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Proceedings of the seventh interim scientific session of the american rheumatism association December 9 and 10 1960 Dallas Texas.

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AMERICAN RHEUMATISM ASSOCIATION
10 Columbus Circle, New York 19, New York
President: EDWARD
F. HARTUNG,
M.D., 580 PARK AVENUE, NEW YORK 21, NEW YORK.
First Vice President and President Elect: JOSEPH L. HOLLANDER,
M.D., 3400
4, PENNSYLVANIA. Second Vice President: HANS
WAINE,M . D . , 25 BENNET STREET, BOSTON 11, MASSACHUSETTS. 0 Secretary TreasI4rW: FELIX
E. DEUkRTINI, 622 WEST 1 6 8 STREET,
~ ~
NEW YOHK 32, NEW YORK
Executive Secretary: GERARDW . SPEYER,10 COLUMBUS CIRCLE, NEW YORK 19,
SPRUCE STREET, PHILADELPHIA
NEW YORK.
Proceedings of the Seventh Interim Scientific Session
of the American Rheumatism Association
December 9 and 10, 1960
Dallas, Texas
Abstracts of Papers Presented i t n d Read by Title
EDITOH:
INDUCTION OF A
u’ilhrl
s.
Clwk
RHEUMATOII) FACTOR-LIKE
SUBS” A N C E
IN RABBITS
John L. Ahriizzo ciwl Cli(ir1e.y 1,. Christicin, Kcw York, X . Y.
Previous studies have indicated that aniinals innoculatecl with killed bacteria may
develop a senim factor which, in some of
its properties, resembles the “rheumatoid
factor.” The apparent spccificity of such
factors for ganiiila globulin, in sonie studies,
may relate to its assimilation of gamma globulin by the bacteria used for innoculation
from either cultme media containing seruni
protein or from recent passage of the organisms through animals.
The present report concerns stndies in
rabbits that were injwted intravenously
over ii period of 6 months with a suspension
of formalin-killed E. coli. This organism h x l
been cultured in a niedinm that contained
only salts and gliicose. A high seriiin titer
of E. coli agglutinins developed and after a
period of several weeks a serum f.actor w a s
noted which had the following chariicteristics: (1) It was a heat stable (96 C.-30
inin.) gamma globulin which increased in
titer upon continued innoculation. ( 2 ) It
agglutinated FIT tanned sheep cells, FII
latex particles and sensitized sheep cells.
f Average FI1 tannccl S \ l ( q l cell titer was
1:640in 5 animals immunized for 6: months. )
( 3 ) It ;igglotin;itec\ tannrd sheep cells
“coated“ with ;,ggrc>giited human gamma
globulin but not 7s gamma globulin. ( 4 ) It
precipitatcd with aggregated hmnan gamma
globulin but not with 7s g;imma globulin.
( 5 ) It was absorbed by small amounts of E.
coli. ( 6 ) It was :ibsorbecl by ;I bovinc serum
albnmin-antibavine :~lbuniinprecipitate. ( 7 )
It scdinicntecl to the bottom of the tube
during density gradient centrifugation, suggesting that it is a heavy niatt.rial. (The E.
coli antibody conccntrated in the middle of
the tube. )
Thest. data support the hypothesis that
sustained antigenic stimdation may result
in the production of an antibody-like fxctor
whosc specificit,v i s directcd against g;unma
globulin in ;in iiiiiiiunr aggrcgatctl statc.
The similarity [if this serum factor to tlic
human rheumatoid factor suggests that prolonged exposure to an antigen (living agent
or otherwise) could be operative in the
pathogenesis of riieiimatoiil arthritis.
104
AMERICAS RHEIrhIATIS-M ASSOCIATION
h NEW PATHWAY
OF
SAI.ICYI.ATE. htETABOLL5M: REOL-CTIO~
O Y SAl.lCYl.ATE
Ilaniel XI. Bachnwn, S(;smnmu John
ReLvntly we h w e observed in cultiires of
Nmrrospi;ru c r a w a new hiochernical path-
way of siilicylatr nietaholisni: the rediiction
of salicylate to saligenin ( selicyl alcohol ).
Sodium salicylate was added to liquid cultures of Nrnrospora crussu SY7A in a concentration of 20 mg. per cent. After t i lag
phase of 48 hours, the salicylatr disiippwrd
rapidly from the growth nierliiiin antl a
metabolic product appeared a s shown t;y
iiltraviolet spectrophotometry antl by ascending paper chromatography with britan01, pyritline, saturated sodium chloride, and
amnioniinii hydroxide. The metabolic prodnct ( R i 0.96) g:ive an orange color with
(ind
TO SALLGENM
Rcllr Dragoon, Portland, Oregon
diazotized sulfiinilic acid and ii milrive color
with diiwotized 11-nitroaniline. These properties were identical to those of saligmin. Thc
metabolic produyt was isolatcd from 96 iind
!20 hoiir ciiltiirer by ether extraction and
sitblimation in vacuo. It was positively identified BS saligeniu by me:ins of its melting
point, mixed melting point, iiltr;tviolet extinction coefficient, and infrared spectruuii.
Semiquantitative estimation of the yiAd by
paper chromatography and ultraviolet spectrophotometry indicated one niol of siiligcmin
produced for mch mol of salicylatc. consumed.
Techniques h:ive heen tlevc?loptd lor the
Rheuniatoid arthritis is a chronic inflnmiuatory disewe of self pc-rpetiiating nature
demonstration of chromosoint~ nrinilwr nntl
inorphology at tlie nietaphase stage of inifor which no etiologic agent tias h e m identified. Faniiliul studies have appeitrcd that tosis in priiiiary ciiltnrcs of peripheral hlootl
show sollie increase in ttir: incidmcc of ~ h r leukocytes. Using tliis techniqric*, threc
clinical disease itself kind an even greater rheumatoid arthritis patients of niotlmite
incidence of sero-positive tests for rheuma- iind severe clinical state were stiitlietl. Two
of these were posiiive for rheiniiatoitl fiictor
toid factor. This c!ould imply a primary
genetic cau5e with different degrees of ex- with the latex fixation test; one wiis nogiitive.
pression probably involving a siibtle change The prepmition:; werc. rvahiated for chroniof genetic rather than gross chrtimosomal osome number ant1 morphology following
material. This investigation of the chromothe standard system of noiiienclatiirr of
some number and morphology i n clinical human mitotic chromosomes ;is proposed tit
rheumatoid arthritis patient5 was undertaken
the Conference (in Human Chroinosomes in
iis a necessary introduction to this problrm.
Denver this year. In the patients studied, the
chromosome nilinher and mc~rpholo~y
were
‘Read by title.
similar to the normal poptilation.
sTLn>lESO F T H E HUMAN SYNOVLAL. FLUJII P’HOTKJNS RY STARCH GEL
EI.E(:THOPHORESIS
1. P . Bkirtte unrl K. Schmid, Boston, hl;\ss.
Comparison of the proteins of synoviiil
fluid with those of serum hy free rlectrophoresis demonstrated a high relative concentration of albuinin, and particularly, the
al-globulins and a low relative content cf
the +-globulins in certain joint fluids. In
view of these results, investigations using
starch gel electrophoresis were undertaken
to determine the proteins that account for
the diffurences in the electrophoretic distribution of the synovial h i d and senim proteins.
Starch gel electrophoresis was carried out
on a limited niimber of different joint fluids.
The “normal” synovial Huids exhibited the
most pronoanced differences as compared
with serum: A distinctly higher relative
concentration of one of the two pre-albumin
coniponenb (a,-acicl glycoprotein) was
noted; the relative concentration of the postalubmins, fast +-globulins, and transferrin
was essentially unchanged; however, the relative content of the slow or-globulin was
lower. The patterns of pathological joint
105
AMERICAN RHEUMATISM ASSOCIATION
fluids including the traumatic ones varied between those of “normal” joint fluid and serum.
From the results of this study and earlier
investigations, it is concluded that the in-
THELLPlD FRACTION
O F POI.YVINYI,
verse al/a2-globulin ratio characteristic of
“normal” synovial fluid is mainly due to the
low relative concentration of the slow a2globulin and the marked increase in the relative concentration of al-acid glycoprotein.
SPONGE
BIOPSYCONNECTIVE TISSUE
Giles G. Bole, Saul R o s m n , Willium E . M . Lands and U’illiciin D. Robinson,
Ann Arbor, Michigan
We have previously reported that the
total lipid content of a water washed chloroform-methanol extract obtained from small
polyvinyl implants (0.3 x 1.0 x 1.0 cm) in
guinea pigs was unexpectedly high.
Chemical analysis of the extracts indicates:
( 1) The total extractible material represents
40 to 60 per cent of the tissue dry weight
for microscopically uniform connective tissue 3 to 8 weeks of age. (2) Approximately
half of the extracted material is not completely characterized but appears to be derived from the polyvinyl sponge. While the
sponge used for implantation is essentially
insoluble in the chloroform-methanol, it becomes significantly soluble after implantation.
(3) The characterized fraction represents
25 f. 5 per cent of the tissue dry weight;
50 per cent is phospholipid, 10 per cent
cholesterol, and the remainder is neutral
esters. (4)Fractionation of the phospholipids
ON
OBSERVATIONS
by silicic acid chromatography suggests
that the composition of the phospholipids
changes qualitatively with age of the implant.
Isotopic phosphate was administered in
an attempt to determine the site of origin of
the phospholipids. Following intraperitoneal (50 p c ) injection of PJ2, the specific
activity of the phospholipids in the implants
increased as rapidly as that in a similar
fraction from liver. Local injection ( 5 pc)
into one of six implants in an animal gave
specific activities of the phospholipid fractions of the injected sponge consistently
higher than other tissues tested during a 96hour period. The specific activities at 22
hours were: injected implant 524, adjacent
implants 100, brown fat 136, liver 60. These
data suggest that the phospholipids are
synthesized in situ by the connective tissue
in the implants.
BEHAVIOROF RHEUMA~OID FACTOR
WITH CELLSCOATEDWITH
GAMMAGLOBULINS
BY THE BDB TECHNIQUE
THE
Vincent P. Butler, Jr. and John H. Vaughan, Rochester, New York
The “reactant” gamma globulin in the
FII tanned sheep cell system must be in
aggregated form to be agglutinated by
rheumatoid sera and the inference has been
drawn that rheumatoid factor, therefore,
may have specificity for an aggregated,
rather than unaggregated, form of gamma
globulin.
In other studies, it was recognized that
unaggregated preparations of egg albumin
are quite inefficient in coating tanned cells
for agglutination by rabbit anti-egg albumin
sera; unaggregated egg albumin, however,
is quite effective in coating cells treated
with bis-diazotized benzidine ( BDB ). To
study the pertinence of these observations
to rheumatoid systems, human gamma globulin (HGG) was separated by sodium sulfate
fractionation or preparative ultracentrifuga-
tion in a manner designed to provide aggregate-free and aggregate-enriched preparations. Aggregated HGG was effective in
coating both tanned cells and BDB-treated
cells as shown in agglutination by both rheumatoid and rabbit anti-HGG sera. The unaggregated HGG, like the previously studied
unaggregated egg albumin, was quite inefficient in coating tanned sheep cells, as
judged by subsequent minimal reactivity
with rabbit anti-HGG; tanned cells exposed
to unaggregated HGG also had no reactivity
with rheumatoid sera. Unaggregated HGG
linked to erythrocytes by the BDB method
yielded strong agglutination both by antiHGG and “rheumatoid factor.”
Rabbit gamma globulin has also been
linked to erythrocytes with BDB; reactivity
of these cells closely paralleled that of ‘‘sen-
106
AhIERICAN RHEUMATISM ASSOCIATlON
sitized sheep cells.” Gamma globulin from
certain other animal species, as well as numerous individual HGG preparations, have
“EPITHELIALTRANSFORMATION”
also effectively coated erythrocytes for
rheumatoid agglutinating activity.
I N -4
HUMANSYISOVIAL CUI.TURE
C. William Castor, Ann Arhor, Michigan
Cultures of mammalian “fibroblasts” usually flourish for a limited period in vitro, and
then for obscure reasons cease proliferating
and die. A less common, and equally puzzeling, fate of such cultures is “transformation” of the fibroblasts to cells bearing the
inicroscopic characteristics of epithelial cells.
We have observed such a “transformation”
in a cell line derived from a patient with
rheumatoid arthritis. It has been possible to
compare the cell morphology, chromosomal
characteristics, growth rate, nutritional requirements, and mucopolysaccharide production in this cell line before and after the
“transformation.” Previously stellate and
spindle shaped cells became polygonal and
left their reticular growth pattern to assume
an epithelial configuration with contiguous
cell borders. The original cell line had been
largely diploid with a few hypodiploid
THE
POTENTIATIDN OF S M W DOSESOF COHTII:OSTF:P.OIU
Glenn
OF
\WTH
hlETHANUROSTENOLONE
M . Clark, Stanley Kaplun, Julio Goohur utul Vcvid Mills, Memphis, Tenn.
During the past six months, 15 patients
with rheumatoid arthritis on maintenance
steroid dosage have been treated with Methandrostenolone in an effort t o prevent osteoporosis. Within a few days the patients gave
evidence of marked increase in steroid effect
as manifested by decreased joint pain and
swelling and increase in moonface, acne,
purpura, dyspepsia and euphoria. On decreasing steroid intake to levels from onefourth to one-half of the previous main&TDIES
chromosomal forms, while the transformed
cell exhibited ikirked heteroploidy. Driring
the process of alteration, cell niutiplication
slowed, and then resumed at the previous
rate when the new cell form had emerged.
Although the initial “fibroblastic” cell produced hyaliironic acid in concentrations up
to 20 pg./ml. of medium, and intermittently
yielded medium which gave a mucin clot; in
the transformed cell synthesis of high molecular weight hyalnronate is virtually undetectable. Where the initial cell line performed optimally with CMRL medium 1066
supplemented with human serum, the altered cell is mnch less demanding in its
dietary requiremaits, growing equally well
in the simple Eagle’s basal medium, and in
an undefined mcdiuni composed of lactalbiimin hydrolysate and yeast extract.
tenance dosage, equivalent suppression of
inflammatory activity as measured by Lansbury indexes was maintained. At the new
levels no evidence of increased toxicity was
observed. These findings seem of importance
since it now may be possible to maintain
patients on a smaller dosc of corticosteroid by
adjunctive therapy with an agent which has
been shown to have an anabolic physiologic
effect.
PHOTOELECTRICALLY QUANTITATEI~
!?I1
P R E C l P l T A T l O N BY
RHEUMATOID
SERA’
Elias Cohen and Erzin Neter, Buffalo, New York
By photoelectric, carrier-free quantitation
(Cohen, Neter and Norcross, Am. J . Clin.
Path. 31:607, 1859) the effects of varying
concentration and temperature treatment of
reactants upon the Cohn Fraction II-rhenmatoid factor( s ) precipitation were determined. Increasing concentration of human
‘Read by title.
FII reactant from 0.83 per ccnt to 5 per
cent decreased precipitation with rheumatoid factor(s), analogous to effect of excess
of antigen, or .intibody in precipitin reactions. Human FII-rabbit anti-FII precipitin reaction could not be detected photoelectrically with same concentration of reactants and incubation time, due to excess
FII antigen. Heating 0.83 per cent FII at 63
107
AMERICAN RHEUMATISM ASSOCIATION
C. for 10 minutes before testing, substantially increased precipitation with rheumatoid sera. Freezing and thawing FII also
increased its reactivity with RF [rheumatoid
factor( s)]. Rheumatoid sera "inactivated"
30 minutes at 56 C., prior to testing, gave
greater precipitation than native sera when
mixed with FII reactant. Destrudion of a
labile inhibitor comparable to that reported
by Schubart, Cohen, and Calkins (New
England J. Med. 261:363, 1959) is suggested. Observation of enhancing effect of
SOME
SERUM PROTEIN
inactivation of rheumatoid serum upon precipitation with human FII reactant, but depression of agglutination of rabbit-antibody
sensitized alligator erythrocytes, with inactivated serum versus native serum, concur
with concept of heterogeneity of rheumatoid
factor( s ) . An evaluation-comparison of photoelectric instrumentation for detecting FIIrheumatoid factor( s ) precipitation will be
presented. The nature and significance of
such characterization of rheumatoid factor( s ) will be discussed.
ABNORMALITIESI N P.4TIENTS WITH PROGRESSIVE SYSTEMIC
SCLEROSIS
AND THEIR
RLXATIVES
Josue M . Corcos, Wilkzrn C . Robbins, B m r d Rogofl and Ralph Heimer,
New York, N. Y.
Twenty-eight patients with scleroderma
were examined and their sera were studied
for the following: gammaglobulin levels by
zinc turbidity; rheumatoid factors by latex
fixation and sensitized sheep cell tests; antinuclear antibodies by L. E. preparations
and complement fixation tests with nuclei.
Seven of 27 patients had hypergammaglobulinemia, which was confirmed by paper
electrophoresis. Three patients had low levels of gammaglobulin. Thirteen of 24 patients had a positive latex fixation test, but
only 6 of these had a positive sensitized
sheep cell test. None of the patients' sera
formed L. E. cells, but 9 of 25 sera fixed
complement with calf thymus nuclei.
The families of 19 patients with scleroderma, comprising 78 blood relatives, have
been studied. Of 75 relatives, 28 had abnormal gammaglobulin levels. Of these, 3
were high and 25 were low. Eleven of these
serum samples were checked by paper electrophoresis, and gammaglobulin levels ranging from 400 to 750 mg. per cent were
found. In 6 families, comprising 30 relatives, there were 22 individuals with low or
low normal gammaglobulin levels. Of 54
relatives, 6 exhibited a positive latex fixation
and 3 of these had a positive sensitized sheep
cell test. In one family, comprising 3 members, all exhibited a positive test for rheumatoid factor; one was the propositus with
scleroderma, the second had classical rheumatoid arthritis, and the third member was
asymptomatic. Tests for L.E. factors were
negative in all relatives.
The occurrence of complement fixing antibodies to nuclei in the sera of patients with
scleroderma indicates a relationship to systemic lupus erythematosus. However, the
lack of these antibodies in sera of asymptomatic relatives (previously noted by others
in relatives of patients with systemic lupus
erythematosus), suggests that antibodies to
nuclei are accompaniments of a well-developed disease process, and unlike the rheumatoid factors, do not reflect a heritable abnormality.
The results obtained in this study, furnish
additional evidence for the close relationship
between scleroderma and other systemic diseases of connective tissue. As already suggested in similar studies of rheumatoid arthritis and systemic lupus erythematosus, this
study suggests that scleroderma also occurs
in families in which there exists a genetic
predisposition for abnormalities of immune
systems.
CORRELATIVE
CLINICOPATHOLOCICAI.
FEATURES
IN THIRTY-TWO
PATIENTSw m i
SYSTEMIC LUPUS ERYTHEMATOSUS'
Charles W. Denko and Lee P . Davis, Columbus, Ohio
With increasing information widening our
understanding of systemic lupus erythema"Read by title.
tosus (SLE 1, including the features of clinical, laboratory and pathological findings,
we studied all patients with systemic lupus
erythematosus who have been examined
108
AMERICAN RHEUMATISM ASSOCIATION
post-mortem at the Ohio State University
Hospital, Columbus, Ohio. Material was
available an 22 women and 10 men about
equally divided between steroid treated and
nonsteroid treated groups.
Pathological findings were oorrelated with
clinical signs and symptoms. The average
duration of life in these patients was about
2% years, remaining relatively unchanged
in those receiving steroids. The majority of
patients in both groups, steroid and nonsteroid treated, died of infections with
uremia and acute lupus itself accounting for
others. Elevated leukocyte counts appeared
frequently with leukopenia occurring much
less commonly. Other laboratory features
were evaluated in relation to the morphw
logical changes found in various organs.
Clinical symptoms, such as gastrointestinal
disturbances, mcntal aberration, chest pain
and musculoskeletal involvement were correlated with lupus lesions involving the
esophagus, stomach, small 'and large intestinal, heart, lungs, pleura, pericardium, peritoneum and other organs. Classic lesions
such as those in the spleen and kidney and
nonspecific changes in the lungs atid liver
were noted.
THEEFFECTOF STEROIDSANI) INFECTION
ON TAUHINE
EXCRETION
IN
SYSTEMIC LUPUS ERYTHEMATOSUS'
Charles W.
Columbus, Oliio unrl L?. Irene Pentz, Chicago, Illinois
The aviiilabilih of a method suitsble for
the routine determination of taurine ( 2 aniinoethanesulfonic acid) led us to measure
taurine excretion in patients with systemic
lnpus erytheniatosns (SLE) during treatment
with steroids an2 during nonsteroid treatment. Taurine is the end product of the
metabolism of sulfur containing amino acids
such as cystine. No information on the biochemical alterations in sulfur metabolism in
patients with SLE has previously been reported. A decreasing output of taurine was
noted in patients receiving decreasing dosages of hydrocortisone and dexamethasone.
A markedly different pattern in taurinuria
was found in patients receiving prednisone,
methylprednisone and triamcinolone. Hero
the taurine excretion rose suddenly as the
steroid level was decreased. This action is
thought to reflect the release of a mechanism
suppressing some phase of pituitary activity
followed by return to normal pituitary and
adrenal function.
Patients with systemic lupus erythematosus who had severe acute infections excreted high levels of taurine and did not follow either pattern even though they also
received high doses of steroids. Severe acute
illness in normal subjects was accompanied
by an elevated tnurine output that gradually
diminished as the clinical features of the
illness subsided. These changes accompanying acute infection were thought to reflect
an increased hydrocortisone level in the
blood stream resulting from the illness.
'Read by title.
THEANTI-INFLAMMATQRY .\Cl I V I T Y
OF A CI.UCW0RTICOID
EPIMER~
EUis Dresncr and Edgur S. Cathcurt, Hosten, 14ass.
Stereoisomers of anti-inflanimatory glucocorticoids, such as the lla-hydroxy and
l'l/+hydroxy isomers of cortisol and the
16/3-hydroxy isomer of triamcinolone, are
generally biologically inert, The 16p-methyl
isomer of dexamethasone ( & - m e t h y l - 9 ~
fluoro-prednisolone) has been synthesized,
and its anti-inflammatory properties in rheumatoid arthritis compared with those of
dexamethasone.
Twelve ambulant patients with active, re'Rend by title.
versible rheumatoid arthritis were studied.
After control periods of 8 or 12 weeks, they
were given dexamethasone and the 8-epimer
alternately in paired monthly treatment periods, the dosage of both compounds being
reduced or increased in subsequent paired
periods according to their response. Each
pntient had 3 or 4 paired observation periods on different doses of the steroids (0.54.5 mg. daily), the duration of observation
being 32 to 48 weeks. The results were assessed at the end of each period [Lansbury
( 1959) criteria].
109
AMERICAN RHEUMATISM ASSOCIATIOB
Comparison of the effects of each compound on the systemic and articular indices
and ESR in each patient shows both steroids
affect them in like manner. In the group
as a whole, the 8-epimer appeared to be
somewhat more active than the dexamethasone in lowering both the ESR I42 paired
studies: 8-epimrr better in 15, (Y in 7,
equal in 201 and the articular index (number of joints involved) (8 better in 17, a
in 10, equal in 151, whilst their effect on
the systemic (noiiarticular ) index was about
equal [B better in 11, a in 11, equal in 201.
Side effects of the steroids were indistinguishable, prominent being moonface, facial
erythema, polyphagia, weight gain and cutaneous purpura.
This is the first example of a glucocorticoid of which two epimers are active.
RELATIONSHIP
OF CALCIUM AND HTDROGEN PHOSPHATE INTERACTIOX TO
OF HYDROXYAPATITE FORMATION
I S A MODEL SYSTEM
Harry R . Elden ond Da&
Evidence concerning the nature of interaction between preformed hydroxyapatite
(free or attached to the fiber) or lesser complexes of calcium and hydrogen phosphate
in the nucleation of hydroxyapatite on collagen was previously reported. ( Elden,
H. R., and Howell, 0.S.: Fed. Proc. 19:
142, 1960.) Investigation was made of the
interactions between calcium and hydrogen
phosphate in solution to define the possible
moeities available for initiating nucleation
or precipitation of hydroxyapatite. It was
advantageous to employ the experimental
desiLgn of Watson and Robinson (Am. J.
Anat. 03:25, 1953) who followed the interaction of calcium and hydrogen phosphate
(ion products 9 to 38x10-4) by x-ray diffraction patterns and electron micrographs
at intervals after miring: they noted transition from clear solution (prior to mixing) to formation of clumps ( 1 minute),
a membrane (10-15 minutes) and finally
crystals of hydroxyapatite.
In the present study, it was possible to
follow the same interaction at concentrations
close to the physiological range (ion product 1x10-6) by measurement of turbidity
in the light-scattering photometer, solution
THE &hCHANISM
S . IJor~ell,Miami, Florida
conductivity, pH and composition of the
precipitate.
The logarithm of turbidity during an
induction-period was related to ion-product
with slope of -1.4 when calcium was fixed
and phosphate concentration varied. However, turbidity was independent of ionproduct when calcium was varied at a fixed
phosphate level. After the induction period,
turbidity showed a momentary decrease,
then an increase (inflection period); finally
turbidity decreased slowly due to settling
of the precipitate. Simultaneously, solution
p H and conductance decreased while analyses of the precipitate showed a smooth
continuons increase of calcium and phosphate content fCa/P ratio varied from 1.2
to 1.4).
The following reaction scheme is postulated for this system: (1) C a + + and
HPO;=+ C a m 0 (clumps); ( 2 ) CaHP04-t
hydroxyapatite with bound clusters of Ca+ +
and HPO,= (inflection-period; (3) Hydroxyapatite-accretion with crystal formation ( settling phenomena ) . Theoretical relationship of these findings to mineralization of collagen, in vitro, will be discussed.
SEQUENCE OF MORPHOLOGIC
CHANGES
IN RHEUMATOID
LYMPHNODECELLSGROWN
IN
M ~ L I P O RCHAMBEHS
E
W a h x V. Epstein and Margaret Ross, San Francisco, California
With the demonstration of rheumatoid
factor in human lymph node cells, the need
has increased for methods permitting direct
observation of cell populations under controlled conditions.
The hlillipore Chamber technique of Algire and Weaver has been adapted for the
growth of human lymph node cells obtained from patients with peripheral rheumatoid arthritis and of rabbit popliteal
lymph nodes after stimulation with bovine
serum albumin (BSA).
Node cells are placed into cold PVP
Macrose solution and suspensions of cells
110
AMERICAN RHEUMATISM ASSOCIATION
placed between membranes of Millipore
HA membranes--pore size 0.45 p supported
by a lucite ring. Multiple chambers are
implanted in separate subcutaneous tunnels
in normal rabbits. Paired chambers are removed at regular intervals over a two week
period and the membrane walls are stained
with heniatoxylin-eosin.
Three days after implantation, extensive
cell degeneration is observed together with
the appearance of stellate macrophages.
After five days, chamber membranes show
extensive mitotic activity with colony-like
organization of lymphocytes and polyblasts
as well as advancing sheets of fibroblasts.
By two weeks, sheets of medium and smallsized lymphocytes are observed together
with collections of plasma cells. General
cellular organization is observed with little
further evidence of cellular degeneration.
The protective effect of host cell exclusion permits this technique to be adapted
to the study of cell populations implicated
in the production of rheumatoid factor,
STRUCTURAL
STUDIESOF RHEUMAT~IDFACTOR
BY CHROMATOGRAPHIC
AND ENZYMATICTECHNIQUES
Wallace V. Epstein and Margaret Tan, San Francisco, California
Macroglobulins of high rheumatoid factor
activity as measured by the F 11 tanned
cell heinagglutination technique were isolated by preliminary separation of euglobulin and non-euglobulin proteins using a
polymerized dextran system (Sephadex
C-25). The euglobulin proteins were then
eluted by ionic strength gradient (pH8)
from an anionic exchange resin (DEAE)
and final purification achieved by precipitation in low ionic strength buffer. The product with a protein content of 0.1 mg./ml.
showed a log, titer of 26 and had s , ~ of
approximately 19s and 26s with 19s material as the major component.
The macroglobulins were treated for 16
hours at 37 C. with papain derived from
mercuripapain in the presence of ethylene-
diamine tetracetic acid and cysteine. The
reaction was stopped by the addition of
the sodium salt of para-Chloromercuribenzoate. Dialysis against 0.01 ionic strength
phosphate buffer pH6 produced a crystalline protein product and the supernate
showed a single peak by ultracentrifugation
of approximately S3.5. Fractionation on a
Chl-cellulose cxchange column revealed
three peaks; two were eluted with 0.01 M
pH6 phosphate buffer and one following
ionic strength gradient. The fractions were
also separable by starch gel electrophoresis
at pH5.5. All serological activity in the F I1
hemagglutination test was lost following
enzymatic digestion. No inhibitory activity
toward rheumatoid factor was exhibited by
these fractions.
THEHEATLABILITY
OF RHEUMATOIDFACTOR
AS MEASURED
BY PRECIPITATION
WITH
HUMANF M ~ I OIIo
N
Wallace V. Epstein and Mnrgaret Ross, San Francisco, California
The heat lability of rheumatoid factor doubling dilution technique to changes less
was shown by heating the serum from pa- than 50 per cent of the total serum activity.
tients with peripheral rheumatoid arthritis Further heating of the supernate of the
to 56 C. for 30 minutes. The effect was precipitation system causes a further fall
shown by decreased activity of the heated in hemagglutinating activity; since this
sera in both precipitation reactions with change is proportional to the amount of
human Fraction I1 and agglutination by the Fraction I1 added, the effect appears due
F I1 tanned cell technique. For one serum to the activation of the adled Fraction 11.
Although the Fraction I1 precipitation
the difference mounted to 110 yg. N/ml.
at the point of maximum precipitation, The system is insensitive to small amounts of
associated decrease in hemagglutinating ac- rheumatoid factor in serum, it will permit
tivity as measured by the F I1 hemagglu- a demonstration of changes in the serum
tination test was evident, but not as dra- content of thermolabile and thermostabile
matic, because of the insensitivity of the rheumatoid factors that cannot be made
with any certainty by techniques involving
*Read by title.
doubling dilutions.
11.1
AMERICAN RHEUMATISM ASSOCIATIOIL'
PERIPHERAL NCUROPATHY
IN RHEVMATOID ARTHRITIS
Richard H . Ferguson and Charles H. Slocumb, Rochester, Minnesota
The occurrence of peripheral nenropathy
of several varieties in patients with rheumatoid arthritis h.is been observed with increasing frequency in recent years. A study
has been made at the hlayo Clinic of all
cases of rheumatoid arthritis seen in the
last 10 years in which an isolated nerve
pdsy or peripheral polyneuropathy was
found. The incidence of the various types
of neuropathy was compared with that in
an earlier era. Particular attention was focused on 61 patients who had peripheral
polyneuropathy seen in the last decade, and
data pertaining to clinical, pathologic, laboratory and electromyographic stud:es will
be presented. Classification of this latter
group into foiir categories on the basis of
the type and exttnt of a neurologic involvement offers a clinical means of separating
patients with nezrotizing arteritis from those
in whom the prognosis is better. The possible role of adrenal-steroid therapy in the
pathogenesis and treatment of these states
will bc discussed.
SERUM SUBSTANCES BEHAVING
LIKERHEUMATOII) FACTOR AND
LUPUSFACTOR
IN CHRONIC
PSYCHOSIS*
W. J . Fessel, San Francisco, Calif.
Psychiatric and psychosock1 factors have
been thought by many to be important in
the pathogenesis of certain connective tissue diseases. This report gives some preliminary results of an investigation into the
possibility that connective tissue disease may
be important in the pathogenesis of certain
psychiatric disorders. FII latex and nucleoprotein (N.P.) latex agglutination tests
were made on sera from about 1100 patients, in a State mental hospital, having
various chronic psychoses. 8.2 per cent had
positive FII tests and 1.3 per cent had
positive N.P. tests; the corresponding figures for control sera, from nonpsychotic institutionalized persons, were 1.4 per cent
and zero respectively. Ultracentrifuge analyses of 9 sera showing positive FII tests
demonstrated elevated macroglobulin levels
in 5. Physical examination of the patients
with positive agi?Jutination tests showed the
*Read by title.
following diseases: rheumatoid arthritis 7;
probable systemic lupus 4; Marfan's syndrome 3; Ehlers Ilanlos syndrome 2; retinitis pignientosa 4. In addition, there was
a high incidence of congenital skeletal deforniities. A randomly selected group of 46
psychotic patients having negative FII and
N.P. latex tests also had a high incidence
of congenital skeletal deformities and produced another example of the Ehlers-Danlos
syndrome. The above findings confirm the
fact that rheumatoid arthritis is uncommon
in a psychotic population and suggest that
other connective tissue diseases might OCccisionally be important associations of a
chronic psychosis. The explanation is not
yet clear for the positive FII tests in the
psychotic patients who do not have clinical evidence of il connective tissue disease.
The possibility is being explored that the
psychosis in the latter patients is a defense
mechanism which is alternative to a connectivc tissue diskase.
THETOXOPLASMA
INTRAPERMAL
TFST IN RHEUMATOIDARTHRITIS'
Arnfn E. Good, Ann Arbor, Michigan
Toxoplusmu gondtt, a protozoan infectious
agent of virtually world-wide distribution,
has shown in numerous population studies
a high infectivity rate, varying geographically and increasing with age. Little is
known of the course of acquired chronic,
'Read by title.
subacute, or inapparent toxoplasmosis, but
the following, selected, reported manifestations may also occur during the course of
rheumatoid arthritis: fatigue, myalgia, arthralgia, lymphadenopathy, splenomegaly,
chorioretinitis, pulmonary infiltration and
macroglobulinemia.
To investigate a possible- association be-
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AMERICAN RHEUMATISM ASSOCIATION
tween toxoplasmosis and rheumatoid arthritis, the intradermal test with toxoplasmin,
prepared from peritoneal exudate of mice
infected with toxoplasma, was carried out
in 45 consecutive paticnts with definite or
classical rheumatoid arthritis and in 45 agematched controls, selected at random from
hospitalized patients free from rheumatic
symptoms or stigmata of connective tissue
disorders. Two of the rheumatoid group
and one of the controls were receiving corticosteroids at the time of the skin testing.
Twenty-three (51 per cent) of the rheumatoid group and 28 (62 per cent) of the
controls showed positive intradermal tests.
The hemagglutination test and SabinFeldman dye test, unavailable for this study,
would have lent increased precision or
specificity. The consensus of workers in this
field is that a positive skin test with toxoplasmin is nearly always associated with
dcmonstrable antibody. The cutaneous test
is generally accepted as a useful aid in
population surveys for late detection of
toxoplasma infection.
This pilot study utilizing the intradermal
toxoplasn~intest showed no evidence for a
positive correlation between rheumatoid arthritis and toxoplasmosis.
OF THE E L B ~ W
IN PATIENTS WITH 1~HEUU4TOlDARTHRITISARTHROPLASTY
AN END RESULT STUDY'
1. Paul Harcer~,Jr., New York, N. Y.
This is a report on the end result of 15
arthroplasties of the elbow done at the Hospital for Special Surgery during the past
20 years, the longest being 20 years postarthroplasty, the shortest 3 years, and the
average 11 years. All the patients were Stage
3 or 4, according to the American Rheumatism Association Classification of the process.
Using activities of daily living as criteria,
close to 50 per cent of the patients have
shown improvement. A few of the remain'Read by title.
INHIBiTORS OF
der have shown improved range of motion
hut lack stability. The remainder have frank
instability or refusion secondary to progression of the disease.
The poor results seem to be secondary
to definite progression of disease, although
a few patients have shown instability immediately postoperatively. The best results
are present in those who have had severe
juvenile rheumatoid arthritis. This procedure is usually associated with procedures
on other involved areas in the upper and
lower extremities.
COMPLEMENT FIXATION-AN APPAnEXT NEW CHARACTERISTIC
OF SERUMOF CONNECT~VE
TISSUE
DISEASES
Ralph Heimer, Josue M. Corcos and C u r b Nosenzo, New York, N. Y.
and Jersey City, N. J.
Although it is known that rabbit, guinea
pig and pig sera in moderate dilutions exert
inhibitory effects on complement fixation
reactions, the behavior of human sera in
such reactions has received scant attention.
We have ex'amined the effect of added human serum on a system fixing complement,
and have found that only sera from patients
with certain connective tissue diseases interfered markedly with such reactions.
Our experimental model for testing inhibitors of complement fixation (ICF) was
as follows: Human thyroglobulin and rabbit
anti-human thyroglobulin, fixing 10 to 12
50 per cent hemolytic units of guinea pig
complement, were incubated in the pres-
ence of dilute human serum. Guinea pig
coinplement w.is then added, and after
further incubation the residual complement
was titrated with sensitized sheep cells. Of
over 200 sera tested, only those of patients
with rheumatoid arthritis, systemic lupus
erythematosus and systemic sclerosis contained appreciable amounts of ICF. In a
variety of other diseases, including osteoarthritis, spondylitis and gout, little or no
ICF was found. Moreover, when present,
such activity could be removed partially
with zymosan.
ICF in sera of patients with connective
tissue diseases is heat-stable and not dialyzable. It is absmbed by antigen-antibody
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AMERICAN RHEUMATISM ASSOCIATION
complexes, but not by sheep cells or zymosan. ICF was purified by DEAE cellulose
chromatography, appeared in a fraction containing mostly albumin and alpha-globulins,
and lacking rheumatoid factor ( R F ) activity. Similar fractionation of control sera,
failed to yield ICF. Zone electrophoresis
on starch of purified ICF recovered from
DEAE cellulose resulted in inactive subfractions. On reconstitution of the albumin
and alpha-globulin subfractions ICF activity was recovered. Similar results were
obtained on electrophoresis of whole serum. ICF is therefore a multicomponent
system. Evidence will be presented that
ICF, although reacting with antigen-antibody systems, is neither RF nor an incomplete RF.
It is proposed that ICF together with
RF comprises a unique immunological system characteristic of certain connective tissue diseases. Whether ICF levels reflect the
clinical course of disease or are modifyable
by treatment remains to be elucidated. ICF,
however, promises to provide a new tool
for diagnosis and for study of the etiology
of connective tissue diseases.
THE RELIEF OF INTRACTABLE
ARTHRITICPAINBY EXTENDED
SYMPATHETIC
DENERVATION’
Robert A. Herfort, White Plains, N. Y.
The principal source of disability presented by most patients with chronic arthritis is pain originating in the weight
bearing joints. Over the course of the past
five years, the author has employed a technique of so-called extended sympathetic denervation in 72 patients with advanced painful rheumatoid arthritis and osteoarthritis
of the hips and/c;r knee joints. The surgery
consists of a retroperitoneal resection of the
lumbar sympathetic trunks from the superior margin of the common iliac vessels to
the second lumbar vertebra encompassing
accessory ganglia on the rami communicantes and also decussating fibers in the
retro-aortic prevertebral plexus. The procedure is, at present, carried out bilaterally
‘Read by titlc.
at one sitting. This surgery in middle aged
and elderly arthritic invalids has had no
mortality. In the immediate postoperative
period, the patients exhibit, consistently, relief of arthritic pain with concomitent improvement in ioint mobility and general
functional capacity. The patients have been
followed for periods up to 5 % years after
surgery; the relief of pain and improved
range of motion have been maintained in
67 of the 72 patients. There have been no
undesirable effects noted after extended sympathetic denervntion. Neuropathic disorganization of the affected joints has not occurred.
It is suggested that the principal afferent
pain pathways from the articular surfaces
of the hip, knee and ankle joints traverse
the lumbar sympathetic trunks and plexuses.
“FALSE
POSITIVE”
L. E. CELLPREPARATIONS
IN LEPROSY’
Conrad Herr, New Orleans, Louisiana
Results are reported of 500 L.E. cell
preparations done on blood from 325 l e p
rosy patients hospitalized at Carville, Louisiana. A case of concomitant S.L.E. and
lepromatous leprosy stimulated this survey.
Since the significance of the L.E. cell, the
L.E.-like cell, “the tart-cell,” and the presence of leukoagglutination is still in doubt,
the preparations were classified as Negative; Class A (many typical L.E. cells);
Class B (rare to occasional typical L.E.
cells with or without leukoagglutination or
leukophagocytosis); or Class C (leukoag‘Read by title.
glutination or leukopbagocytosis without
L.E. cells). The preparations, throughout
the study, were done by the two-hour clot
technique.
Correlation with the type and activity
of the patients’ disease; with types of drug
therapy; with concomitant systemic diseases
unrelated to leprcsy; and with the presence
of secondary amyloidosis is attempted. A
survey for cryoglobulins was made, and
their effect on the L.E. cell preparation is
demonstrated.
Only the patient with clinical S.L.E. presented Class A preparations.
All with Class B preparations ( 2 1 cases)
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AMERlCAN RHEUMATISM ASSOCIATION
had lepromatous leprosy. Nine had biologic
false positive serologic tests for syphilis; ten,
abnormal liver functions; five, "reactive episodes of leprosy"; several single cases, diseases unrelated to leprosy; and two, secondary amyloidosis.
Those with Class C preparations (58
cases) formed a more homogeneous group
which could not be distinguished from the
average patient population. However, all
but one had lepromatous leprosy, and one
carries the only recorded diagnosis a t Carville of concomitant leprosy and rheumatoid
arthritis.
The implications of this work are discussed. Histochemical staining and studies
to demonstrate autoimmune antibodies are
in progress.
TRANSFER
OF AUTOIMMUNE
NEPHROSIS
IN RATSBY MEANSOF LYMPHNODECEL.LS
E w l y n V. Hem, Charles T.Ashworth unrl -Vowis Zif, Dallas, Texas
Recent studies have demonstrated that
nephrosis may be induced in rats by the
intraperitoneal injection of rat kidney homogenate with Freund's adjuvant. The disease
is characterized by proteinuria, hypoproteinemia, hypercholesterolemia and hyperlipemia; histological studies have shown a
membranous gloinerulonephritis. The method of induction suggests an autoimmune
basis for this condition. Hemagglutination
and precipitin tests have not demonstrated
serum antibody. The possibility that cellular
antibody was coiicerned in this phenomenon
was accordingly investigated by attempting
transfer of the disease to immunologically
tolerant recipients by means of lymph node
cells.
A total of 28 rats were pretreated neonatally with spleen cells from the prospective donors. When lymph node cells from
10 nephrotic donors were injected intra-
venously into 10 prepared recipients, chemical and histological evidence of nephrosis
was observed in 9 of the latter. No abnormalities were noted in any of 18 prepared
recipients who received cells from nonimmunized animals or animals injected with
Freund's adjuvant alone. Successful transfer
was accomplished in only 1 of 8 recipients
who had been pretreated neonatally with
spleen cells from rats other than those providing the donor lymph node cells. Nontolerant recipients of lymph node cells from
nephrotic rats did not develop disease.
Immunofluorescent and electron microscopic as well as histological studies have
confirmed the transfer of the rat nephrosis.
These experiments provide evidence for the
role of cellular antibody in the renal lesion
produced in rats by immunization with kidney tissue with Freund's adjuvant.
GREATEREFFECTIVENESS
OF SMALLDOSES
OF CHLOROQUINE
IN THE
TREATMENT
OF RHEUMATOLD
ARTHRITIS*
William K . lshmuel a d R. W. Pnyne, Oklahoma City, Oklahoma
Chloroquine phosphate was administered
to 40 patients with rheumatoid arthritis in
doses of 500 mg. daily (Group .4)and 250
mg. daily (Group B ) for periods of from
3 to 36 months.
Of the 18 patients receiving the larger
doses of Arden (Group A ) , the following
subjective responses were noted: Grade
1-0; Grade 2--5; Grade L 5 ; Grade 46. Toxicity requiring discontinuance of
medication occurred in 5 of these patients.
The average time of beginning response to
-
'Read by title.
--
nicdication was 6 weeks.
The smaller dose of .4ralen was wed in
the treatment of 24 patients (Croup B )
with the following subjective responses:
Grade 2-18; Grade k 5 ;
Grade 1-1;
Grade 4-2.
Toxicity necessitating discontinuance of medication occurred in only one
of these patients. Beginning response to
medication averaged three months.
Effects of the two dosage schedules on
body weight loss and on serum protein
pnlysaccharide ratio further indicated reduced toxicity nnd greater therapeutic usefulness of the lower dose schedule of Aralen.
115
AMERICAN RHEUMATISM ASSOCIATION
CHOLESTEROL-ASSOCIATED
PROTEINS
PRECIPITABLE
FROM RHEUMATOIDPLASMA
‘WITH CHONDR~ITIN
SULFATE
Grace P. b b y , Durham, N. C.
Levels of acid mucopolysacharides in the
euglobulin fraction of plasma from patients
with rheumatoid and nonrheumatoid inflammatory disease increase nonspecifically in
parallel with the euglobulin fraction. (Kerby, 1958). The present study explored the
proteins remaining in the supernatant plasma (after isoelectric precipitation of euglobulin) which were then precipitable by
adding chondroitin sulfate.
Salt concentration and pH were important in determining amount of protein additionally precipitated, as noted by Badin
and Schubert ( 1955). In adidtion, plasma
dialysis itself increased later precipitability
of a protein fraction from the supernatant
plasma with chondroitin sulfate. This effect
was reversed by adding to dialyzed plasma
(prior to euglobulin precipitation) a pressure dialysate of plasma. The factor involved resisted heating to 100 C but not
incineration.
The amount of biuret-positive material
(related to mg. of standard albumin) precipitable by chondroitin sulfate from the
supernatant fraction of plasma under rigidly
controlled conditions was significantly ( p <
.01) greater in rheumatoid plasma (371 f
175 (S.D.) mg. per 100 ml. plasma) than
in either nonrheumatoid inflammatory (225
f 160) or noninflammatory 176 f 117)
plasma. The material from rheumatoid plasma, diluted to serum equivalence, reacted
with FII-coated latex particles in about o n e
half of the instances. Its cholesterol content
(6.94 -t 5.66 mg. per 100 of plasma) far
exceeded that from inflammatory (1.82 +1.94) or nonidammatory (1.66 -I- 1.24)
plasma. The ratic of cholesterol to biuretpositive material in the fraction showed a
high correlation with plasma cholesterol in
the two non-rheumatoid groups ( p < .01)
but not so in tht rheumatoid group ( p >
.1).
The protein additionally precipitable with
chondroitin sulfate after isoelectric removal
of euglobulins offers opportunity for study
of factors determining aggregation of globulins, of particular interest since the material
precipitable from rheumatoid (as compared
to nonrheumatoid inflammatory and nonidammatory) plasma was significantly increased and in some instances reacted with
FII-coated latex particles.
ONEPOSSIBLE
MECHANISM
IN THE UPTAKEOF SEROTONIN
BY HUMANPLATELETS“
Grace P . Kerby and S. M. Taylm, Durham, N. C.
Enhancement of dye reduction by human
platelets in the presence of serotonin and
a heat-stable, nondialyzable plasma factor
or factors (“5HT redox effect”) has been
shown to be correlated with increased uptake of serotonin by intact platelets. Investigations into the cause of the increased
uptake revealed that serotonin was capable
of solubilizing calcium phosphate in vitro,
suggesting some type of complex formation.
Prior mixing of serotonin with Mg++ did
not affect serotonin uptake by platelets, nor
did disodium calcium EDTA. Disodium
EDTA enhanced both 5HT redox effect and
uptake. However, prior mixing of serotonin
with Mg-++ followed by chelation with di‘Read by title.
sodiuni EDTA diminished both 5HT redox
effect and uptake of serotonin by intact human platelets in the presence of plasma.
The results suggested the possibility that,
in the presence of plasma, serotonin which
was free to combine as a complex with a
divalent cation was taken up by human
platelets as a complex with a divalent cation
bound to a nondialyzable plasma factor.
It is evident from studies previously reported by others (e.g., Weissbach and coworkers, 1958, 1960) that factors which
appear important in the uptake of serotonin
by platelets in vitro vary with the in vitro
conditions imposed. Resultant data cannot
at this time be assumed to relate to in vivo
events.
116
AMERICAN RHEUMATISM ASSOCIATION
OSTEOARTHROSIS:
UNUSUAL,MANIFESTATION’
Harry F. Klinefeltez, Jz. and John I € . Y(irdIq, Baltimore, Maryland
Degenerative changes in the inetacarpophalangeal joints of the hands is exceedingly
rare. A 68-year-old carpenter showed siich
changes as well as involvement of the left
hip. The hand joints were treated by capsulotomy and resection of the osteophytes;
pathological examination of the tissue
showed only degenerative changes in the
cnrtilage, with fibrous tissue reaction, but
no inflammation. Marked improvement followed surgical treatment.
Clinically, the patient was thought to have
rheumatoid arthritis, hut there was no supporting evidencv for this diagnosis, either
radiologically, serologically, or hematologically. The benign course of his disease is
characteristic of osteroarthrosis.
“Read by title.
STUDIES OF A WALDENSTHOM-TYPE hIACROGLOBLI.IN WITH
RHEUMATOIDFACTORPROPERTIES
Julius Kritzmun, Henry G . Linkel, John McCarthy and Robert C . Mellms,
Boston, Mass. and New York, N. Y.
A macroglobulin obtained from the blood
of a patient with typical Waldenstrom’s
macroglobulinemia has been shown to have
serologic properties similer to rhriunatoid
factor. In other respects (behavior in the
iiltracentrifuge, reaction with rabbit antimacroglobulin serum, cryoprecipitation, electrophoretic mobility and carbohydrate content), it was a typical macroglohiilin. An
important difference between it and rheumatoid factor is its pH optimum of about
6.0, whereas tht. optimum for rheumatoid
factor’s serologic activity is ahout 8.6. This
difference in pH optimum affects both
the precipitin reaction with aggregated
gamma globulin and the agglutination of
vcarious test particles. The macroglohiilin
AN ATTEMPT
TO CHARACTEHIZE THF
was demonstrated in the cytoplasm of spleen
‘and lymph node cells by the use of fluorescent aggregated gamma globulin. Extracts
of splenic tissue (obtainsd at autopsy) had
the same serologic activity as the serum
factor. The morphology of the cclls of origin of this protein is to be described in
detail.
The patient from whom this protein was
obtained had no joint symptoms at any
time. The resenihlance of his cellular proliferation to that seen in other diseases
where proteins are elaborated that brhave
like antibodies C
t J gamma globulin suggest‘s
d hroadcr significance of the rheumatoid
factor phenomenon than a pathogenetic role
in iirthritis.
ANTIGENIC
SPECIFICITS O r THE
L. E. CELL FACTOR
P. J. Lachmann, Cambridge, England
The failure to produce the L.E. cell factor
by immunization of experimental mimals
has made it neccssary to rely solely on the
analysis of the lupus Phenomenon as given
by human lupus sera for information on its
antigenic specificity. A two-stage indirect
L.E. cell test has been devised for this
purpose. This technique allows the initial,
immunological stage of the lupus phenomenon to bc studied independently of the
later changes, and can be u s e d for exprinients involving either variation in the substrate particle or inhibition of the reaction.
It has been shown that liipus inclusion
can be formed not only from a variety of
cell nuclei but also from sperm heads. Inhibition experiments have shown that efficient inhibition of the lupus phenomenon
is given by DNA-nucleoproteins only in
their native state. Denaturation of the nucleoproteins or degradation with DNAase
or papain destroys their inhibitory activity.
The results of such experiments have lent
support to the concept that it is the “backbone” configuration of DNA-nucleoproteins
( both DNA-histone and DNA-protamine)
in their native state that carries the relevant
antigenic determinants.
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AMERICAN RHEUMATIShl ASSOCIATION
A
SPECIFIC
ENZYMATIC
SPECTnOPH01.0hiETRIC METHOD TO I>ETERMlNE
HOMOGENTISIC
ACID IN BIOLOGICAL
MATERIAL
B . N. La Du, V. G . Zunnmi, L. Laster and J . E . Seegmiuer, Bethesda, Maryland
Methods currently available for the estimation of homogentisic acid depend upon
its reducing properties and therefore lack
both specificity and precision when applied
to biological material. We have developed
a specific enzymatic method utilizing purified homogentisic acid oxidase to convert
homogentisic acid to maleylacetoacetic acid.
The ultraviolet absorption of the latter acid
is measured spectrophotometrically at 330
mp. The method is specific for homogentisic
acid since no other compounds are known
to be oxidized by homogentisic acid oxidase,
and as little as 1 or 2 micrograms of homo-
gentisic acid can be measured accurately.
After suitable deproteinization and extraction, honiogentisic acid can also be determined in tissues, such as cartilage, s k i n and
synovial fluid.
Plasma levels of homogentisic acid and
the variation during a single day have been
determined for nlcaptonuric patients. .4ttempts to detect a partial defect in the
ability to metabolize homogentisic acid in
heterozygous carriers of alcaptonuria are being made and some results of these studies
will be presented.
IN V m o FORMATION
OF 7s AND 19s GAMMA
GLOBULINS
BY TISSUES
FROM
NOFMALSUBJECTS AKD PATIENTS
WITH RHEUMATOIDARTHIWITS
Howard I. Levene, Edward C . Franklin and G. Jeunette Thorhecke, New York, N . Y.
Recent studies using fluorescein labelled
antibody techniques have demonstrated the
presence of 7s and 1% gamina globulins
in lymphoid tissues from normal and diseased human siibjects. These studies however could not distinguish between prodnction and storage of these proteins by the
tissues. In order to answer this question,
experiments have been perfonned to demonstrate the incorporation of C,, labelled
amino acids into gamma globulins by human lymphoid tissues in tissue culture.
Specimens of lymph node m d splenic tissue from 13 normal subjects and 3 patients
with rheumatoid arthritis were incubated
in a medium cop,taining ovalbumin and a
mixture of amino acids, Some of which were
labelled with C,,. After a 24 hour period
of incubation, the supemates were twice
absorbed with n diphtheria toxoid equine
antitoxin precipitate, following which gamma globulins, were specifically precipitated
with rabbit antisera against 7s or 19s
gamma globulins. The radioactivity of the
specific precipitate was compared to that
nonspecifically absorbed from the same culture fluid by the second absorption precipitate.
7s gamma globiilin formation was d s
tected in 13 of 28 culture tubes prepared
from specimens from 13 normal subjects
and in 3 of 9 tubes from 3 patients with
rheumatoid arthritis. 19s gamma globulin
fonnation was found in 7 of I0 tubes from
three patients with rheumatoid arthritis but
was never detected in 22 tubes prepared
with tissues from normal subjects.
Attempts to detect formation of rheumatoid factor by the F-I1 tanned cell agglutination technique and by measuring the
specific absorption of radioactivity to heat
precipitated human gamma globulin were
unsuccessful.
THEPRODUCTTON
OF 19s ANTIBODIES
IN ADULTSAND PREMATURE INFANTS
Joseph LoSpalluto, William M i l k , Jr., Chester Fink and Barbara Dorward, Dallas, Texas
Normal individuals, premature infants,
and patients with a variety of diseases have
been immunized with a mixed typhoidparatyphoid vaccine with a view toward
studying
duced in
obtained
course of
the size of the antibodies
response to these antigens.
after either one injection
three injections of vaccine
proSera
or a
were
118
AMERICAN RHEUMATISM ASSOCIATION
fractionated by anion exchange chromatography on DEAE cellulose. The 7s and
19s Containing fractions were examined for
antibody activities with standard agglutination tests.
In 23 individuals (group l ) , including
patients and normal subjects, who had received three weekly injections of the mixed
vaccine for the first time, the chief respcnse
three weeks after the initial injection was
the production of a preponderance of 19s
antibodies against each of the antigens.
Small amounts of 7s antibodies to three of
the four antigens given were found. Antibodies to Typhoid 0, the fourth antigen
administered, were consistently found in the
19s fraction only.
In another group, consisting of 32 individuals who had undergone previous immunization (group 2), the major response
observed was the formation of 7s antibodies. Comparison of the antibody levels
in &e 7s and 19s fractions in this group
has indicated that, although 19s antibody
levels are present, the 7s antibodies predominate.
Further studies on those individuals who
had produced a preponderance of 19s antibodies (group 1 ) has indicated that after a
three month period there is an increase in
the production of 7s antibodies with little
change in the titers of 19s fraction. In a
group of premature infants, however, an
almost complete conversion from the production of 19s to 7s antibodies occurred
within a three month period.
The results obtained indicate that the
earliest response to the antigens administered in this study has been the production
of 19s antibodies and that this is followed,
in time, by production of 7s antibodies.
Preliminary studies on a relatively small
group of patients with rheumatoid arthritis
suggest that conversion from 19s to 7s
antibody production requires somewhat
longer periods. Additional data, however,
are required.
DEPOLYMERIWTIONOF HYALURONIC
Acm BY BACTERIAL
HYALWRONIDASE:
THE QEAV-4GE OF THE GLYCOSIDIC
LINKAGE
Julio Ludowteg, Sir@ Vennesland and Albert Dwfman, Chicago, Ill. and
San Francisco, California
Hyaluronic acid is a straight chain heteropolysaccharide containing alternating units
of N-acetyl glucosamine and glucuronic acid
linked by glycosidic bonds [J.A.C.S. 76,
1753 (1954)l. Bacterial hyaluronidase degrades hyaluronic acid to a disaccharide
containing a double bond in its uronic acid
moiety [J.B.C. 279, 13 (1956)l. The enzymic degradation of hyaluronic acid may take
place by two different types of hydrolysis
that can be distinguished by carrying out
the reaction in a medium containing H,O*e.
Thus, the bond may be broken initially between C-1 of N-acetylglucosamine and oxygen or between C-4 of glucuronic acid and
oxygen. In the former case, the 1sOH from
the medium will be taken up by the C-1
of the N-acetylglocosamine moiety of the
hal product. In the latter case, the 18OH
of the medium would not appear in the
disaccharide product,
The results of degrading hyaluronic acid
with purified hyaluronidase from C1. Welchii
in a medium containing H201*shows that
the reaction does not involve any incorporation of label in the unsaturated disaccharide above the amount which occurred
as the result of exchange reaction unrelated
to the enzymatic degradation.
Polarimetric studies were also made of
the depolymerization of hyaluronic acid by
bacterial hyaluronidase. These studies show
that the reaction product first released must
have the same 't3-configuration at the reducing group as hyaluronic acid itself.
The above mentioned results provide
strong support for Meyer's conclusion [J.B.C.
219, 13 (195e)I that the reaction catalyzed
by bacterial hyaluronidase should be formulated as a simple elimination reaction.
SPONTANEOUS
A c m SEPTIC BURS^*
Andre March-,
Roberte S h i m , David S. Howell and Haroey E. Brown. Jr.,
Miami. Florida
119
AMERICAN HHEUMATISM ASSOCIATION
Attention is called to this entity because
of its apparent rarity. In this small series
(7 patients) all were young-to-middle aged
males without evidence of chronic internal
medical diseases. Involvement was confined
to the prepatellar or olecranon bursae. Organisms responsible for the infection were
identified as staphylococcus aureus in 6 patients and diplococcus pneumoniae in one.
Two patients had been under treatment
with adrenocortical steroid derivatives by
outside physicians who had labelled the
condition “gouty arthritis.” Six patients were
cured by antibiotics alone, administered according to, in vitro, sensitivity studies. One
patient required an incision and drainage
in addition to antibiotics.
Possible causes of acute septic bursitis
and the relationship of the present data to
the rising frequency of staphylococcal infections in the community will be discussed. Failure to recognize this illness may
result in its mismanagement.
‘Read by title.
IN RHEUMATOID
ARTHRI-IIS
PELVO-SPONDSLITIS
William Martel and Icun F. D u f , Ann Arbor, Michigan
Involvement of the pelvic bones in ankylosing spondylitis is well known but it is
not widely recognized that erosions frequently occur at the ischial tuberosities,
symphysis pubis and femoral trochanters in
otherwise classic rheumatoid arthritis. The
sacroiliac joints and cervical spine iue frequently abnormal in these individuals, but
the radiographic RS well as clinical features
STUDIESos
differ from ankylosing spondylitis.
There is a tendency among rheumatologists to underscore the clinical differences
between rheumatoid arthritis and ankylosing
spondylitis. Roentgen features of the pelvospondylitis in rheumatoid arthritis often
seems to support this differentiation. However, in some patients both conditions seem
to coexist.
URINARY
HYDROXYPROLINE
E . Meilman and M. Lrriuetsky, New Hyde Park, New York
Comparative studies of “bound” (or peptide) hydroxyproline excreted in urine have
been undertaken as part of a study of in
vivo collagen metabolism to compare excretion patterns in various connective tissue disorders.
Urine specimens (24 hour) were collected
from the following subjects after 24 to 72
hours of a hydroxyproline-free diet: a normal adult male, an adult female with
sclerodenna, an adult male with scleroderma, an adult male with Marfan’s syndrome, three postpartum females, and an
adult female with lupus erythematosus. In
addition, a urine from the normal adult was
collected after a normal diet,
Amino acids and peptides present in the
urine were isolated by batchwise adsorption onto and subsequent elution from
Dowex 50 ( 2 b 5 0 mesh). The eluates were
concentrated by flash evaporation and aliquots of the concentrates were used to
charge 1 x 1-50 cm. Dowex 50 (200-400
mesh) columns which were then eluted
using a p H and K+ ion gradient. Effluent
fractions were separated in a fraction collector; ninhydrin and hydroxyproline assays
were run on alternate tubes in order to 13rate hydroxyproline peptides.
T h e elution pattern showed 4 to 5 welldefined hydroxyproline peaks. The pstterns
obtained from the patients studied showed
remarkable qualitative similarity, with some
quantitative differences being noted in the
relative amounts of certain hydroxyproline
fractions. Free hydroxyproline was low,
varying from 0 to 0.3 mg. per 24 hour smple. The normai individual on a regular
diet showed 1.5 mg./24 hr. of free hydroxyproline; the postpartum women on a hydroxyproline-free diet showed 1.5-2.5 mg./
24 hr. on the third postpartum day, gradually diminishing over the fourth and fifth postpartum days. Bound hydroxyproline was
present in a range of 30 to 80 mg. per day,
the largest amounts being present in the
Marfan’s patient.
A detailed investigation into the nature
and composition of the isolated peptides is
in progress.
120
AMERICAN RHEUMATISM ASSOCJATION
DETECTION
OF CELLULAR
RHEUMATOIDFACTOR
WITH FLUORESCENT
IMMUNE
COMPLEX
Robert C. MeUors, Adam Nowoslawski, Leonhard Korngold and Beth L. Sengson,
New York, New York
In previous work (J. Exp. Med., 110, 875,
1959) fluorescein-labeled aggregated human gamma globulin was demonstrated to
be a sensitive reactant for the detection of
rheumatoid factor in frozen sections of
synovial membranes and lymph nodes obtained from patients with active rheumatoid
arthritis. Similar observations have now
been made with fluorescent immune complex and are reported herewith. The starting preparatory material was rabbit antiserum against bovine serum albumin. An
immune precipitate was formed at antigen
equivalence with fluorescein-labeled bovine
serum albumin and, after thorough washing, was converted to a soluble complex by
addition of labeled antigen at two times
equivalence. With this fluorescent immune
complex, cellular rheumatoid factor was detected in synovial and lymph node biopsies
obtained from adults and children with
active rheumatoid arthritis. Plasma cells in
the synovial membranes and germinal-center
cells, and rarely plasma cells, in the lymph
nodes contained cellular rheumatoid factor
demonstrable with the fluorescent complex.
In the identical tissues, cells of similar kinds
but in more abundant number also contained rheumatoid factor detectable with
fluorescein-labeled aggregated human gamma globulin. Comparable sections of normal and pathological tissues obtained in
diseases other than rheumatoid arthritis did
not stain with the fluorescent immune complex.
EXCRETION
AND STORAGE
OF MUCOPOLYSACCHARIDFS
IN HURLER’S
SYNDROME
Karl Meyer and Philip Hoffman, New York, N.Y.
In Hurler’s syndrome, two mucopolysaccharides, chondroitin sulfate B ( ChS-B) and
heparitin sulfate (hep. S.), are excreted in
the urine and stored in various organs. In
the majority of the cases studied, between
60 and 80 per cent of the urinary mucopolysaccharide is ChS-B and 40 to 20 per
cent hep. S. The mucopolysaccharides of
the organs vary greatly in type and quantity. Liver shows a great predominance of
hep. S. over ChS-B in five out of six cases,
while in spleen the ratio is reversed. In kidney and brain, the two polysaccharides are
present in approximately equal concentration. In two cases, the polysaccharides of
rib cartilage and bone were isolated and
characterized. In both cases, ChS-B was
isdlated from the cartilage and bone, while
the presence of hep. S. was questionable.
The major component was a mixture of
STUD^ OF
TKE
ChS-A and C (60 to 80 per cent) with C
predominating, and keratosulfate (12 to 30
per cent). Sections of these tissues showed
broad intensely stained collagen bands. It
is suspected that the abnormal presence of
ChS-B induces, in these organs, abnormal
fibrous tissues. Another striking feature in
the disease is the generalized involvement
of large and small arteries. This is of special interest since ChS-B and heparitin sulfate are normally major constituents of
aorta and probably of other blood vessels
and both mucopolysaccharides increase with
age andlor arteriosclerosis. The primary lesion of Hurler’s syndrome probably lies in
an abnonnal synthesis of ChS-B, which in
turn may cause a secondary release of the
chemically unrelated hep. S. presumably
from mast cells.
MECHANISM
OF AWORPTXON
AND SENSITIZATION
OF TANNED
SHEEP CELLS
WITH H
W GAMMA GLOBULIN
Irwin Oreskes, Jacques M. Singer and Bernard Liebennan, Brooklyn, N.
Tanned sheep cells adsorb human gamma
globulin in amounts proportional to the concentration of the added HGG. Logarithmic
plots of amounts adsorbed verses amounts
Y.
added are linear, corresponding to a Freundlich type of adsorption isotherm. Maximum
adsorption is about 1,500,000 HGG mole
cules per red cell. Under conditions of the
121
AMERICAN RHEUMATISM ASSOCIATION
FII S.C. procedure, and at a sensitizing
amount of 1,000 PgN, one red cell adsorbs
about 500,000 HGG molecules. When the
volume of the system is doubled, the number of molecules adsorbed per cell at 1,000
pgN added HGG decreases to 350,000. At
saturation each HGG molecule occupies
4,000 A2 of red cell surface. This is the
same area occupied by an HGG molecule
adsorbed in a monolayer to a 0.8 p diameter
latex particle. Both 7s HGG and aggregated
THEEFFECTOF NUTRITION
ON
HGG are adsorbed to tanned sheep cells,
and the corresponding isotherms are very
similar. However, only aggregated HGG is
capable of sensitizing tanned sheep cells
for reaction with rheumatoid factor. Maximum and constant titers were obtained with
tanned cells sensitized with 37.5 to 6,000
PgN HCG, even though the amount of
protein adsorbed increased continilally in
this range.
EXPERIMENTAL
“ADJUVANT DISEASE”
Carl M . Pearson, Shennan A4eUinkoff, Fae D . Wood, and Om01 Eshelman,
Los Angeles, California
Periodically within the past several years
evidence has appeared from various laboratories for the nutritional dependence of
( a ) increased bacterial resistance or SIISceptibility of a test animal, ( b ) the caliber
of antibody production, or ( c ) the susceptibility of an experimental disease such
as allergic encephalomyelitis.
Experimental “adjuvant disease” has
been produced in about 90 per cent of our
laboratory rats by the administration of
Freund‘s adjuvant and has been the subject
of previous reports. Its most constant feature is a polyarthritis, but often associated
also are a balanitis, an intis, a chronic dermatitis and mild diarrhea. The disease has
been postulated to be the result of a delayed hypersensitivity reaction to a component of the acid-fast bacillus in the adjuvant.
In the present study, utilizing over 200
animals, the disease was found to be quite
susceptible to dietary influences. Hence,
when weanling rats were maintained for 6
weeks prior to inoculation with adjuvant on
a diet which contained 50 per cent protein
(lactalbumin) as well as all dietary supplements, they were mildly resistant to “adjuvant disease”; on 80 per cent protein they
were significantly resistant; m d on 98 per
cent protein (and 2 per cent fat) they were
resistant at the highly significant level, Animals on high carbohydrate diet showed no
variation from control groups but those on
a high fat diet (corn oil) showed a greater
susceptibility, both in incidence and severity of various lesions. The animals on the
highest protein diet gained less weight than
did the other groups but appeared otherwise healthy. The inoculation sites healed
promptly and in none of the 40 animals in
this group was the arthritis more than mild
in degree.
It is postulated that dietary factors, notably protein or amino acid composition in
the present seric-s, may modify enzymatic
mechanisms within the body to the extent
that the normal mechanics of the delayed
hypersensitivity xesponse are grossly altered.
PREPARATION
AND PROPERTIES
OF HYALURONIC
Ac~D”
Ward Pigmun, S. Rizoi and Howard L. Holley, Birmingham, Ala. and New York, N. Y.
Roseman, Watson, Duff and Robinson
( 1955) have used an electrodeposition method for the isolation of hyaluronic acid from
a number of sources. The method has been
described more recently (1959) by Balazs
and Sundblad.
In the present work, the method has been
*Read by title.
applied to the preparation of hyaluronic
acid from bovine synovial fluid. After five
repetitions of the process, products were
obtained in yields of about 20 per cent
which appeared to be free of proteins and
only slilrhtly degraded relative to the original
synovial fluids. The intrinsic viscosity in 0.2
M pho.rphate buffer was about 60. The use
of 5 to 10 per cent ethanol or a small amount
122
AMERICAN RHEUMATISM ASSOCIATION
of tolue-ie in the solutions decreased the degradation and offered other advantages.
Purifiid hyaluronic acid products could
be stored as lyophilized powders or gels at
-10” if free of salts. With 20 per cent
ethanol present, solutions could be kept at
room temperature.
THEDEGRADATION
OF HYALUHONIC
ACIDBY SmuM PROTEINS*
Ward Pigmun, S. Rizci and Howard L. Hollcy, Birmingham, Ala. and New York, N. Y.
A hyaluronidxse-like action of blood serum, previously reported in the literature,
has been found to result from the depolymerizing action of certain blood serum fractions. Whole blood serum, serum proteinfractions, and other selected proteins have
been studied for their degrad:itive effect on
highly polymerized hyaluronic acid at p H
7.3 and 4.2. Degradations were assessed by
irreversible decreases of viscosity. It was
found that at pH 7.3 Cohn serum-fractions
IV-4 (-globulins) and V (albumin) and
other selected proteins caused a slow degradation. hole serum and fraction II-1,2
were found to have no effect at pH 7.3, but
at pH 4.2 they produced appreciable degrdation. The degradation by most serum
proteins had a pH optimum at about 3.0,
but was extensive at p H 4.2. The so-called
serum hyaluronidase, active at weak acidities, seems, thus, to be a nonspecific action
of several serum fractions and of other proteins.
Wead by title.
STUDIESON SERUMHAPTQGLOB~N
I N EXPERIMENTAL
DISORDERS
OF CONNECTIVE
TISSUE
Leslie Roberts, Paolo S . Mombelloni and Piwfranco Crosti, Chicago, Illinois
Serum haptoglobin ( H p ) was determined
by a colorimetric modification of Jayle’s “activation’’ method in guinea pigs and rabbits
before and after the induction of several
different types of connective tissue alteration. In guinea pigs, the experimental lesions
and the results obtained were as follows:
( 1 ) Guinea pigs with carrageenin granulomas had average Hp values about four
times higher than those of normal controls;
( 2 ) acutely scorbutic guinea pigs had average Hp values eight times higher than those
of normal controls; (3) scorbutic animals
bearing carrageenin granulomas had soniewhat lower values than the preceding group,
at five times the normal level; ( 4 ) pair fed
controls to the scorbutic animals, receiving
a 2.5 mg./day supplement of ascorbic acid,
had a significantly higher Hp value than the
normal controls.
In rabbits, the normal serum Hp is significantly higher than in guinea pigs. ExperiSTUDIESOF
TEE
mental procedures and results were as follows: ( 1) Biweekly subcutaneous injection
of turpentine leading to the production of
amyloidosis induced high titers of Hp. After
three months of treatment, values levelled
off, but in some cases, a second rise occurred after 4-S months. ( 2 ) I.V. injection
of papain in young rabbits produced the
well known cartilage changes and also a
very marked rise of Hp reaching a maximum
between 18 and 50 hours after treatment.
The average level then was four times the
initial value but may reach 7.5 times. ( 3 )
I.V. hyaluronidase injection produced a
moderate rise in Hp while injections of saline, ovalbumin, trypsin and chymotrypsin
induced no signilkant changes.
The possible relation of serum Hp to the
different types of connective tissue changes
obtained in the present experiments will he
discussed.
RABBITANTIBODIESWHICH SENSITIZERm BLOODCELLSFOR
AGGLUTINATION
BY RHEUMATOID
FACTORS
John H . Rockey and Hennj G . Kunkel, New York, New York
Observations from a number of laboratories have indicated that the sheep red cell
agglutinating and hemolytic antibodies in
rabbit antisera represent a mixture of the
high and low molecular weight types. However, little specific information is available
123
AMERICAN RHEUMATISM ASSOCIATION
about the characteristics of the rabbit antibody responsible for the sensitization of red
cells for agglutination by rheumatoid factors.
Separation of these two classes of antibodies
was accomplished by the use of zone electrophoresis, sucrose density gradient preparative ultracentrifugation and DEAE column
chromatography. Clear differences in their
capacity to sensitize cells in subagglutinating
titer for agglutination by rheumatoid factors
was demonstrated. The low niolecular
weight antibodis possessed the capacity in
high degree. Thr liigh molecular weight antibodies completely lacked this capacity. Observations on the use of antibody from individual rabbits of differing globulin allotypes and on rabbit incomplete antibodies
will be described. The rabbit incomplete
antibody was shown also to be of the low
molecular weight type.
DECREASED
RENAL EXCRETION OF URIC ACID AS
A
CAUSEOF HYPERURICEMIA
IN GOUT
J. E . SeegmiUer, Arthur I . Grayzel, Lois V . Liddle and Candace Pluto, Bethesda, Maryland
The cause of the hyperuricemia of gout
has been under investigation in recent years
with the use of isotopically labeled uric acid
and its precursors. An excessive production
of uric acid was found in a substantial portion but not in all of the gouty patients
studied. Seven of 16 gouty subjects showed
an incorporation of isotopically labeled
glycine into urimry uric acid that was indistinguishable from normal. Correction for
an increased extrarenal disposal of injected
uric acid-2-ClJ present in two subjects left
5 patients in whom no evidence of increased
uric acid production could be found by either glycine incorporation or urate turnover
values.
Renal function studies showed that the
group of gouty subjects who did not overproduce uric acid had urate/inulin clearance
ratios significantly lower than those found in
the group of “overproducers” of uric acid
or in a group of normal control subject. in
whom an increased uric acid production and
hyperuricemia was produced by the administration of ribose nucleic acid.
It seems unlikely that a single metabolic
defect common to all gouty subjects could
cause both an excessive production of uric
acid in some patients and a decreased excretion of uric acid in others. We are left then
with the view that the hyperuricemia of
gout is the result of a variety of metabolic or
physiological disturbances. Although excessive production of uric acid may be the
major factor contributing to hyperuricemia
in the majority of gouty subjects, a decreased
renal excretion D f uric acid can also be the
primary factor in producing a hyperuricemia
in the absence of excessive uric acid production.
TI~E
PRODUCTION
OF RENALDISEASE
EXPERIMENTAL STRF.PTOCOCCALINFECTIONS:
IN THE WHITEMOUSE
John T.Sharp, Detroit, Michigan
It has been reported that about 60 per
cent of male, Webster, white mice inoculated with more than 108 chains of live,
group A streptococci (strain D58, type 3)
developed albuminuria, hypoalbuminemia,
generalized edema, and histological changes
in the kidneys. These studies were extended
to examine daerences in host responses.
Thirty-seven female Webster mice infected
with more than 108 chains of D58 coccus
remained edema free. Only 1 of 24 male
CSI/BL mice and 5 of 46 male Hau-M mice
similarly infected developed edema.
Studies of dose response of male Webster
mice revealed that only 3 of 45 animals inoculated with between 107 and 10s chains
of streptococci developed edema, whereas
121 of 232 animals challenged with more
than 108 chains became edematous. Albumin
was observed in pooled urine samples as
early as the 3rd day of infection and was
excreted in largest amounts in those groups
in which a high proportion of animals later
developed edema (2.89 to 23.7 mg.!mouse/
day). Animals that did not develop edema
either excreted no albumin in their urine or
only small amounts !1.31 mg./mouse/day).
Serum albumin of 13 edematous animals
averaged 13.2 per cent of serum proteins.
In animals without edema, serum albumin
was 29.2 per cent of serum proteins for 32
streptococcus infected animals, 29.8 per cent
124
AMERICAN RHEUMATISM ASSOCIATION
for 14 E. coli infected animals, 24.2 per cent
for 4 staphylococcal infected animals and
39.2 per cent for 9 animals inoculated with
heat-killed organisms or sterile susppnsions.
It is concluded that male mice of the
Webster strain are more likely to develop
kidney disease when inoculated with large
numbers of group A streptococci (strain
PRESENCE OF
D58) than female Webster inicr or inalv
mice of other strains studied. This renal disease is manifested by the development of
albuminuria, hypoalbuminemia, and edema
in many instanccs. It is believed that these
manifestations reflect a glomendar lesion
induced by the streptococcal infection.
“SERDLOGICALLY ACTIVE MACROGLOHULINS”
IN SERA OF SOME
WITH ACTIVE PULMONARY
TUBERCULOSIS
Jucyues 31. Singer, Francisco A.!
PATIENTS
Perdta, Harold U.Lyom and Charles Id.Plotz,
Brmklyn, N.Y.
Sera from 220 cases of active pulmonary
tuberculosis were examined with the FII
latex particle procedure. It was found that
38 of these sera were positive, although none
of these cases e-xhibited clinical evidence of
rheumatoid arthritis. In addition, 12 sera out
of the 38 gave :a positive serological test for
syphillis. Presence of macroglobulins in FII
latex particle positive sera was deniocstratc.d
by analytical ultracentrifugation, DEAE cel-
lulose column chromatography, and density
gradient ultracentrifugation. Mercaptoethan01 treatment resulted in loss of FII laiex
particle activity. FII latex particle positive
sera and an equal number of matched negative sera were examined by paprr electrophoresis and livm function tests. No correlation was found between clinical activity and
serological activity, protein variations and
liver function tests in tuberculosis.
THEACTIONOF GRISEOFULVIN
IN ACUTEGOUTY
ARTHRITIS
Roberte Slonim, David S . Howell and Hnrliey E . Brown Jr., Miami, Florida
A fungistatic antibiotic, griseofulvin, was
observed to have a resemblance to colchicine in regard to structural formulae, interference with purine nietabolism and cell division. The agent was used to treat 22 acute
attacks of gouty arthritis in 20 patients. Four
to 12 Gm. of griseofulvin were administored
over a 48 hour period. Complete remission
of symptoms was obtained in 12 to 24 hours
without any definite toxic reactions, and
stiffness, pain, crythema, swelling and heat
decreased greatly or disappeared during this
period in 14 patients. Of the 6 patients who
had a slight or moderate response to griseofulvin, 5 had previously received colchicine
in adequate dosage without improvement.
Serum levels of griseofulvin in 5 patients indicated peaks of 6 to 9 pg. per cent at 18
hours.
If the antiarthritic response is produced
by colchicine and griseofulvin at the same
biochemical sites, certain similarity of their
chemical structure may provide a clue to
the active groups. However, a less specific
antiphlogistic action of the drug remains to
be excluded.
SERUM GLYCOPROTEIN
ABNORMALITIES
IN WHIPPI~E’S
DISEASE,INCLUDING
A FAMILY
STUDY
Mary Betty Steum, Baltimore, Maryland
There are conflicting data regarding the
character and significance of the increased
serum glywproteins reported in Whipple’s
disease, and family investigations have been
limited to siblings with this disorder. T h e
glycoprotein abnormalities in a proven case
of Whipple’s disease were recorded serially
over a 15-month period, during which the
‘Read by title.
clinical activity varied and corticosteroids
were not administered. Chemical and electrophoretic data were also obtained on 12
family members.
In the index patient, seromucoid, total
protein-bound hexosamine, and the hexosamine:protein ratio were all strikingly increased. Most of the glycoprotein elevation
was accounted for by increased seromucoid.
Electrophoretically, the elevation in the al-
1%
AMERICAN RHEUMATISM ASSOCIATION
pha-1 carbohydrate-rich globulin persisted
throughout the observation period; the increase in the alpha-2 glycoproteins was more
variable; and transient hypergammaglobulinemia was not associated with a concomitant increase in gammaglobulin glycoprotein.
Although the elevations of the glycoproteins
were maximal when the disease was clinically active, abnormally high levels continued during spontaneous remission of symptoms.
Serum protein and glycoprotein determinations were normal in 10 (2 male, 8 female)
of the 12 relatives studied. One brother, suspected clinically of having Whipple’s disease,
had persistent mild elevation of the alpha-1
globulin and a transient increase in the carbohydrate-rich components. The patient’s
only son had an “alpha3 globulin” electrophoretic pattern as the only abnormality;
the four daughters had normal findings.
DISSOCIATION
AND REASSOCIATION
STUDIES OF A 19s hlAC:ROGLOBUI.IN
RHEUMATO
FACTOH
~
YHOPEH~TES
WITH
Thomus B. T o m s i and Henry G . Kiinkt.1, New York, N. Y.
Human 19s macroglobulin of both normal
and pathological sera can be dissociated by
means of sulfhydryl compounds into 7s subunits. Recently certain well d d n e d 19s antibodies including Rh saline agglutinins, isohemagglutinins, and cold agglutinins have
been found to dissociate in a similar manner.
In all well defined instances dissociation results in a complete loss of biological activity.
Rheumatoid factors which resemble this class
of antibodies loses on dissociation their ability to precipitate aggregated gamma globulin
as well as their activity in the latex and
sensitized sheep cell tests. Despite partial
reaggregation to 19s material following removal of the reducing agent, in none of the
reported cases has biological activity been
regained.
A 19s macroglobulin has been encountered recently in the serum of a patient with
the diagnosis of Waldenstom’s macroglobulinemia which behaved like a rheumatoid
factor in a variety of ways, including its
ability to give a precipitin reaction with ag-
gregated gamma globulin. This macroglobd i n was isolated and dissociated to 7s subunits in .1 Methylmercaptan. Renioval of
the sulfhydryl reagent resulted in reassociation into a heterogeneous group of higher
polymers, of which the major constituent was
19s. Dialysis against iodoacetamide completely prevented reassociation.
Quantitative precipitin analyses performed
on the native, dissociated, iodoscetamide
treated, and reaggregated products indicated
a complete loss of precipitin activity in the
dissociated and iodoacetamide treated samples. However, the reaggregated product
showed a return of about 90 per cent of the
activity of the original preparation. This return of activity could be blocked in large
part by reassociation in the presence of dissociated heterologous inacroglobulins. The
results are interpreted in terms of the structure of the macromolecule. They suggest
that the high molecular weight configuration
is of primary importance in determining the
activity of this molecule.
OF THE NUCLE~PR~TEIN-REACTIVE
GAMMAGLOBULINS
IN
QUANTITATIVE
ESTIMATES
SYSTEMICLUPUSEHYTHEMATOSUS
Ahan&
S. Tounes, C . R. Steuart, Jr. a d dbrahnin G. Osler, Baltimore, Mary!and
As a basis for immunological studies in
systemic lupus erythematosus ( SLE ) and
related disorders, effort has been directed
toward developiilg a method for quantitative
estimations of gamma globulins which r.=act
with aucleoprotein. Particulate suspensions
of calf thymus niicleoprotein are reacted with
dilutions of test serum. Particles are recovered by centrifugation and thoroughly
washed in isotonic saline. The amount of
gamma globulin bound to these particles is
then estimated by means of a quantitative
complement fixation reaction with a constant
quantity of rabbit anti-human gamma globulin serum. Results are expressed as equivalents of gamma globulin nitrogen by reference to a calibration curve established under
identical conditions with known amoiints of
human gamma globulin.
In patients with SLE, whose sera induce
AMERICAN RHEUMATISM ASSOCIATION
L.E. cell formation> the quantity of equivalent gamma globulin ranges from approximately 10 to 40 micrograms nitrogen per
ml. (reproducible within 10 per cent). In
these sera, uptake of gamma globulin by
nucleoprotein is associated with loss of antinuclear antibody as defined by a fluorescent
anti-globulin technique.
Values for normal sera range from 0.5 to
3 micrograms nitrogen per ml. Some rheumatoid and hyperglobulinemic sera give
EVALUATIONOF
THE
AGGLUTINATIDNS
IN
values above the normal range, occasionally
as high as those for SLE sera. In a preliminary series these reactive sera also give a
positive FII latex test in high titer. In contrast, SLE sera have shown negative FII
latex tests. Further studies are underway to
expend these observations in order to clarify
the nature of interaction between gamma
globulin and nucleoprotein in SLE as compared to other disease states.
SENSITIZED
HUMANCELL, SENSITIZED
SHEEP CELL AND THE LATEX
INDIVIDUALS,
IN PATIENTS WITH ~ U M A T O I DARTHRITIS A N D
PATIENTSWITH OTHER DISEASES
NORMAL
M. V. Wuller, B. Decker, E . C. Toone, W. R . 1rby und N . E l . Cur?/, Richmond, Virginia
This study was designed to ( 1) evaluate
individuals with “false positive” reactions
for rheumatoid factor, ( 2 ) compare the
SHC with the better known latex and SSC
agglutinations and (3) correlate the three
types of rheumatoid serological reactions
with various clinical features of rheumatoid
arthritis. The SHC tests were done by the
method of Waller and Vaughan, the SSC
tests by the method of Heller, and the FII
latex agglutination was done with commercial reagents ( Hyland Laboratories ) . The
study was carried out on a total of 500 patients which included 340 normal individuals, 87 patients with rheumatoid arthritis
and 73 patients with other diseases.
There were 14 (49;) positive reactors
detected among the 340 seemingly normal
individuals chosen from among blood bank
donors and a prenatal clinic. The SHC agglutination was positive in 3 per cent, the
latex in 3 per cent and the SSC in 0.3 per
cent. Nine of the 14 reactors were studied
in greater detail and 5 had either suggestive
family histories, mild rheumatic symptoms or
hypergammaglobulinemia. No abnormalities
were found in the remaining 4. A detailed
serological and clinical study of the normal
individual with the highest titre of rhewnatoid factor (SHC-2048,SSC-256), followed
bi-monthly for two years, will be presented
in detail.
In 87 cases of rheumatoid arthritis, the
rheumatoid factor was found to be present
in 89 per cent; in 82 per cent by the SHC;
in 86 per cent by the latex; and in 86 per
cent by the SSC agglutinations. Positive
agglutinations for rheumatoid factor were
significantly more common in stage 111-IV,
classic-definite rheumatoid arthritis but could
not be correlated with age, duration of disease, nodules, sex or race.
These studies indicated that ( 1) the rheumatoid factor may be present in normal
persons, (2) the SHC method, as used here,
parallels the latex agglutinations, both mc-thods being more sensitive and less specific
than the SSC agglutination, ( 3 ) positive agglutinations for rheumatoid factor were more
common in classic-definite, stage 111-IV rheumatoid arthritis.
OF DEEP SOMATIC
PAIN*
A QUANTITATIVE
MEASURE
B. Berthold Wolfl and Murray E . Jumik, New York, N. Y.
Pain threshold as well as tolerance and reaction to pain may form good bases for the
prediction of success in the rehabilitation of
the chronic arthritic patient, but an adequate
objective and quantitative e.xperimenta1
method is required for this. A modified ver‘Read by title.
sion of Lewis’s and Kellgren’s technique was
followed by injecting hypertonic saline intramuscularly to produce deep somatic pain,
which is subjectively described as a poorly
localized, long-lasting and unpleasant dull
ache, starting several seconds after injection,
increasing in intensity during 10 to 45 seconds, and persisting at least 30 seconds. In
127
AMERICAN RHEUMATISM ASSOCIATION
this investigation, using the verbal responses
of 14 normal subjects, it was found that ( a )
the best technique consisted of using 25
gauge 1%inch hypodermic needles, inserted
intramuscularly at an angle of 45" through
the anesthetized skin in rosettes of 4 each,
so that in the muscle the points of the needles were about 1 inch apart; ( b ) intradermal injection of procaine satisfactorily abolished the perception of cutaneous pain; and
that ( c ) volume of saline and rate of injection were important variables in the production of deep somatic pain. However, when
tested with isotonic saline, a quantity of 0.1
or 0.2 cc. injected slowly satisfactorily minimized error due to volume or pressure. In 10
subjects the mean deep somatic pain threshold of the gastrocnemius on injection of 0.1
cc. of hypertonic saline at &minute intervals
was found to be 2.34 NaCl, with a median
of 2.5% NaCl and a standard deviation of
-C 0.69 per cent NaCl. In another experiment with 7 subjwts the mean deep somatic
pain threshold of the gluteus maximus on
injection of 0.2 cc. of hypertonic saline at
2-minute intervals was found to be 4.5 per
cent NaCI, with a median of 5.0% NaCl and
a standard deviation of 1.61% NaC1. In addition 2 chronic arthritic patients in a pilot
study described the experimentally produced
pain as similar to their arthritic pain. The
subjective deep somatic pain responses,
measured as percent concentration of saline,
are also meaningful psychophysically, because they range from zero at an isotonic
level to 100 per cent at 4 per cent NaCl
for the gastrocnemius and at 8 per cent NaCI
for the gluteus maximus. Therefore, these
results suggest that the hypertonic saline
technique is a satisfactory objective and
quantitative method for the measurement of
deep somatic pain.
ADMINISTRATION
OF TRIAMCINOLOXE
ACETORIDE AND TRIAMCKNOLONE
DIACETATE
IN THE TREATMENT
OF RHEUMATOID ARrHIuTlS: h E L I M I N A R Y &PORT*
INTRAMUSCULAR
Jack Zuckner and Robert H. Ramey, St. Louis, Missouri
The acetonide and diacetate derivatives
of triamcinolone were injected intramuscularly into patients with rheumatoid arthritis.
There were 73 injections, 44 with triamcinolone acetonide and 29 with triamcinolone
diacetate. The dose per injection was 100
mg. The maximum duration of benefit was
49 days after an acetonide injection and 50
days after the diacetate form, averaging 18.1
and 15.4 days, respectively. Eighteen of 21
patients receiving triamcinolone acetonide
and 15 of 19 receiving triamcinolone diacetate had satisfactory responses. (The latter
was defined as greater than 50 per cent improvement for a period of seven or more
days. ) Such improvement followed approximately two-thirds of injections with either
derivative. When compared to hydrocortisone acetate injected intramuscularly in 100
and 500 mg. doses, the triamcinolone prepsrations gave greatly superior results, particularly in reference to duration of effect.
Fifteen patients were receiving other steroids orally at the time the intramuscular injections were administered. In about half
these individuals the close of the oral steroid
could then b e lowered, and in several patients, the total of the oral plus the parenteral
dose was less than the total oral dose given
previously.
Toxic reactions were as follows: euphoria,
2 patients; leg cramps, 2; nausea, vomiting,
anorexia, and weakness, 3. The duration of
the study did not allow long term evaluation
of toxicity.
*Read by title.
--
-
-
A.R. A. N E W S
ABSTRACT DEADLINE FOR 1961
ANNUAL MEETING Is
Those wishing to present papers at the
1961 Annual Meeting (June 22 and 23,
Hotel Roosevelt, New York City) should
send ten copies of abstracts (maximum
length: 250 words) to the Program Chairman, Dr. R. W. Lamont-Havers, Room 1700,
10 Columbus Circle, New York 19, N. Y.
128
The Program Committee will make special
efforts to select for the meeting’s plenary
sessions a relatively large number of clinical
papers, if their quality merits acceptance.
Abstracts of all papers on the program and
a selective list of those to be read by title
will be published in the journal, ARTHRITIS
A N D RHEUMATISM. Ten minutes are allowed
to each speaker, followed by a five-minute
discussion period.
There may be a series of three specialized
concurrent sessions. The Chairman of each
of these sessions will report a summary of
the proceedings to the plenary sessions so
that everyone in the audience will have an
opportunity to inform himself of the highlights of all sessions.
Among other plans is a panel discussion,
jointly sponsored by the A.R.A. and the
American Academy of Orthopedic Surgeons,
on “Reconstructive Surgery of the Shoulder,
Elbow and Knee.”
For the first time, the Program Committee will select a Westhoff Memorial Lecturer. This lecture, to be given for a series of
annual meetings, is named in memory of the
late Mrs. Anna H. Westhoff of St. Petersburg, Florida, who bequeathed a substantial
part of her fortune to the American Rheumatism Association.
Dr. John Talbott, editor of the J o u m l of
the American Medical Association, will give
a luncheon address on the subject of “Trials,
Triumphs and Thwartways of a Medical
Editor.”
The annual business meeting has been
scheduled for the afternoon of June 23.
To bridge the gap between the A.R.A.
meeting and the annual convention of the
American bledical Association (June 26-30),
the New York Rheumatism Association is
planning a postgraduate seminar on arthritis
and rheumatism at the Roosevelt Hotel, Saturday, June 24 and Sunday morning, June
25.
The New York Rheumatism Association
will also be host to A.R.A. members and
their wives at a reception Thursday evening,
June 22, at the Hotel Roosevelt’s Terrace
Room.
Members will receive room reservation
forms several weeks before the meeting,
Printed programs will be distributed to the
membership early in June.
A limited number of rooms will ;dso be
A.R.A.
NEWS
available at Olin Hall, residence hall for
the Cornell Medical School, 69th Street
and York Avenue. Accommodations ($3.00
per day per person) are available for both
men and women. A few double rooms are
also available. Olin Hall is within convenient
distance from the Roosevelt Hotel.
Room rates at the Roosevelt Hotel range
from $8.50 to $1850 for singles, and $13.50
to $23.50 for doubles.
For the entertainment of members and
their wives, the Local Arrangements Committee and the Ladies’ Sub-committee have
set aside a hospitality room, immediately
adjoining the Roosevelt’s Grand Ballroom,
site of the plenary sessions. Information on
shopping, sightseeing and entertainment
will be available at the Hospitality Room.
A block of 50 tickets for the Loew-Lerner
Broadway musical, “Camelot,” has been
secured. Out-of-town members who wish to
obtain tickets for Friday, June 24, will be
given preference. To reserve tickets, send
your reservation and check to the American
Rheumatism Association, 10 Columbus Circle, New York 19, N. Y. All tickets are
priced at $8.05.
DALLAS INTERIM MEETING
A total of 36 papers was presented at the
Seventh Interim Scientific Session of the
A.R.A. in Dallas, Texas, December 9 and
10, 1960. The meeting was attended by
nearly 300 physicians, more than half of
these A.R.A. members.
A panel discussion on lupus nephritis
was moderated hy Dr. Halsted R. Holman.
Panel members were Drs. Robert M. Kark,
Lawrence E. Shulman, Conrad L. Pirani
and Howard Worthen.
At the business meeting, 115 new applicants were elected to membership. This
brings the total of new members gained
during the last twelve months to 226.
Among distinguished visitors from abroad
were Professor Franpis Coste, of Paris,
France, President, International League
against Rheumatism, and Dr. Oswald Savage, of London, England, Secretary of the
Empire Rheumbtism Council. Professor
Coste, as guest of President Dr. Edward F.
Hartung, attended not only all scientific sessions but also the all-day meeting of the
A.R.A.’s Executive Committee.
The Dallas educational television station,
A.R.A. NEWS
KEFU, devoted a half-hour program to the
subject of arthritis. Participating in this
round-table discussion were Drs. Edward F.
Hartung, Morris Ziff, Howard C. Coggeshdl.
R. W. Lamont-Havers and MI. Robert
Straws, President, North Texas Chapter,
Arthritis and Rheumatism Foundation. The
Foundation’s Dallas Chapter helped to underwrite part of the cost of thtb scientific
sessions and presented President Hartnng
with a ten-gallon hat.
POSTGRADUATE COURSES
FROM MARCH-MAY 1961
Among postgraduate courses on rheumatic disorders scheduled for the next few
months are two at New York University
Medical Center and one at the IJniversity
of Michigan, Ann Arbor.
A special coiirse for the experienced clinician and research worker, under the direction of Drs. Cuirier McEwen and Edward
F. Hartung, will be given March 13 through
March 17, 1961 at New York University
Medical Center. Tuition will be $100. For
application, write to the Office of the Dean,
New York University Post-Graduate kledical School, 550 First Avenue, New York 16,
N. Y.
The course is designed for those physicians who are already familiar with the
fundamental data essential to an understanding of arthritis and related disorders.
In general, applicants should have had five
or more years of experience in an arthritis
clinic or its equivalent. Standard differential
diagnosis and treatment will not be covered.
Particularly stressed will be newer concepts
of etiology, newer research technics and
recent advances in our !,asic knowledge of
what underlies these disorders.
Among the guest lecturers will be Drs.
Ward Pigman, New York; Jerome Gross,
Boston; C. William Castor, Ann Arbor;
129
Philip H. Henneman, Jersey City; Maciyn
McCarty and Alexander Gutman, both of
New York.
The same Center will hold another course
for general practitioners May 15 through
19, 1961. Clinic and bedside teaching will be
stressed. Tuition is $85.
A postgraduate course in rheumatology is
being offered April 24, 25 and 26, 1961 by
the University of Michigan, Department of
Postgraduate Medicine, at the University
Hospital, Ann Arbor.
The course consists of three days of instruction on the newer coiicepts of diagnosis
and management of rheumatic disease, including lectures, case demonstrations, as
well as consideration of laboratory aids and
physical therapy.
Dr. Richard H. Freyberg, Professor of
Clinical Medicine, Cornell Univeriity, will
participate as guest lecturer in the program.
The prograni and application forms may be
obtained from Dr. John hl. Sheldon, Director, Department of Postgraduate Medicine,
University Hospital, Ann Arbor, Michigan.
NEW BIBLIOGRAPHY AVAILABLE
The first three issues of The National
Foundation’s new bibliography on Arthritis
and Related Diseases contains abstracts of
192 articles from 91 journals. Of these, 124
articles were from 46 American journals,
and 68 from 45 foreign journals. .4.R.A.’s
official journal, ARTHXUTIS AND R H E m ~ A T I s M ,
contributed the largest number ( 3 2 ) from
any one journal.
The monthly biblography is distributed
to 3,500 professional individuals and institutions, including medical and associate
professional schools, voluntary and governmental agencies. In addition, it i5 sent to
651 libraries in this country and abroad,
and to 370 medical journals.
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