31
males and 69 females) were reviewed and the patients recalled for clinical and laboratory studies. At study, ages
ranged from 3 to 43 with a mean of 17; age of diagnosis
ranged from 1 to 16 with a mean of 7; disease duration
ranged from 1 to 40 with a mean of 11.3. Six patients had
died.
ARNOLD GOLDENBERG
and NAOMI
Fifty-two percent of the patients had active disease, and
48% were in remission for at least 6 months. Patients were
classified by type of onset (first 3 months of disease) as
mono-, pauci-, poly- or anarticular; they were also classified as to final pattern of joint involvement. Of 37 patients
with monarticular onset, 21 (51%) developed polyarticular
disease. There was no significant difference between the
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
451
ABSTRACTS
sexes in respect to either type of onset or final classification.
However, of 23 females with monarticular onset 7 remained
monarticular, whereas, none of the 14 males with monarticular onset remained monarticular (P<O.Ol). This difference was not a reflection of disease duration. Four of 31
males and only 2 of 69 females had died (P<0.10).All
deaths were related to the disease.
There was no significant difference in remission based on
either sex or type of onset. However, patients with a final
classification of monarticular or pauciarticular arthritis had
a higher incidence of remission than those with polyarticular disease (P<0.01). Furthermore, those patients with
monarticular onset who developed polyarticular disease had
a similar incidence of remission as those who were polyarticular from onset.
C 3 was depressed (<I00 mg%) in only 4 of 91 patients.
The mean C 3 was not affected by sex, number of active
joints, activity of disease or duration of remission. There
was a significantly higher incidence of antinuclear antibodies (ANA) in undiluted sera in females (53 of 65) than in
males (15 of 27) (P<O.OI). Of 20 patients with ANA titers
> 1 : 16 only 1 was male (P<0.05).Neither presence or titer
of ANA correlated with disease activity.
T h e data suggest that males tend to develop polyarticular
disease even if their onset is monarticular, and that patients
with polyarticular disease have a lower incidence of remission. On the other hand, ANA were more common in females, and neither ANA nor serum C3 levels could he correlated with disease activity.
Role of Increased Vascular Permeability in Am yloid Formation
RVSSELL T. SCHULTZ,
T h e Veterans Administration Hospital, Oklahoma City, Oklahoma
Deposition of light chain fragments of immunoglobulins
in tissues or immunologic tolerance as mechanisms for
amyloid formation do not provide a satisfactory explanation
for the different clinical types of amyloidosis, or the particular organ localization of amyloid lesions. T h e possible
role of increased vascular permeability in amyloid formation was evaluated in the following experiments.
Amyloidosis was induced in C57 bl/lOJ mice by either
daily of 5 day/week injections of casein. Vascular permeability was assessed by injecting the animals intravenously
with radiolabeled proteins (albumin, IgG, fibrinogen).
sacrificiny the animals within 1 to 30 minutes following injection and then determining, by autoradiography, the degree to which the labeled proteins had entered the amyloid
lesions. Strikingly increased vascular permeability was
found in relation to all amyloid lesions, and appeared tc
increase progressively with the age of the lesion. Definite
antibody formation to casein confined to the y, subclass
of immunoglobulin could be demonstrated by radioimmunoelectrophoresis in all groups of animals, even those with very
advanced lesions. Increased vascular permeability in the
absence of lesions. Both circulating and insolubilized plasma proteins, as shown in previous studies, would appear
to he an integral part of the amyloid lesion as seen by light
microscopy. Complete immunologic tolerance to casein is
not present in these experimental models. Ample opportunity for the deposition and degradation of plasma proteins
exists in amyloid lesions, and this observation may account
for the finding of light chain fragments in some amyloid fibril preparations.
Arthropathy in Sickle Cell Disease
H . RALPH SCHUMACHER,
Hospital of the University O f Pennsylvania, RONALD ANDREWS and
Philadelphia, Pennsylvania
Joints may be involved in sickle cell disease by aseptic
necrosis, septic arthritis, gout. osteomyelitis and possibly
hemarthrosis. Children can get the hand-foot syndrome.
Occasional reports allude to other unexplained joint effusions, but none of these effusions have been studied. This
report describes 5 patients who presented with effusions
(with varying amounts of pain and warmth) associated with
sickle cell disease. Septic arthritis or unrelated rheumatic
disease was initially considered in all. All patients had SS
disease. Ages ranged from 6 to 23. Two had had previous
hand-foot syndrome. One had a previous septic joint and 4
had a past-documented bone infarcts. Knees were involved
in 4 patients and an elbow in I . Four attacks were coincident with other evidences of painful crises, but in only I case
452
GEORGE MCLAUGHLIN,
was there adjacent bone tenderness. All patients had fever
of lOOto 101".
Synovial effusions were yellow-yellow brown, clearslightly cloudy, viscous, 2 to 30 ml in volume, with 100 to
800 WBC with predominantly mononuclear cells. There
were no crystals and all cultures were negative. There were
sickled RBC in I effusion. Arthroscopy in the 1 patient with
a previous septic joint showed only rouqhening of the cartilage.
Needle synovial biopsies were performed in 4 knees.
These showed slight lining cell proliferation, rare inflammatory cells and, in 3 patients, obliterated small synovial
vessels. lntraluminal RBC in an epon-embedded section
were sickled. By electron microscopy in 2 patients, occa-
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
sional vessel lumens were packed with necrotic cells, platelets and RBC. T h e RBC in 1 patient were shown to contain
acid phosphatase in vacuoles. There was thickening of vascular basement membrane and perivascular fibrosis. Dense
material consistent with hemoglobin was seen in some
lumens and endothelial pinocytic vesicles.
Most effusions lasted from 4 to 7 days. The patient with
previous infection had persistent effusion over 1 year. No
treatment was required other than rest and aspiration. Patients received analgesics, oxygen and intravenous injections
for the associated crises. No joint x-ray changes developed;
3 patients showed periosteal elevation in adjacent bones
that may represent cortical infarcts, but we have so far
documented no adjacent medullary infarcts. Synovial or
periarticular thrombosis may be the cause of these noninflammatory synovial effusions.
Phagocytosis of Calcium Pyrophorphat. Crystah by Polymorphonucle~rLeukocytes:
Sequentla1 Electron Microscopic Observations
H . RALPH SCHUMACHER,
Hospital of the University of Pennsylvania and PAULDINC PHELPS, Philadelphia, Pennsylvania
Phagocytosis of monosodium urate crystals by polymorphonuclear (PMN) leukocytes in vitro results in electron
microscopic evidence of P M N leukocytes degranulation,
phagolysosome membrane dissolution and cell death. This
report describes a study of the sequence of changes following mixing of calcium pyrophosphate dihydrate crystals
(CPP) and PMN leukocytes. Since synovial fluid cells in
pseudogout have easily demonstrable phagosomes, while
those in gout often do not, it has been suggested that C P P
may not produce the same damage after phagocytosis.
Synthetic C P P crystals were added to human leukocytes
separated from heparinized blood by dextran sedimentation. Aliquots were fixed in one-half strength Karnovsky's
paraformaldehydeglutaraldehyde and osmium tetroxide at
intervals of 3 , 8 , 3 0 and 120 minutes. All crystals were
typical C P P by compensated polarized light and x-ray diffraction. Election microscopy showed that, crystals were
clumps of 400 to 600A rods with a central hole from which
some of the rods may have been dislodged during sectioning; others were lucent with dense borders as with urates,
and the remainder were uniformly dense or foamy as seen
with C P P of human origin.
At 3 minutes, 2% of cells had phagocytized crystals. All
crystals (or residual holes) were in intact phagosomes. By 8
minutes, only 5% of leukocytes had phagocytized crystals
(compared to 15% with urates). Dense bodies and granular
material from dense bodies had emptied into the phagosomes, some of which were distended. Occasional phagosoma1 membranes in this and subsequent specimens were
damaged by the mechanical dislodging of the crystals.
Phagosomal membranes around intact crystals were generally intact. By 30 minutes, 12%ofcells contained crystals.
At 2 hours, 22% of the P M N leukocytes contained crystals
(compared to 50% with urates). Crystals were in large and
small phagosomes with still few membrane defects; only
10% of the P M N leukocytes were necrotic. Controls not
exposed to crystals had 8% dead cells. Thirty-three percent
of cells had been necrotic after 2 hours incubation with
urates. Considering only cells with identifiable crystals,
23%were necrotic after CPP, compared to 71 70after urate.
These results show less avid phagocytosis of these C P P
crystals despite using larger doses of crystals than with
urates. After phagocytosis, dense bodies emptied into
phagosomes, but there were only scattered phagosomal
membrane defects, and the dramatic sequence of increasing
cell necrosis that was seen with urates was not seen with
CPP.
Milgrbm-Type Antiantibody in Matched Serum and Pleural or Synovial Fluids
CHARLES w. SEWARD,
Washington University School of Medicine, LEWIS P. PARKER and c. KIRK OSTERLAND,
St. Louis, Missouri
It has been reported that pleural and synovial fluids from
nonrheumatoid patients contain rheumatoid factor (RF)
with sufficient frequency to cast doubt on the diagnostic
usefulness of such serology. Rheumatoid factor serology was
reexamined to determine the nature of these reported reactivities. Matched serum and fluid samples from 65 patients
with a variety of diseases were R F titered in four standard
systems: latex particles sensitized with human y-globulin
(Hu-latex) or bovine 7-globulin (Bov-latex), sensitized
sheep cell agglutination (SSC) and human O+ red cells
coated with incomplete anti-Rh antibody (SDC). Significant titers in nonrheumatoid fluids were as follows: 0/31
synovial positives in any of the four systems; 2/24 pleural
fluids by Hu-latex, 0/24 Bov-latex and SSC, but 5/24
pleural fluids by SDC. These coincided with the identification of significant, often remarkable, SDC titers of matched
sera in 1 of 31 synovial and 10 of 24 pleural fluid patients.
Further study of these SDC agglutinating substances in
nonrheumatoid fluids and sera showed them to be resistant
to heat inactivation (56" C, 2 hours), and not inhibited by
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
453
ABSTRACTS
pooled Frll nor aggregated human y-globulin, a pattern of
reactivity characteristic ofMilgrom-type antiantibody, This
was distinctly different from results obtained in rheumatoid
sera and fluids which showed the highest concurrent titers
in Hu-latex and S D C (10 of lo), with the S D C reactivities
inhibited by both heatinq and addition of the y-globulin
that many
to the test system. It seems,
RF-positive, nonrheumatoid effusions and sera on discriminatory testing utilizing the SDC and inhibition technics c o n t a i n identifiable RF-like reactivity distinctly
separable from classic RF.
’IJbstances
Double-Blind, Cross-Over Study of lntraarterial Reserpine and Saline in Scleroderma
ROBERT
c. SIECEL,Stanford University School of Medicine, and JAMES F.
Recent uncontrolled studies demonstrate that intraarterial reserpine is clinically effective for either primary or
secondary Raynaud’s phenomenon and associated digital
ulcers. Since oral reserpine increases capillary blood flow
and intraarterial reserpine alters the pattern of digital heat
loss in Raynaud’s toward normal, clinical benefit from reserpine is thought to be due, to increased capillary blood
flow. However, reported rlinical responses of up to 7
months from a single intraarterial reserpine injection are
longer than might be expected from pharmacologic properties of this drug.
T o test whether the clinical benefits were solely due to
the documented physioloTic effects of reserpine on digital
blood flow, a double-blind, cross-over study comparing the
effect of intraarterial saline and reserpine on the frequency
of Raynaud’s phenomenon and healing of digital ulcers was
carried out. Sixteen patients (12 female, 4 male, average age
42.5) with the diagnoses of progressive systemic sclerosis
(12) or mixed connective tissue disease (4) were injerted in
the most severely affected arm with intraarterial saline or
1.0 mg reserpine randomly assigned. Indications for treat-
FRIES, Stanford, California
ment were severe, painful Raynaud’s phenomenon or
failure of digital ulcers to show signs of healing after 2 to 3
months of conservative therapy. Subsequent injections were
administered at an average of 8-week intervals dependinq
on symptoms. A total of 50 treatment courses were given
with positive responses obtained at some point in 10 of 16
patients. T h e initial randomized injection resulted in often
dramatic clinical improvement in 5 of 9 patients who received placebo and 2 of 7 with reserpine. Effects were generally noted in both the injected and noninjected arms. Responses similar in quality and duration were observed with
both treatments. Duration of response varied from 1 week
to over 7 months in 1 patient who received placebo. T h e
average duration of response was 6 to 8 weeks. Side effects
of dizziness, headaches and nausea were observed in both
groups with similar frequency. Local pain at the injection
site occurred in 2 patients after reserpine injection. Twelve
of 26 treatment courses with saline and I 1 of 24 with reserpine resulted in favorable responses. Patient expectation
and variable natural history of disease both appear to be
important factors in clinical responses previously attributed
to intraarterial reserpine alone.
Demonstration of Microtubular Inclusions in Cell Cultures of Systemic lupus
Erythematosus (SLE) Skin
H . ARTHUR SILVERMAN, University
of Tennessee College of Medicine, ROBERT w. C H A N D L E R , NANCY N I E L A N D and
HASHIMOTO, Memphis. Tennessee
Inclusion bodies consisting of microtubules resembling
the nucleocapsids of paramyxoviruses have been described
in various tissues in SLE. In a n attempt to grow cells containing such inclusions, skin punch biopsies of 4 patients
with SLE were cultured. Cells taken from partial monolayers at 7 to 9 weeks were fixed and embedded for electron
microscopy. Of the 4 specimens, 2 showed inclusions in both
the tissue and about 90%of cultured cells; 1 was positive in
tissue and showed an occasional positive rultured cell, and
the fourth was negative in both tissue and cultured cells.
T h e frequency of inclusions in positive and actively multiplying cultures did not diminish with multiple subpassages.
Control biopsies of patients with dermatomyositis, rheumatic heart disease and primary amyloidosis were negative
in both tissue and cultured cells.
454
KEN
T h e inclusions in cell culture were similar to those in tissues, except that they were found free in the cytoplasm
rather than associated with the endoplasmic reticulum,
and apparent buds of cell membranes containing inclusions
were noted more frequently. T h e inclusion-positive, cultured cells were fibroblasts and appeared healthy. One inclusion-positive culture, whose growth rate markedly
diminished at 3 to 35 months, is still being maintained
and shows inclusions after 12 months in rulture. T h e other
two positive cultures continue to contain inclusions at 5 and
6 months, and the negative ones have remained so for 6 to 8
months. In addition, light microscopic studies at 7 to 9
weeks revealed that approximately 30% of cells grown on
coverslips contained 0.5 to 1 .O micra-sized, intracytoplasmic
inclusions which stained blue-purple with hematoxylin-
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
eosin and positive with the methylgreen-pyronine method
for ribonucleic acid.
T h e fint demonstration of the propogation of inclusions
within SLE fibroblasts in cell culture suggests that they re-
sult from either an intrinsic cellular defect o r an infectious
mechanism rather than a circulating factor. T h e in vitro
method offers the possibility of isolation and more definitive
laboratory analyses of these inclusions.
Transynovial Molecular Exchange in Arrhriris
PETER
A. SIMKIN,
University of Washington School of Medicine, Seattle, Washington
The local warmth of arthriticjoints and the higher protein concentrations of their synovial fluid are thought to reflect increases in vascularity and capillary permeability of
inflamed synovial membranes. Little is known, however,
about the pathophysiology of these small vessel changes.
This study examines the effects of inflammatory disease on
the exchange of small molecules between synovial fluid and
blood.
Transynovial exchange of tritiated water (THO), urea,
urate, glurose and total protein was studied in 26 normal
volunteers, 15 patients with definite or classic rheumatoid
arthritis (RA), and 19 patients (non-RA) with a variety of
other joint diseases (gout, DJD, psoriasis, etc). T h e knee of
each subject was irrigated until returns were clear and then
injected with 30 ml of saline, including 2 pCi of T H O .
No. of
patients
Normal
RA
Non-RA
26
15
19
THO
Periodic samples were taken over the next 35 minutes, and
constants for the rate of equilibration between blood and
synovial fluid of each solute were calculated from plots of
solute concentration against time. Constants for the three
groups (percent equilibrated/min f SE) are listed below.
Analysis of these data shows. a ) Transynovial exrhange
of small molecules is not altered in RA despite a marked
increase in protein exchange; b) Exchange rates for all molecules increase significantly in non-RA patients; c ) Rates
of glucose equilibration are significantly impaired in RA
patients (P< 0.01); and d ) There is a highly signifirant
difference (P< 0.005 for T H O , urate and glucose) between
RA and non-RA patient groups.
These findings provide evidence of significant differences
in the microvascular system of inflamed synovial membranes.
Urea
3.6 * 0.3
3.6 A 0.4
6.4~ 0 . 4 5.0 A 0.5
4.9 * 0.2
4.7 A 0.4
Urate
Glucose
Protein
* 0.2
2.1 A 0.2
3.2 i 0.3
3.0+ 0.2
1.9 * 0.4
3.5 + 0.4
0.04 + 0.00
0.13 + 0.01
0.15 i 0.02
2.4
The Disrriburion of HL-A Antigens in Black Patients wirh Systemic Lupus
Eryrhematosus (SLE)
PETER STASTNY,
T h e University of Texas Southwestern Medical School. Dallas, Texas
Two previous studies (Waters el al, 1971; Grumet et al,
1971) have sqgested that an association may exist between
HL-A antigens and predisposition to develop SLE. In the
present work the distribution of HL-A antigens in BlackAmerican patients with SLE has been examined. T o determine the effect of the ethnic background, HL-A antigens
were determined in 60 Black-American patients and 100
patients of European descent attending the outpatient clinic. They were typed for 22 HL-A antigens using a lymphoryte microcytotoxirity technic. Gene frequencies were calculated from the phenotypes by the gene counting method of
maximum likelihood and genetic distances (f values) were
calrulated as desrrihed by Cavalli-Sforza. Significant differences were observed between Caucasians and Blacks for
the first (P< 0.0005) and the second segregant series
(P< 0.025). The overall genetic distance (f value) was
0.037.
Forty blark patients with SLE and 40 matrhed controls
were then similarly tested. In addition, for ronfirmation.
typing of 25 patients with S L E was also done by platelet
complement fixation. Two antigens were found more frequently in the group with SLE: antigen W19 of the first
series defined by serum TH-Li was positive in 21 patients
with SLE and only in 3 controls; in the second series HL-A
5 was twice as frequent in patients with SLE. T h e overall
difference for the first segregant series (taking into account 8
antigens tested for) was highly significant (P< 0.005). T h e
overall difference for the second series (14 antigens) was not
significant. T h e genetic distance (f value) between black
patients with S L E and black controls was 0.032. These results again demonstrate a correlation between transplantation antigens and predisposition for development of SLE.
T h e antigens increased in this group of black patients,
however, were different from those previously found in
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
455
ABSTRACTS
factors that appear to play a role in the development of this
disease.
other populations. The observations on these genetic
markers in SLE may offer new tools to evaluate the genetir
Controlled Trial of Azathioprine, Cyclophosphamideor Placebo in Lupus Nephritis
ALFRED D. STEINBERG, T h e
National Institutes of Health, KENT w. SALISBURY, NORTIN M. HADLER, GEORGE H .
and JOHN L. DECKER, Bethesda, Maryland
Twenty-five patients (23 females and 2 males) with systemic lupus erythematosus, diffuse glomerulonephritis on
biopsy and clinically active renal disease were hospitalized
for a 10-week, double-blind therapeutic study. Patients
randomly received azathioprine, cyrlophosphamide or
placebo starting at a dose of 3.0 mg/kg/day orally. T h e
dose was adjusted on the basis of white blood cell count; it
could he lowered or raised to 4.0 mg/kg/day. Concurrent
corticosteroid therapy up to 30 mg/day prcdnisone was
permitted for control of extrarenal symptoms. T h i s
averaged 17.9 mg/day for patients treated with azathioprine. 19.7 for cyclophosphamide and 23.4 for placebo, and
was held steady. T h e table lists the number of patients
showing improvement (+) or deterioration (-) in each
measure for each treatment during the 10 weeks. Net
change indicates number imporved (+) -number worsened
Azathioprine
(N = 6)
MYERS, JR
(-)/total measures.
Cyclophosphamide (average dose: 1.66 mg/kg/day)
preferentially reduced lymphocytes, whereas, azathioprine
(average dose: 1.93 mg/kg/day) preferentially reduced
neutrophils. Both (cyclophosphamide > azathioprine) reduced IgM more than IgC or IgA. During the 10 weeks. 2
patients treated with placebo were withdrawn because of
deterioration and 1 azathioprine for drug allergy. T o date,
4 patients have died: 1 patient treated with cyclophosphamide of pneumorystis carinii, 2 placebo of intracranial
hemorrhaqe during renal deterioration and I placebo of
pneumonia during subsequent azathioprine treatment. We
conclude that treatment with both azathioprine and cyclophosphamide were superior to placebo during the 10week study. Long-term data is not yet sufirient for a choice
between treat ments.
Cyclophospharnide
(N = 8)
Measure
+ -
+
-
Creat i nine clearance
Urine sediment
Proteinuria
Serum complement
Anti-DNA antibodies
Extrarenal disease
3
2
1
1
4
0
3
6
2
3
4
0
1
0
1
0
0
0
Net change
+ 5/36
2
2
0
1
0
1
+ 16/48
Placebo
(N = 4)
+
-
4
3
0
2
2
0
3
4
5
1
0
6
4/66
RNA- DNA Hybridization Studies in Chronic Porcine Erysipelothrix Arthritis
CHARLES R. STEINMAN, Mount
Sinai School of Medicine,
KONRAD
T h e arthritis induced in pigs by Eryszpelothnx zntidzosa
has many similarities to human rheumatoid arthritis. One
of these is the inability to consistently isolate an organism
from synovium during the later stages of active disease, even
though, in the animal model, the causative agent is known.
Since inability to culture the organism does not exclude
its presence, a means of identifying it independent of cultural or immunologic technics seemed desirable. T h e
method used here is based on an adaptation of the wellknown technic of RNA-DNA hybridization. Tritium-
456
mu and
CHARLES L. C H R I S T I A N ,New
York, New York
labeled RNA specific for E insidiosa is prepared in vitro
using RNA polymerase and E insidiosa DNA as template.
DNA is extracted from diseased synovium and then
examined for the presence of E insidiosa-specific base sequences by its ability to hybridize with E inszdiora RNA.
By use of appropriate controls and model experiments the
specificity of the method can be demonstrated and its sensitivity determined. Under conditions which can detect lo-’
pg E insidiosa DNA in the presence of 2 0 p g porcine
DNA, no specific hybridization is detectable in the synovial
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
preparations. Further, initial results obtained under conditions which can detect IO-'to
wg E insidiosa DNA per
20% porcine DNA also appear to show no specific hybridization. This latter sensitivity corresponds roughly to
one organism per 100 to 1000 porcine cells, and is sufficient
to distinguish between D N A preparations from mouse tissues acutely infected with E znszdzosa from those infected
with Listeria monocytogenes. When intact organisms are
added to tissue in these amounts the DNA prepared from
them shows about the expected hybridizability.
It is therefore concluded that in the diseased synovium
there are no more E insidiosa organisms that can be currently detected by this method. This is consistent with results obtained by cultural methods, and has pathogenetic
implications. T h e nucleic acid hybridization approach has
potentially broad applicability to problems related to persistent infection.
Response of Synovial Fluid and Peripheral Blood Lymphocytes to In Vitro Stimulation
JOAN A. STRATTON,
University of California at Los Angeles, and JAMES 8 . PETER, Los Angeles, California
Previous work suggests that peripheral blood lymphocytes from patients with rheumatoid arthritis have altered
reactivity. T h e purpose of this study was to compare the in
vitro responses of peripheral blood and synovial fluid lymphocytes of patients with rheumatoid arthritis. Simultaneous samples of peripheral blood and synovial fluid were
drawn from the patient. T h e peripheral blood lymphocytes
were purified on a Hypaque-ficoll density gradient and
synovial fluid lymphocytes by passage through a nylon wool
column. Both preparations were thoroughly washed. In
other experiments the response of peripheral blood lymphocytes passed through nylon wool columns was not
grossly altered. T h e purified, 95% viable (trypan blue exclusion) lymphocytes were cultured in the absence and
presence of optimum concentrations of mitogen (phytohemagglutinin-P, concanavalin A and pokeweed mitogen).
After 3 days the supernatants were tested for cytotoxic
activity (5'Cr release from L cells) and the lymphocytes
for proliferation ()H thymidine incorporation).
All of the patients examined showed normal proliferation
and good cytotoxin production of peripheral blood lym-
phocytes in response to the three mitogens. None of the
synovial fluid lymphocytes examined demonstrated any
proliferation in response to mitogen stimulation. However,
cytotoxin production, although less than that of the corresponding peripheral blood lymphocytes, was invariably
present.
Incubation of peripheral blood lymphocytes in autologous synovial fluid for 1 hour failed to diminish either the
proliferation o r cytotoxin production. Addition of
peripheral blood lymphocytes to synovial fluid lymphorytes, and vice versa, neither augmented nor decreased the
response to mitogen stimulation.
These results demonstrate that although the peripheral
blood lymphocytes of patients with rheumatoid arthritis
respond to stimulation with mitogens in vitro, the response
of synovial fluid lymphocytes is deficient. T h e synovial fluid
lymphocytes could already be maximally stimulated or
perhaps the receptor sites are covered. It is of interest that
despite the lack of proliferation the synovial fluid lymphocytes are capable of producing cytotoxin, a fact which again
emphasizes that cytotoxin production is not absolutely dependent on mitogen-enhanced proliferation.
Intermittent Arthritis Following Rubella Vaccination- Two- Year Followup
GEORGE R . THOMPSON,
Wayne County General Hospital, Eloise, Michigan, JOSEPH J . WEISS, JOAN L.
BRACKETT, Eloise and Detroit, Michigan
We previously reported the occurrence of acute arthritis
in 40 children following vaccination with HPV77-DK/I 2
rubella vaccine. T o determine if any sequelae had occurred,
we conducted a 2-year, follow-up examination. Thirty-nine
of the original 40 children were studied.
Eighteen of the 39 children (46%) had intermittent arthritis or arthralgias. Definite recurrent attacks of arthritis
were present in 1 1 children (28%), and possible recurrences
in 7 (18%). T h e knee joint was most commonly involved (14
of I8 or 78%). T w o children had recurrent wrist involvement; 1 ankle involvement and 1 polyarticular. There was
slight female predominance (11 of 18). In 16 children the
recurrence was in a joint involved in the original attack. No
SHILLIS and ROBERT G .
relation to antecedent infections. trauma, etc was present,
nor was there any periodicity of attacks; weeks or months
intervened between exacerbations. Characteristically, the
child would awaken with inability to use the involved joint
because of pain and stiffness. ,Joint swelling was uncommon. This picture would last from a few hours to 5 days
with gradual clearing. There was no permanent joint
damage or impairment following a recurrence.
Complete blood count and tests for rheumatoid factor, Creactive protein and total hemolytic complement were
normal; serum protein electrophoresis and immunoglobulins were unremarkable. Two children had positive antinuclear antibody tests (speckled pattern); neither child had
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
457
ABSTRACTS
recurrent arthritis. Sedimentation rate was elevated in 8
patients, 5 with attacks. The geometric mean rubella hemagglutination-inhibition titer for the entire group was
1:343. For those with definite recurrence, mean titer was
1294; possible recurrence: 1 :313; no arthritis: 1:383.
These differences are not statistically significant. Antibody
titers for rubeola, polio Types I, I1 and 111 were similar in
all groups, and in the expected range.
This study indicates that the arthritis occurring in some
children following rubella vaccination may be recurrent for
over 2 years. However, the attacks are of brief duration and
clear without residual impairment. The etiology of these
attacks is not established. In view of their favorable outcome, conservative therapy is recommended.
Induction of Polymorphonuclear leukocyte Chemotaxisb y Gammaglobulin, Rheumatoid
Factor and Their Complexes
TERESA WAGNER,
University of Rochester School of Medicine and Dentistry, GEORGE ABRAHAM and JOHN BAUM, Rochester,
New York
In this study we are demonstrating the roles of IgG (native and aggregated), rheumatoid factor (IgM) and the
complexes produced by their interaction on the induction of
chemotactic factor(s) from human complement.
Cohn fraction I1 (DIFCO) is cleared of aggregates by
high-speed centrifugation. After aggregation of IgG by
heating, reactive aggregates are prepared by the sodium
sulfate method of Christian. The rheumatoid factor used is
sephadex G-200 column purified monoclonal IgM. Human
complement from 1 individual is used in virtually all the
studies. Polymorphonuclear leukocytes are concentrated
from the blood of normal volunteers. Chemotaxis is
measured as the chemotactic index using our modification
of the Boyden method.
In experiments using complement plus IgG in concentrations ranging from 0.2 to 20 mg/ml, no consistent relationship with these concentrations is found in the values of
the chemotactic index. The average chemotactic index is not
significantly different with native (563.6) or aggregated
(564.9) IgG. At all concentrations there is significantly less
chemotaxis when complement is not present, (native IgG,
70.7; aggregated IgG, 73.6). Rheumatoid factor (IgM) plus
complement is moderately active with a chemotactic index
of 273.6. However, the complex (aggregated I@ + RF) is
inhibitory (compared to IgG alone) with a chemotactic indexof 181.3.
These results indicate that complex formation (IgG +
RF) is not necessary for the induction of chemotactic factor(s) which bring polymorphonuclear leukocytes into a
joint as part of the inflammatory reaction. This study confirms earlier work by others of competition by rheumatoid
factor for the site where complement fixation occurs on aggregated or altered IgG. This is shown by the decrease in
activation of chemotactic factors by the complex. However,
the ability of the separate components (I@ and IgMrheumatoid factor) to activate chemotactic factor(s) from
complement appears to more than make up for the inhibitory effect of the IgG-rheumatoid factor complex. Thus, the
major mechanism for activation of chemotactic factor(s) is
the same in both factor-positive and factor-negative rheumatoid arthritis.
Studies on the Infectious Etiology of Human Rheumatoid Arthritis
JOHN R. WARD,
University of Utah College of Medicine, BARRY c. COLE and CHARLES B.
The present studies were undertaken to examine recent
claims that: a) Mycoplama fermentans is associated with
rheumatoid arthritis (Lancet 2:277, 1970) and b) an agent
in rheumatoid synovium can be transferred to fetal mice and
chicken embryos resulting in joint lesions upon birth of the
animals (Nature 223:646,1969; Arch Intern Med).
In a blinded design, 15 rheumatoid and 7 nonrheumatoid
synovial tissue specimens removed at surgery were collected
in sterile tissue culture media. Homogenates of these tissues
were transferred to a variety of mycoplasma and T-strain
media, and also to blood and thioglycollate media. Fractions
prepared by sucrose density gradient centrifugation of the
suspensions were also transferred to mycoplasma media.
Aliquots of each suspension were injected intraperitonealy
into 5 pregnant mice and subcutaneously into 15 10-day-old
458
SMITH,
Salt Lake City, Utah
chicken embryos. Newly born animals were examined for
joint lesions.
All tissues examined failed to yield growth of mycoplasmas. Density gradient centrifugations failed to produce additional isolates. Bacteriologic cultures produced diphtheroids from 2 nonrheumatoid specimens only. In newborn
mice, lesions consisting of erythematous areas on one or
more digits and occasional transient joint swellings were
observed by examination with a dissecting microscope.
These were observed in 13% of animals, and no difference
in severity or frequency was detected between groups injected with rheumatoid and nonrheumatoid tissues. In
newly hatched chicks, occasional paralyses of legs, curly
toes, red feet and stiff joints were observed. However, no
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
difference in severity or frequency of lesions was evident
between embryos injected with saline, rheumatoid and
nonrheumatoid synovial tissue. Thus, the present study fails
to confirm the association of mycoplasmas with rheumatoid
tissues, and provides no evidence for a transmissible aqent
to newborn mice or chicken embryos.
Antibodies to Double-Stranded RNA (DSRNA) in Human Sera: Detection, Incidence and
Relation to Disease Activity in Rheumatic Diseases
ROBERT D. W A R R E N ,
University of Missouri Medical Center, PATRICK H.HENRY,
Columbia, Missouri
The Farr (NH4),S04 technic is a sensitive method to
determine serum antibodies to DSRNA or native DNA
(DSDNA). Addition of (NH4),S04 to a mixture of radioactive DSRNA in the form of ''C-polyinosinicpolycytidylic acid (Poly 1:C) and serum yields a precipitate
which contains radioactivity if the nucleic acid is bound by
antibody. In this study we determined the incidence of antibodies to DSRNA in rheumatic diseases and assessed the
test's usefulness in monitoring disease activity. Optimal
conditions were determined by various concentrations of
Poly I:C, antibody (serum), percent saturation (NH4),S 0 4 , length of incubation time at 37O and molarity of buffer. T h e final experimental conditions were 10 pliter
serum, 0.34 fig Poly 1:C (sp act = 2800 dpm/ pg), 80 pl of
.05M Tris buffer (pH 7.8) and incubation for 2 hours at 370
followed by a 20 to 24 hour incubation at 4". T h e antigen:
antibody complex was precipitated with 70% (NH4),S04
(final conc = 35%) and the precipitate was washed with
3.5% (NH4)2S04 and dissolved for liquid scintillation
counting. T h e amount of input Poly I:C recovered i n the
precipitate was expressed as the percent radioactive binding. The range of binding in sera of 35 normal controls was
WILLIAM
s. I R V I N and CORDON
C. S H A R P ,
0 to 6.570, with a mean of 2.3% (SD = f 1.5). Values > 3
SD (6.8% binding) from the mean were designated as abnormal. Sera from 25 patients with active systemic lupus
erythematosus (SLE) showed a range of 1.8 to 20% with a
mean binding of 9.7% (SD +5.0); 79% showed > 6.8%
binding. Sixteen patients during clinical remission had a
binding of 0 to 8% (mean: 2.570,SD +2.6); 1 of those had
> 6.8% binding. All patients studied serially showed a decline in percent binding as disease activity subsided. Sera of
2 of 7 patients with scleroderma, 1 of 13 with rheumatoid
arthritis and 2 of 8 with mixed connective tissue disease had
binding > 6.8%. That the antibodies to DSRNA are distinct from the antibodies to DSDNA was demonstrated by
studies employing 'H-KB cell DSDNA, Poly I:C and sera
with known to antibodies to both DSDNA and DSRNA.
There was no significant decrease in percent bindinq of
either nucleic acid when incubated together. Our modification of the DSRNA binding technic allowed us to demonstrate elevated percent binding in 79% of patients with active SLE. As disease activity decreased the percent binding
returned to within the normal range in 15 of 16 patients.
This test may be useful in the serial evaluation of patients
with SLE.
Nitroblue Tetrarolium (NBT) Dye Reduction b y Peripheral Leukocytes from Parienrs with
Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE)
MARLIN E. WENGER,
University of Michigan, and GILES C. BOLE, Ann Arbor, Michigan
Phagocytic activity measured by spontaneous in vitro
reduction of NBT using rheumatoid or SLE neutrophils
(PMN) has been reported to be normal. The effect of steroid
therapy on N R T dye reduction remains controversial. Re-
cent reports indicate that N B T dye reduction is increased in
bacterial infections.
Using the qualitative method of Park et ui, the followinq
results were obtained:
Mean
Group
Control
RA
SLE
Total untreated
> 10 mg prednisone
Infected (2)
< 10 mg prednisone
+
No. of
patients
14
19
12
4
7
Age
24-60
24-65
15-38
1
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
+ SE NBT positive P M N k u rnm
Percent
5.4 i 0.76
5.5 + 0.73
3.4 f 0.79
1.3 + 0.58
4.1 1.00
2.5
7.0
A bsol Ute
259 i 52
253 + 34
169 L 53
54 i 36
161 + 39
115
565
459
ABSTRACTS
In addition to P M N activity, a high percentage ( + 6 5 % )
of normal, rheumatoid and S L E peripheral monocytes reduced dye. T h e mean decrease in dye reduction observed in
patients with SLE was further examined using a spectrophotometric method. Patients with SLE on suppressive
doses of prednisone had a mean optical density (OD)/107
phagocytes ( P M N + monocytes) of 0.310 + 0.047,
whereas, 8 healthy adult controls had a mean OD of 0.533
f 0.068. Untreated patients had values of 0.281 to 0.325
OD units. One patient with SLE on 80 mg prednisone with
culture proven septic arthritis, had a low qualitative and
quantitative N B T test. Untreated, infected or steroidtreated patients with SLE had lower mean qualitative and
quantitative N B T dye reduction than control or patients
with RA, suggesting the presenceof a cellular defect in these
cases. In interpreting N B T dye studies, the reducing activity of peripheral blood P M N and monocytes must be simultaneously evaluated.
Partial Suppression of Autoimmune Disease of NZB/NZW Mice by Malarial Infection
JOHN
c. WIIELTON, T h e ,Johns Hopkins Hospital,
HAROLD D. E I C A I N G E R ,L A W R E N C E E. SHCLMAN
TOWNES.
and
ALEXANDER
s
Baltimore, Maryland
An unexpectedly low incidence of systemic lupus erythematosus (SLE) in Africans residing in Nigeria has been
reported. Malaria, endemic in that area, was postulated as
an environmental factor contributing to this low incidence.
Malarial infection in NZB/NZW mice (the animal model
of SLE) was reported to block the emergence of both antinuclear antibody and proteinuria. All infected mice survived 1 year, in contrast to the expected mortality of 75 to
100% (Greenwood et al, 1970).
The present study was undertaken to confirm these observations and elucidate the mechanism by which malarial
infection blocks the autoimmune disease. Twenty NZB/
NZW mice were infected with Plasmodium beryhei yeolii
at 1 month of age. Serial observations of antinuclear antibody, proteinuria, hematocrit and C:oombs’ test were compared with those in 20 control NZB/NZW mice. T h e mean
peak of parasitemia in infected mice was 15.8% infected
erythrocytes. The mean duration of parasitemia was 17
days. O u r data showed that the disease was significantly
delayed, but not blocked, by infection. Both onset of proteinuria and appearance of antinuclear antibody were delayed by an average of 2 months. Mortality was delayed by
an average of 3 months. At 12 months, Coombs’ antibody
had appeared in 10% of infected mice, in contrast to 30% in
noninfected controls; mortality was lower in infected mice
(50%),than in controls (75%).
Immunization with sheep erythrocytes (SRBC) before,
durinq and after malarial infection was performed in 3
groups of NZB/NZW mice. Sheep erythrocyte antibody
responses were compared with noninfected immunized
NZB/NZW and DBA mice. Both infected and noninfected
NZB/NZW mice developed higher SRBC antibody responses than DBA mice. There was no significant difference
between infected and noninfected NZB/NZW mice. These
data suggest that the suppression afforded by malarial infection is not mediated by nonspecific blockade of the humoral immune system. Other mechanisms are being explored.
Synovectomy of the Proximal lnterphalangeal Joint of the Finger in Rheumatoid Arthritis
ALAN H . WILDE, T h e
Cleveland Clinic Foundation, Cleveland, Ohio
Synovectomy of the proximal interphalangeal joint of the
finger in rheumatoid arthritis has been performed in an effort to relieve pain and prevent the Boutonniere deformity.
This operation has been performed on 121 joints in 39 patients. The longest followup is 59 months (average: 34.2).
Thirty-four patients had classic rheumatoid arthritis persisting into adulthood. There was a mean of 8.6 years duration of joint symptoms prior to surgery with a range of 2
to 37 years.
Recurrent synovitis of the proximal interphalangeal joint
occurred after synovectomy in 30.8%. There was an average
of 4.1 degrees lost in the proximal interphalangeal joint
postoperatively. After synovectomy, 78.7% of the patients
stated that they had no pain, 13.5% felt they were improved
460
and 7.5% felt their pain was the same as preoperatively.
None stated that they had more pain postoperatively than
preoperatively. In the follow-up period, radiographic joint
surface erosions healed in 17.6% of cases, there was no
change in 44.7% and erosions progressed in 37.6%.
Postoperatively, radiographic joint space narrowing increased in 2870, and there was no change in 7 1.1%.
Postoperative complications are as follows: there were no
new Boutonniere deformities; there was postoperative
wound sepsis in 3 cases, 3 suture granulomas, 1 neuroma, 3
hematomas and 3 with marginal wound necrosis.
Synovectomy of the proximal interphalangeal ,joint of the
finger in rheumatoid arthritis appears to be an effective
procedure for prevention of the Boutonniere deformity.
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
Diminished Response of SLE LymphocytesStimulated with Anti-lG Antisem
RALPH
c. WILLIAMS,JR. University of New Mexico School of Medicine, and OVE J. MELLBYE, Albuquerque, New Mexico
Lymphocytes from patients with connective tissue disorders are of growing interest, since they may be one of the
prime mediators of tissue damage in these diseases. As a
corollary to previous work from this laboratory on possible
differences of RA and SLE lymphocytes from those of
normal subjects, we studied lymphocyte. responses to stimulation with anti-+, A and M antisera in vitro. Correlations of lymphocyte stimulation were made with relative
percentages of peripheral blood B cells as defined by immunofluorescent staining. of surface lymphocyte immunoglobulin determinants. A group of 20 normals were compared with 12 patients with active RA and 10 with SLE.
No difference in lymphocyte stimulation with anti-+, A or
M antisera was noted when normals were compared to patients with active RA. In contrast, a significant degree of
hyporeactivity to stimulation with anti-Ig antisera was
noted among the lupus group. T h e markedly diminished
lymphocyte stimulation particularly with anti-+ and M
antisera in L E patients was significant at the P < 0.005
level.
Some correlation was noted in individual SLE lymphocytes between low percentages of peripheral blood B cells,
as defined by lymphocytes showing demonstrable surface
6,
A or M and the lack of normal stimulation by anti-Ig
antisera. However, no clearcut correlation was detectable
among patients with RA or normals between lymphocyte
surface immunoglobulins and degree of stimulation using
antiimmunoglobulin antisera.
The marked diminution of lymphocyte stimulation in
peripheral blood lymphocytes from patients with SLE infers that either the cells have already been turned on by
other mechanisms, or that basic cellular hyporesponsiveness
in this test system is a feature of the disease itself.
Studies of T and 8 Cells in Patients with Rheumatoid Arthritis (RA) and Systemic Lupus
RALPH c. WILLIAMS, JR, University of
New Mexico School of Medicine, JAMES R.
MEUBYE, Albuquerque, New Mexico
T h e immune system of lymphocytes may be divided into
two groups of cells: a) B cells: involved in antibody formation and showing surface immunoglobulin determinants,
and b) T cells: lacking detectable surface immunoglobulin
but committed to functions of cellular immunity. In this
study relative percentages of peripheral blood B lymphocytes were studied using direct immunofluorescence with
anti-+, A and M antibody purified by immunoabsorbent
columns. Rabbit antisera prepared against pooled human
thymocytes obtained from 20- to 30-week human abortions
were used as a source of T-cell specific antiserum. These
antisera were absorbed with cells from patients with chronic lymphatic leukemia (CLL) used as a presumptive source
of B cells showing 80 to 90% cells with surface immunoglobulins. Five serial absorptions of 1 ml of T-cell antisera
using 60 x lo6CLL cells produced antisera which after the
third, fourth and fifth absorptions still reacted with 60 to
80% of normal peripheral blood lymphocytes, 100% fetal
thymic cells and 90% of cells from a patient with adult acquired agammaglobulinemia. This absorbed T-cell an-
DEBORD, RONALD P. MESSNER
and OVE J.
tiserum was used to estimate percentages of peripheral
blood T cells by immunofluorescence.
Normal lymphocytes studied from 40 individual donors
showed slightly higher total percentages of B cells than
those of an equal number of rheumatoid sub,jects
(P< 0.01). Values for percentages of T cells in normals
ranged between 55 and 95%. T-cell percentages in 12 patients with RA ranged between 15 and 90%; however, no
essential difference in T-cell fluorescent pattern or percentage could be detected. In general, studies using a small
group (6 patients) with SLE showed the same range of Tcell percentages as did those of the normal control group.
This approach has not defined basic differences among
T-cell populations between patients with RA and SLE. Of
considerable interest, however, was the finding in several
patients with connective tissue disease of a discrepancy between the total percent of lymphocytes identified as T + B
76 to 77% and 10070, whereas the totals of T + B cells
among normals ranged from 85 to 103%.
Nasal Septa1 Perforation in NongrarnulomarousRheumatic Disease
ROBERT F. WILLKENS, Harborview Medical
Center, and JOSEPH
Atraumatic nasal septal perforations are frequent occurrences in patients with Wegner’s granulomatosis, but rarely
W. WALIKE, Seattle, Washington
seen in patients with other rheumatic disorders. We have
analyzed our experience with 9 patients with nasal septal
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
461
ABSTRACTS
perforation and rheumatic disease other than Wegners, and
found they have common abnormalities which might, in
part, account for the septal perforation.
A variety of disorders were represented in this group:
rheumatoid arthritis (RA), psoriatic arthritis (PsA), systemic lupus erythematosus (SLE) and scleroderma (PSS).
Common clinical manifestations included arthritis,
Raynaud’s phenomenon and epistaxis. Also noted were leg
ulcers, pulmonary abnormalities and peripheral neuritis.
High titer of rheumatoid factor were present in patients
with RA, and antinuclear factors were present in RA as
well as SLE and PSS. High serum levels of I& were
common.
Biopsies of the paraperforated mucosa and cartilage were
obtained from 6 patients, and control biopsies were ob-
tained from 2 patients with intact septa and SLE. Sections
showed chronic inflammatory nongranulomatous change
with minimal vasculitis. Fluorescent antigammaglobulin
staining failed to show immune complex deposition in vascular endothelium. Vascular endothelial proliferative
changes were suggestive of reaction to local ischemia.
Serum and blood viscosity were normal, in 2 of 3 patients
so evaluated, and distinctly elevated in 1 patient. This patient expired of a massive epistaxis, and was found to have
extensive amyloid deposition.
These patients illustrate that nasal septal perforation is
probably not a chance occurrence in patientswith rheumatic disease and may be related to ischemic vascular change
due to primary vascular involvement, increased blood viscosity and/or immune complex deposition along a poorly
phagocytable surface.
Assessment of Disease Activity in Systemic lupus Er ythematosus (SM)
JOHN B. WINFIELD,
University of Virginia School of Medicine, CAROLYN M. BRUNNER, JOHN s. DAVIS IV and WILLIAM M.
O’BRIEN, Charlottesville, Virginia
A comparison was made of laboratory data from groups
of outpatients with stable SLE classified independently on
clinical grounds alone as having active or inactive disease.
Systemic lupus erythematosus was considered active if one
of the following was present: CNS dysfunction, fever, arthritis or persistent arthralgia, typical rash, serositis, anemia or leukopenia. (Active nephritis was not a criterion for
classification in Group 1.) Active (Group 1) and inactive
(Group 2) groups were subclassified on the basis of a) past
history of biopsy-proven lupus nephritis, or b) its absence.
Laboratory data from a blood sample obtained at the time
of clinical examination included: complement (CHI,, nl =
34 to 48), ESR (Westergren), cryoglobulins, ANF, antiDNA by counter-immunoelectrophoresis using calf thymus
DNA and Farr Assay with “C-DNA from human KB cells
and Ouchterloney analysis of SLE sera against RF-IgM
(RF reactivity). For quantitative data, means and standard
deviations are given below in parentheses. Significance of
differences was determined by F test.
Group 1 differed significantly from Group 2, and Group
Group
No. of
patients
1
la
lb
2
2a
2b
20
10
10
30
16
14
462
l a from Group 2a for C’H,,, ESR and %-DNA binding
(P= .01). Group l a differed from Group 1b both for CH,,
(P = .0018) and I4C-DNA binding (P = ,0169). Significantly different I4C-DNA binding was not noted between
Groups 1b and 2b (P = .27). The only difference between
Groups 2a and 2b was in ESR (P= .01). Rheumatoid factor reactivity was positive in 1 of 20 Group 1 patients and in
10 of 30 Group 2 patients. Antinuclear fluorescence, quantity of cryoglobulins and anti-DNA, as determined by CIE,
were higher in patients with active SLE, and especially in
Group l a patients, but significance of differences could not
be determined for these semiquantitative data.
Conclusions were: a) Assessment of disease activity in
SLE by history and physical examination correlates closely
with abnormality of laboratory parameters, although for
individual patients there is considerable variability; b) High
“C-DNA binding and low CH5, may be sensitive indicators
of nephritis in active SLE; and c) Reactivity with RF occurs
more frequently in clinically inactive SLE.
CHw
24(11)
15 (6)
29 (9)
38(14)
37 (17)
40 (8)
ESR
86 (35)
88 (37)
80 (31)
47 (24)
52 (24)
32 (17)
DNA binding
No.
%
45
64
33
21
22
23
31
27
24
19
19
20
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
ABSTRACTS
Problems of Methotrexate Administration
PAUL YOUNG and TOM R A R D I N , J R ,
Methotrexate has been given to 129 patients with rheumatoid arthritis (RA) for 6 to 24 months. Liver, kidney and
bone marrow function were normal before treatment. A
priming dose of 3 times the maintenance dose of one tablet
(2.5 mg)/20 kg/wk was given for 3 weeks. Then the
maintenance dose was given once weekly. This is much
lower than the usual maintenanre dose of 10 to 20 tablets
per week used in psoriasis. Early in the study patients in
Group A who received methotrexate without folic acid had
a high incidence of toxicity. With repeated courses of
methotrexate the toxicity occurred earlier and was more
severe. Some patients in Group A were added to other patients who were shown below as Group B. These patients
received the same dose of methotrexate for 1 I weeks.
Then methotrexate was omitted for a week while tablets of
folic acid 5 mg t.i.d. were given for 7 days. In Group C, the
folic acid was given for a week before methotrexate. Then
methotrexate was given for 7 weeks and folic acid for 1
week. Comparison of the incidence of granulocytopenia ocrurrinq in Group A and Group B showed x 2 = 4.72
Asheville, North Carolina
(P< 0.05). Comparison of Group A and Group C showed
x2 = 125.3 (P < 0.0001). T h e less frequent elevation of
S G O T in Group C did not reach statistical significance
in these sample sizes.
T h e results of this study are consistent with the finding-s
of low serum folate by Dwyer, Kendall and Hawkins (Ann
Rheum Dis, Nov 1971) which they considered due to
malahsorption. This could be expected to predispose the
patient with RA to methotrexate toxicity. We found that
methotrexate is indeed a particularly toxic drug- for the patient with RA. This toxicity was reduced by pretreatment
and intermittent treatment with oral folic acid. Injections of
folic acid might have been more effective. Parenteral
methotrexate might result in less liver toxicity. Methotrexate should he given only under strict laboratory control.
As an additional precaution methotrexate and folic acid
should he dispensed and not prescribed. In light of our experience, it would seem wise to administer folic acid hefore
methotrexate and then after every 7 weeks of methotrexate
therapy.
Patient group
No. of
patients
Granulocytopenia
below 2000 (YO)
SGOT elevated
above 40 (%)
A
B
C
82
95
49
27 = 34
17 = 18
1=2
10 = 15
4 = 15
2=4
Sequential Needle Biopsy in the Evaluation of Known Rheumatoid Arthritis
P A I Y YOCNC., GEORGE LACY,
Iom MCCLOUD and TOM R A R D I N , JR, Asheville, North Carolina
Needle biopsies from four locations in each knee showed
less variability between different lorations than is usually
anticipated. It seemed that sequential biopsies might help to
evaluate the efticacy of different treatments given to groups
of patients, and help to resolve problems of management in
individual patients. Biopsies were taken before and after
intraarticular injections of nitrogen mustard (HN2) 1.0
mg/ml mixed with 0.4 mg/ml dexamethasone phosphate,
given 3 times 1 0 days apart. Biopsies taken 10 days after the
third injection showed less than half as murh inflammation
as before treatment biopsies in 29 of 40 patients. Biopsies
hefore and after intravenous injection of H N 2 in 8 patients,
intraarticular cyclophosphamide in I2 patients and intraarticular methotrexate in 24 patients showed no appreciable change.
In another group of patients, intraarticular methotrexate, given every 7 days for 3 injections into the ri,ghehl
knee only alternating with intraarticular HN2 into both
knees every 10 days for 3 injections, showed necrosis of the
synovium in both knees in 7 of 8 patients receiving three injections, 3 of 6 patients receiving two injections and 0 of 2
patients in whom toxicity limited therapy to one set of injections of each drug. Open biopsy of cartilage and capsule
in 6 knees showed no undesirable effects. Postinjection
therapy consisted of gold, oral cyclophosphamide and oral
methotrexate. Needle biopsy 6 to 12 months after intraarticular HN2 and methotrexate treatment in 4 patients
showed that in 3 the previously hyperplastic synovium had
been replaced with a flat layer of lining cells on dense hylanized connective tissue. Concurrent use of oral methotrexate and relatively low-dose oral cyclophosphamide (1 .O mg/
kg/day) produced definite improvement in biopsies and
clinical findings without striking toxicity in 68 patients
treated for 6 to 12 months. Photomicrographs of biopsies
before and after treatment in each of these groups will be
shown. Sequential needle biopsy was of unique help in the
Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972)
463
ABSTRACTS
management of individual patient’s problems. Four examples will be shown.
T h e procedure appears to offer a new dimension in drug
evaluation and patient management which warrants further
study. T h e purpose of this paper is to suggest a wider use of
sequential needle biopsy, and is not intended to advocate
any of the treatments mentioned, which at this time must be
considered as strictly investigational. They are mentioned
only to illustrate the benefits of evaluation by sequential
needle biopsy.
The Apparent Elimination of Chloroquine Retinopathy
PAUL YOUNG, TOM RARDIN, JR, BETTIE LANKFORD, AUGUSTA YOUNG, GEORGE WHITE
and JOHN
FRY,
Asheville, North
Carolina
T h e fear of chloroquine retinopathy, and particularly
delayed chloroquine retinopathy, has led many rheumatologists to abandon antimalarials. We constructed a Retinal
Threshold Tester ( R T T ) which rapidly and reliably
measures the function of the paramacular retina. A rarefully done visual field to 2 mm Red on the B&L Auto-Plot
Projection Tangent Screen (2RPTS) gives very similar information. If this is done prior to antimalarial therapy, the
2RPTS can apparently select patients in whom antimalarial therapy is reasonably safe and exclude those with a
high risk of retinal toxicity. There is a high incidence of
asymptomatic retinal malfunction in patients with RA,
which seems to predispose to chloroquine retinopathy. We
did R T T and 2RPTS tests on 146 patients with RA under
age 65 and free of all eye disease. Abnormalities were found
in 64 patients (44%). Several of these patients had definite
improvement of the 2RPTS visual field when the RA was
successfully treated with gold and/or cyclophosphamide.
Several examples will be shown. T h e retinal damage of
chloroquine retinopathy appears to be due to a combination
of the effect of RA on the retina plus antimalarial therapy.
Perhaps sequential 2RPTS tests should be done on patients
receiving all new drugs used in RA, as this disease appears
to predispose the retina to d r q toxicity
464
Our 1967 survey of 461 patients who had not been tested
before antimalarial therapy, showed asymptomatic scotomas to white in 10, scotomas to red in 16 and abnormal retinal function in 147; a total of 173 (38%). In our 1971
survey of 341 patients who had been tested prior to and
every 3 months during antimalarial therapy with the R T T
and the ZRPTS. we found 0 scotomas to white, 0 scotomas
to red and 68 (19%) with mild reversible retinal toxicity.
All were detected far short of the irreversible damage accompanying asymptomatic scotoma to white, which may
progress to retinal damage after antimalarial are stopped. It
appears to be this stage of toxicity which leads to the socalled delayed chloroquine retinopathy as described by
Burns (N Engl ,J Med, 1966). This sequence of events was
observed in 9 of the 10 patients in the 1967 survey with
asymptomatic scotoma to white. However, it has not occurred in a single 1 of our other 235 patients in whom antimalarial therapy has been stopped for early retinal toxicity.
We have had 0 scotomas to white, 0 retinal lesions and 0
symptoms of visual damage since 1967. During these 5
years we have treated an average of 409 patients each year
with antimalarial therapy.
Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972)
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