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Proceedings of the Sixth Interim Scientific Session of the American Rheumatism Association December 1959.

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AMERICAN RHEUMATISM ASSOCIATION
10 Columbus Circle, New York 19, New York
President: CHARLEY
J. SMYTH,M . D . , 4200 EAST OTH AVENUE, DEKVER 20, COLORADO.
First Vice President and President Elect: EDWARD
F. HARTUNC,M.D., 580 PARK
AVENUE, NEW YORK 21, NEW YORK.
Second Vice President: DONALD
C. GRAHAM,
M.D., 34 ANNESLEY AVENUE, TORONTO, ONTARIO, CANADA.
Secretarg Treasurer:
FELIXE. DEMARTINI,
M.D., 622 WEST 1 6 8 STREET,
~ ~
NEW YORK 32, NEW YORK.
Executive Secretary: GERARDW. SPEYER,10 COLUMBUS CIRCLE, NEW YORK 19,
N E W YORK.
Proceedings of the Sixth Interim Scientific Session of
the American Rheumatism Association
December 11, 1959
Henry Ford Hospital, Detroit, Michigan
EDITOR:
William S. Clark
EDITORIAL ASSISTANT:
Alargnret Walsh
PLENARY
SEssIow-Presiding:
1.
DR. CHARLEY SMYTH
STUDIESOF ALCAPTONURIA:
A GENETICSTUDYOF 47 CASESOCCURRING
IN EICIIT
GENERATIONS
OF EIGHTINTERRELATED
DOMINICAN
KINDREDS
Robert Austin A4ilch, Baltimore, Md.
Previous investigations have generally concluded that the alcaptonuria syndrome
is inherited as a simple, recessive character. A number of studies have appeared,
however, which have suggested that the alcaptonuria gene may either behave as a
dominant in a “restricted range of variation,” or may appear to be an incompletely
penetrant dominant. Accordingly, three families, including those initially reported
by Osler, by Smith, and by Milch and Milch have been followed through additional
generations. The family reported by Pieter in 1925, and generally conceded to be
the only apparently unequivocal instance of dominance in the literature (other than
some recent insufficiently followed rases reported from Czechoslovakia) has been
found to be directly related to that reported by us in 1951.
A family study has been conducted of all known relatives of the two initially
reported kindreds. Data have been obtained of individuals in eight generations of
eight highly interrelated kindreds residing in the Dominican Republic and in various
other areas of North and Central America. Despite apparent, though spurious dominant transmission occurring in four successive generations of one kindred and despite a significantly high incidence of concomitant herniae and mental disease in
several kindreds, the observed data support the hypothesis that, in this family stuay
as in other less extensive pedigrees, the alcaptonuria syndrome is inherited as if in
consequence of a rare, simple, autosomal recessive allele.
238
239
AMERICAN RHEUMATISM ASSOCIATION
DISCUSSION OF PAPER 1
DR. JOSEPH BUNIM (Bethesda, hld.): I
wish to congratulate Dr. Milch on this illuminating family study. We appreciate the
tenacity and perserverance it required to
complete the pedigree of 8 generations,
especially since to do so involved traveling
to a foreign country. The results go far
toward resolving the controversy as to
whether the defective autosomal allele in
alcaptonuria is inherited as recessive or
dominant. The author himself, if he will
pardon this personal reference, embodies
a welcome mutation from orthopedics to
genetics.
2. INDUCTION OF POLYARTERITIS NODOSA
BY TRANSPLANTATION OF PITUITARY GLANDS
OF POLYARTERITIC
RATSINTO CEREBRAL HEMISPHERES
O F HOSTRATS
James C. Perry,
Milwa,dee, Wis.
Investigations carried on over the past five years have shown that polyarteritis
nodosa may be induced in male rats by administering subcutaneous injections of
estradiol proprionate for 30 days followed by injection of FSH (follicle-stimulating
hormone) for another 30 days. The vaculitis, however, does not develop until 4 to 6
months after the cessation of treatment.
In addition to extensive involvement of mesenteric, testicular and other arteries
a majority of treated animals have been found to have neoplastic pituitary and
adrenal glands. Evidence supports the inference that the tumorous pituitaries are
primarily responsible for the development of the vasculitis. The disease does not develop in treated hypophysectomized animals. In intact treated animals, evidence
indicates that tumorous pituitaries induce neoplasia in the adrenals which in turn
are productive of the arteritis.
To test the validity of this inference pituitaries from polyarteritic animals were
transplanted into the cerebral hemispheres of untreated rats. To date polyarteritis
nodosa has appeared in 50 per cent of host animals. The neoplastic transplants have
proliferated in the brain, host adrenals have become neoplastic, and mesenteric,
testicular, splenic and other arteries have become polyarteritic. Many animals, both
hosts and experimental donors, exhibit concomitant arthritic and rheumatoid reactions particularly in the hind feet and ankles.
DISCUSSION OF PAPER 2
DR. JEROME ROTSTEIN
(New York, N.
Y . ) : Dr. Perry, could you please tell me
what prompted you five years ago to give
a course of pituitary injections to cause the
syndrome?
DR. PERRY: Yes, doctor. Previous to that
time I had been using estrogen in various
experiments and as a result of using estrogen I noted, as we all will, particularly extensive atrophy in the seminiferous tubules.
I thought if we gave FSH we might restore
the normal condition and so we proceeded
to give the FSH injection. Obviously, there
was some restoration, but in order to determine whether there would be any residue
of injured testicular cells remaining, we
decided to autopsy animals at periodic in-
tervals after the cessation of injections, so
we more or less stumbled on the polyarteritis as a result of that.
DR. CURRIER MC EWEN
(New York, N. Y ) :
May I ask Dr. Perry if this picture ever
develops if he uses only one of the agents.
Have any animals been treated with other
hormones, as for example, corticosteroids,
in a similar way and has anything like this
occurred?
DR. PERRY: First of all we have run
several controls in which we gave only
estrogen and allowed them to continue after
treatment. I have not observed any evidence
of polyarteritis, 6 to 7 months after treatment. It is possible that it might develop,
AMERICAN RHEUMATISM ASSOCIATION
presuming that the secretion FSH is stimulated somewhere along the line of that
post-treatment period. Secondly, as to the
administration of other corticosteroids, Selye
and his workers, of course, have administered desoxycorticosterone in connection
with high salt and protein diet and they got
polyarteritis or periarteritis, as he calls it,
and, of conrse, that occurs or develops
r‘ither rapidly, in about 31 days. I feel
that in our animals we are causing thc
disease hy the development of soine endo-genons factors which are most likely incream1 corticosteroids from the tnniorons
adrenals.
1111. CONHAD L. P l H A N l (Chicago, 111. ) : Dr.
Perry, are these animals hypertensive and/or
do they develop renal insufficiency?
DH. PEKHY: As far iis we can answer that
question it seems that some of them do
develop hypertension-not
all of thein.
Renal insufficiency is a n accompanying
phenomenon certainly. There are numerous
changes in the kidney which would indicate
that tlierc are rather definite, although not
cxtensivc, pathological changes.
(New York, N. Y. ) : Arcs
there any species differences? Does this
iipply to any particular strain of rats? Does
this occur only in old rats?
1 ) ~ . CHARLES HAGAN
usually iise aniiiials that are about 6 months
to one year old when we start treating them.
Dn. JOSEPH E. WARREN (Pittsburgh, Pa. ) :
These experiments suggest that a rebound
phenomenon is a critical part of the pnthophysiology involved. After initial omission
of the estrogen and FSH therapy, have yon
had occasion to continue the estrogen
therapy for several months and see whether
it inhibits the appearance of periarteritis?
DR. PEHRY: We have not carried it on
for several months. We have given estrogen
after FSH and we have found that orrliniirily we do not get too much polyarteritis
with the estrogen alone but if we give the
FSH again after the estrogen we (lo get
;I very pronounced polyarteritis.
im. wAiuxN: And always with an interval
of several months after omission of the provocative therapy?
DH. m n w : Yes. In that case it would be
about 2 or .3 inonths after the omission.
n i ~
MAKHY BAHTFELO (New York, N. Y.) :
Have yon been able to examine the pituitaries of patients who died of polyarteritis
nodosa?
1x1.
PEHHY:
You ineaii our experimental
aniinals?
n ~ P’EHHY:
.
Well, we have used three
different strains, Sprague-Dawley, Holtzinan ancl a nondescript hooded group, ancl
we have obtained it in all strains used. We
I)R. HAHTFELU:
DH. PEnrw:
NO, humans?
No, I have not done that.
3. PITUITARY-ADHENAL FUNCTION
IS PATIENTS \VITH RHEUMATOIDARTHRITIS
FOLLOWING
STEROIDTHERAPY
J. Willium Meukin, Mariu S . Tantongco, Jecin Crabbe, Theodore B. Bayles,
and Don H . Nelson, Boston,
mass.
Sixteen patients with rheumatoid arthritis from whom steroid therapy had been
withdrawn were studied with respect to their adrenal and pituitary ( A C T H secretion) function. Six similar patients who h a d never received steroids were likewise
studied.
Adrenal function was estimated by the urinary excretion of 17-hydroxycorticosteroids ( 17-OHCS) and 17-ketosteroids ( 17-KS ) before and after standard
infusions of ACTH. Pituitary function was estimated by the plasma A C T H level
aiid the urinary excretion of 17-OHCS a n d 17-KS following the administration of
an inhibitor of adrerial 11-B hydroxylation (Ciba, SU 4885).
In the six patients never treated with steroids, adrenal function was low or sub-
241
AMERICAN RHEUMATISM ASSOCIATION
normal. In 3 of 4 patients with adequate SU 4885 tests, pituitary function appeared
intact. In one patient, a measurable (i.e., elevated) plasma ACTH was found.
Nine of the 16 steroid-treated patients showed 17-OHCS responses to ACTH
below the average range for the nonsteroid-treated group, and all showed responses
below t h e mean response in healthy subjects. Following SU 4885, plasma ACTH
was measurable in six of the steroid group, four of whom h a d adrenal responsiveness
to exogenous A C T H below the mean of t h e nonsteroid-treated group.
Thus, the debility of rheumatoid arthritis may be associated with a decline in
adrenal function which is aggravated by the suppressive action of steroid therapy.
I n contrast, ACTH secretion by the pituitary is better preserved and recovers more
quickly following steroid suppression. These observations suggest that the use of
. mav
. maintain adrenal function.
periodic A C T H administration during steroid therapv
DISCUSSION OF PAPER 3
UH. WILLIAM HOBINSON
(Ann Arbor,
Mich.): I would like to ask what was the
average interval between cessation of
steroid therapy and the carrying out of
these tests? We were told of an interval of
3 months for one subject. I would be particnlarly interested in whether the six snbiects on whom you gave detailed information had somewhat different intervals than
the other 10.
i m . MEAKEN: I cannot recall the average
interval figure bnt it varied from 3 weeks
to 17 months in the six subjects with measurable plasma ACTH and from 1 week to
18 months in the other ten.
DK. CIiAnLEY SMYTH (Denver, Col. ) : Are
you recommending now that every patient
who is on long-term steroid therapy receive
ACTH and, if so, at what interval and in
what dosage?
regarding the comment on ACTH, for good
and welfare. I wonder whether it is based
on observations of actual periodic or coinbined administration of ACTH? I wonder
what really is accomplished by combined
therapy or by the periodic administration
of ACTH in patients who are going to be
put back on corticosteroids. It means interrupting a procedure which is being carried
out, a very ineddlesonie process-taking
a
patient off corticosteroids and after giving
ACTH again putting the patient into a deficiency and periodically doing that. I wonder whether these recommendations are
based on a series of patients or whether
they are deductive from the data reported.
I am looking for practical information.
DH. MEAKEN: I did not quite clearly get
your question, sir?
DR.
STXINBROCKER:
Are your comments
on the benefits of combined or intermittent
DR. MEAKEN: I am really not in a position to recommend the interval and the
dosage. I think Doctor Young’s group, which
published a paper in 1957 has done some
work on this, but it would seem that anybody who received 50 mg. of cortisone or
the equivalent for a period of longer than
2 weeks probably should receive intramuscular ACTH, perhaps in a dose of 100
iinits twice a week, if it is anticipated that
they are going to be withdrawn from
steroids. If you anticipate that they are
going to be on steroids for the remainder
of their life, obviously it is not necessary
to maintain their adrenal function.
im. OTTO STEINBROCKER (New York, N.
Y. ) : I would just like to bring up a question
use of ACTH with corticosteroid therapy
based on a series of patients actually so
treated or, are they deduced from the claka
presented?
UH. MEAKIN: No. I only suggested that
intramuscular ACTH be given to people,
while they are getting the steroid, in a low
enough dosage to maintain their adrenal
function. I was suggesting 100 units intramuscularly twice a week based upon the
studies of Dr. Young and his colleagues published in 1957.
UR. STEINBROCKER: I would like to inquire
into your answer again, if I may. This is
just for clarification. Have you found from
any direct clinical observation on the concur-
242
AMERICAN RHEUMATISM ASSOCIATION
rent administration of ACTH, while patients
are getting corticosteroids, that it makes a
contribution to the patient’s symptoms,
ACTH level, and so on?
DR. MEAKIiv: Not personally, no. 1 am
only transposing from what I studied in
these patients with respect to their pituitary and adrenal function which demonstrate that the pituitary remains intact but
the adrenal does not. In association with
the studies done by other people, showing
that they can maintain adrenal function by
giving periodic ACTH during steroid therapy, I am suggesting that this is a reasonable thing to do because during steroid
therapy it would appear that there is a
decline in adrenal function which may he
irrevocable, while the decline in pituitary
function is transitory.
DR. CHARLES RAGAN (New York, N. Y.):
In our opinion, several qualifications should
be made to each of the statements made in
the summary of this paper. In our experience, adrenal suppression as measured by
the rise in serum levels of ll-hydroxycorticosteroids following the intravenous administration of ACTH is not uniformly observed. By and large, it is dependent on
dose hut there are numerous exceptions to
this. If a patient is receiving small doses of
steroids, say under 500 mg. per day, we
have been, in general, unable to find any
evidence of adrenal unresponsiveness. If
the patient is receiving big doses of steroids,
we have not found that p a r e n t e d ACTH
for 2, 3 or even 4 days, has made their
adrenals responsive. W e have also noted
the development of sensitivity to a given
species ACTH when we have attempted to
use intermittent corticotropin during longterm steroid administration.
DR. HUGH A . SMYTHE (Toronto, Canada) :
Your data would suggest that the adrenal
unresponsiveness is the same in patients not
treated with steroids as in the pcitients
treated with steroids, particularly if you
take out your 76 year old lady from the
steroid group. Therefore, it would follow
that you should also treat your nonsteroid
patients with intermittent ACTH as well.
mi. MEAKIN: Yes. I did not present
sufficient data perhaps in these slides to
show that there was a small difference in
the group and we really do not have a big
enough group to argue it successfully. With
16 steroid-treated patients, 9 of these patients showed adrenal hypofunction much
below that of the nonsteroid-treated group,
but there were a number in the steroidtreated group which showed surprisingly
good adrenal function.
DR. PAUL IfOLBROOK (Tucson, Ariz. ) : In
support of Dr. Ragan we find that patients
on long-term therapy with cortisone or
cortisone-like substances do not respond to
corticotropin so long as they continue on
maintenance doses of the steroid. It may
require days or weeks after discontinuing
the steroid before response to corticotropin
is obtained. It would appear that nothing
is accomplished by using corticotropin so
long as steroid therapy is continued.
DR. MEAKIN: Certainly not if you anticipate keeping them on steroids for the remainder of their lives.
4. ANKYLOSING
SPONDYLITIS
AND RHEUMATOII)
DISEASE:
A STUDYOF
VISCERALLESIONS
Bruce Cruikshank, Boston, Mass.
Autopsy protocols a n d microscopic sections from over 200 cases of ankylosing
spondylitis h a v e been studied. This material was collected in collaboration with the
MRS Group for Study of the General Effects of Radiation (Director, W. M. CourtBrown). The findings have been cornpared with those i n 100 cases of rheumatoid
disease previously studied. The diagnosis of spondylitis is based upon clinical radiographic d a t a i n the majority of patients. N o examples of rheumatoid granuloma
were detected a n d the incidence a n d type of visceral lesions encountered in
243
AMERICAN RHEUMATISM ASSOCIATION
spondylitis are quite different from those seen in rheumatoid disease. Details of the
various lesions found i n spondylitis will be given.
DISCUSSION O F PAPER 4
DR. c u n R m n MCEWEN (New York, N.
Y.): May I ask Dr. Cruikshank if he will
say a word more about the aortitis in these
patients. You list it as a cause of death,
in what way did it cause death? Was it
related to aortic insufliciency and heart
failure?
DR. C n u x s H A N K : Yes. These patients all
died of congestive cardiac failure. They had
a long period of compensated cardiac disease and like so many others of the spondylitis aortitics they went suddenly into left
ventricular failure and died.
DR. DONALD c. GRAHAM (Toronto, Canada) : Dr. Cruikshank, your slide indicated
that there were 48 deaths recorded with a
diagnosis of either leukemia or aplastic
anemia. Had all those patients had radiation therapy?
DR. CRUIKSWANK:
yes.
DR. ELAM c. TOONE (Richmond, Va.): Dr.
Cruikshank, I should like to ask two questions. One, what is the incidence of females in your autopsied cases of spondylitis
generally and the incidence of females in
the cases of aortitis specifically. The second
would be the results of the STS reactions
since the pathological lesion is so closely
related to that of syphilitic aortitis.
DR. CRUIKSIIANK: I do not think I can
answer the first two. This analysis was done
as a very provisional thing and it is by no
means final. I have not looked into questions of sex incidence. Certainly the aortitics
were all males. I would just be guessing on
the proportion of females in the whole
autopsy population. Electrocardiographic
findings were very similar to those that have
been reported by previous workers in
spondylitic aortitis.
DR. ELAM c. TOONE: Possibly you misunderstood me. I meant the serological tests
for syphilis.
DR. CRUMSHANK: I should explain, many
of these patients died in hospital of intercurrent disease. Their spondylitis would be
diagnosed at anytime between 1935 and
1954 and many of them would have radio
therapy for their spondylitis a t anytime between 1935 and 1940, so that serological
tests certainly were not done in a great
many of them.
DR. CHARLEY SMYTH (Denver, Col.):
Serological tests for syphilis?
Dn. CRUMSHANK: I beg your pardon.
Serological tests for syphilis were all negative.
DR. CHARLES RAGAN (New York, N. Y. ) :
I would like to ask Dr. Cruikshank one
question and then make one comment. The
question concerns the frequency of pericarditis in the patients with spondylitis and
the comment was the confirmation of Dr.
Smyth’s statement that we should stop calling spondylitis a similar disease to peripheral joint rheumatoid arthritis.
Dn. CRUIKSHANK: 1 do not think 1 can
answer your question. Pericarditis certainly
occurred in a number of the uremia and I
could not give you any accurate information
about that. It occurred among some of the
rheumatics. I do not think it was picked up
apart from that.
DR. RAGAN: What was the frequency Of
pericarditis in your patients with peripheral
joint rheumatoid arthritis?
DR. CRUIKSHANK:
I do not know. If I
might make one other comment in connection with this question of syphilis, there
were among the autopsy series, 3 patients
with ruptured aortic aneurisms. These all
were sero-negative and the aneurisms were
all in the descending thoracic aorta. They
were not in the abdominal aorta where the
arteriosclerotic aneurisms are and being
244
AMERICAN RHEUMATISM ASSOCIATION
sero-negative one wonders if these too were
examples of spondylitic aortitis.
spondylitic group and in the Boston City
control series?
DH.
LAWRENCE
SHULMAN
(Baltimore,
M d . ) : What were the sex ratios in the
DR. CRUIKSHANK:
I cannot tell you. It
may well be important.
5. THEAPPEARANCE
OF HYPERGAMMAGLOBULINEMIA,
POSITIVESEROLOGIC
REACTIONS
FOR RHEUMATOIDARTHRITIS
AND COMPLEMENT
FIXATION
HEACTIONSWITH
TISSUECONSTITUENTS IN THE SER4 OF RELATIVESOF PATIENTS
WITH SYSTEMIC
LUPUSERYTFTEMATOSUS
H . Holmun und H . R. Deicher, New York. N . Y.
A total of 57 relatives from 18 families of patients with S.L.E. have been examined.
Eleven of the 57 relatives have distinct hypergammaglobulinemia. Another 10 have
high normal or slightly elevated serum gamma globulin levels. Individuals in 5 of
the families have symptoms, serological reactions, and x-ray evidence of rheumatoid
arthritis. Asymptomatic individuals in at least three additional families have at least
one of the rheumatoid serologic tests positive. Positive complement fixation reactions
have occurred in members of only one family.
Individuals in 10 of the 18 families show definite serological abnormalities; in
many instances, the majority of relatives studied in a family had abnormalities.
An additional family has been studied in which the maternal grandmother and
mother died of S.L.E. and the granddaughter has had arthralgia, hives and a positive
L.E. cell preparation.
The incidence of these abnormal reactions in the relatives of patients with S.L.E.
appears to be considerably higher than in the general population, though completel!
satisfactory studies have not yet been done in the latter group. Hyperganimaglobulinemia has been the most prevalent abnormality in the relatives in this study.
Rheumatoid serologic reactions, as identified by agglutination of Rh positive human
red cells, occurred in 14 per cent of the relatives as compared with 6 per cent of
the normal population.
The presence of asymptomatic persons with serologic abnormalities and of persons
with clinical rheumatoid arthritis in the families of patients with S.L.E. raises the
possibility that a familial tendency for an immunologic abnormality exists which
may manifest itself as S.L.E., rheumatoid arthritis, or unusual serologic reactions
without accompanying symptoms.
DISCUSSION OF PAPER 5
m. GEHALD RODNAN (Pittsburgh, Pa. ) :
\Ve too have been interested, as I imagine
many people have been, in this sort of study,
m d would like to compliment Doctors Holinan and Deicher on a very exciting paper.
IVe can add our own observations on approximately 60 close relatives of 15 more
patients with S.L.E. I may say simply that
our observations closely parallel those of
Iloctors Holman and Deicher in the finding
a very high percentage (approximately 25
per cent ) of hypergamniaglobulinemia
among the parents, siblings, and children
of the original cases. We find a number of
indivitlnals with false-positive serologic
tests for syphilis, a number with positive
latex fixation reactions, and at least 2 with
positive L.E. preparations. There is one
other feature which we have noted on a
number of the L.E. cell preparations, concerning which I would appreciate some
discussion from the floor, and that is a
high incidence of erythrophagocytosis in
slides which are otherwise negative as far
a s the L.E. cells are concerned.
DH. CHARLEY
SMYTH (Denver, Col.):
Thank you Dr. Rodnan. I think it is good
to get this kind of evidence into our proceedings. Do you want to answer this
AMERICAN RHEUMATISM ASSOCIATION
question first and we will move to the next
discussant?
DR. HOLMAN: I can only say that we have
not noticed erythrophagocytosis. However,
we do the L.E. cell preparations by the
Snapper-Nathan method and it is a little
hit difficult to judge erythrophagocytosis in
these preparations.
DH. SMYTH: If anyone has an answer to
this who wishes to discuss it please remember the original discussant’s question.
n ~ i.h L P H HEIMER (New York, N. Y.):
How do you explain the high incidence of
positive rheumatoid serological reactions
and the low incidence of complement fixation reactions?
DR. HOLMAN: I do not have an explanation for it. It seems to be real, and, mind
you, we used a complement fixation method
which is not the most sensitive. We use 2
units of complement and we might conceivably be missing low level reactions. But
it is perfectly clear that in the relatives of
the patients with S.L.E., the rheumatoid
5erologic tests far outweigh those of S.L.E.
We have one family that I did not mention
in which without any doubt grandmother
and mother had S.L.E. and teen age granddaughter has now been seen by us and
has symptoms that might suggest S.L.E.
She has had one positive L.E. cell preparation. This might add another to the positive
S.L.E. reactions but they are far outnumbered by the serologic tests for rheumatoid
arthritis.
DH. M O H H I ~ SIEGEL (Brooklyn, N. Y . ) :
This fall we began a similar study of family
members in connection with a long-term
epidemiological study of lupus and our results thus far have not been so rewarding
as those reported by Dr. Holman. At present we have preliminary results on 34 members from 10 families in which there were
cases of lupus in 9 families and a case of
rheumatoid arthritis in the tenth, and so
far we have had a direct Coombs’ reaction
in a 5 year old child whose mother had
rheumatoid arthritis and also a direct
Coombs’ reaction evidenced with the L.E.
factor in the sister of a woman with lupus
and finally evidence of hypergammaglobu-
245
linemia in the sister of another patient with
lupus. Thus far we have had no positive
serologic reactions for syphilis and our
latex slide reaction has been negative in
all of our cases. Unfortunately, we do not
have all of our results of paper electrophoresis and other tests that we are doing.
1x4. CARL PEARSON (Los Angeles, Calif. ) :
I was wondering whether in some of the
families where the child has shown evidence
of lupus and the mother has had some
clinical illness, has the mother developed a
disease such as nephritis during pregnancy?
We have recently seen a family in which
2 children, ages 17 and 19, have lupus
with positive L.E. cell and lupus complement fixation tests in which the mother
now has lupus nephritis after a 20 year
illness. She also has L.E. cells and a positive complement fixation test and had episodes of “nephritis” with each of her two
pregnancies.
DH. HOLMAN: No. Except for the grandniother-mother-granddaughter, relationship
we have not had that. However, we have
not specifically looked for it amongst thc
parents of the patients with S.L.E. \i7e had
R 33 per cent over-all figure of peopIe with
a t least one abnormality in this preliminary
study. Amongst the parents alone, the incidence is 41 per cent. If we looked carefully for what you are asking for, we might
he able to find it.
UH. MOHRIS ZIFF (Dallas, Tex.): When
we first studied this problem four or five
years ago, we found about 20 per cent positive tests in relatives of rheumatoid arthritis
patients. When we corrected for those who
had joint symptoms, there remained about
16 per cent positives among the asymptomatic individuals. These asymptomatic relatives who comprised 80 per cent of the
group barely had enough rheumatoid factor
to demonstrate. We found the factor by
direct agglutination only in two of thew
individuals. The rest were shown to have
it by the inhibition test. We thought about
the possibility of hypergammaglobulinemin
being present in the relatives and determined gamma globulin by paper electrophoresis, but we found no evidence of hypergainmaglobulinemia among them. They
had very low titers of a rheumatoid factor-
246
AMERICAN RHEUMATISM ASSOCIATION
like substance, and little more. In other
words it is not as intense a reaction in
relatives of rheumatoid patients (except in
the case of symptomatic relatives) as it
appears to be in relatives of patients with
systemic lupus erythematosus.
m. HOLMAN: I think we might turn your
point around and say that perhaps when
we analyze all of our available relatives
with the care that you did, we may reduce
the surprising incidence somewhat. To date,
however, only one of the relatives seen
complained voluntarily of symptoms of
rheumatoid arthritis, and the diagnosis had
bcen made two weeks before we saw her.
The remainder who had symptoms would
admit to them only under questioning.
Therefore, we did not include symptoms in
the group of abnormalities. With regard to
the gamma globulin levels, I agree that
this is a very difficult problem. We are in
the process of standardizing the zinc turbidity method we use. For purposes of
showing liypergammaglobulinemia in this
study we included as significant only values
that were more than 3 standard deviations
away from the mean of the people who
were the normals. The normals in this
case do not yet include identical families.
DR. HARRY BARTFELU (New York, N. Y.):
Assuming the cause might be hypergammaglobulinemia, an altered globulin, primary
or secondary, can you tell us whether the
pstients also had a rheumatoid arthritislike syndrome especially where their relatives had rheumatoid arthritis?
IIR. HOLMAN: The answer is no. Neither
did the propositi, that is the patients with
S.L.E., have a clinical picture of rheumatoid arthritis, nor did any appreciable number of them have positive rheumatoid serologic reactions. A minority did. I do not
know how to generalize this in terms of the
inimunobgic abnormality except to say
that we have not yet identified what is
wrong immunologically with our patients.
These data permit the inference that their
immunologic handicap may antedate the
clinical disease by many years ( a point first
suggested by Moore from the Hopkins
studies on false positive Wassermann reactions), that it may have a genetic basis,
and that it may manifest itself as one of at
least two diseases, S.L.E. or rheumatoid
arthritis.
CONCURRENT
SESSION“A”-Presiding:
DR. WILLIAM ROBINSON
6. SOMECOMPONENTS
OF BOVINE
NASALCARTILAGE
Jerome Rotstein and Maxwell Schzihert, New York. N . Y .
Malawista and Schubert evolved a rapid a n d efficient way of dissolving the
chondromucoprotein portion of bovine nasal cartilage, which accounted for 42 per
cent of the total product. A method of dissolving the remaining nonchondromucoprotein portion through the use of superheating under pressurc followed by tryptic
digestion has now been developed. After dialysis, which removes GO per cent of
the dissolved product, the anionic polysaccharide is first precipitated with hexamine
cobaltic chloride a n d from this solution a fraction ( F ) rich i n neutral polysaccharides
subsequently is precipitated with ethanol.
The oxidation by periodic acid of F a n d of chondroitin sulphate has been compared. Within 20 minutes the oxidation of F is virtually complete, whereas that of
chondroitin sulphate is insignificant. After 24 hours the oxidation of chondroitin
sulphate is still not great. These results suggest that in the PAS reaction, as used i n
the standard histological technic, it is the polysaccharide that is stained.
F is hydrolysed in 4 N HC1 a t 100 for 6 hours, m a d e u p to 0.1 N HCl a n d passed
through a Dowex 50, mesh 100-200, resin column, which previously has been
equilibrated with 0.1 N HCI. Then the column is washed with 0.1 t o 1 N HCI.
The 0.1 to 0.3 N HCl eluates contain mainly neutral sugars, a hexosamine and a
hexuronic acid, while the 0.5 to 1 N HC1 eluates a r e mainly amino acids. The 0.1
t o 0.3 N HC1 eluates have been chromatographed in three solvent systems a n d the
chromatographs stained with three different stains. Five neutral sugars (mannose,
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AMERICAN RHEUMATISM ASSOCIATION
galactose, glucose, fucose a n d ribose), one hexosamine (Slucosamine) a n d one
hexuronic acid (glucuronic acid in its lactone form) a r e identifiable.
DISCUSSION OF PAPER 6
DR. L. ROBERT (Chicago, Ill.): I would
like to ask Dr. Rotstein whether he had
made a similar analysis of the dialysate.
We made very similar studies of carrageenin tissue produced in guinea pigs and
found rather important neutral polysaccharide fraction in our dialysate. W e found
some evidence of the presence of glycopeptide. Do you think that your dialysate
contains similar material.
DR. ROTSTEIN: Thank you very much for
the comment. We are chromatographing
the dialysate now and determining the
neutral sugar and amino acid content. On
one analysis, the dialysate contains onethird of the total hexosamine. It is high in
nitrogen, with, a high amino acid content
and it is lower than either of the fractions
in reducing substances by the anthrone
method.
DH. KARL MEYER (New York, N. Y.): I
wonder whether you observed any kerato
sulfate fraction in either your acidic fraction or in the so-called neutral fraction?
I ask this because we have recently shown
the kerato sulfate from cornea as well as
from human cartilage, to contain up to 2?h
per cent of a material, which we have not
yet identified but which is definitely a
component of this material migrating with
the same mobility.
DR. ROTSTEIN: Yes. Thank you very much
for the comment, Dr. Meyer. We have not
made an analysis but hope to do so in the
future.
DR. H. R. CATCHPOLE (Chicago, Ill.) : We
are all familiar with the very brilliant staining of the cartilage with PAS. We are not
doubting the conclusion to any great extent.
I wonder whether you could give us an
idea of the relative amounts of chondroitin
sulfate and of the neutral heteropolysaccharides of your tissue. Because it would
seem that possibly the overwhelming component of cartilage would be a chondroitin
mucoid and if it gave any type of reaction
with the PAS it might readily show up in
the histological preparation.
DR. ROTSTEIN: Thank you for the comment. The analysis of the original cartilage
is about 49 per cent of chondroitin sulfate,
that includes the 42 per cent that Dr.
Schubert gets out of his fraction and the
7 per cent or so which is more tightly
bound, that I get out of my fraction. We
believe that if the chondroitin sulfate accounts for any, it accounts for a very small
amount of the staining reaction. Recently,
Leblond's laboratory has come oiit with
several papers which have confirmed what
we have done and we have confirmed what
he has done. You can take chondroitin sulfate, any amount that you want, and you
can use periodic acid for 24 hours and the
uptake, or the oxidation, is very small. It
occurs, I am sure, over a period of time,
but the 1,2 glycol groups are almost inaccessible in the uronic acid, and are not
oxidized by the periodic acid. I do not
think that it accounts for any satisfactory
portion of the staining technic.
DR. MATTHEWS (Chicago, Ill.) : The content of chondroitin sulfate in bovine nasal
septa1 cartilage is about 20 per cent as Dr.
Schubert has reported. You did not answer
the question as regards the content of
neutral carbohydrate or glycoprotein.
DR. ROTSTEIN:
I am awfully sorry. The
content is about 12 per cent of neutral
sugars in bovine nasal cartilage.
7. ISOLATION
OF HYALURONATE
FROM HUMANSYNOVIAL
FLUIDBY
ZONE ELECTROPHORESIS
David Hamerman, and John Sandson, New York, h'. Y.
Hyaluronate (Hy.) was precipitated from human synovial fluid by addition of
acetic acid a n d 50 to 60 per cent of the proteins present coprecipitated (J.Clin.
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AMERICAN RHEUMATISM ASSOCIATIOX
Invest. 37~57,1958). Hy. was separated from proteins by moving boundary electrophoresis (Platt et al. Arch.Riochem. 64152, 1956), but was not recovered and
analyzed. Hy. was obtained by repeated electroconvection of pooled fluids in a yield
of 70 per cent or less with a ratio mg. protein/mg. Hy. of .13 (Roseman et al.,
abst. 2nd Interim Session 1955). In the present study, normal human svnovial fluid
(1 Gm.) was diluted and dialyzed in an acetate buffer (T/2 .07ij, pH 5.3) and
applied to a block of polyvinyl chloride. After electrophoresis at 2 C. for 14 hours
a t a potential gradient of 8.7 volts/cm., or 21% hours a t a gradient of 5.5 volts/cm.,
the block was cut into segments starting at the anodal end. The contents of the
segments were eluted and analyzed for hexuronic acid (as a measure of Hy.) and
protein. Segments nearest the anode contained Hy. and protein. Hy. was recovered
in a yield of 80 to 90 per cent. Eluates containing Hy. were pooled. The ratio of
mg. protein/mg. Hy. in 5 experiments was .024, .032, .032, .062 and .lo. The
isolated Hy. contained less than 1 per cent of the proteins present in the whole
synovial fluid. Protein in 2 samples of isolated Hy. was identified as a gamma globulin by double diffusion in agar. Neither orosomucoid nor albumin which migrated
just behind Hy. could be detected in the isolated Hy. by immunologic methods. Hy.
isolated from 4 synovial fluids was pooled, precipitated with alcohol and analyzed:
hexuronic acid 33 per cent, protein 3.5 per cent, nitrogen 3.1 per cent. The presence
of gamma globulin in the Hy. zone is surprising and worth further study. (The
authors are grateful to Dr. L. Korngold and Dr. E. Franklin for the agar diffusion
studies. )
DISCUSSION OF PAPER 7
1x3. WAHU PICMAN (Birmingham, Ala. ) :
\Ve enjoyed hearing this very interesting
paper. One specific question is why did
yo11 chose pH 5.4 for your work?
1)H. SANDSON: We performed experiments first at pH 8.6 and then at pH 6.5.
These studies were reported at the Federation Meetings this year. At neither of these
pH’s were we able to get good separation
of hyaluronate from albumin. We had to
lower the pH to 5.4 in order to separate
hyaluronate completely from orosomncoid
:ind albumin.
1x3. PIGMAN: This system is a very complex system and we now have quite a bit
of data. The composition of association
products of hyaluronic acid with the protein depends markedly upon the pH. In
the very low pH range, the clotting range,
the composition of the much clot also v a r h
with the pH. Although all of the hyaluronic
acid precipitates with the clot, the composition of the protein part changes, althougli
the major protein seems to be the albumin.
As you get above the clotting range you
get complexes, usually precipitates of hpaluronic acid and the beta and gamma
globulins. As the pH increases above pH 7,
with high inolecular weight hyaluronic
acids, soluble complexes of the albumin
are formed. This type of complex is tlic
explanation of the Pi peak that we reported
earlier. The Pi component is a complex of
albumin with high molecular weight hyaluronic acid, but it does not seem to be
formed from partially depolynierized hyaluronic acid. Roseman a d \Vatson have
prepared hyaluronic acid at pH 7 in phosphate buffer by electrodialysis. We use this
method ordinarily now and have improved
it some. In 3 or 4 repetitions of the process
we can free the hyaluronic acid completely
of protein just as they reported. I helievr
this method is the best one for preparinq
hyaluronic acid. In summary, I would likr
to make the point again that complexing
such as that you observed is highly d:pendent upon the pH and upon the stntc
of polymerization of the hyalnronk acid.
IIH. SANDSON: In this regard it is of interest
that vr7hen hyaluronate was separated from
normal synovial fluid by zone electrophoresis at pH 8.6 it also contained protein
identified as gamma globulin by immunological technics. Gamma globulin was
present not only in the hyahronate isolated
at pH 5.4 bnt also in the prodiict obtained
nt pH 8.6.
m. SAUL ROSEMAN (Ann Arbor, Mich. ) :
First, I think I better thank Dr. Pigman for
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AMERICAN RHEUMATIShl ASSOCIATION
the free ad and secondly, since our work
has been quoted to some extent here, perhaps I had better make a comment on it.
In the first place, I should apologiye for
the fact that this work has never been
published in detail. This is due to two
reabons, one is that we are naturally lazv
and, secondly, that we conducted joint
studies with Dr. Balazs at Boston and his
group trying to characterize these products
in a physicochemical sense. The electrodeposition method yields hyaluronate from
5ynovial fluid, vitreous humour, umbilical
cord, cock‘s comb and a variety of 0 t h
sources. As near as we can determine by
direct analysis, which has its limitations,
these materials are protein-free. As near a5
Dr. Balazs can determine by his various
physicochemical technics, these materials
are undegraded. Now Dr. Pigman has discussed the problems of interaction with protein. When the work which was presented
here today was presented at the Federation
Meetings last spring it was stated, I believe,
you can correct me if I’m wrong, Dr.
Hamemian, that there were 2 hyaluronate
fractions of synovial fluid. One was obtained
protein-free and the other, where the dati
looked very good, would seem to be c~
protein-hyaluronate complex. Based on wh7t
you said this morning and based on what
Dr. Pigman has stated, it sounds like the
complex idea is more or less out the window, and the question now is whether the
residual 2 or 3 per cent protein, the gamma
globulin, which you found in your preparation, is due to a similar type of complex.
According to what Dr. Pigman said yon
should completely remove the protein by
clectrophoresis at different pH’s. That is a
question. The rest was a statement.
DH. SANDSON: In answer to Dr. Roseman’s
question, we have not yet performed electrophoresis of the isolated hyaluronate at various pH’s. We do plan to perform such
studies. In regard to the other comments
it should be stated that it was not the purpose of our present study to prepare proteinfree hyaluronate. The technic involved only
one electrophoretic procedure. Hyaluronate
was separated from most of the proteins
of normal human synovial fluid by a single
electrophoresis at p H 5.4. Electrodeposition
on the other hand must be repeated many
times if hyaluronate is to be obtained protein-free. Each additional electrodeposition
decreabes the amount of hyaluronate recovered and possibly increases the risk of
degradation. At times only 60 per cent of
the hyaluronate may be recovered. In April
1959, Dr. Hamerman and I reported at the
Federation Meetings that hyaluronate could
not be completely separated from albumin
by zone electrophoresis a t either p H 8.6
or 6.5. By performing the zone electrophoresis at pH 5.4 instead of p H 8.6 or
6.5, hyaluronate can be completely Teparated from the adjacent protein zones, i.e.
orosomucoid and albumin. This means that
protein contamination did not occur in the
hyalnronate zone at p H 5.4, and increase5
the significance of the protein found in the
hyaluronate zone. Did I answer your question?
DR. ROSEMAN: I think so, but with reference to the yield of material obtained by
electrodeposition, I will just make this onc
comment. One always worries whether any
method applied to natural material will
yield a product which is not degraded. In
fact, Dr. Ropes brought this up when we
first presented these data 4 years ago and
I can only answer that nobody knows that
any method is that good. Physicochrinical
data indicate no degradation. In terms of
the yield, when you say 60 per cent you
are referring to the yield at any step. On
the other hand, it has been possible to
increase the yield to 90 to 95 per cent by
using longer periods of time during the run.
Furthermore, reworking of the material
which did not “deposit” in the initial run
also gave a hyaluronate gel in the same
yields. This material appeared to have the
same physicochemical properties as thc
first gel.
DH. PIGMAN: I would like to qualify Dr.
Roseman’s statement to one extent. There
is considerable danger of degradation of
hyaluronic acid by his procedure. This is
why I was disturbed about pH 5.4. As the
p H decreases, the degradation increases.
It is possible to avoid this but one should
consider that hyaluronic acid can be easily
degraded under these conditions.
.
UR. SANDSON: I think it is likely that by
using p H 5.4 we may have slightly degraded the hyaluronate but what interests
11s most about this study is not whether
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AMERICAN RHEUMATISM ASSOCIATIOX
the product is native or minimally degraded,
but rather that a t p H 5.4 the isolated
hyaluronate contains a small amount of
gamma globulin. The nature of the inter-
action between hyaluronate and gamma
globulin that causes them to migrate together electrophoretically has not yet been
established.
8. THESIALICACIDS
Saul Roseman, and Donald G. Comb, Ann Arbor, Mich.
The sialic acids a r e a group of substances, derived from neuraminic acid, which
a r e widely distributed in vertebrate tissues. The substances are components of
mucins, mucoproteins a n d mucolipides.
Recent studies were concerned with the structure a n d intermediary metabolism
of the compounds. As a result of this work, the following results were obtained:
( 1 ) The enzymatic synthesis of certain of the sialic acids; (2) evidence that the
acetylhexosamine moiety of the sialic acids was N-acetyl-D-mannosamine; ( 3 ) the
recognition of mannosamine a s a naturally occurring hexosamine; (4)the enzymatic
synthesis of N-acetyl-D-mannosamine by two routes; ( 5) the isolation of a nucleotide
containing N-acetyl-neuraminic acid.
DISCUSSION OF PAPER 8
IIH. KARL MEYER (New York, N. Y . ) : I
do not want to discuss this paper. I want
to ask one question. I wanted to state here
this is one of the most brilliantly executed
series of studies reported by Dr. Roseman
and his collaborators in recent years. Now
you left it
the one question I have-and
open-is
whether this uridine phosphate
sislic acid is alpha or beta. Apparently, as
far as I know all of them described so far
have been alpha so it is possible this
might be beta.
First, we do not know.
Secondly, we have not really looked yet,
and the reason is that the entire characterization was done on 10 mM when it
was first isolated. However, Dr. Jourdian,
whose name I mentioned before has been
working on improving the method of isolation and has altered it and .has stabilized
the compound. He has managed to obtain
the compound in sufficient quantity so that
DR.
ROSEMAN:
we should be able to do polarimetric studies
and hope to answer this question.
IJR. F. LINKER (New York, N. Y.) : I have
two questions. Do you think there is any
significance that this is a monophosphate,
while the UDP derivatives are diphosphates.
Second, is there an amino acid associated
with this? In other words, is it at all similar
to the nncleotide muramic acid derivatives
which were found in bacteria?
DR. ROSEMAN: Let me take one question
at a time. We think it is very interesting,
in fact this is a unique compound as you
point out. In finding only one phosphate
group we hope that this has something to
do with the way it is formed and we have
ideas about this but no evidence. In terms
of the amino acids, there are trace contamination of amino acids on these chromatograms. There is no significant amount in
the purified material.
9. THEPARTICIPATION
OF LYSINE
IN MINERALIZATION
OF COLLAGEN
Harry R. Elden, Harriette C . Schapiro, and David S . Howell, Miami, Flm
An important unanswered question i n mineralization of osteoid matrix is the pathway by which calcium a n d hydrogen phosphate ions of lymph become incorporated
as hydroxyapatite o n collagen fibers. Solomons and Irving ( Biochem. J. 68:499, 19.58)
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AMERICAN RHEUMATISM ASSOCIATION
suggested that the r-amino group of collagen-incorporated lysine might nucleate the
deposition of the mineral phase. The current study was designed to ascertain at what
stage of the mineralization process, in vitro, lysine might be reactive.
Optical absorption measurements a t 225 millimicrons were determined on solutions of lysine mixed with hydrogen, sodium, calcium, phosphate and chloride ions.
Continuous-variation analysis of the absorption of these solutions showed that the
predominant association was for hydrogen ions with -COO- and -NH, to form
-COOH and -NH,+, respectively. Analysis of the viscosity data for solutions of
lysine mixed with sodium, calcium, phosphate, and chloride ions failed, also, to
show interaction.
Hydroxyapatite was prepared and suspended in phosphate and acetate buffers a t
p H 7.0. Analysis of the transmission-turbidity data showed positive deviations when
lysine was mixed with suspension. Glycine failed to show this evidence for interaction. Positive deviations of light scattered a t 45 were observed when hydroxyapatite was mixed with hide powder collagen (suspended in 10 molar urea) and
gelatin (pH 7.0).
From these and other data it seems likely that, under these experimental conditions, hydroxyapatite might attach in a preformed state to the fibers rather than
by step-wise addition of ions. The turbidimetric technic employed here may prove
a useful adjunct in the study of bone mineralization.
DISCUSSION OF PAPER 9
DR. M. K. KEECH (Leeds, England) : Have
you any evidence for the presence or absence of mucopolysaccharide in your soluble collagen?
DR. ELDEN: No. There most probably is
some mucopolysaccharide in the collagen
obtained from rat-tail tendons by reconstitution, and it was for that reason that
we went to working with the model system,
using amino acids to see what functional
groups known to be in collagen could react with hydroxyapatite.
DR. MATTHEWS (Chicago, Ill.): It is not
clear to me how, from the slide scattering
at 45 you can interpret very much about
the interaction between the solubilized
apitate and the soluble collagen.
DR. ELDEN: The 2 constituents which we
mixed were shown to give a linear intensity
to light scattered with concentration upon
mixing. If no interaction occured one would
expect simply addition of the intensity. The
evidence for interaction was that our
measured values were either positive in
the case of lysine and collagen or they were
negative in the case of the glutamic acids.
In all cases which we studied there is interaction. The experimentally measured values
deviated from that which you would expxt
to be simply mixing.
DR. MATTHEWS: The collagen in solution
was relatively unstable to many conditions,
temperature and other possibilities of denaturation. What is the nature of this
interaction?
DR. ELDEN:
The point we were trying to
arrive at first was to find out if an inter-
action exists using turbidometric methods.
The only thing I can state about the nature
of the interaction is that certain groups
known to be in collagen when attached to
hydroxyapitate in small molecules also
showed this evidence for interaction. Beyond that I have not any idea of what it
is that holds these two together.
DR. I. ORESKES (New York, N. Y.): My
question is somewhat repetitive. I think
that in your light-scattering data the maximum deviation was at 50 per cent by
weight of each of the 2 products. If I read
that correctly, that is a 1 to 1 ratio. Does
this suggest anything to you?
DR. ELDEN: The difficulty is interpreting
whether it is a 1 to 1 ratio. The x-axis
really represents ratios of volumes of sus-
Ah4ERICAN RHEUMATISM ASSOCIATION
pensions of hydroxyapatite and suspensions
of collagen and I do not know what the
moler ratio of these 2 would be.
(New York, N. Y.) : Untloribtedly there is now excellent experimental evidence for the participation of
collagen and maybe of the r-amino groups
of lysine in the process of calcification.
There is, however, still no explanation why
osseous tissue calcifies while connective
tissue o f other origin, which is supposed to
contain the same collagen, does not. As far
iis I know one cannot solubilize collagen
from bone but what one can do, of course,
is to get gelatin from bone. If you repeated
your experiments with bone would you expect any difference? Obviously yoii have
DR. KARL MEYER
not clone this-otherwise
reported it.
you would have
I ~ R . ELDEN: Perhaps I could speculate
going back to the data of Solomons ant1
Irving. Their study was to deinineralizc
soft and hard cartilage or the collagen obtained in soft and hard cartilage. In the soft
cartilage they were able to free ?4 of the camino groups by demineralizing with DDTA.
In the hard cartilage they could free 100 per
cent of the r-amino groups. Now I would
expect if we took some of the collagen from
soft cartilage of which one-third is lilockcd
by something there perhaps woiild lie ;i
difference in the trirbometric data olitainetl
by making these 2 types collagen with
hydroxyapatite.
10. THENUCLEICACIDSOF GROWING
CONNECTIVE
TISSUE
John L. Decker, Seattle, Wash.
T h e inability of the scorbutic animal to synthesize collagen a t a normal rate
directs attention to the ribomcleic acid content of proliferating connective tissue
i n sciirvy. Earlier data garnered from the carrageenill granuloma suggested that the
RNAjDNA ratio was reduced in scurvy and, thris, that the scorbutic error might
lie in deficient RNA production.
T w o methods of connective tissue sampling have been combined; polyvinyl
sponges were soaked in 1 per cent carrageenin before being implanted subcutaneously in the guinea pigs. More rapid tissue proliferation was found than in s a1'mesoaked sponge implants and the biopsied sponges contained no extraneous tissue
formed prior to the experimental procedure. After suitable periods in normal and
scorbutic animals, amino acid determinations were made on total tissue hydrolysates
and hydrolysates of collagen extracts of the sponges. Aliquots were appropriately
extracted for RNA and DNA determinations with orcinol a n d diphenvlamine
respectively.
The nucleic acid content of the tissue did not differ between the scorbutic and
the normal, one series of experiments disclosing RNA/DNA ratios of 1.8 a n d 1.7.
respectively. The hydroxyroline content of the salt and acid extracts a s well as of
the gelatin was reduced both 11 a n d 11 days after the start of ascorbic acid eliminntion. I t was concluded that, with the methods used, there was no relationship between the RNA mexwred and the collagen synthetic activity of the tissue.
1)ISCUSSION OF PAPER 10
DH.
WILLIAM
ROBINSON
(Ann Arbor,
\ l i d ) . ) : Dr. Decker, would you feel that
your observations are in line with the gen-
c n l hypothesis that the production of
precursors of collagen in perhaps soluble
forin, may be quite normal in scurvy but
the actual formation of the collagen fibril
is the point which is defective?
1 ) 1 ~ l)ECKEI{:
1 do not know ahout the
fibril, Dr. Robinson. I think it is conktent
with the notion that hydroxylation of tlit.
proline takes place after peptidc I)ontl
fonnation and this is an event occiirring
after separation from the RNA.
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AMERICAN RHEUMATISM ASSOCIATION
11. THEDEPLETION
OF CARTILAGE
MATRIXBY ACUTE HYPERVITAMINOSIS
A
IN THE
RABBIT
Jack Potter, Gerald Weissmann, Robert T . McClziskey, and Lewis Thomas,
New York, N . Y.
The depletion of cartilage matrix produced by papain in rabbits has been simulated by the induction of hypervitaminosis A. The gross changes included a curious
curling of the distal third of the rabbits’ ears within a few days. W h e n 1 minion
units of vitamin A i n corn oil were administered intraperitoneally to rabbits daily,
lesions were produced in ear, tracheal, epiphvseal a n d articular cartilage resembling
those produced by papain protease. A loss of matrix was noted, associated with a
loss of basophilic a n d metachromatic staining of cartilage throughout the animal.
While these changes occur, chondroitin sulfate-like material producing turbidity
with hexaminecobaltic chloride appeared in the blood a n d urine. Rabbits given S3:
a n d then treated with vitamin A demonstrated a significant rise of nondialysable
S35activity in blood and urine as compared to control animals. At 48 a n d 72 hours,
appreciable amounts of serum S35 activity were precipitable by cobalt. The treated
groups showed profound loss of radioactivity over epiphyseal a n d articular cartilage
by autoradiography. This was correlated with the presence in extracted ear chondromucoprotein of only half the Ss5 activity of controls. T h e probability of vitamin
A causing dissolution of preformed cartilage matrix in the rabbit will be discussed.
DISCUSSION OF PAPER 11
I)R. CAM. PEARSON (Los Angeles, Calif.) :
As I recall from some of Dr. Thomas’
original work he felt that cystine accentuated the papain effect of destruction of
chondroitin sulfate. Is this thc case when
you use vitamin A?
DR. POTTER: W e have not decided that
particular point Dr. Pearson. The situation
i7 that cystine accelerates the proteolytic
activity of papain in vitro. If you put an
isolated piece of cartilage in a solution of
papain it is broken down more rapidly if
there is also cystine present. On the other
hand if you injected into an intact rabbit,
papain and cystine, you get much less
effect than if you inject an inactivated
preparation of papain. This has to do, I
think, with the distribution in the rabbit
and how the material gets from ths circulation in the cartilage. We have not tried
the effect of cystine with vitamin A. Dr.
Thoinas has been working with Dr. H. B.
Fell in England on bone rudiment7 grown
in tissue culture. In this situation there is
‘in enhancement of the effect of vitamin
A by papain and vice versa, in prolonged
cultures. I think cystine did not make niuch
difference in this particular situation. Cys-
tine is one of a possible variety of donors
of siilfhydryl groups and I think they have
not tried any other. It is true th,jt in vitro
t h e is an acceleration.
UR. JOHN VAUGHAN (Rochester, N. Y.): I
gather in looking at your radiograms that
once the mucopolysaccharide is calcified
it is not susceptible to release by vitamin
A. I wonder if this was actually so. Have
you had any calcium studies done on your
normals?
DR. POTTER: It is true that intense subepiphyseal image in the vitamin A-treated
rabbit is actually in the region of thcartilagenous c o r c ~ of trabeculae which
forms probably during the period after the
administration of S35 and vitamin A. We
1,ave done no studies on calcium.
DR. M A T l H E W s
(Chicago, 111. ) : The
chondroitin sulfate protein complex. a\ we
havc shown, evists in cartilage as a very
high molecular unit of 4 million and is
‘iggragated to give particles that iggragatr
up to 50 million. As a consequence this may
be niaintained in the cartilage withoiit
being able to diffuse out as we recently
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AMERICAN RHEUMATISM ASSOCIATION
reported in April at the Federation Meetings, the polysaccharide moiety and the
protein moiety appear to be formed as a
biological unit in the tissue and reproducing the true metabolic unit. Now there is
an alternative explanation of the results
that you obtained in that the interference
of vitamin A may be in the synthesis of
this unit, that is, there may be some interference in the joining of the mucopolysaccharide with protein so that it is not then
a breakdown but an interference in synthetic mechanism.
UR. POTTER: Thank you, Dr. hfatthews.
We feel that, from our studies in the rabbit, there probably is some breakdown. Dr.
Fell and Malanby, and more recently, Dr.
Thomas with Dr. Fell, have done some
studies on embryonic rudiments grown in
the presence of 9 5 and then transplanted
to medium containing vitamin A to test this
possibility, and they concluded that there
was some inhibition or similar effect upon
synthesis of chondroitin sulfate or chondromucoprotein in the presence of excess
vitamin A. We looked at this data again
and we are not too sure that this is conclusive.
DR. DAVID HOWELL
(Miami, Fla.): Will
you comment on the specificity of your
serum test with hexaminocobaltic chloride
and possibly other sulfated mucopolysaccharides perhaps giving a similar reaction?
Yes, this is true. Some other
sulfated mncopolysaccharides will. Heparin
will complex with hexaminecobaltic chloride. We think that in this experimental
situation the material is probably chondroitin sulfate. In fact we tended to use the
term “chondroitin sulfate-like material,” but
that was such a mouthful that we just use
chondroitin sulfate. There is other indirect
evidence; the fact, that the S35 disappeared
from cartilage and was present in increased
amounts in the precipitated material suggests that, at least in this experiment, the
test was fairly specific. We would not care
to extrapolate beyond this a t all.
DR. POTTER:
(Dallas, Tex.): How
does the rate of disappearance of metaDR.
MORRIS
ZIFF
chromatic material produced by vitamin A
compare with the rate which is observed
on steroid administration?
DH. POTTER: I could not be sure about
that. I think that steroid administration
takes several days or weeks. With vitamin
A it is a matter of 72 hours to reach the
maximum depletion, although it may be
apparent as early as 24 hours after the
first injection.
DH. ZIFF: One would assume that this is
an inhibition of synthesis on steroid administration.
DR. POTTER:
Yes.
DR. ZIFF: If the rates are fairly comparable, the vitamin A effect might be siniilar.
DR. POTTER:
I think the vitamin A effect
is more rapid.
DR. ROSENBERG: Do you know whcther
the serum in sulfated polysaccharide or the
urine sulfated polysaccharide are sensitive
to hyaluronidase?
DR. POTTER: The precipitated niatcrial
was tested with hyaluronidase in one experiment and it was sensitive. I think that
is right, Dr. Weissinan?
DR. WEISSMANN: The material appearing
in serum after vitamin A administration was
precipitated by hexaminecobaltic chloride
was shown to be metachromatic when isolated. This was not subjected to hyalnronidase treatment. However, similar precipitates following papain have been suhjected
to hyaluronidase and have been susceptible.
However, this has not been testcrl for
specifically in the vitamin A animal. The
papain-provoked material does move as a
chondroitin sulfate in chromatograms.
DR. ROSENBEBG: Was that bacterial or
testicular hyaluronidase?
DR. WEISSMAN: It was testicular hyaluronidase. We used turbidity technics and
it abolished turbidity almost completely.
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AMERICAN RHEUMATISM ASSOCIATION
CONCURRENT
SESSION“B-Presiding : DR.
E. P. ENGLEMAN
12. THECAUSESOF STIFFNESSIN PATIENTS
WITH CONNECTIVE
TISSUE
DISORDERS
Verna Wright and Richard J. Johns, Baltimore, M d .
(Introduced by Lawrence E. Shulman)
Joint stiffness has been measured quantitatively and qualitatively in precise
physical terms in normal subje-ts and patients with connective tissue disorders. By
niodification of an apparatus previously described a sinusoidal motion was imposed
passively on a metacarpophalangeal joint over a wide range of amplitude (0 to 1.74
radians) and frequency (0.003 to 2.86 cycles per second) of rotation, and the torque
resisting this imposed motion measured and related to these parameters.
From these data it has been shown that elastic and plastic stiffness are the important components of over-all joint stiffness. \%cous stiffness accounts for less
than 10 per cent of stiffness in normal and damaged joints, and frictional stiffness for only 1 per cent in badly damaged joints studied.
Plastic stiffness has been measured by the difference in torque recorded with
increasing frequency of rotation, and by studying stress relaxation. The importance
of plasticity is emphasized by the fact that the torque produced a t a given position
of a joint is due not so much to the position as to the direction from which the
joint has come.
Studies in the cat to determine the relative importance of structures causing
joint stiffness have shown that the concept of a joint capsule as a distinct entity is
fallacious. Elastic and plastic stiffness is due to the apposition of tendons around
the joint, and in small part to the skin.
It is emphasized that this method measures objectively what the patient experiences subjectively as stiffness a t the joint.
DISCUSSION OF PAPER 12
m.JOSEPH E. WARREN (Pittsburgh, Pa.) :
Have you had the opportunity to measure
diurnal variations in stiffness?
DR. WIGHT:
We have been most interested in the observation of diurnal variations of stiffness. The original stimulus to
this study was the fact that we observed
marked diurnal variations in the strength of
grip in both normal subjects and in patiexts with rheumatoid arthritis. Both
groups were weak in the morning with iucreising strength over a 3 hour period.
Strength then remained static during the
day and fell toward night time. Preliminary
observations would suggest that the morning stiffness of rheumatoid arthritis may
well be a phenomenon associated with
muscles rather than with the joint.
DR. SIDNEY COBS (Pittsburgh, Pa.): You
say that this stiffness may be associated with
muscles rather than joints, but if I understood your interpretation of these diagrams
correctly, when you disconnected the
muscles from the joint you lost most of the
stiffness in this phenomenon. Where do
you make this distinction between morning
stiffness and this kind of stiffness?
DR. WRIGHT: Our suggestion is that tendons contribute to joint stiffness in two
ways. First, the tight apposition of the
tendon to the joint is responsible for much
of the stiffness which we are measuring.
Second, the fact that the tendon is connected to the muscle also contributes to
the stiffness which we are measuring. Other
experiments which we have done have
shown that both muscles and joint structures contribute to this stiffness. For instance, in patients with myotonia congenita
or Parkinsonism there is increased stiffness
due to muscle factors without changes i n
the joint itself. On the other hand, we have
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AMERICAN RHEUMATISM ASSOCIATION
produced changes limited to the joint, for
ruample, local cooling or the local injection of saline in which stiffness was increased without altering muscle factors.
Thus, it would appear that both the
13.
INHIBITION
OF
passive pull on muscles as well as
structures themselves contribute
stiffness which we are measuring,
neither is so pronounced as to
changes in the other.
HEXAMINECOBALTIC
CHLOHIDE-DNA CLOTS
PATIENTS WITH DLE
HY
the joint
to tlic
and that
ohwnre
SERA
OF
Gerald Weissmnnn, New Ymk, N. Y.
A new, rapid method for the deinonstration of u reactant to DNA in the seiiini of
patients with D.L.E. has been devised while studying the reaction of DNA with
hexaminecobaltic chloride. This polyvalent cobalt salt has the property of forming
easily visible, strand-like clots of D N A in concentrations as low as 50 micrograms
of DNA per ml. W h e n normal serum is added to DNA, the clot still forms following the addition of hexaininecobaltic chloride. The serum of many patients with
D.L.E., however, inhibits such clot formation. T h e test is performed b v mixing 100
micrograms of highly polymerised, protein-free DNA in 0.4 ml. of water with 0.5
ml. of test serum. The mixture is incubated for 30 minutes a t 37 C. and 0.2 ml. of
‘1 0.5 per cent w/v aqueous solution of hexaniiiiecobaltic chloride is added foi
production of the clot. Pancreatic DNAse prevents clot formation, while protamine
or histone inhibit the r e x t i o n . T h e reactant can be absorbed from serum with whole
guinea pig nuclei, b u t not with cartilage powder. Clot formation was inhibited b\
23 of 36 DLE sera, 7 of 27 sera from patients with possible DLE or systemic RA,
1 of 28 classical RA sera, a i d 6 of 132 miscellaneous sera from hospitalized patients
This test will be correlated with results obtained by L.E. cell method. and fluorescent antibody studies. (ilbbreviations used: D.L.E., disseminated lupus ervthematorus; DNA, desoxyri bosenncleic acid; DNAse, desox\iribonuclease; RA, R h e m n a t d
Arthritis. )
IIISCUSSION OF PAPER 13
UR. WALLACE EPSTEIN (San Francisco,
Calif. ) : Is DNA added to the serum prior
to the addition of histone in the histone inhibition procedure?
UH. WEISSMANN: Let me explain this. I
did not make myself clear. The test is very
\imply run by adding to aqueous DNA,
normal serum to which histone has been
added and incubated for half an hour. In
other words histone has been added to
norinal serum in another tube. This mixturc was then added to DNA in aqueous
solution and the same was done with protamine. However, as you know, if you increase the concentration of these you will
get the DNA precipitate anyway so yon
have to adjust this rather carefully.
UR. EPSTEIN: What does not seem clear
is whether histone alone can react with
the constituent of L.E. serum which reacts
with DNA to form the clot.
1)R. WEISShlANN: 1 do not know how to
approach this directly. Because histone i \
prone to react with DNA itself we went on
to use protamine and streptomycin and the
mtiinalarials. These have similar propertie\
to histone, we tested other basic compounds.
By the way, I may add that these results
can be obtained when you take streptomycin
sulfate as the commercial preparation and
dilute it in water. You can substitute that
for lrevaminocobaltic chloride.
UH. GEOHGE FHlOU (New Haven, c O 1 i l 1 . ) :
Do you have any idea whether thir activity
you are dealing with is a gamma glohnlin?
DR. WEISSMANN: One of the troublrs witli
this test is that unlike some of the others
it is very difficult to isolate something that
does not happen. One way this may 1x1
done, of course, is to put serum on starch
block electrophoresis and see where thik
factor comes out. W e were very lucky: in
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AMERICAN RHEUMATISM ASSOCIATION
the first 2 elutions it came out in the gamma
globulin region. This was at the origin and
we were very happy. Unfortunately, this
has not been our experience with recent
elutions. The fact is, it does not seen1 to
be present in the euglobulin precipitates,
while it is present before, but not after
incubation with human white cells that
make the L.E. phenomenon happen in a
disc method. The disc methods are not
very reproduceable phenomena either and
it is really quite difficult to try to identify
any similarity. The next method, of course,
that we can use is column chromatographic
elution and then to see whether we can
elute the factor this way. I certainly cannot
answer your question as to whether they
are identical or not, as I said the trouble
with this method for the isolation of a
“factor” is that it is very difficult to isolate
something that doesn’t happen and that is
actually what we are measuring in this
technic.
IJR. JOKN DECKER (Seattle, Wash.) : Were
the supernatants positive for L.E. producing
factor? After you formed a colt in this
case where the clot was not formed.
1x1. WEISSMANN: No. The norinal serum
forms the clot, the lupus serum does not
form a clot. That is the whole problem.
There is no supernatant to test. What we
have done is this, we have tested the supernatant of euglobulin precipitates, for example, that we have gotten from positive
sera and, of course, they are not active in
the L.E. cell test. I may add that this test
does correlate quite nicely with the work
Dr. Phythyon in our school has been doing
using Dr. Friou’s method, and in 28 sera
that Dr. Peter Miescher has tested for his
conglutinin reaction.
DH. METRO OGRYZLO (Toronto, Canada) :
To enlarge on that question, you say that
there is no supernatant, but if you add
DNA you do get a clot, then will that supernatant do it?
UR.
WEISSBIANN:
No. When you add
DNA to normal serum and then the cobalt
salt, there is a clot. This supernatant naturally has no L.E. cell factor activity. When
you add DNA to lupus serum only in a
very few will you get precipitate overnight.
Yon will get precipitate that is probably
akin to the type of precipitates that Dr.
Holman has been working with. There is
no greater precipitate in those tubes left
overnight when you add cobalt than when
you do not. In other words the point about
this lupus serum after addition of cobalt salt
is that there is no clot, no supernatant,
nothing to measure what has happened
visibly. That is the trouble with working
through at this level.
DR. HARRY BARTFIELD (New York, N. Y.):
Maybe I can add a word to answer your
question. This test is similar in some respects to the LA1 test that Stanley Lee has
devised in New York. This is the leukocyte
inhibition test in which thymus or ageing
white blood cells plus quinacrine are agglutinated by normal serum but not by
lupus serum. He has identified the substance in lupus serum that inhibits the
agglutination with the L.E. cell factor as
far as his work is concerned.
DR. WEISSMANN: I want to comment on
that last one. Stanley Lee’s quinacrine system is a very remarkable one but it again
tests for the same factor, which reacts with
whole cell, this is not then extracted nucleoprotein or DNA, this is just washed whole
cell material. Our test, instead is proposed
as a means of studying serum fractions
obtained from columns, from chromatograms and from other factors for simple
protein-free DNA reactant activity. It is not
proposed as a measure of the total factors
that react with the total cell nucleus.
14. HEMAGGLUTINATION
STUDYOF SERUMFACTORS
RELATEDTO L.E.
CELLFORMATION
Wallace V. Epsteiri nnd Rondd C. Lee, Sun Francisco. Calif.
Antinuclear factors of differing specificity h a v e been detected in sera obtained
from patients having systemic lupus erythematosus, rheumatoid arthritis, or rarely
other diseases by a variety of laboratory technics. That serum constituent or con-
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AMERICAN RHEUMATISM ASSOCIATION
stituents responsible for the formation of L.E. cells is designated L.E. cell factor
and is thought to combine with intact nucleus or the nucleoprotein derived from
such nuclei.
A hemagglutination test utilizing tannic acid treated sheep erythrocytes exposed
to a saline extract of calf thymus nucleoprotein (N.P. extract) was used in this
study. Sera from patients having systemic lupus erythematosus and capable of
producing L.E. cells were able to agglutinate such coated cells u p to dilutions of
1: 100,000. No obvious relation was found between the F I1 hemagglutination titer
of these same sera a n d the NP-E hemagglutination titer. Sera from patients having
Stage 111-IV rheumatoid arthritis, but negative L.E. cell preparations failed to
produce positive NP-E hemagglutination.
The hemagglutination system can be inhibited by nucleoprotein and the nucleoprotein extract, b u t not by deoxvribonucleic acid derived from the same nucleoprotein nor by high concentrations of human gamma globulin (Fraction 11).
Nucleoprotein and the nucleoprotein extract were also capable of inhibiting L.E.
cell formation.
I t would seem, therefore, that this hemagglutination system measures the L.E.
cell factor distinct from the DNA reactive constituent of lupus factor.
DISCUSSION OF PAPER 14
DR. ROGER L. BLACK (Rockville, Md.):
Had you done any work on rheumatoid with
positive L.E. tests?
DR. JOSEPH BUNIM (Bethesda, Md. ) : Dr.
Epstein, do you get hemagglutination in
rheumatoid sera using dilutions of less than
100,000?
No. V7e have excluded this
group from this study. Considerable confusion still exists as to whether such patients
have rheumatoid arthritis or lupus erythematosus. We do plan to study this group,
however.
DR. EPSTEIN:
DR. JACQUES M. SINGER (New York, N.
Y . ) : How many lupus sera did you get with
this technic?
Dn. EPSTEIN: Are you referring to the
hemagglutination system using cells coated
with gamma globulin?
DR. BUNIM:
DR. EPSTEIN: We use an initial serum
dilution of 1:28 in all of our test systems.
DR. B u N m s :
DR.
Yes.
How about undiluted serum?
EPsmIN: Approximately 40.
Since lupus sera apparently
contain a family of antibodies againTt nuclei, nucleoprotein, DNA and histone, it
would b e helpful to know which of the
antigens were present. We have tested
several L.E. sera against sheep cells sensitized with DNA, but were not able to
obtain positive results.
DR. SINGER:
DR. EPSTEIN: At present we do not know
the constituent or constituents of a saline
extract of nucleoprotein which coat the cells
and react with L.E. serum. The sensitivity
of this system will allow us to test relatively pure nucleoprotein constituents for
inhibitory ability.
DR. EPSTEIN: We do not use undiluted
serum because of nonspecific clumping of
cells in undiluted serum. Our controls are
consistently negative when a 1:28 dilution
is used.
DR. MORRIS ZIFF (Dallas, Tex.): I know
you have found a marked increase of vascular disease in patients with F I1 titers that
are very high. Do you have data showing
the influence of a positive L.E. test in patients with high titers of factor (over
56,000) on the incidence of vascular disease?
DR. EPSTEIN: I do not have information
on the incidence of positive L.E. hemagglu-
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AMERICAN RHEUMATISM ASSOCIATION
tination titers in sera having high F I1
hemagglutination titers. In our previous
study of sera of high F I1 titer, we did
find a significant incidence of positive L.E.
cell preparations. We would hope to follow the clinical course of patients whose
sera is strongly positive in both of these
tests.
UR. ZIFF: I have the impression that if
the patient has a titer of over 56,000 by
the tanned cell test and also has a positive L.E. cell test, he has a very high
probability of having vascular disease. That
is why I asked.
DR. EPSTEIN: The four positive F I1
hemagglutination titers in this group of
L.E. sera do not seem remarkably high
when compared to that frequently found
in patients having rheumatoid arthritis with
marked vascular involvement.
DR. ENGLEMAN: I would certainly share
your impression, Dr. Ziff.
15. EXPERIMENTAL
PRODUCTION
OF AN L.E.-LIKEDISEASE
F . W. McCoy, N . Bigley and M . C. Dodd, Columbus, Ohio
Alterations simulating lupus erythematosus were produced in rabbits injected
intramuscularly with glucose incorporated in Freund adjuvant. These changes were
evidenced by the appearance of antiglobulin positive red cells, hyperglobulinemia,
particularly of the gamma globulin fraction, the development of anticomplementariness in these sera, and increases in serum DNA. Positive complement fixation tests
were noted with these sera and DNA and with rabbit liver nuclei in a percentage
of the animals. Moreover, skin hypersensitivity to hexoses and hexose-linked
materials was demonstrated. The most marked skin reactions were to glucose, 2deoxyglucose and uridine diphosphoglucuronic acid. At sacrifice, the kidneys, lungs
and the areas around the organs revealed marked nuclear alteration with many
areas of “smudging” and changes histologically compatible with hematoxylin bodies.
The histochemical reactions of these nuclear changes were similar to those observed in material from patients dying with D.L.E.
DISCUSSION OF PAPER 15
UR. CARL PEARSON (Los Angeles, Calif.):
Did you use adjuvant without the glucose?
DR. MCCOY:
DR. PEARSON:
YeS.
It doesn’t happen?
UR. MC COY:
It doesn’t happen with
Freund’s adjuvant alone.
sacrifice him, except now we are sacrificing
them at regular intervals in an effort to
make time studies. Not all of the animals
will show changes and some will show it
very beautifully in many organs. Most frequently we find changes in the nuclei in
the heart first, and more constantly there.
DR. HARRY BARTFELD (New York, N. Y. ) :
Were the changes that you found in rabbits
self-prepetuating or limited?
DR. OTTO STEINBROCKER (New York, N.
Y.): I would like t o ask Dr. McCoy
whether he had occasion to permit some
of the animals to go unsacrificed for a while
to see whether the phenomenon disappeared?
DR. MC COY: There are several things that
happen. One is that the outcome of any
particular rabbit per se is a very guarded
thing. He may expire very rapidly. The next
time one may survive a considerable length
of time and unless he gets ill we do not
DR. MC COY: It will disappear I think. We
have animals that have gone 120 days, and
for all practical purposes we just sacrifice
them to see what we could find and it is
not always present. It is a temporary affair
and unless it is really severe, if you can
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AMERICAN RHEUMATISM ASSOCIATION
use such a term, the animal does survive.
Incidcntly, one of the experiments we did,
which is very difficult to explain, hut does
happen is that if you would inject one of
thcse animals with an enzyme-like betaglucuroniclase he simply goes to pot. He
just passcs out wtihin an hour or so. It is
a n example of just having an enzymatic
dissolution of his cells and they demonstrate
considerable pathology.
DR. JOHN VAUGHAN (Rochester, N. Y.):
Dr. McCoy, you have implied that there is
mtibody formation to glucose. What criteria
have you used to indicate that there actually is antibody formation? I think that the
only thing that you have done is skin tests
in animals which I suppose are relatively
sick at the time the skin tests are done. I
would like to know whether animals injected with anything else that might make
them somewhat ill might show skin tests
andogous to this. Have yon set up controls
of this sort? Is it possible that the concen-
tration of glucose you have used is at a
very hypertonic level, relative to the cells?
m. hi(’ COY: In answer to your last que+
tion first, thc skin tests were made with
0.01 per cent solution. In regard to the first
question, we only tested animals made ill
by our cqerimental procedurc.
MomRATon:
Did you
Vaughan’s first question?
answer
Dr.
DR. M C COY: As far as the skin test is
concerned we will admit that a demonstration of antigen-antibody reactions is sometimes beset with difficulties. The best cvidence that we can submit at this time for
it being of this nature is the fact that in
a few animals we can passive transfer
something and this I think, would tend to
make you feel that it was either an antibody or complex that produces an interinediate or delayed type of sensitivity reaction.
AND TUMOR
REGRESSIONIN RATS
16. MYCOPLASMAL
( P P L O ) POLYARTHRITIS
Russell S. Jones, E . Virgil Howcird, and John R. Ward, Salt Lake City, Utuh
A sporadic polyarthritis was observed in Sprague-Dawley rats bearing MurphyStrirm lymphosarcoma but did not develop in normal controls. Mycoplasma arth1 itidis ( pleuropneumonia-like organisms, PPLO) were cultured from the joint lesions
and from tumor tissue.
Rats bearing lymphosarcoma were treated with tetracycline and were considered
“PPLO-free” as these organisms could not be recovered by culture. PPLO were
transmitted to experimental animals by tumor cell transplants (such cells contained
PPLO culture) or by injection of broth cultures of PPLO isolated from joint lesions
or from the tumors. Necrosis of tumors ~ 7 a sproduced by the intravenous injection
of Klebsiella pneumoniae polysaccharide.
Rats injected with PPLO a n d bearing Murphy-Sturm lymphosarcoma tumors
developed a severe polyarthritis. This arthritis occurred with regularity in animals
whose tumors either regressed spontaniously or as a result of polysaccharide injection. If tumor regression or necrosis did not occur, arthritis was not observed.
“PPLO-free” animals did not develop arthritis despite tumor regression. “PPLO-free”
tumor-bearing rats with spontaneous tumor regression developed polyarthritis within
2 to 4 days following injection of PPLO.
The data indicate that the significant conditions for the production of polyarthritis
in these tumor-bearing rats are: (1) the presence of Mycoplasma arthritidis; and
( 2 ) the concurrent or prior necrosis of tumor tissue. A host-response to the necrotic
tumor appears important since rats with recently regressed tumors, b u t without
remaining necrotic tissue, have enhanced susceptibility to arthritis on injection
of PPLO.
DISCUSSION OF PAPER 16
UH. HARliY B A R r w x D
(New York, N. Y. ) :
1 assume you mean the animal must have a
double injury. The production of some un-
known antibody to the regressive tuinor
that may be causing this regression and
acting casually with PPLO. Is this antibody
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AMERICAN RHEUMATISM ASSOCIATION
;I macroglobulin? If you do a sheep cell or
latex test in rats does it give a positive test?
UH. JONES:
The presence of tumor-necro-
\is has in some way modified the host so
that the PPLO, nonpathogenic in the normal or tumor-hearing rat, induced polyarthritis. Whether this is due to a change
in the PPLO or in the host or both is not
clear. The present study clearly indicates
that growth of PPLO in intact tumor is not
necessary for the development of polyarthritis. The exact nature of the host
changes are also uncertain. Antibody or
cytotoxins against the developing tumor is
one possibility. Antibody against the PPLO
perhaps formed within the lymphosarcorna
itself, or entering the tuinor after the injury
by bacterial polysaccharide is another aspect requiring further study. Sheep cell or
latex fixation tests have not been performed
on rats in the group reported although Dr.
Ward has done some in a separate study.
UH. BARTFELU: What about regression of
the tumor with agents other than polysaccharide?
DR. JONES: We have only studied tumor
regression occurring spontaneously and that
induced by bacterial polysaccharide. The
polysaccharide per se in the tumor-free
control did not predispose to the PPLO
arthritis.
17. DIRECT
IDENTIFICATIONOF GROUPA STREPTOCOCCI
HY
FLUORESCENT
a4NTlSERUM METHOD
Andrew Huuos, and George J . Frioii, West Haven, Conti
Detection of beta hemolysis in blood agar plates is well recognized as an inadequate means of identification of Group A streptococci, although under epidemic
conditions the error introduced by this procedure is small. Under nonepidemic conditions, such as usually exist in civilian populations, a large percentage of beta
liemolytic streptococci isolated are found to belong to other groups. The specific
precipitin test identification of streptococci is relatively time consuming for use
in small laboratories and yields results only after two or three days’ delay. Use of
this procedure has been less than would be desired for adequate laboratory support
of rheumatic fever prevention programs.
A serologic method allowing immediate identification of group A strep tococci is
described. Group specific anti-A rabbit serum was prepared and its globulin fraction labeled with fluorescein isothiocyanate. When beta hemolytic streptococci
were incubated with this fluorescent antiserum and thoroughly washed with buffer,
green staining of the organisms was observed with fluorescence microscopy. After
a1,sorption with killed group C and G organisms, the reaction was specific for
group A in tests with a small number of stains of known serologic group. The serum
thus prepared has maintained its potency for several months.
Ninety freshly isolated strains of beta hemolytic streptococci were tested independently by the method described and the conventional precipitin test. All of 69
organisms identified as group A by precipitin tests were correctly identified with
fluorescent antiserum. Of 21 strains not identified as group A by the precipitin test,
none were positive with the fluorescent technic.
These results indicate that the fluorescent antiserum has the same specificity as
the precipitin test and may provide a simple method for rapid serologic identification
of group A streptococci.
DISCUSSION OF PAPER 17
PHYSICIAN: Dr. Friou, did you run any
studies on patients that had received penicillin for 10 days?
DR. FRIOU:
We have not done that. The
only clinical studies that we liave done were
on patients who had a culture obtained
which was positive before treatment. I
think one area where there may be problem
in using this for direct smears may be thc
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AMERICAN RHEUMATISM ASSOCIATION
occasional patient who has very few organisms present; this may be true in some
patients after treatment.
DR. MELVIN KAPLAN (Cleveland, Ohio) :
Dr. Friou, I think perhaps one ought not
conclude that one is staining the Group A
carbohydrate on these cells despite the fact
that it is on the periphery of the coccus,
probably corresponding with cell wall. I
wonder if you have any data as to the
specific antigen or antigens detected by
fluorescence?
DR. FRIOU: I did not mean to emphasize
that the doughnut appearance means that
there is selective staining of the cell wall.
It may simply be a matter of diffusion into
the cell, but is of interest because of the
location of the group specific polysaccharide
in the cell wall. The antiserum was made
by the usual method for preparing group
A antiserum. It gave a strong precipitin
test with group A polysaccharide, and was
negative with other similar extracts. Such
serum is generally considered to react only
with polysaccharide in extracts, but other
intracellular group specific antigens may
exist. This possibility was not investigated,
but we would be interested in the information that you apparently have on this subject.
DH. KAPLAN: I might make this little
comment: in our studies, anti-A grouping
sera absorbed with purified A carbohydrate
still give a staining reaction which is specific
for group A streptococci, indicating that the
fluorescent method is not directly related
to the precipitin reaction used for grouping
streptococci.
PLENARY
SEssroN-Presiding
: DR. CHARLES RAGAN
18. CEI,LUI.AR
ORIGINOF RHEUMATOIDFACTOR
R . C. Mellors, R. Heimer, J. Corcos, and L. Korngold, New York, N . Y.
The presence of proliferative and exudative inflammation of the synovial membrane and the existence in the plasma of unusual macroglobulins comprising the
rheumatoid factor are characteristic features of rheumatoid arthritis. The cellular
sites of formation of the factor and its relevance to the synovial inflammation are
unknown. The reactant in certain serological tests for rheumatoid factor is thought
to be aggregated human gamma globulin. Labeling of this reactant with fluorescein
was found not to destroy its serological reactivity and provided a sensitive and
specific reagent (or stain) for the microscopic detection of rheumatoid factor in frozen tissue sections. The synovial and certain other tissues obtained surgically from 10
patients with classical active rheumatoid arthritis and strongly positive serological
tests for rheumatoid factor have been studied by this means. Cellular rheumatoid
factor was detected in each specimen of rheumatoid synovitis. Cells in lymph node5
obrained from a patient with rheumatoid arthritis also contained rheumatoid factor.
In the synovial membranes plasma cells in various stages of maturation and comprising prominent constituents of the submesothelial inflammatory exudate contained
rheumatoid factor. In lymph nodes “large pale cells” in the germinal centers of
lymphoid nodules and internodular plasma cells contained rheumatoid factor.
Rheumatoid factor was not found in normal and pathological control tissues obtained
from patients without rheumatoid arthritis and with concomitant negative serological
tests for rheumatoid factor.
Fluorescent antibody stains for 7 S and 19 S human gamma globulin were also
used in this investigation. Evidence for the differential function of cells was seen
in lymph nodes obtained from a patient with rheumatoid arthritis. Approximately
8 out of 10 germinal centers contained 7 S and/or 19 S gamma globulin whereas
about 1 out of 10 centers contained 19 S, which corresponded closely with the
occurrence and distribution of rheumatoid factor.
263
AMERICAN RHEUMATISM ASSOCIATION
DISCUSSION O F PAPER 18
DR. MORRIS ZIFF (Dallas, Tex. ) : I would
like to congratulate Dr. Mellors on this
excellent paper. Do you have any observations about the presence of the factor on
the endothelial lining of vessels or in perivascular tissue?
DR. MELLORS:
To date I have not seen
any deposition of rheumatoid factor in
blood vessel walls. I do have material on
an individual with rheumatoid arthritis,
positive L.E. tests, and some evidence of
necrotizing vasculitis in which normal 7 S
gamma globulin is deposited in the necrotizing arterial lesions; but rheumatoid factor is not there or if it is it is hidden or
hound up somehow.
DR. ZIFF:
Even in nodules?
ER. MELLORS: We have studied rheumatoid nodules in some number. The central
necrotic eosinophilic zone of a rheumatoid
nodule does not contain detectable rheumatoid factor. The palisading cells around
that center zone do not contain rheumatoid
factor. However, when one examines blood
vessels in the fibrous connective tissue in
which the rheumatoid nodule is arising,
this comprising its third or outer zone, one
may see rheumatoid factor in plasma cells
that form perivascular cuffs.
IXI. ZIFF; This is what I was getting at.
We had noticed that phenomenon too,
DR. JOSEPH BUNIM (Bethesda, Md.) : Dr.
Mellors, I wonder if you had occasion to
examine the lymph nodes of adults, or
especially of children, who have unquestionable rheumatoid arthritis, but who have
a persistently negative rheumatoid factor
reaction in the serum?
DR. h f u L o R s : I did mention the recent
experience with an adult, clinically classified by Dr. Freyberg’s staff as probable
rheumatoid arthritis, who had negative latex
fixation tests and whose synovial biopsy of
knee did show cellular rheumatoid factor.
Rr. Freyberg may wish to make a comment
on this particular situation. We hope with
the cooperation of his staff to carry out
more studies of this type of case.
DR. RICHARD FREYBERG
(New York, N.
Y.): I think Dr. Bunim was asking about
the studies in children. These are being
started; we do not have results to report
at this time. The excellent demonstrations
of rheumatoid factor in plasma cells that
Dr. Mellors has shown you were found in
patients who had high titers of rheumatoid
factor in the blood, shown by latex fixation.
The question that arises in the minds of all
of us is; can it be demonstrated that plasma
cells in subsynovial tissue are making this
factor before the sheumatoid factor can be
detected in the plasma of the patient?
Studies of this question are underway and
this one patient that Dr. Mellors referred
to was studied just last week and the finding of the rheumatoid factor in the plasma
cells in the inflamed joint capsule, when
the factor was not found in the blood,
strongly suggests that this special tissue
staining technic might allow diagnosis or
confirmation of the diagnosis of rheumatoid
arthritis before the rheumatoid factor can
be demonstrated in the blood. Studies of
this question are being continued.
DR. JOHN SHARP (Boston, Mass.) : Have
you had the opportunity to study the
lymphoid tissues from patients who have
so-called rheumatoid factor but not rheumatoid arthritis, such as, sarcoid, leprosy
or even syphilis?
DR. MELLORS: Due to the kindness of Dr.
Kunkel and Dr. Chase of Rockefeller Institute we have been able to study portions
of a lymph node and t w o spleens from
individuals with positive F I1 tests for
rheumatoid factor. According to Dr. Kunkel
these individuals had a diagnosis of sarcoidosis. The sections did not contain
demonstrable cellular rheumatoid factor.
However, those tissues had been in storage
for a long time, and I mention this only
as a preliminary experience that must be
enlarged upon before any conclusions can
be made.
264
AMERICAN RHEUMATISM ASSOCIATIOS
JJH. JOHN VAUGHAN (Rochester, N. Y . ) :
llr. Mellors’ study has certainly given u s
the sort of thing we have been looking for.
Howevcr, I think it is important to eniphasize one or two points. First of all, Dr.
Alellors has labeled aggregated fraction 11
mtl demonstrated this to fix to cells. It is
something that needs further demonstration. Attempts to make isolated cell suspensions make antibodies in tissue cnltnrr
are necessary before we really can be sure
that the rheumatoid factor is being formed
here. Everybody, including ourselves, who
has looked at isolated tissues have been
unsiiccessful in demonstrating formation of
rhenmatoid factor in vitro. I would suspect
that this is a manifestation of our inadecliiacies rather than indicative that Ilr.
14ellors’ data does not demonstrate formation at the sites that he has pointed out t:,
11s. There is another point. This is thnt
there are several criteria for indicating
whether or not a substance is rheumatoid
factor. That is, we like to think of interaction with rabbit gamma globnlin as well
:IS
human gamma globulin. I wonder
whether control studies should not be included to indicate that this material follows
the pattern of interaction one would expect
of it. I would like finally to have Dr. Mellors comment on one more point. I gather
the plasma cells in the villus projections
of the synovia were stained and yet it was
not the plasma cells in the lymph nodes
that were emphasized hnt the pale cells in
the middle of a lymphoid follicle. I wonder
why this needs elaboration.
DH.
MELLOHS:
The Russell bodies which
I have illustrated containing rheiimatoid
factor are an integral part of the structural
make up of those plasma cells, as can bc
shown by electronmicroscopy. The Rnssell
bodies arise in close relation to the ribosomes or microsomes of the plasma cell
which are the constituents of the cell that
are concerned with the fabrication of protein molecules. I think that this is strong
cvidence for cellular origin of the factor.
There are other relevant morphological data
that I cannot relate because of the shortness
of the hour, Concerning specificity, thr
staining reaction for rheumatoid factor is
completely abolished by first treating the
tissue section with unlabeled antibody
against 19 S human gamma globnlin. In
other words, if antibody against 19 S
gamina globulin is first layered over rherimatoid factor, there is no subsequent staining with dye-labeled reactant for rheumatoid factor. Concerning lymph nodes thr
large pale cells of the germinal centers
contain rheiimatoid factor as do also sonic
of the plasma cells which occupy the intrrnodnlar, medullary spaces.
I)R. NORMAN COOPER (New York, N. Y . ) :
Do the germinal centers which contain the
rheumatoid factor lack 7 S gamma globulin
and vice versa?
DR. MELLORS: Yes.
19. GAMMAGLOBUIANCOMPLEXES
IN
TEE
P.4TIEXTS WITH RHEVhf ATOID
SERAOF CERTAIN
ARTHRITIS
IIenry G. K i r n k d , H u g h H. Fudenherg, and H .
New York, N . Y.
J.
Afua2ler-Eherhurd,
Six patients with severe rheumatoid arthritis have been encountered who showed
series of unusual components by ultracentrifuge analysis of whole serum, euglobulin and gamma globulin fractions. These proved to be complexes with S rates of 9
to 17 S a n d where readily disspciated in urea a n d acid buffers to simple 7 S
miits. In three of the sera these complexes made u p the dominant portion of the
gamma globulin fraction. The distribution of the complexes was markedly affected
by t h e addition of ordindrv 7 S gamma glohulin causing a shift to components
of smaller size (10 S) .
The complexes were devoid of rheumatoid factor activity bnt were assoriated
with high titers in the rheumatoid factor serological reactions. Howevrr, they
showed strong inhibition capacity. Complexes between these components nntl
rheumatoid factors were observed giving S rates as high a s 28 S. The significance
a n d distribution of these complexes remains unclear because no sensitive method
‘t
983
AMERICAN RHEUMATISM ASSOCIATION
for their detection is as yet available. Some evidence for a relation to “spontaneous
precipitates” was obtained. The possibility exists that t h e complexes represent the
7 S counterparts of the 19 S rheumatoid factors combined with ordinary p i n m a
globulin, although simpler polymers are not ruled out.
DISCUSSION OF PAPER 19
I>H. CHARLES CHRISTIAN (New York, N.
Y.): Two questions. One, could you make
m y correlation between the presence of
these intermediate complexes and the activity of the rhenmatoid arthritis. The other
question, you mentioned a c1issoci:itio:i with
acid buffers or urea, do these reassociate
when you bring them back to isotonic conditions or back to normal pH?
1x3.KUNKEI.: I do not think that we could
distinguish anything about the clinical
coiirse of these cases anymore than we have
been able to distinguish anything about the
course in relation to the rheumatoid factors.
Some were relatively benign rhenmatoid
arthritis; some were extremely severe. Therc
seemed to be some correlation with age,
that is, the cases tended to occur in younger
individuals. Four of the ten were in the
age group below 25. The second question
regarding reassociation: this certainly occurs. We have carried out experiments following urea dissociation: after dialyzing
away the iirea the complexes reformed.
DR. RALPH HEIMER (New York, N. Y.) :
What proof do you have that the 17 S
material or any high sedimenting materials,
let us say 11 S or 15 S, are not rheumatoid
factors themselves? As I will show later
on in the afternoon, I have isolated a 17 S
compound with rheumatoid factor activity.
The second question, have you done electrophoretic experiments to show that these
aggregates are really gamma globulin?
Thirdly, what is your evidence for stating
that these aggregates are dissociated into
7 S monomers? From the pictures which
you have shown, there was a limited amount
of 7 S gamma globulin present to start
with and I was wondering whether perhaps these monomers could be other than
7
s.
DR. KUNKEL: I thing that there is no
question that these are not 19 S or even
17 S rheumatoid factors, because the most
striking characteristic of these materials is
their dissociation. They just melt away in
buffers a5 high as p H 5.5 with any buffer
that is used and this is certainly not a characteristic of rheiimatoid factors JS we know
them. Regarding the exact S rate of the
dissociated material, we have calculatecl
this in a large number of experiments and
it is clearly between 6.5 and 7 S. We have
been very anxious to know whether at
least the active portion of the fraL;ment is
classical 7 S gamma globulin and has thc
properties of a classical antibody. One
possibility that was raised in the presentation is that perhaps this is rheumatoid factor of the 7 S type. We know that antibodies occur frequently of both the high
niolecuIar weight, and the low inoleciilar
weight class. If the rheumatoid factors are
antibodies, one might expect to find low
molecular weight types in addition to the
usual high molecular weight materials. The
complexes were clearly defined as gaiiim I
globulins by a variety of electrophorztic as
well as immunological technics.
DR. MORRIS ZIFF (Dallas, Tex. ) : We have
seen three such sera. All three were from
men in their 60’s. Two had rheumatoid
arthritis and one was a relative of two
rheumatoid arthritics who himself had
liver disease. All had very high sensitized
sheep-cell titers and all had positive SIA
tests, as well as globulins sedimenting iii
the range 11 S to 13 S. When brought to
a p H of 3.5, in two cases all of the extra
heavy component apparently turned into
7 S gamma globulin and one a large fraction of it did so. When Dr. Lospalhito
chromatographed the sera on IIEAE cellulose in one case, the 13 S component was
eluted with the 7 S gamma globulin and
that fraction showed F I1 precipitating
activity. Electrophoretically, they all had
gamma globulin spikes and these spikes
were, as far as we could tell, due to the
extra component. All had very high total
protein concentration, about 10 per cent,
and positive SIA tests. All patients were
regarded in the beginning as examples of
macroglobulinemia of the Waldenstrom
266
AMERICAN RHEUMATISM ASSOCIATION
type in association with rheumatoid arthritis. On ultracentrifugation, one sees a
good deal of heavy protein but the ultracentrifugal heavy peaks are not discrete.
I would assume that this type of patient
can be picked out by doing SIA tests on
all sera with fairly high rheumatoid factor titers.
DR. HARRY BARTFIELD (New York, N. Y. ) :
You mentioned that these materials have
a relation to spontaneous precipitates. What
relation do you mean?
DR. KUNKEI.: Well, in the first place, in
several of the sera very large amounts of
precipitates formed directly on standing,
more than with our other rheumatoid arthritis sera. Then when we isolated these
complexes they proved to be most insoluble.
Even more so than the rheumatoid factor
complexes and we had difficulty in some
cases keeping them in solution. Also by
using Dr. Christian’s criteria of prccipitation on dilution 10 times in saline, these
had that characteristic very strongly in a
number of instances.
OF THE SENSITIZEDSHEEP-CELL
20. STABILIZERSAND INHIBITORS
AGGLUTINATION
REACTION
E. C. Franklin, New York, N . Y.
Euglobulin fractions from most normal sera prevent the agglutination of sensitized sheep cells by rheumatoid sera. Similar fractions from rheumatoid sera fail
to do so. Starch zone electrophoresis of euglobulins demonstrated two peaks with
gamma and alpha-2 globulin mobilities and small amounts of beta globulin. Inhibitory activity was associated with the gamma and alpha-2 globulins and occasionally with the beta globulins. Inhibitory activity of pseudoglobulins was found
in the same fractions and never with albumin. Following separation of euglobulins
by density gradient ultracentrifugation into a top ( 7 S ) , middle (19 S ) and pellet
fraction (aggregates), inhibitory activity was associated with each.
Pure 7 S gamma globulin failed to precipitate with rheumatoid factor and did
not inhibit. Inhibitory and precipitating activity was increased by heating a t 56
to 63 degrees C., and was due to aggregates.
The alpha-2 globulin which prevents agglutination was stable a t 56 degrees C.
for two hours, and a t 63 degrees C . for one hour. Its stabilizing activity was primarily associated with the top and middle fractions following gradient centrifugation, but could not b e identified with any specific protein of the alpha-2 globulins.
Although the euglobulins from rheumatoid sera and sera from certain patients
with liver disease failed to inhibit they contained an alpha-2 globulin inhibitor
activity, but accurate quantitation proved difficult. The importance of the inhibitor
is illustrated by the failure of the euglobulins of 5 patients with agammaglobuh e r n i a and certain patients with niultiple myeloma to inhibit. Possible mechanisms of action of the inhibitor and alpha-2 stabilizer and their clinical significance
will be discussed.
DISCUSSION OF PAPER 20
DR. WALLACE EPSTEIN (San Francisco,
Calif.): Do the sera of individuals thought
to have rheumatoid arthritis show significant
inhibitory activity when the direct tests
for rheumatoid factor are negative? Have
you studied the sera obtained from cases
of juvenile rheumatoid arthritis or arthritis
associated with psoriasis?
DR. FRANKLIN: We examined 2 sera from
patients with juvenile rheumatoid arthritis
whose euglobulins did not agglutinate sensitized sheep cells and whose euglobulins
also failed to inhibit. In both instances were
we able to demonstrate inhibitory activity
once the fractions had been isolated. This
may be in part due to the fact that these
AMERICAN RHEUMATISM ASSOCIATION
fractions have been further treated, and
consequently altered during the procedure.
I would like to emphasize again that accurate quantitation of inhibitory activity
is exceedingly difficult.
stabilize the unsensitized or sensitized particle so that no reaction can take place in
the presence of the factor. The term competitor is preferable to inhibitor when applied to the reactivity of latex particles.
DR. JACQUES M. SINGER (New York, N.
Y.): I shoidd say that we ought to distinguish between inhibitors and competitors. Plasma fractions other than fraction
I1 do not act as an inhibitor for the rheumatoid factor, but they may compete for
the surface of the latex particle and thus
DR. FRANKLIN: I would agree with you
Dr. Singer, and I would think that Dr.
Ragan’s suggestion that these alpha-2
globulins and similar substances be called
stabilizers rather than inhibitors is a very
good one.
21. THE BEHAVIOROF GROWINGSYNOVIALCELL CULTURES
C. William Castor and Florence F . Fries, Ann Arbor, Mich.
Previously reported experiments revealed similar rates of hyaluronic acid
synthesis in synovial cell cultures derived from four normal persons and one
rheumatoid patient. Data are now available concerning the behavioiir of fifteen
cell “lines,” derived from normal synovial tissue in ten instances, rheumatoid synovium in three cases, and from single examples of pigmented villonodular synovitis
and traumatic synovitis.
Monolayer cell cultures were grown on glass in a semisynthetic medium and
subcultured with the aid of 0.25 per cent trypsin. Cell “lines” were subcultured
as often as nineteen times and survived up to nine months in vitro. The parameters
cf the cellular system measured included cell counts, total culture mass and total
culture protein. Supernatant medium was analyzed for mucopolysaccharides,
lactic acid and residual hexose.
Cellular proliferation was accompanied by synthesis of increasing amonnts of
acid mucopolysaccharide. In older cultures sporadic formation of fibers with staining characteristics resembling collagen was noted. During rapid proliferation over
90 per cent of the hexose consumed could be accounted for as lactic acid. Mucopolysaccharide synthesis could account for only 0.5 to 3.0 per cent of the hexose
utilized. The rate of mncopolysaccharide synthesis in these cultures was similar,
in spite of differences in the age and health of the donor. In older cultures lactate
formation tended to lag behind hexose consumption, a finding which may be
related to the observed intracellular deposition of glycogen. Under the conditions
of these experiments, neither insulin nor testosterone induced clear cut alteration of
the parameters measured.
(No discussion followed paper 21.)
22. THE SIMULTANEOUS
PRODUCTION
OF ACID MUCOPOLYSACCHARIDES
AND
COLLAGEN
BY FIBROBLASTS
IN TISSUE
CULTURE
L. Myrton Gaines, Jr., Baltimore, kld.
The production of acid mucopolysaccharides has been previously demonstrated
in tissue cultures of synovia and other tissues which are wholly or partly of
mesenchymal origin. Although fibroblasts are presumed to be the cells which
synthesize these mucopolysaccharides, this presumption rests primarily on mor-
268
AMERlCAN RHEUMATISM ASSOCIATIOX
phologic criteria which are often invalid in tissue culture. These investigations are
designed to obtain evidence concerning the type of cell capable of mucopolysaccharide synthesis. Since there is considerable evidence that the fibroblast is
required for collagen synthesis, studies demonstrating simultaneous synthesis of
collagen and mucopolvsaccharides will be presented as evidence of functional
identity of cell type.
Heart and skin explants from 11 day old chick embryos were cultured with
standard technics in natural media. Fibers were demonstrated by reticnlin staIns
within 5 days of growth. At specified intervals cultures were terminated. Collagen
content was determined by extraction of gelatin, hydrolysis and measurenient of
hydroxyproline. Acid miicopolvsaccharides were quantified by u turbidimetric
in ethod .
There was an 100 per cent increase in collagen content after 10 days of growth;
up to 300 per cent after 13 days. In the same cultures synthesis of acid mucopolysaccharides of comparable magnitude, 1.00 per cent or greater over control values,
was demonstrated. The predominant cell type in these cultures was a spindleshaped cell with long cytoplasmic processes: i.e. morphologically, a fibroblast.
Therefore, additional evidence is provided to support the concept that fibroblasts
participate in the synthesis of acid mucopolysaccharides as well as of collagen.
1)ISCUSSION OF PAPER 22
I)R. JEHOME Rors’rEIN (New York, N . Y.):
\Vhat is the sensitivity of your hydrouyprolinr method?
I)H. CAINES:
Plns or minus 3 per cent
DK. HOTSTEIN: How many micrograms per
miIliIiter can you detect?
DR. GAINES:
Micrograms?
IIR. ROTYTEIN:
DR. GAINEs:
Yes
N. Y.): There is another way of checking
whether the fibroblast is in the state of
differentiation, this is chromosome counting and I wonder very iiiucli whether
chromosomes have been counted at variotis
stages in your preparation because if these
varied this would suggest that some process of dedifferentiation has taken plnce.
This is what everyone is so worried about,
whether collagen or acid mucopolysaccharide are produced by the same cell in
vivo, in the differentiated cell.
Down to about 4 micrograms
DR. GAINES: Chromosome counts were not
done. This is an unanswered problem, T
think.
per milliliter.
CONCURRENT SESSION
“C’’-PreSid’hg :
DH. JOHN VAVGHAN
23. PROPERTIES
OF AGGREGATEDH U M ~ G
N4
~
GLonuLn
~
4
Charles L. Chrbtiun, New York, N . Y
The serological systems in which rheumatoid arthritis sera react can be divided
into two general categories: ( 1) immune sensitized systems, i.e., sensitized sheepcell agglutination, Rh-sensitized agglutination, and the reaction with immune
precipitates; and ( 2) nonimmune sensitization, i.e., a variety of systems utilizing
Cohn fraction I1 ( F 11), where specific immune affinity of gamma globulin foi
antigen is not required. In the latter reactions, evidence has been presented
which suggests that the reactive material in F I1 and other preparations of humair
gamma globulin (HGG) consists of molecular aggregates of 7 S gamma globnlin.
The present report concerns a comparative study of aggregated HGG and non-
;ui
269
AMERICAN RHEUMATISM ASSOCIATION
aggregated HGG with respect to electrophoresis, serum complement ( C' reactivity, a n d inflammatory responses in skin.
Aggregated HGG formed a sharp electrophoretic boundary (moving boundary
i-echnic) which migrated slightly faster than t h e peak of nonaggregated HGG.
Aggregated HGG was intensely anticomplementary, a n d preliminary evidence
indicated that specific C (component inactivation) resembles immune decomplementation. In vivo denomplementation of guinea pigs could b e accomplished by intravenous administration of aggregated HGG. Intracutaneous injection of aggregated HGG
resulted in a n inflammatory reaction.
Aggregated HGG, then, resembles immune aggregates with respect to (1) reactivity with the rheumatoid factor, ( 2 ) C' inactivating properties, a n d ( 3 ) inflammatory cutaneous responses. It is suggested t h a t t h e primary specificity of the
rheumatoid factor is directed towards antigen-antibody complexes, a n d that the
artificially aggregated HGC, required in the various F IT tests simulate5 immnne
aggregation.
I>ISCUSSlON OF PAPER 23
DR. ABRAHAM OSLEH (Baltimore, Md. ) : I
think it is very encouraging that the results
reported by Dr. Christian are entirely in
conformity with those which we have obtained, approaching the problem from a
slightly different point of view. ,4s some of
you may know it has been possible to take
native human gamma globulin and aggregate it by heat and this aggregated gamma
globulin possesses all of the properties th:it
Dr. Christian has mentioned, including the
ability to induce a weal and flare reaction
in human skin as well as in the guinea pig
and rat. It is further of interest that the
aggregated gamma globulin has enormously
greater anticomplementary properties and it
remains for further work to determine the
more precise relationship between uptake
of complement by aggregated gamma globnlin and the biological effects of these
materials.
DH. WALLACE EPSTEIN (San Francisco,
Calif.): I am sure Dr. Christian would
agree with a word of caution concerning
the intrinsic irritating effects of heated
gamma globulin. The aggregated globulin
may have the ability to imbibe fluid and
irritate tissue at the site of injection. Further controls would seem necessary before
a direct parallel to the tissue damaging
effects of antigen-antibody systems arc
established.
DR. CHRISTIAN:
I agree with Dr. Epstein's
recommended caution. The amounts rcquired are infinitely small and I think Dr.
Osler has had experience with lesser concentrations than I. The immediate response
is rather striking and I think could not be
related to an oncotic or osmotic effect. In
addition we have used denatured human
sernni alhnmin without a rcsponse.
DR. 11. J . MULLER-EBEHHARD (New York,
N. Y . ) : We are very interested in the problem of interaction of gamma globrilin aggregates with complement components.
Recently, we were able to isolate one of the
complement components of human serum
which turned out to be one of the two
subcomponents of the classical third component of complement. This protein has
the interesting feature of being physically
changed on inactivation which occurs either
dnring aging of serum or by interaction with
substrate. With the aid of immunoelectrophoresis this physical change can readily
be detected. When we incubated serum with
soluble gamma globulin aggregates at 37
degrees C. we found that this component
of complement was inactivated and physically changed as it was on interaction with
immune precipitates. As this protein is part
of c', it is safe to conclude that the reaction with gamma globulin aggregates also
involved the first, fourth and second component of complement.
I)H.
JACQUES
M. SINGEH
(New York, N.
Y.): From the work of Ishiazakal which
has confirmed your findings, the aggregated
human gamma globulin obtained by heating produced an immediate skin reaction in
normal guinea pigs and fixed complement
whereas aggregated gamma did not havc
either activity. In our laboratory rheumatoid
270
factor was not inhibited by bovine gamma
globulins. The term “aggregated gamma
globulin” probably needs further clarification.
DR. OSLER: I quite agree with Dr. Singer’s
remarks and rise to point out that bovine
gamma globulin lacks two properties which
seem to be necessary for the biological
tissue damage. One, it does not fix complement and secondly, bovine gamma globulin
does not stay fixed to the skin of the experimental animal, the guinea pig.
DR. HENRY KUNKEL (New York, N. Y.):
I would like to ask Dr. Christian if he
thinks the mechanism of the skin sensitivity
is perhaps related to the aggregates uniting
with complement components. In addition,
whether he has seen any greater skin sensitivity in a patient that has a high titer of
rheumatoid factor and whether the interaction of the rheumatoid factor and aggre-
AMERICAN RHEUMATISM ASSOCIATION
gates would also show skin sensitvity. Is
it possible to differentiate the two types
of reaction, that with complement, if that
is the cause in the normal, and that with
rheumatoid factor in the case of the rheumatoid arthritic?
DR. CHRISTIAN: The possible role of complement in mediating some of the tissue
damage in hypersensitivity is an attractive
one. I cannot go beyond speculating that
the cutaneous inflammatory properties might
be related to the interaction of this material
with complement. Dr. Osler has done a
considerable amount of work which suggests that complement may mediate anaphylactic response. In answer to the other
question, in rheumatoid patients that we
have tested by injection with this material
there has not been any quantitative variation from the response of normal subjects.
1. Ishiazaka, I., and Ishiazaka, K.: Proc.
Soc.Exper.Biol.& Med. 101:845, 1959.
24. A PRECIPITINFOR AGGREGATEDHUMANFRACTION
11, PRESENTIN NORMAL
HUMANSERA,AND WITH PROPERTIES
SIMILARTO THOSEOF THE: FIHST
COMPONENT
OF COMPLEMENT
Angelo Taruntu and Howard S . Weiss, Irvington, N . Y. and hlew York, N . Y.
Normal human sera (NHS), mixed with preparations of aggregated human
serum fraction I1 (AHF TI) were found to yield a precipitate, whose formation
could be inhibited by excess AHF 11. Quantitative determinations of the precipitates obtained using equal amounts of NHS and increasing quantities of AI-IF 11,
revealed three zones, characterized by increasing, maximal, and decreasing
precipitates and by NHS precipitin excess, equivalence, and AHF I1 precipitinogen
excess in the respective supernatants. This system thus shows formal analogies with
classical precipitating antigen-antibody systems and with the rheumatoid factor.IHF I1 system.
Previous exposure of the NHS to unrelated antigen-antibody complexes, but
not to antigen or antibody alone, inhibited the ability of the serum to precipitate
with AHF 11, pointing to a similarity in behavior between serum complement (C)
and this normal precipitating factor.
Fractional inactivation of complement components revealed that: ( 1) Heatinactivated serum, lacking the first and the second components of complement
(C, and CZ),
and the supernatant of NHS dialysed against dilute acid buffers
(lacking C‘l and C 3 ) do not precipitate with AHF 11. ( 2 ) The redissolved precipitate of NHS dialysed against dilute acid buffers (lacking C’, and C’4), the
zymosan-inactivated serum ( lacking C’3) and the hydrazine-inactivated serum
(lacking C’4) still precipitate with AHF 11, although the recovery of precipitating
activity is not complete.
These data indicate that normal human serum contains a factor which behaves
like a precipitin when mixed with AHF 11. This factor behaves like the first
component of complement in a number of significant ways, and might prove to
be identical with it.
27 1
AMERICAN RHEUMATISM ASSOCIATION
DISCUSSION OF PAPER 24
DR. H. J . MULLER-EBERHARD (New York,
N. Y . ) : I would like to say that we can
essentially confirm Dr. Taranta’s results,
especially those obtained with the various
reagents for the detection of complement
components. We have looked into the ultracentrifugal composition of precipitates obtained from such reagents and we found
that they were all essentially alike. We
were able to isolate the serum protein presumably responsible for the formation of
these precipitates and it turned out to be
a 10.7 S component. In a highly purified
form it still was able to cause precipitation
of soluble gamma globulin aggregates. The
possible relationship of the first component
is presently under investigation.
DR. TARANTA: Thank you, Dr. Muller,
for your confirmatory findings. Dr. Edward
Franklin has done an ultracentrifugal analysis of our redissolved precipitates and preliminary results indicate a major component
of about 9 S.
DR. WALLACE EPSTEIN (San Francisco,
Calif.): We have not found a strict correlation between the rheumatoid factor content
of serum and the total nitrogen precipitated.
Am I correct in believing that 3 mg. of
nitrogen added to serum resulted in 1.8
mg. total nitrogen precipitated?
DR. TARANTA: No. All milligrams and
fractions thereof in this slide are milligrams
of protein and not of nitrogen.
DR. EPSTEIN: How great a variation in the
amount of protein precipitated is found in
sera of differing rheumatoid factor content
as measured by other systems, and have
you found this precipitation system volume
dependent?
DR. TARANTA: In regard to the first question, only a limited number of quantitative
precipitin tests with rheumatoid sera have
been done and in these few the precipitate
obtained with quite markedly greater than
the one obtained with normal human sera.
With regard to the second question, we
have studied the effect of the reaction
volume on the amount of precipitate, not
through the whole range of the precipitin
reaction but at the point of maximal pre-
cipitation only, and we found with a 20fold increase of volume, a very slight decrease of precipitate, which is unlike the
early reports on the rheumatoid factorhuman fraction I1 reaction.
DR. HENRY KUNKEL (New York, N. Y . ) :
Last year we were interested in the problem of why some hepatitis sera show
positive precipitin reactions with aggregated
gamma globulin. W e would get a marked
precipitate with the hepatitis sera but they
would not give positive tests by some of
the classical serological tests. We noticed
that this factor was heat labile and was
increased considerably over a definite
amount of similar material in normal serum.
Precipitin-type curves could be obtained
with both normal and hepatitis sera when
purified aggregates were added. I think
that in some of the rheumatoid factor tests
this component can interfere. It apparently
does not in the latex test as it is usually
run but in the F I1 tanned cell test hepatitis sera which had a lot of this factor, which
is heat labile, did give positive reactions.
DR. E. COHEN (Buffalo, New York): In
using photoelectrically detected precipitation reactions of the F I1 gamma globulin
and rheumatoid sera we found that we
get the same shape of curves that you do.
However, where we use heat aggregated
globulin or F 11, if we heat the patient’s
serum, that is, inactivate it a t 56 C. for
3 minutes, we get an enhanced precipitation. I believe in one of your slides using
your formalin treated aggregated globulin
you found that that was not the case. Do
you have any comment?
DR. TARANTA: Was this work done with
normal human sera? Or with pathological
sera?
DR. COHEN: With rheumatoid sera we
have this increase which is the point that
I wanted to make, where with your normal
sera you have a decrease in activity.
nn. TARANTA: Of course a possible inhibitory effect of some complement component on at least one serological test of
rheumatoid arthritis has been described.
272
AMERICAN RHEUMATISM ASSOCIATIOIV
The pheiioinenon you describe may be similar to the one already known. On more
general groimds it is possibly pertinent to
rcmembrr that complement has been shown
b y Maiircr and others to delay precipitation
of antigen-mtibody aggregates and, thercfore, perhaps a time factor is involved here.
In all thc precipitin tests that we have done
refrigeration followed incubation at 37
tlegreos C. and refrigeration was carried out
for four days and maybe just the time fac-
25.
COMPOSITION
OF
tor inay have caused a difference. I would
like also to thank Dr. Kunkel for his comment and although I have no direct information on the heat labile positive rheiiiliatoid test that has been found by you a n d
by a number of other investigators in known
rheumatic disease and in relatives of rheumatoid patients, the hypothesis that thc
factor( s ) responsible for this reaction mny
be the same as that found in normal sera
is an intriguing one.
F O R M E D BETWEEX RIIEUMATOIU SERA
DYE-LABELLED
F I1 REACTANT
PHEClPlTATES
.4ND
Melvin €I. Kaplun, Mary L o r i Sirch!j wid Murie hleyeseriwz, Cleoeland, Ohio
Human fraction I1 was labelled with p-arsanilic acid by diazotization, or witli
1iuorescein-isothiocyaiiate by thioureide linkage, and used as reactant in the precipitation reaction with rhenmatoid sera. The rnolar ratio of label to gamma globulin was 7 to 1Fj for the arsanilic tag, and approximately 1.0 for fluorescein; at
these levels reactant activity was not inhibited, while quaiititation by photometry
or fluorimetry was adequate. Precipitates were analyzed for total N and for
reactant gamma globulin N; rheumatoid factor N was then calculated by difference.
The proportion of the total precipitate due to reactant always exceeded that
of rheumatoid factor, and was markedly increased as a result of heating reactant
:i.t S8 C. or 63 C., 10 min., prior to admixture. The ratio of labelled reactant N to
rheumatoid factor N in the precipitate showed a progressive increase throughout
the precipitation curve, the absolute values of these ratios varying with the conditions of aggregation of the labelled F 11 preparations. The amount of rheumatoid
factor N precipitated could not be equated with the total rheumatoid factor content of the serum, inasmuch as the serologic activity of the supernatants in the
inhibition zone showed considerable variability of behavior among the sera tested.
Most sera showed persistingly positive latex tests in this zone, with negative sensitixed sheep cell tests, in a few sera, both tests persisted positive; in others, both
tests became negative.
While the course and composition of the rheumatoid curves with labelled reactant bore similarities to classical antigen-antibody reactions, the relative importance
of other factors which might exert influence on these reactions, such as polydispersit).
of aggregates in the F I1 preparations, and varying association activities of individual rheumatoid factors, remains unknown.
Using these dye-labelled reactants it has been possible to demonstrate b!l i n inunofluorescent technics the presence of rheumatoid factor within germinal follicle
cells, plasma cells and Russell-body containing cells. These findings are consistent
with an antibody origin id function for rheumatoid factor.
DISCUSSION OF PAPER 25
(New York, N.
im. JACQUES M.
Y . ) : Because of the variable amounts of
aggregated material in different lots of
gamma globulin, a family of precipitation
curves can be obtained with the same rheuiliatoid serum. The shape of these precipitation curves will vary from the precipitin
type to the flocculation type. The precipitation curve presented by you does not necessarily represent a specific antigcn-antibody reaction. Many protein-antiproteii1
systems have been described such as thc
reactions of chicken whole egg-white witli
anti-conalbnmin sera. milk whey proteins
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AMERICAN RHEUMATISM ASSOCIATION
with antibeta lactoglobnlin sera, hiunan
gammal globulin with antiganima2 globulin sera, and clenaturcd ovalbumin with
antinative ovalbumin sera. All these are
charactcrizcd hy the type of curves discussed here.
1113. KAPLAN: Thank you very much for
your comment, Dr. Singer. We agree with
you entirely that this precipitation curve is
not specific for antigen-antibody systems
alone. Very similar curves have been
described in mathematical terms by Freundlich, Langmuir and many others, for certain
adsorption isotherms, which are, of course,
nonimmune. One cannot, therefore argue,
from the curve showing a changing ratio
of reactant to rheumatoid factor in the
precipitates that an antigen-antibody reaction has been proved. However, classical
antigen-antibody reactions do exhibit such
hehavior. We interpret these data as consistent with an immune hypothesis, without
necessarily excluding an alternative one.
The finding of such changing ratios in thc
rheumatoid factor-rcactant system, has
given a firm basis for approaching the question of the sites of origin of rheumatoid
factor using labelled reactant. It may be
recalled that, in studies carried out in
collaboration with Dr. J. H. Vaughan, and
reported at the Conference on Host Response Mechanisms in Rheumatoid Arthritis
(Atlantic City, May 2, l959), we described
binding of rabbit gamma globulin by germinal follicle cells and plasma cells in lymph
nodes biopsied from patielits with rheumutoid arthritis. In an attempt to confirm this
finding, labelled reactant F I1 was cniployed, as reported today.
These two bodies of data, from composition of precipitates and from cellular localization of rheumatoid factor, lead 11s to thc
interpretation that rheumatoid factor is
probably autoantihocly.
UH. HARRY BARTFIELD (New York, N. Y.):
This question was raised earlier when Dr.
h4ellors spoke. Before you can postulate the
antigen-antibody theory for your work you
will have to explain why you get a similar
result in sero-positive nonrheumatoid disease. Do you think they have the same
antigenic insult?
1)H. KAPLAN: YOLU’ (lUeStiO11, Ill‘. Bartfield, probably cannot be answered as yet.
Whether rheumatoid factor itsclf or the
factor in nonrheumatoid individuals which
behaves identically to rheumatoid factor,
occur in response to similar antigenic insults, is a question for which we should all
like an answer. How this factor is stimulated, might perhaps be approached by tissue culture studies, as suggested by Ilr.
Vaughan earlier in these sessions, or by
studying the response of susceptible individuals to antigen-antibody precipitates.
Beyond this, I have no comment, except
to agree that further study of the mechanism of production of these factors is needed.
26. STABILITY
AKD GAMMAGLOBULIN
BINDINGSTUDIES
LATEXPARTICLES
ON POLYSTYRENE
Irwin Oreskes and Jacques Singer, New York, N . Y.
Sinall ainouiits of protein e q d to 34 molecules/particle will agglutinate the
polystyrene latex particles whereas larger quantities serve t o protect. The latter
effect is strongly pH dependent. The stability of protein coated particles is a function of pH and ionic strength, with the least stability observed near the I.E.P. of
the protein. Increasing salt concentration promotes agglutination except near the
1.E.P. where stability is observed at intermediate salt concentrations. This effect is
analogous to “salting in.”
The stability of particles in the absence of protein is a fuiiction only of ionic
strength. In t h e pH range 4 to 9 agglutination WJS observed a t u = 0.30 a n d abovc.
In the pH range 1 to 3 agglutiwation was observed a t u = 0.01 and above.
Rinding of protein by latex particles was studied by determining total proteins on
supernatants after adsorbing with latex particles. The binding data was plotted
x c o r d i n g to the expression r,’A = K (N-r) where r equals amount bound; A eqnals
274
AMERICAN RHEUMATISM ASSOCIATION
equilibrium supernatant concentration, K equals binding constant and N equals
maximum amount that can be bound. The d a t a when plotted could be resolved into
two straight lines. They may be interpreted as meaning that the proteiii forms
niultilayers on t h e surface of the latex particles, with different binding constants
for each layer. The mean value of N1 was 0.170 mg. p r o t e i d m g . latex for particles
2200 A diameter. This corresponds to 3800 protein molecules i n the first layer per
particle, a n d 4010 A! surface area per protein molecule. F o r 8020 A diameter
particles N, was 0.043 mg. protein mg. latex. This corresponds to 48,000 protein
molecules in the first layer a n d 4230 A 2 surface area per protein molecule.
DISCUSSION OF PAPER 26
DR. SIDNEY COBB (Pittsburgh, Pa.): I
would like to congratulate the author on a
brilliant piece of basic work in this area
which will be of importance to all of 11s
as we continue our work. It seems to me
that there is one unknown which I hop?
we can smoke out and I wonder if you
have succeeded in doing this. The fact that
you have not mentioned it suggests that
perhaps you have been as unsuccessful as
have we. I think perhaps as a group we
may be able to force the issue. We are
dealing here, not with just latex particles,
but with latex particles which are held in
suspension with some emulsifying agent.
This is a secret of Dow Chemical Company
and before we can really understand this
system we must know the nature of this
emulsifying agent and the part that it
plays in the system.
DR. JOHN VAUGHAN (Rochester, N. Y. ) :
This is a very pertinent question. I do not
think I was aware that there was a
stabilizer of that sort and I would like to
add to this question whether you know that
the gamma globulin solutions that you are
using really are homogeneous in respect
to the type of protein that is being absorbed
out. There were no aggregates. Do we know
the relative roles of aggregated and nonaggregated protein?
DR. ORESKES: With regard to the first
question. We are aware of the fact that
there is present in latex suspensions an
emulsifying material but we have been
unable to find out from Dow what it is. I
should have mentioned that we attempted
to remove this material by prolonged washing and dialyzing of the latex particles prior
to these experiments. With regard to the
homogeneity of the protein, inhibition procedures in our laboratory indicated the
presence of only trace amounts of aggregated material. From the point of view of
adsorption, if we were successful in obtaining 95 per cent or better pure 7 S, we
have assumed that the relative weight of
the aggregated material would not be too
important. On the other hand, I might
mention one experiment of a preliminary
nature in which we deliberately heat aggregated gamma globulin and determined
the binding constant. We did find that
aggregated material bound some 5 times
more strongly to latex than did the presumably non-aggregated material that we
used.
DR. JOHN VAUGHAN: This would make it
very important then if you had 5 per cent
aggregated material. It really would be quite
important.
DR. QRESKES: Then it would have more
weight than we originally thought. However, since the protein used was twice
centrifuged at 40,000 rpm and the sediment discarded, it is very likely that the
amount of aggregated material remaining
was well under 5 per cent.
27. THE DIFFERENT
RHEUMATOIDFACTORS
IN THE SERUMOF ONE
HYPOGAMMAGLOBULINEMIC
PATIENT WITH RHEUMATOID
ARTHRITIS
Ralph Heimer, Edith R. Schwarts, and Richnrd H . Freyberg, hTewYork, W . Y.
The serum of a patient with classical rheumatoid arthritis a n d a history of infreq u e n t infections contained only 260 mg. per cent gamma globulin, b u t exhibited
275
AMERICAN RHEUMATISM ASSOCIATION
high titers of rheumatoid factor (SSC = 1:3,500,latex fixation = 1:112,000). On
starch block electrophoresis the serologically active material was found mainly in
the beta globulin fraction. Five distinct macroglobulins belonging to the rheumatoid
factor complex were isolated from this serum by the following technics.
The serum was absorbed with sheep erythrocyte stroma sensitized with rabbit
hemolysin. Removal of the macroglobulins reactive with sensitized sheep cells was
thereby accomplished. On elution from the stroma a large amount of 19.7 S and
some 27 S material was recovered. On electrophoresis these macroglobulins migrated
a t a significantly higher rate than human gamma globulin. Approximately 0.03
niicrograms of this material sufficed for a positive SSC or latex fixation test.
The macroglobulins which failed to adhere to sensitized sheep erythrocyte stroma
were fractionated by DEAE cellulose chromatography. Three additional macro~) of~ 17.5
~ S,,
globulins were thus found, having sedimentation coefficients ( s
22.2 S and probably 19.7 S respecively. These three macroglobulins exhibited no
activity toward sheep cells sensitized with rabbit hemolysin. With latex particles
sensitized with human gamma globulin they were ten times less active than the
niacroglobulins which adhered to the Sensitized sheep erythrocyte stroma.
The results of the isolation experiments were confirmed by ultracentrifugation of
a concentrate of this patient’s serum. Components sedimenting a t approximately
27 S, 22 S, 19 S and 17 S were seen. The 27 S and the 17.5 S macroglobulins
appear to be minor and infrequently occurring components of the rheumatoid factor.
The nonidentity of some macroglobulins was established by quantitative analysis
of amino acid content. The 19.7 S macroglobulin and the 17.5 S macroglobulin exhibited large differences in the tyrosine and cysteine content, but no significant
differences were found in the quantities of the other amino acids.
DISCUSSION OF PAPER 27
CR. ROBERT F. WILLKINS (Seattle, Wash. ) :
Dr. Heimer, I would like to know whit
form of elution you use from your DEAE
column. Is it gradient or is it stepwise?
DR. HEIMER:
It is stepwise elution.
DR. WILLKINS:
Do you think that this
possibly could produce some artifactual
peaks?
DR. HEIMER: Actually when we get to
pH 5.5, we can change the ionic strength
of the eluent from .03 molar to .05 molar
and so on, and each eluting solution runs
through a number of tubes. I arn quite
sure that the possibility mentioned by you
could occur. However, I have rerun these
eluted materials on an identical column and
found the same distribution and approximately the same sedimentation constant on
a complete rerun of isolated material. I fed
quite confident that the DEAE column is
reasonably reproducible and does not produce artifacts. Furthermore, I think that
the demonstration of these materials in the
whole serum also indicates that the column
is fairly reproducible.
DR.
EDWARD
FRANKLIN
(New York, N.
Y.): I wonder if you feel that this 22 S
component that you have isolated is the
same as the one that has been previously
reported and, if so, I would like to know
if it was stable at acid pH and in 4 to 6 M
urea which are known to dissociate the 22 S
complex.
DR. HEIMEH: I have not done much work
with this compound. I merely was able to
get a sedimentation constant of it. I would
like to have degraded it with urea, but because of the small yield 1 have not been
able to do this. The other thing that I
think is important in this is that this 22 S
compound only reacted with latex particles
and human gamma globulin and not with
sheep cell? sensitized by rabbit antibody.
In a way, this might suggest some of the
findings that you have reported earlier,
where you found that the correlation between the height of this 22 S peak and the
amount of sensitized sheep-cell activity
does not always agree. Thus there probably
are 22 S compounds which react with
sensitized sheep cells, and we are not saying at this point that all 22 S compounds
are simply of the macroglobulin I1 type.
~
376
DR. JACQUES M. SINGER (New York, N.
Y.): I would just like to mention a discrepancy between a very high agglutination
titer and the ultracentrifuge pattern in one
of our patients. In this instance the titers
were as follows: F I1 L.P., 1 million; SSC,
50,000; S.H.C.-D, 50,000. However, there
were only a small amount of 19 S and 22 S
components. The clinical diagnosis in this
patient is osteoarthritis.
DR. HARRY BARTFIELD (New York, N. Y.) :
I want to point out that many years ago
George Heller, who did very outstanding
work in our field, pointed out that rheumatoid factor was heterogeneous and it might
also be a beta globulin as well as a gamma
globulin.
Yes, as a matter of fact, I
might comment that we are very indebted to
Heller's work not only for this, but he was
really one of the first to show that one could
absorb only portions of the rheumatoid
facior with sensitized sheep cells; and he
actually inferred from these experiments
the heterogeneity of these factors.
DR. HEIMER:
DR. JOHN VAUGHAN (Rochester, N. Y . ) :
I cannot allow this session to come to a
close without making one or two remarks
myself. I think somewhere along the line
here we have to come to grips with the
definition of what is the rheumatoid factor.
If the rest of you are as confused as I am
by the barrage of macromolecules which
have been presented to us, then I find myself in good company. I rather think that
we could and should come to such a definition and that this might well be in terms
of the reaction of the rheumatoid factor
with the 7 S gamma globulin. Doctor
Franklin and Kunkel's group have shown
the resulting complex so nicely. This is the
way this material circulates in the blood
stream. We are given the fact that many
other aggregates may occur under certain
circumstances in a certain few patients with
rheumatoid arthritis and other diseases. We
have to find some way to know whether
these have just the same biological significance as does what we originally thought
of as the rheumatoid factor. We have to
AMERICAN RHEUMATISM ASSOCIATIOY
know which end of the interaction we actually are viewing when examining any
one of these isolated materials. I think we
have also to face very carefully the problem of whether we are sometimes getting
factitious fractions of a substance which
biologically might be a single unit, but
chemically can be separated into numerous
sub-units by such things as step-wise gradient elution, and so on. As originally defined,
this was a substance capable of causing
aggregation of cells which are coated with
antibody, and we have certainly gotten
used to thinking of this as a primary reaction with gamma globulin. I think somewhere in this vicinity we have to come to
grips with the situation and decide what
is and what is not rheumatoid factor.
DR. HEIhiER: First of all, I am not averse
to accepting new information, either in this
field or in any other field. I think that we
must not overlook that as more people
work in a given field, more diverse information will b e obtained. I am not at all
sure whether I wonld accept your very
restricted definition of rheumatoid factor.
On the other hand, if I were to accept
your definition, I would have to point out
to you that only approximately 30 per cent
of the individuals having high serological
titers for rheumatoid factor would qualify
as having rheumatoid factor. The other 70
per cent do not have the 22 S component,
as Kunkel and his associates have demonstrated. Certainly, because of the insensitivity of the ultracentrifuge, one would have
to find another tool to demonstrate the 22
S component in minor quantities. However,
one has to search for a broader definition
of the rheumatoid factor, in order to satisfy
currently available data. Such a definition,
to my mind, should place the emphasis on
serological reactivity with the various agglutinating agents, such as antigen-antibody
complexes and/or aggregated human gamma
globulin, and ignore as much as possible
the size or charge of the reacting protein
molecule. Thus, if 27 S and 30 S macroglobulins are encountered with the requisite
serological activity, I see no objection to
calling them rheumatoid factors. We do
not know whether our 27 S component
really had serologic activity because we did
277
AMERICAN RHEUMATISM ASSOCIATION
not obtain it free of 19 S material. I doubt,
however, that this unusual component is
a part of the complement system. These high
molecular weight proteins are bona fide
28. OBSERVATIONS
proteins, and one does not have to assume
that they are aggregates of a monomeric
rheumatoid factor. Their chemistry, however, needs to be studied.
ON SEROLOGICALLY
POSITIVE
hTONRHEUMATOI1) SERA
Joseph Lospalluto, Bnrbarn Dorward and Morris Zif, Dallas, Tex.
Properties of nonrheumatoid sera which precipitate with Fraction I1 were studied
by chromatography on carboxymethyl cellulose. The following sera were examined:
cirrhosis 2, infectious hepatitis, 1; cryoglobulinemia, 1; rheumatoid arthritis, 1.
Fractions were tested by the sensitized sheep cell (SSC), Rh+ , tanned cell, and
latex agglutination tests and by F I1 precipitation.
Activity was demonstrated in one or more of the fractions eluted at pH 6.0, 6.5,
and 7.0 in all cases. The two cirrhosis sera were positive only in the pH 7 fraction;
all tests but the SSC were positive. The hepatitis serum reacted similarly, but the
pII 6 fraction was also positive. In contrast, a11 three rheumatoid serum fractions
gave positive tests including SSC activity in the pH 6.5 fraction. Thus, the rheumatoid serum showed SSC activity at pH 6.5, while the liver disease sera showed no
activity of any type at this pH, but had activity similar to the rheumatoid serum
in the pH 6 and 7 fractions.
In the case of the cryoglobulinemia serum, which contained a 22 S component,
all tests but the SSC were positive at all three pH’s. Investigation disclosed that the
properties of the cryoglobulin were related in an interesting manner to a reaction
between fraction I1 and a 19 S macroglobulin. Anion exchange chromatography of
the cryoglobulin yielded approximately equal amounts of 7 S and 19 S gamma
globulin. Neither alone was a cryoglobulin. Recombination of the 19 S with the 7 S
fraction from the patient’s serum or from a normal serum resulted in a reversibly
precipitating ciyoglobulin. The 19 S component gave positive reactions with all
tests but the SSC, and precipitated irreversibly with heated fraction 11. These results suggest that cryoglobulinemia sera be tested with heated fraction I1 to determine whether this type of cryoglobulin is present.
The above observations support the conclusion that the agglutination reactions of
iionrheumatoid sera are due to proteins chromatographically distinguishable from
the SSC factor, the latter usually being found only in rheumatoid sera.
DISCUSSION OF PAPER 28
DH. CHARLES RAGAN (New York, N. Y.):
Did the cryoglobulin go into solution with
an excess of 7 S gamma?
not used high enough concentrations. Perhaps something of the order of 10 to 1
should be tried.
DR. LOSPALLUTO: No, but as a matter of
fact, in the whole serum from which cryoglobulin was isolated there is an excess of
7 S gamma globulin. If you recall in the
original pattern shown with whole serum
the 7 S component was rather high. In the
titration I have used as much as a 2-fold
excess of 7 S over 19 S. It would be interesting to see if an increased 7 S could cause
the complex formation in solution. I have
DR. HENRY KUNKEL (New York, N. Y.):
It is certainly a very interesting phenomenon that Doctors Lospalluto and Ziff have
found regarding this cryoglobulin. I wonder
whether yau have any thoughts on why this
does not happen more often. The rheumatoid factor combines with 7 S gamma
globulin to give rise to the 22 S complex,
as far as we know, in all rheumatoid sera
giving positive serological tests. Why should
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AMERICAN RHEUMATISM ASSOCIATION
precipitation just happen in this instance
and not with the others? Is there anything
particular about this rheumatoid factor that
you have isolated that makes it precipitate
and the others do not under the same
conditions?
OR.
LOSPALLUTO:
I really do not know
what differences there are. There seems to
be one intriguing possibility; since the 22 S
component exists in whole serum, one might
expect that when the cryoglobulin fraction
is prepared in a manner similar to that used
to precipitate euglobulin from rheumatoid
serum, one would expect to get some 22 S
complex in the precipitate. In this case the
22 S complex seems to dissociate more
readily since it cannot be demonstrated in
the dissolved cryoglobulin. Now, ii could
be that there is a very critical t-mp-rature
range over which the complex exists or it
could also be that the complex formation
is dependent upon the 7 S gamma globulin
content. Other than that there appears to
be no specific difference between this
cryoglobulin and rheumatoid factor except
for the sheep cell reaction.
DR. JOHN VAUGHAN (Rochester, N. Y . ) :
Did you tell us, Dr. Lospalluto, what was
the clinical condition of this patient?
DR. LOSPALLUTO:
I do not know too much
C O N C U R R E N T SESSION
about that, but I have been informed that
this is a patient with renal tubular acidosis
and she is currently being studied as that
type of patient. I have been assured that
there is no evidence of arthritis of any kind.
UR. VAUGHAN: This would then be the
first sortie of the rheumatologists into the
renal tubules.
m.
CHARLES CHRISTIAN
(New York, N.
Y.): Were the three main chromatographic
fractions with activity heat stable?
DR. LOSPALLUTO: Well, these sera were
heated prior to chromatography. Now
whether there was loss of activity on heating I do not know. We did not test the
serum before it was subjected to chromatography, mainly because we had anticipated
doing sheep cell agglutination reactions on
the fractions.
DR. VAUGHAN: Are your chromatographic
fractions reconcentrated to original serum
concentration?
DR. LOSPALLUTO:
The ones that are
shown here were not. Those are tested on
just samples of the peaks and in some instances we also combined whole peaks and
retested.
“D’-Presiding:
DR. JOSEPH J. B U N I M
29. RELAPSING POLYCIIOVDRITIS; A UNIQUE SYNDROMEuSU.4LLY INCLUDING
A POLYARTHRITIS
Carl M . Pearson and Henry hl. Kline, Los Angeles, Calif.
T w o cases of a unique clinical syndrome have been studied i n some detail within
the past few months and provide the subject of this report. The striking feature is
a relapsing acute or subacute inflammation of several or many cartilaginous structures of the body. Those most prominently affected are the external ears or the
nasal cartilage, which after several inflammatory episodes may collapse or soften
perceptibly. Prominent also in this syndrome is t h e recurrent appearance of a
migratory polyarthritis with effusion, presumably d u e to inflammation of t h e articular
cartilages. When the arthritis is the initial manifestation it closely resembles rheumatoid arthritis a n d this diagnosis has been made in more than one-half of the 9 cases
reported to d a t e i n the literature.
Other areas of chrondritis include the larynx, the trachea a n d bronchi, which may
collapse causing suffocation, sternocostal cartilage dissolution with the developinent
of a flail chest wall, and deafness, presumably due to involvement of the middle ear
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AMERICAN RHEUMATISM ASSOCIATION
structures. The only noncartilaginous tissue involved is the sclera and recurrent
scleritis is commonly seen.
Pathologically the process is an acute chondritis with morphological loss of
cartilage basophilia, lysis of cartilage a n d the presence of a large number of plasma
cells and a few eosinophiles.
A third case has shown some features of this syndrome in addition to the presence
of a diffuse vasculitis similar to that seen i n polyarteritis nodosa.
The etiology is unknown b u t the relapsing nature of the disorder a n d the significant relief that has been achieved with corticosteroid therapy strongly suggests the
presence of a peculiar hypersensitivity state.
T h e differential diagnosis will be considered in some detail since there a r e similarities to rheumatoid arthritis, Reiter’s syndrome, polyarteritis, Wegner’s granulomatosis, Cogan’s syndrome and others.
DISCUSSION OF PAPER 29
DR. JACK POTTER (New York, N. Y.) : Dr.
Pearson, I would like to ask two questions,
one regarding the pathogenesis of this condition. Many people have seen, as we hnve
on a number of occasions, changes in cartilage adjacent to inflammatory foci. For
example, inflammation of the subcutaneous
tissues of the ear, which is often associated
with local depletion of matrix of cartilage
in which there has been some leakage of
papain injrcted via the marginal vein in
rabbits. The second point is really rather
facetious! With regard to etiology, it is of
some interest that the first reported case
of polychondritis was in a gentleman connected with the brewing industry, because
papain has been widely used in that industry for many years as a material for clearing
the beer. It is conceivable that if he drank
a lot of that stuff he might get the papain
when it is freshly added, and that some of
the enzyme might pass into the circulation.
DR. PEARSON: I might talk to the second
point first. This is a very interesting one because Prof. Jaksch-Wartenhorst first suggested th3t perhaps the cause in his case
might have been related to the high intake
of beer, some 5 liters a day, that his patient
drank. This is certainly an interesting
thought. It seems to us from some of the
pathologic studies that the basic defect is
a degeneration of cartilage and that the inflammation seems to be secondary, if we
can reason from biopsy material. This has
been our general impression in several
biopsies. Otolaryngologists not uncommonly
see a cellulitis of the ear but rarely do they
see such flabbiness of the ear and marked
softening following acute or recurrent in-
fections inflammation. Hence it seems to
me that this polychondritis is a rather
specific condition. Many of the cartilages
throughout the body degenerate. This is
somewhat similar to Dr. Potter’s observations about the effects of vitamin A and
papain.
DR. POTTER: At N.Y.U. we have been examining the ears of most of our patients
as they come to the service and something
fairly amazing has happened, again
prompted by Dr. Thomas’ observations of
soft ears in rabbits, patients with the
nephrotic syndrome rather dramatically
have the softest and flabbiest ears one
would ever want to feel, and this is something that is not generally appreciated.
Pediatricians say they have seen this, but
this seems to be a genuine thing in the
adult as well and is not associated with
hypoalbuminemia, nor is it associated with
hypercholesterolemia per se, but seems to
be an almost specific physical finding in
the nephrotic syndrome. We have seen 4
such cases now, one was discovered as we
walked around the ward to get normal
controls and this patient was supposed to
have congestive heart failure with incidental three plus proteinuria. He turned
out to have the nephrotic syndrome diagnosed by chondromalacia. I would suggest
that chondromalacia in the pathology of
the ear is a manifestation of systemic cartilage injury to be sought for more readily.
I think you have stimulated this line of
thought nicely.
DR. CURRIER MC EWEN (New York, N. Y.):
I want to ask Dr. Pearson whether the help
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AMERICAN RHEUMATISM ASSOCIATION
your patients got from corticosteroids was
limited to improvement in evident inflammation. The worsening effects of corticosteroids on his rabbits with droopy ears
which Dr. Potter has mentioned suggest
that corticosteroids could lead to cartilagenous collapse. I wondered, therefore,
whether your patients had any difficulty in
respiration when they received corticosteroids?
DR. PEARSON: Usually when the inflammation is mild or moderate in degree and
is in such an area that one can watch it, as
the ear, the nose, the sclera or the uveal
tract, small or moderate doses of prednisone
satisfactorily inhibit an inflammatory response and the ear or other structure seems
to stay in a quiescent phase. We have not
30. EFFECTOF FASTING
ON
given larger doses of corticosteroids. Our
second patient, who seems to have complete
inhibition of the visible components of her
disease, as far as we can tell, has had a
progressive respiratory tract disease, especially in the trachea. In other cases advanced
tracheal and laryngeal disease has been
seen at autopsy which pathologically reveals a progressive invasion by granulation
tissue. This may be a response which is
secondary to the disorder of cartilage and
we have just begun to consider whether it
would be appropriate to give her a larger
dose of corticosteroid. We have found on
several occasions that discontinuance or reduction of the corticosteroid dosage has
caused a relapse. Hence it seeins very
definitely that we are inhibiting the active
process by the use of these hormones.
URIC I\CID EXCRETION BY THE KIDNEY
D. D . McCarthy and A4. A. Ogqzlo, Toronto, Cenada
It has long been known that fasting is accompanied by a reduction in the excretion
of uric acid in the urine and an elevation of the uric acid in the serum, in nongouty
patients.
Six patients, three of whom had gout, were fasted for periods u p to nine days.
In all patients, the serum uric acid rose from previously established base lines to
values from 53 to 171 per cent above the mean control values. Coincident with this
was an abrupt fall in the urine uric acid, the 24 hour excretion amounting to onlv
23 to 32 per cent of the previous mean urine uric acid excretion. The 24 hour uric
acid clearance decreased sharply, reaching values only 14 to 24 per cent of the
mean uric acid clearance during the control periods.
Maintenance of previous steroid therapy or addition of sodium chloride to the
fast did not affect the uric acid retention. Breaking the fast with pure fat increased
slightly the retention of uric acid. However, breaking the fast with the previous
control diet, a high purine diet, carbohydrate or protein diet, was associated with
a return of the serum uric acid to mean control values within six days, and a return
of the urine uric acid excretion and uric acid clearance to mean control values within
48 io 96 hours.
Many biochemical changes were followed during the study, but those which
correlated most directly with uric acid excretion were a fall in urine pH and C 0 2
C.P. and an increase in ketone bodies and ammonia nitrogen in the urine.
The results of the study suggest that dietary factors may influence the renal
tubular mechanism for regulating the excretion of uric acid.
DISCUSSION OF PAPER 30
DR. CURRIER MC EWEN (New York, N. Y. ) :
I was surprised that a patient’s basic uric
levels decreased when the fast was broken
with a high purine diet. About 1935, following up some work of Dr. Maxwell
Lockie’s, we gave some gouty patients very
high fat diets and very high purine diets.
On the purine diets the uric acid levels
rose strikingly and reached a high level
sooner than they did on the high fat. However, in your patients when you went from
one to the other, the uric acid level fell.
The only difference I could see between
what you did and our study was that we
did not give one on top of the other. We
would interrupt a period of high fat with
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AMERICAN RHEUMATISM ASSOCIATION
a course of high carbohydrate before starting the high purine.
DR. MC CARTHY: On that particular patient it was just high fat.
On the high fat then, when
that was stopped and then high carbohydrate given and then the high purine, but
I believe that the thinking had been a t the
time that a high fat, coming from Lenox’s
work in epilepsy, that the factor in fasting
was essentially the same as the high fat
diet, which is the reason I link those two
together.
DR. MC EWEN:
DR. J. EDWIN
SEEGMILLER
(Bethesda,
Md.): If we consider just what the body
is using during the fasting stage, we must
conclude that it is largely fat. So as far
as the body is concerned then, fasting is
the metabolic equivalent of a high fat diet.
Therefore, Dr. McEwen’s data would, in
a sense, he comparable. Have you made
any estimation as to just how much purine
was in this high purine diet?
DR. MC C A m H Y : Yes. The estimation of
uric acid content in the diet was approximately 7 3 mg. a day. You realize that this
is a moderate purine diet and these results
are going to he repeated using a high purine
diet.
PHYSICIAN: I would like to know if you
have observed any clinical symptoms in any
of the patients in the course of variation
in their blood levels with this type of diet,
whether you precipitated an acute attack.
We had the experience of seeing a patient
whom we were sort of starving to reduce
for surgery for an ulcer and on a diet of
very low caloric intake, he suffered a severe
attack of gout with a very high uric acid
level.
DR. MC CARTHY: We had one patient, the
first fasting patient that was done who had
an acute attack of gout on his second day
of food following the fast as the serum uric
acid was falling. Our other gouty patients
and nongouty patients did not flair up with
any form of acute arthritis. We did protect
our other 2 with cholchicine.
PHYSICIAN:You showed the ketosis to
continue with this high fat diet, hut this
was reversed with the high protein. Did
that diet consist of carbohydrate in addition
to protein?
DR. MC CARTHY: No. The high protein
was a diet made up of skimmed milk
powder, which had electrolytes, sugars and
all carbohydrates removed. It was a pure
protein diet that the patient had.
DR. MC EWEN (New York, N. Y.): I do
not remember the exact number of patients
in the series I spoke of before-5
or 6, I
think. In all of them, as I recall, at least
one attack of gouty arthritis came on during their high fat diets. The attacks came
more promptly when the high purine diet
was taken.
DR. DAVID HOWELL (Miami, Fla.): I
would like to ask your opinion regarding
the mechanism of this phenomenon. It is
characteristic of weak organic acids to he
excreted more poorly in urine of low pH,
just as it is characteristic of weak bases to
be excreted more poorly at a high urine
pH. I wonder if there is any data on urinary uric-acid excretion as affected by
respiratory acidosis or metabolic acidosis
induced by ammonium chloride?
DR. MC CARTHY:
We have not carried out
any such procedure. There are various reports I understand in the literature which
will indicate this. There is one report indicating that a patient with diabetic acidosis
does not exhibit this situation, although
they have both acidosis and ketosis. In our
patients the urinary pH tended to fall
from about 5.5 to 5 during the fast and
fat periods. There was no more dramatic
fall than that.
31. PHYSIOLOGICAL
DISPOSITION
AND URICOSURIC
EFFECTOF
OF OXYPI~ENBUTAZONE
(G-29701)
A
KETO ANALOGUE
J. E . Seegmiller, Peter G. Dayton and John J . Bzwns, Rethesda, Md.
and New York, h7. Y.
Previous studies have shown that the urinary metabolite of phenylbutazone, oxyphenbutazone (Metabolite I ) , has potent antirheumatic effects, but little uricosuric
282
AMERICAN RHEUMATISM ASSOCIATION
activity, in gouty subjects. However, addition of a keto group to this molecde,
thereby making it more acidic (pKa 2.3) yields a compound which has very potent
uricosuric effect but very little antirheumatic activity. It produced a marked uricosuria in all eight gouty subjects in whom it was used, including three subjects with
severe renal dysfunction. No evidence of toxicity was found with short term
administration.
Studies of its physiological disposition showed a half life of eight hours compared
with the half life of 72 hours of the parent compound, oxyphenbutazone. The uricosuric activity is correlated with the low pKa and the lack of antirheumatic activity
seems to be correlated with its more rapid excretion. The half life of eight hours
suggests that this compound may provide a more prolonged uricosuric action than
either sulfinpyrazone (half life 3 hours) or zoxazolamine.
DISCUSSION OF PAPER 31
DR.CURRIER MC EWEN (New York, N. Y.) :
May I ask Dr. Seegmiller if there were any
unpleasant effects, such as gastrointestinal
irritation?
DR. SEEGMILLER: We have noted no ill
effects at all, but the longest period of
32.
ARTHRITIS
administration in these studies that we
have just reported was a period of just 4
days and the patients tolerated it very
well. One patient has received the drug for
as long as 3 weeks without evidence of
toxicity.
ACCOMP.4NYING
ULCERATIVE
COLITIS
Currier McEwen and Clnire Lingg, New York, N . Y.
Clinical, laboratory and radiological features of 52 patients with ulcerative colitis
and musculoskeletal symptoms are reviewed. In 8 patients the musculoskeletal complaints were minor and were considered to be unrelated to the colitis. One patient
had chronic, deforming arthritis and a positive latex fixation test, and was considered
to have rheumatoid arthritis. In the remaining 43 patients the joint disease was
considered to be distinct from rheumatoid arthritis and related in some way to the
colitis.
In 29 patients, arthritis was confined to the peripheral joints. Knees and ankles
were those most frequently involved, but any joints might be affected. Asymmetrical
involvement was common. Arthritis usually was subacute and subsided within n
few weeks or months, leaving no clinical or radiological residua. Spondylitis clinically
and radiologically indistinguishable from usual rheumatoid ( ankylosing ) spondylitis
occurred in 14 patients, and 7 of them also had involvement of peripheral joints
other than hips and shoulders. Sheep erythrocyte agglutination was equivocally
positive in only one patient.
The relationship to rheumatoid arthritis and ulcerative colitis will be discussed.
DISCUSSION OF PAPER 32
DR. Humi SMYTHE (Toronto, Canada) :
An even more neglected field is the arthritis
of Crohn’s disease. We have not gone over
our material in detail yet, but at least 5
out of some 32 cases of Crohn’s disease
have typical ankylosing spondylitis and
others have peripheral joint involvement.
This raises the very important point that
six conditions: gonococml infection, nonspecific urethritis, accompanied by Reiter’s
syndrome, epidemic dysentery accompanied
by Reiter’s syndrome, chronic ulcerative
colitis, Whipple’s disease and Crohn’s disease, are all accompanied by a very similar type of peripheral polyarthritis, running
a very similar course and with a similar
AMERICAN RHEUMATISM ASSOCIATION
incidence of spondylitis. Surely, there is
some common mechanism involved in these
cases.
PHYSICIAN: I think the high incidence
you have in spondylitis is probably a reflection of the approach to the problem and
the method of selection. We analyzed 180
cases of ulcerative colitis looking for these
rheumatic complaints and found 17 patients with the arthritis of ulcerative colitis
which you so ably described and in one
other we found ankylosing spondylitis. I
think that some very convincing evidence
suggests, however, that there is relationship
between theqe two conditions. I would like
to ask one other question, that is, in the
patients whom you saw with ankylosing
spondylitis, did you find any who had the
clinical pattern of the peripheral joint
manifestation with the arthritis of ulcerative colitis was a fleeting type of arthritis
or was it the arthritis that you usually get
with peripheral arthritis associated with
ankylosing spondylitis per se?
DR. M C E W E N : My comments start with
the previous question; I should have mentioned that there was one patient in this
group who had regional enteritis. Some
believe, of course, that regional enteritis
and ulcerative colitis probably are the same
thing. Whether that is so or not, I should
283
have listed that one patient in a sepirate
category. As regards the other point, there
was nothing strange about the selection of
these patients. They were not picked up
on the basis of examination of gastrointestinal x-rays, as in Dr. Zvaifler’s study.
These were merely patients with ulcerative
colitis who developed joint symptoms.
Neither did selection from Veteran’s Administration hospitals increase the numbcr
of male patients and thus account for the
number of cases of ankylosing spmdylitis.
Only two of these patients are from a
Veterans Administration hospital. The rest
are from general hospital services. There
was one other point--the peripheral arthritis of these patients with spondylitis is in
every way similar to the pzripheral arthritis
in patients who have what our A.R.A.
classification calls rheumatoid spondylitis
and which I prefer to call ankylosing
spondylitis. The permanent changes occurred in hips and shoulders and we did
not find permanent changes in any of the
other joints. As the slide showed, there
were some examples of fleeting involvement in other joints. I should mention too
in connection with Dr. Smythe’s comment
about ileitis, that I have had the opportunity to read a manuscript by Dr. Denys
Ford describing arthritis in patients with
ulcerative colitis and regional enteritis,
and joint disease in both appeared to be
similar.
33. RHEUMATOID
SEROLOGICAL
REACTIONS
IN PATIENTSWITH SJ~GREN’S
SYNDROME
WITH OR WlTHOUT RHEI~MATOID
ARTHRITIS
K. J. Bloch, M. J . Wolal, 1. I. Ship, R. B . Ogleshy and J. J. Bunim, Bethesda, h!d.
The diagnosis of Sjogren’s syndrome is based on the triad of keratoconiunctivitis
sicca, xerostomia a n d rheumatoid arthritis. T h e syndrome has also been described
i n patients who h a d scleroderma or periarteritis rather than rheumatoid arthritis.
Twenty-one women with ocular a n d oral manifestations of the syndrome were
studied. The diagnosis was supported by objective findings of filamentary keratitis.
abnorrnal staining of the brrlbar conjunctiva, diminished tear a n d salivary flow a n d
radiographic patterns of sialectasi5 demonstrated by secretory sialography. Biopsy
of the salivary glands i n 10 patients revealed extensive lymphocytic infiltration,
acinar atrophy and, i n some, epi-myoepithelial islands. Eight patients h a d Sjogren’s
s! ndrome a n d rheumatoid arthritis, 3 h a d possible rheumatoid arthritis, 2 h a d
scleroderma a n d 8 had Sjogren’s syndrome without connective tissue d i s e x e .
Hypergammaglobulinemia was prejent in 73 per cent. Twenty patients had a
positive bentonite flocculation test a n d of these 19 w e r e positive i n the sensitized
sheep-cell agglutination test. “Antinuclear” antibodies in significant titers were
demonstrated by the indirect Coomb’s technic in 13 patients; the L.E. cell preparation
254
AMERICAN RHEUMATISM ASSOCIATION
was positive in two. Five patients had positive direct Coombs’ tests and three had
elevated titers of antibody to thyroglobulin.
Density gradient ultracentifugation demonstrated that the serum constituent
reactive in the BFT was a macroglobulin. Analytical ultracentrifugation revealed
serum components with a sedimentation constant of approximately 2.2 in 4 of 7
patients with Sjogren’s syndrome and rheumatoid arthritis, 1 of 2 with Sjogren’s
syndrome and scleroderma and 1 of 5 with Sjiigren’s syndrome alone.
The unusual immunological reactivity of patients with Sjiigren’s syndrome combined with the similarity in the appearance of the histological changes in the salivarv
glands in Sjogren’s syndrome and those of the thyroid in Hashimoto’s disease, suggests that an autoimmune mechanism may be responsible for the changes in the
salivary and lacrimal glands.
( N o discussion followed paper 33.)
34. EVALUATION
OF ANTIRHEUMATIC DRUGPOTENCY IN A DOUBLE-BLIND
OF PATIENTS
WITH RHEUMATOID
ARTHRITIS
STUDY
R. Slonim, I . Kiem, P. Vaughan and D . Howell, Miami, Fla.
A deterrent to adequate evaluation of antirheumatic drugs in patients with
rheumatoid arthritis is lack of information on the sensitivity of various clinical parameters of improvement. One hundred ten patients with definite rheumatoid arthritis
hv American Rheumatism Association criteria were carefully screened with 31
found suitable for a modified dose-response study. After a two week period of
placebo at the onset, patients were assigned by a random numbers table to group
A (19 patients) and B (12 patients). Group A received Medrol, 4 mg. daily
throughout a 3 month period. Concurrently, group B received in successive 2 week
intervals, 4, 8, 12 and 16 mg. daily. The patients were examined twice weekly and
data were obtained following a double-blind technic. Significance of results was
analyzed by the Wilcoxen-White Two Sample Rank Method, and modified doseresponse curves were constructed for each parameter.
None of the parameters listed below distinguished group A from B at the 8 mg.
dosage level; duration of morning stiffness, alone, differentiated the two groups,
with group B on the 12 mg. dosage (.01 P .001); with group B on 16 mg. of Medrol
daily physician’s comprehensive evaluation (.01 P .001) and duration of morning
stiffness were successful in making the distinction between groups ( 01 P .001).
Number of joints tender (.07 P .06), tenderness on joint motion (.06 P .05), grip
( .08 P .07) and distance between acromioclavicular and first carpometacarpal
joints ( . 2 P . l ) were less sensitive methods under these experimental conditions.
Implication of the results in relation to designing a multiclinic drug trial will be
discussed.
( N o discussion followed paper 34. )
EDWARD
€IARTUNG
INSTALLED AS 1960-61
PRESIDENT OF A.R.A.
Dr. Edward F. Hartung, of New York,
was installed as 1960/1961 President of
the American Rheumatism Association at
the Association’s annual meeting in
Hollywood-by-the-Sea, Florida, June 10,
1960. Formerly the Association’s First
Vice-president and President-Elect, Dr.
Hartung succeeds Dr. Charley
, ”T. Smvth,
of DenGer.
The new President’s affiliation with
the A.R.A. extends over many decades.
He was accepted as a member of the
Association’s forerunner, the American
AMERICAN RHEUMATISM ASSOCIATION
285
Association for the Study and Control of papers to be presented is September
of Rheumatic Diseases, in the early 26. Ten copies of all abstracts are to be
nineteen-thirties and presented his first mailed to Dr. Morris Ziff, The Univerformal paper at the June 1934 organiza- sity of Texas Southwestern Medical
tional meeting in Cleveland, Ohio. The School, Department of Internal Medicine,
A.R.A. dates its origin from this meeting. 5323 Harry Hines Boulevard, Dallas 35,
Graduated from Columbia University Texas, Chairman of the Program Comin 1922 with the degrees of A.B. and mittee. Dr. Howard C . Coggeshall, also
M.D., Dr. Hartung esixblished his of Dallas, serves as Chairman of the
arthritis clinic at the New York Post- Arrangements Committee.
All members are invited to attend the
Graduate Medical School and Hospital
(now the University Hospital of the meeting of the Texas Academy of InNew York University School of Medi- ternal Medicine on Saturday, December
cine) in 1924. Jt was one of the first 10, at the same hotel. Current plans
such specialized clinics in the city and provide for a special arthritis panel disthe whole country. He was Director of cussion on Saturday morning, with leadthe Division of Arthritis for many years, ing A.R.A. members participating as
as well as Chief of the Arthritis Clinic, panelists.
Bellevue Hospital-Fourth Division.
RHEUMATISM REVIEW
His post-graduate courses in arthritis
COMMITTEE
at the old New York Post-Graduate
Medical School were the only courses
The A.R.A.’s Rheumatism Review
consistentlv given year after year from Committee reports that it would greatly
these early days to the present. Many facilitate the committee’s work in collectearly A.R.A. members received their ing all reprints relative to arthritis,
first formal instruction in this field rheumatism and connective tissue and
through these courses.
allied diseases, if A.R.A. members would
From June 1955 to June 1959, Dr. automatically send two copies of all
Hartung served the Association as its future publications in English to the
Secretary-Treasurer. He is also a found- Editorial Office, c/o Dr. Charley J.
ing member and past President of the Smyth, 4200 East Ninth Avenue, Denver
New York Rheumatism Association and 20, Colorado.
served on many of its committees as
The mailing of all reprints for review
well as the A.R.A.’s.
as soon as they become available would
At the present time, Dr. Hartung is save much time, effort and expense.
Associate Professor of Clinical Medicine Members are urged to notify their
at the New York University School of secretaries to adhere to this procedure.
Medicine. In this capacity, he is very
DR. J. J. BUNIM TO GIVE
active in teaching Post-Graduate MediHEBERDEN ORATION
cine, in clinical research and in private
practice.
Dr. Joseph J. Bunim, 1958/1959
President of the American Rheumatism
INTERIM SCIENTIFIC SESSION
Association, and Clinical Director, NaTO BE HELD IN DALLAS
tional Institute of Arthritis and Metabolic
DECEMBER 9, 1960
Diseases. has been asked to give the
The Seventh Interim Scientific Session 1960 Heberden Oration in eondon,
of the American Rheumatism Association England, this fall.
will be held at the Sheraton-Dallas Hotel
Predecessors in this honor have been
in Dallas, Texas, Friday, December 9, Drs. Philip S. Hench, Robert Stecher,
1960.
Walter Bauer and Charles Ragan, all
The deadline for receipt of abstracts past presidents of the Association.
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