Proceedings of the seventh interim scientific session of the american rheumatism association December 9 and 10 1960 Dallas Texas.код для вставкиСкачать
AMERICAN RHEUMATISM ASSOCIATION 10 Columbus Circle, New York 19, New York President: EDWARD F. HARTUNG, M.D., 580 PARK AVENUE, NEW YORK 21, NEW YORK. First Vice President and President Elect: JOSEPH L. HOLLANDER, M.D., 3400 4, PENNSYLVANIA. Second Vice President: HANS WAINE,M . D . , 25 BENNET STREET, BOSTON 11, MASSACHUSETTS. 0 Secretary TreasI4rW: FELIX E. DEUkRTINI, 622 WEST 1 6 8 STREET, ~ ~ NEW YOHK 32, NEW YORK Executive Secretary: GERARDW . SPEYER,10 COLUMBUS CIRCLE, NEW YORK 19, SPRUCE STREET, PHILADELPHIA NEW YORK. Proceedings of the Seventh Interim Scientific Session of the American Rheumatism Association December 9 and 10, 1960 Dallas, Texas Abstracts of Papers Presented i t n d Read by Title EDITOH: INDUCTION OF A u’ilhrl s. Clwk RHEUMATOII) FACTOR-LIKE SUBS” A N C E IN RABBITS John L. Ahriizzo ciwl Cli(ir1e.y 1,. Christicin, Kcw York, X . Y. Previous studies have indicated that aniinals innoculatecl with killed bacteria may develop a senim factor which, in some of its properties, resembles the “rheumatoid factor.” The apparent spccificity of such factors for ganiiila globulin, in sonie studies, may relate to its assimilation of gamma globulin by the bacteria used for innoculation from either cultme media containing seruni protein or from recent passage of the organisms through animals. The present report concerns stndies in rabbits that were injwted intravenously over ii period of 6 months with a suspension of formalin-killed E. coli. This organism h x l been cultured in a niedinm that contained only salts and gliicose. A high seriiin titer of E. coli agglutinins developed and after a period of several weeks a serum f.actor w a s noted which had the following chariicteristics: (1) It was a heat stable (96 C.-30 inin.) gamma globulin which increased in titer upon continued innoculation. ( 2 ) It agglutinated FIT tanned sheep cells, FII latex particles and sensitized sheep cells. f Average FI1 tannccl S \ l ( q l cell titer was 1:640in 5 animals immunized for 6: months. ) ( 3 ) It ;igglotin;itec\ tannrd sheep cells “coated“ with ;,ggrc>giited human gamma globulin but not 7s gamma globulin. ( 4 ) It precipitatcd with aggregated hmnan gamma globulin but not with 7s g;imma globulin. ( 5 ) It was absorbed by small amounts of E. coli. ( 6 ) It was :ibsorbecl by ;I bovinc serum albnmin-antibavine :~lbuniinprecipitate. ( 7 ) It scdinicntecl to the bottom of the tube during density gradient centrifugation, suggesting that it is a heavy niatt.rial. (The E. coli antibody conccntrated in the middle of the tube. ) Thest. data support the hypothesis that sustained antigenic stimdation may result in the production of an antibody-like fxctor whosc specificit,v i s directcd against g;unma globulin in ;in iiiiiiiunr aggrcgatctl statc. The similarity [if this serum factor to tlic human rheumatoid factor suggests that prolonged exposure to an antigen (living agent or otherwise) could be operative in the pathogenesis of riieiimatoiil arthritis. 104 AMERICAS RHEIrhIATIS-M ASSOCIATION h NEW PATHWAY OF SAI.ICYI.ATE. htETABOLL5M: REOL-CTIO~ O Y SAl.lCYl.ATE Ilaniel XI. Bachnwn, S(;smnmu John ReLvntly we h w e observed in cultiires of Nmrrospi;ru c r a w a new hiochernical path- way of siilicylatr nietaholisni: the rediiction of salicylate to saligenin ( selicyl alcohol ). Sodium salicylate was added to liquid cultures of Nrnrospora crussu SY7A in a concentration of 20 mg. per cent. After t i lag phase of 48 hours, the salicylatr disiippwrd rapidly from the growth nierliiiin antl a metabolic product appeared a s shown t;y iiltraviolet spectrophotometry antl by ascending paper chromatography with britan01, pyritline, saturated sodium chloride, and amnioniinii hydroxide. The metabolic prodnct ( R i 0.96) g:ive an orange color with (ind TO SALLGENM Rcllr Dragoon, Portland, Oregon diazotized sulfiinilic acid and ii milrive color with diiwotized 11-nitroaniline. These properties were identical to those of saligmin. Thc metabolic produyt was isolatcd from 96 iind !20 hoiir ciiltiirer by ether extraction and sitblimation in vacuo. It was positively identified BS saligeniu by me:ins of its melting point, mixed melting point, iiltr;tviolet extinction coefficient, and infrared spectruuii. Semiquantitative estimation of the yiAd by paper chromatography and ultraviolet spectrophotometry indicated one niol of siiligcmin produced for mch mol of salicylatc. consumed. Techniques h:ive heen tlevc?loptd lor the Rheuniatoid arthritis is a chronic inflnmiuatory disewe of self pc-rpetiiating nature demonstration of chromosoint~ nrinilwr nntl inorphology at tlie nietaphase stage of inifor which no etiologic agent tias h e m identified. Faniiliul studies have appeitrcd that tosis in priiiiary ciiltnrcs of peripheral hlootl show sollie increase in ttir: incidmcc of ~ h r leukocytes. Using tliis techniqric*, threc clinical disease itself kind an even greater rheumatoid arthritis patients of niotlmite incidence of sero-positive tests for rheuma- iind severe clinical state were stiitlietl. Two of these were posiiive for rheiniiatoitl fiictor toid factor. This c!ould imply a primary genetic cau5e with different degrees of ex- with the latex fixation test; one wiis nogiitive. pression probably involving a siibtle change The prepmition:; werc. rvahiated for chroniof genetic rather than gross chrtimosomal osome number ant1 morphology following material. This investigation of the chromothe standard system of noiiienclatiirr of some number and morphology i n clinical human mitotic chromosomes ;is proposed tit rheumatoid arthritis patient5 was undertaken the Conference (in Human Chroinosomes in iis a necessary introduction to this problrm. Denver this year. In the patients studied, the chromosome nilinher and mc~rpholo~y were ‘Read by title. similar to the normal poptilation. sTLn>lESO F T H E HUMAN SYNOVLAL. FLUJII P’HOTKJNS RY STARCH GEL EI.E(:THOPHORESIS 1. P . Bkirtte unrl K. Schmid, Boston, hl;\ss. Comparison of the proteins of synoviiil fluid with those of serum hy free rlectrophoresis demonstrated a high relative concentration of albuinin, and particularly, the al-globulins and a low relative content cf the +-globulins in certain joint fluids. In view of these results, investigations using starch gel electrophoresis were undertaken to determine the proteins that account for the diffurences in the electrophoretic distribution of the synovial h i d and senim proteins. Starch gel electrophoresis was carried out on a limited niimber of different joint fluids. The “normal” synovial Huids exhibited the most pronoanced differences as compared with serum: A distinctly higher relative concentration of one of the two pre-albumin coniponenb (a,-acicl glycoprotein) was noted; the relative concentration of the postalubmins, fast +-globulins, and transferrin was essentially unchanged; however, the relative content of the slow or-globulin was lower. The patterns of pathological joint 105 AMERICAN RHEUMATISM ASSOCIATION fluids including the traumatic ones varied between those of “normal” joint fluid and serum. From the results of this study and earlier investigations, it is concluded that the in- THELLPlD FRACTION O F POI.YVINYI, verse al/a2-globulin ratio characteristic of “normal” synovial fluid is mainly due to the low relative concentration of the slow a2globulin and the marked increase in the relative concentration of al-acid glycoprotein. SPONGE BIOPSYCONNECTIVE TISSUE Giles G. Bole, Saul R o s m n , Willium E . M . Lands and U’illiciin D. Robinson, Ann Arbor, Michigan We have previously reported that the total lipid content of a water washed chloroform-methanol extract obtained from small polyvinyl implants (0.3 x 1.0 x 1.0 cm) in guinea pigs was unexpectedly high. Chemical analysis of the extracts indicates: ( 1) The total extractible material represents 40 to 60 per cent of the tissue dry weight for microscopically uniform connective tissue 3 to 8 weeks of age. (2) Approximately half of the extracted material is not completely characterized but appears to be derived from the polyvinyl sponge. While the sponge used for implantation is essentially insoluble in the chloroform-methanol, it becomes significantly soluble after implantation. (3) The characterized fraction represents 25 f. 5 per cent of the tissue dry weight; 50 per cent is phospholipid, 10 per cent cholesterol, and the remainder is neutral esters. (4)Fractionation of the phospholipids ON OBSERVATIONS by silicic acid chromatography suggests that the composition of the phospholipids changes qualitatively with age of the implant. Isotopic phosphate was administered in an attempt to determine the site of origin of the phospholipids. Following intraperitoneal (50 p c ) injection of PJ2, the specific activity of the phospholipids in the implants increased as rapidly as that in a similar fraction from liver. Local injection ( 5 pc) into one of six implants in an animal gave specific activities of the phospholipid fractions of the injected sponge consistently higher than other tissues tested during a 96hour period. The specific activities at 22 hours were: injected implant 524, adjacent implants 100, brown fat 136, liver 60. These data suggest that the phospholipids are synthesized in situ by the connective tissue in the implants. BEHAVIOROF RHEUMA~OID FACTOR WITH CELLSCOATEDWITH GAMMAGLOBULINS BY THE BDB TECHNIQUE THE Vincent P. Butler, Jr. and John H. Vaughan, Rochester, New York The “reactant” gamma globulin in the FII tanned sheep cell system must be in aggregated form to be agglutinated by rheumatoid sera and the inference has been drawn that rheumatoid factor, therefore, may have specificity for an aggregated, rather than unaggregated, form of gamma globulin. In other studies, it was recognized that unaggregated preparations of egg albumin are quite inefficient in coating tanned cells for agglutination by rabbit anti-egg albumin sera; unaggregated egg albumin, however, is quite effective in coating cells treated with bis-diazotized benzidine ( BDB ). To study the pertinence of these observations to rheumatoid systems, human gamma globulin (HGG) was separated by sodium sulfate fractionation or preparative ultracentrifuga- tion in a manner designed to provide aggregate-free and aggregate-enriched preparations. Aggregated HGG was effective in coating both tanned cells and BDB-treated cells as shown in agglutination by both rheumatoid and rabbit anti-HGG sera. The unaggregated HGG, like the previously studied unaggregated egg albumin, was quite inefficient in coating tanned sheep cells, as judged by subsequent minimal reactivity with rabbit anti-HGG; tanned cells exposed to unaggregated HGG also had no reactivity with rheumatoid sera. Unaggregated HGG linked to erythrocytes by the BDB method yielded strong agglutination both by antiHGG and “rheumatoid factor.” Rabbit gamma globulin has also been linked to erythrocytes with BDB; reactivity of these cells closely paralleled that of ‘‘sen- 106 AhIERICAN RHEUMATISM ASSOCIATlON sitized sheep cells.” Gamma globulin from certain other animal species, as well as numerous individual HGG preparations, have “EPITHELIALTRANSFORMATION” also effectively coated erythrocytes for rheumatoid agglutinating activity. I N -4 HUMANSYISOVIAL CUI.TURE C. William Castor, Ann Arhor, Michigan Cultures of mammalian “fibroblasts” usually flourish for a limited period in vitro, and then for obscure reasons cease proliferating and die. A less common, and equally puzzeling, fate of such cultures is “transformation” of the fibroblasts to cells bearing the inicroscopic characteristics of epithelial cells. We have observed such a “transformation” in a cell line derived from a patient with rheumatoid arthritis. It has been possible to compare the cell morphology, chromosomal characteristics, growth rate, nutritional requirements, and mucopolysaccharide production in this cell line before and after the “transformation.” Previously stellate and spindle shaped cells became polygonal and left their reticular growth pattern to assume an epithelial configuration with contiguous cell borders. The original cell line had been largely diploid with a few hypodiploid THE POTENTIATIDN OF S M W DOSESOF COHTII:OSTF:P.OIU Glenn OF \WTH hlETHANUROSTENOLONE M . Clark, Stanley Kaplun, Julio Goohur utul Vcvid Mills, Memphis, Tenn. During the past six months, 15 patients with rheumatoid arthritis on maintenance steroid dosage have been treated with Methandrostenolone in an effort t o prevent osteoporosis. Within a few days the patients gave evidence of marked increase in steroid effect as manifested by decreased joint pain and swelling and increase in moonface, acne, purpura, dyspepsia and euphoria. On decreasing steroid intake to levels from onefourth to one-half of the previous main&TDIES chromosomal forms, while the transformed cell exhibited ikirked heteroploidy. Driring the process of alteration, cell niutiplication slowed, and then resumed at the previous rate when the new cell form had emerged. Although the initial “fibroblastic” cell produced hyaliironic acid in concentrations up to 20 pg./ml. of medium, and intermittently yielded medium which gave a mucin clot; in the transformed cell synthesis of high molecular weight hyalnronate is virtually undetectable. Where the initial cell line performed optimally with CMRL medium 1066 supplemented with human serum, the altered cell is mnch less demanding in its dietary requiremaits, growing equally well in the simple Eagle’s basal medium, and in an undefined mcdiuni composed of lactalbiimin hydrolysate and yeast extract. tenance dosage, equivalent suppression of inflammatory activity as measured by Lansbury indexes was maintained. At the new levels no evidence of increased toxicity was observed. These findings seem of importance since it now may be possible to maintain patients on a smaller dosc of corticosteroid by adjunctive therapy with an agent which has been shown to have an anabolic physiologic effect. PHOTOELECTRICALLY QUANTITATEI~ !?I1 P R E C l P l T A T l O N BY RHEUMATOID SERA’ Elias Cohen and Erzin Neter, Buffalo, New York By photoelectric, carrier-free quantitation (Cohen, Neter and Norcross, Am. J . Clin. Path. 31:607, 1859) the effects of varying concentration and temperature treatment of reactants upon the Cohn Fraction II-rhenmatoid factor( s ) precipitation were determined. Increasing concentration of human ‘Read by title. FII reactant from 0.83 per ccnt to 5 per cent decreased precipitation with rheumatoid factor(s), analogous to effect of excess of antigen, or .intibody in precipitin reactions. Human FII-rabbit anti-FII precipitin reaction could not be detected photoelectrically with same concentration of reactants and incubation time, due to excess FII antigen. Heating 0.83 per cent FII at 63 107 AMERICAN RHEUMATISM ASSOCIATION C. for 10 minutes before testing, substantially increased precipitation with rheumatoid sera. Freezing and thawing FII also increased its reactivity with RF [rheumatoid factor( s)]. Rheumatoid sera "inactivated" 30 minutes at 56 C., prior to testing, gave greater precipitation than native sera when mixed with FII reactant. Destrudion of a labile inhibitor comparable to that reported by Schubart, Cohen, and Calkins (New England J. Med. 261:363, 1959) is suggested. Observation of enhancing effect of SOME SERUM PROTEIN inactivation of rheumatoid serum upon precipitation with human FII reactant, but depression of agglutination of rabbit-antibody sensitized alligator erythrocytes, with inactivated serum versus native serum, concur with concept of heterogeneity of rheumatoid factor( s ) . An evaluation-comparison of photoelectric instrumentation for detecting FIIrheumatoid factor( s ) precipitation will be presented. The nature and significance of such characterization of rheumatoid factor( s ) will be discussed. ABNORMALITIESI N P.4TIENTS WITH PROGRESSIVE SYSTEMIC SCLEROSIS AND THEIR RLXATIVES Josue M . Corcos, Wilkzrn C . Robbins, B m r d Rogofl and Ralph Heimer, New York, N. Y. Twenty-eight patients with scleroderma were examined and their sera were studied for the following: gammaglobulin levels by zinc turbidity; rheumatoid factors by latex fixation and sensitized sheep cell tests; antinuclear antibodies by L. E. preparations and complement fixation tests with nuclei. Seven of 27 patients had hypergammaglobulinemia, which was confirmed by paper electrophoresis. Three patients had low levels of gammaglobulin. Thirteen of 24 patients had a positive latex fixation test, but only 6 of these had a positive sensitized sheep cell test. None of the patients' sera formed L. E. cells, but 9 of 25 sera fixed complement with calf thymus nuclei. The families of 19 patients with scleroderma, comprising 78 blood relatives, have been studied. Of 75 relatives, 28 had abnormal gammaglobulin levels. Of these, 3 were high and 25 were low. Eleven of these serum samples were checked by paper electrophoresis, and gammaglobulin levels ranging from 400 to 750 mg. per cent were found. In 6 families, comprising 30 relatives, there were 22 individuals with low or low normal gammaglobulin levels. Of 54 relatives, 6 exhibited a positive latex fixation and 3 of these had a positive sensitized sheep cell test. In one family, comprising 3 members, all exhibited a positive test for rheumatoid factor; one was the propositus with scleroderma, the second had classical rheumatoid arthritis, and the third member was asymptomatic. Tests for L.E. factors were negative in all relatives. The occurrence of complement fixing antibodies to nuclei in the sera of patients with scleroderma indicates a relationship to systemic lupus erythematosus. However, the lack of these antibodies in sera of asymptomatic relatives (previously noted by others in relatives of patients with systemic lupus erythematosus), suggests that antibodies to nuclei are accompaniments of a well-developed disease process, and unlike the rheumatoid factors, do not reflect a heritable abnormality. The results obtained in this study, furnish additional evidence for the close relationship between scleroderma and other systemic diseases of connective tissue. As already suggested in similar studies of rheumatoid arthritis and systemic lupus erythematosus, this study suggests that scleroderma also occurs in families in which there exists a genetic predisposition for abnormalities of immune systems. CORRELATIVE CLINICOPATHOLOCICAI. FEATURES IN THIRTY-TWO PATIENTSw m i SYSTEMIC LUPUS ERYTHEMATOSUS' Charles W. Denko and Lee P . Davis, Columbus, Ohio With increasing information widening our understanding of systemic lupus erythema"Read by title. tosus (SLE 1, including the features of clinical, laboratory and pathological findings, we studied all patients with systemic lupus erythematosus who have been examined 108 AMERICAN RHEUMATISM ASSOCIATION post-mortem at the Ohio State University Hospital, Columbus, Ohio. Material was available an 22 women and 10 men about equally divided between steroid treated and nonsteroid treated groups. Pathological findings were oorrelated with clinical signs and symptoms. The average duration of life in these patients was about 2% years, remaining relatively unchanged in those receiving steroids. The majority of patients in both groups, steroid and nonsteroid treated, died of infections with uremia and acute lupus itself accounting for others. Elevated leukocyte counts appeared frequently with leukopenia occurring much less commonly. Other laboratory features were evaluated in relation to the morphw logical changes found in various organs. Clinical symptoms, such as gastrointestinal disturbances, mcntal aberration, chest pain and musculoskeletal involvement were correlated with lupus lesions involving the esophagus, stomach, small 'and large intestinal, heart, lungs, pleura, pericardium, peritoneum and other organs. Classic lesions such as those in the spleen and kidney and nonspecific changes in the lungs atid liver were noted. THEEFFECTOF STEROIDSANI) INFECTION ON TAUHINE EXCRETION IN SYSTEMIC LUPUS ERYTHEMATOSUS' Charles W. Columbus, Oliio unrl L?. Irene Pentz, Chicago, Illinois The aviiilabilih of a method suitsble for the routine determination of taurine ( 2 aniinoethanesulfonic acid) led us to measure taurine excretion in patients with systemic lnpus erytheniatosns (SLE) during treatment with steroids an2 during nonsteroid treatment. Taurine is the end product of the metabolism of sulfur containing amino acids such as cystine. No information on the biochemical alterations in sulfur metabolism in patients with SLE has previously been reported. A decreasing output of taurine was noted in patients receiving decreasing dosages of hydrocortisone and dexamethasone. A markedly different pattern in taurinuria was found in patients receiving prednisone, methylprednisone and triamcinolone. Hero the taurine excretion rose suddenly as the steroid level was decreased. This action is thought to reflect the release of a mechanism suppressing some phase of pituitary activity followed by return to normal pituitary and adrenal function. Patients with systemic lupus erythematosus who had severe acute infections excreted high levels of taurine and did not follow either pattern even though they also received high doses of steroids. Severe acute illness in normal subjects was accompanied by an elevated tnurine output that gradually diminished as the clinical features of the illness subsided. These changes accompanying acute infection were thought to reflect an increased hydrocortisone level in the blood stream resulting from the illness. 'Read by title. THEANTI-INFLAMMATQRY .\Cl I V I T Y OF A CI.UCW0RTICOID EPIMER~ EUis Dresncr and Edgur S. Cathcurt, Hosten, 14ass. Stereoisomers of anti-inflanimatory glucocorticoids, such as the lla-hydroxy and l'l/+hydroxy isomers of cortisol and the 16/3-hydroxy isomer of triamcinolone, are generally biologically inert, The 16p-methyl isomer of dexamethasone ( & - m e t h y l - 9 ~ fluoro-prednisolone) has been synthesized, and its anti-inflammatory properties in rheumatoid arthritis compared with those of dexamethasone. Twelve ambulant patients with active, re'Rend by title. versible rheumatoid arthritis were studied. After control periods of 8 or 12 weeks, they were given dexamethasone and the 8-epimer alternately in paired monthly treatment periods, the dosage of both compounds being reduced or increased in subsequent paired periods according to their response. Each pntient had 3 or 4 paired observation periods on different doses of the steroids (0.54.5 mg. daily), the duration of observation being 32 to 48 weeks. The results were assessed at the end of each period [Lansbury ( 1959) criteria]. 109 AMERICAN RHEUMATISM ASSOCIATIOB Comparison of the effects of each compound on the systemic and articular indices and ESR in each patient shows both steroids affect them in like manner. In the group as a whole, the 8-epimer appeared to be somewhat more active than the dexamethasone in lowering both the ESR I42 paired studies: 8-epimrr better in 15, (Y in 7, equal in 201 and the articular index (number of joints involved) (8 better in 17, a in 10, equal in 151, whilst their effect on the systemic (noiiarticular ) index was about equal [B better in 11, a in 11, equal in 201. Side effects of the steroids were indistinguishable, prominent being moonface, facial erythema, polyphagia, weight gain and cutaneous purpura. This is the first example of a glucocorticoid of which two epimers are active. RELATIONSHIP OF CALCIUM AND HTDROGEN PHOSPHATE INTERACTIOX TO OF HYDROXYAPATITE FORMATION I S A MODEL SYSTEM Harry R . Elden ond Da& Evidence concerning the nature of interaction between preformed hydroxyapatite (free or attached to the fiber) or lesser complexes of calcium and hydrogen phosphate in the nucleation of hydroxyapatite on collagen was previously reported. ( Elden, H. R., and Howell, 0.S.: Fed. Proc. 19: 142, 1960.) Investigation was made of the interactions between calcium and hydrogen phosphate in solution to define the possible moeities available for initiating nucleation or precipitation of hydroxyapatite. It was advantageous to employ the experimental desiLgn of Watson and Robinson (Am. J. Anat. 03:25, 1953) who followed the interaction of calcium and hydrogen phosphate (ion products 9 to 38x10-4) by x-ray diffraction patterns and electron micrographs at intervals after miring: they noted transition from clear solution (prior to mixing) to formation of clumps ( 1 minute), a membrane (10-15 minutes) and finally crystals of hydroxyapatite. In the present study, it was possible to follow the same interaction at concentrations close to the physiological range (ion product 1x10-6) by measurement of turbidity in the light-scattering photometer, solution THE &hCHANISM S . IJor~ell,Miami, Florida conductivity, pH and composition of the precipitate. The logarithm of turbidity during an induction-period was related to ion-product with slope of -1.4 when calcium was fixed and phosphate concentration varied. However, turbidity was independent of ionproduct when calcium was varied at a fixed phosphate level. After the induction period, turbidity showed a momentary decrease, then an increase (inflection period); finally turbidity decreased slowly due to settling of the precipitate. Simultaneously, solution p H and conductance decreased while analyses of the precipitate showed a smooth continuons increase of calcium and phosphate content fCa/P ratio varied from 1.2 to 1.4). The following reaction scheme is postulated for this system: (1) C a + + and HPO;=+ C a m 0 (clumps); ( 2 ) CaHP04-t hydroxyapatite with bound clusters of Ca+ + and HPO,= (inflection-period; (3) Hydroxyapatite-accretion with crystal formation ( settling phenomena ) . Theoretical relationship of these findings to mineralization of collagen, in vitro, will be discussed. SEQUENCE OF MORPHOLOGIC CHANGES IN RHEUMATOID LYMPHNODECELLSGROWN IN M ~ L I P O RCHAMBEHS E W a h x V. Epstein and Margaret Ross, San Francisco, California With the demonstration of rheumatoid factor in human lymph node cells, the need has increased for methods permitting direct observation of cell populations under controlled conditions. The hlillipore Chamber technique of Algire and Weaver has been adapted for the growth of human lymph node cells obtained from patients with peripheral rheumatoid arthritis and of rabbit popliteal lymph nodes after stimulation with bovine serum albumin (BSA). Node cells are placed into cold PVP Macrose solution and suspensions of cells 110 AMERICAN RHEUMATISM ASSOCIATION placed between membranes of Millipore HA membranes--pore size 0.45 p supported by a lucite ring. Multiple chambers are implanted in separate subcutaneous tunnels in normal rabbits. Paired chambers are removed at regular intervals over a two week period and the membrane walls are stained with heniatoxylin-eosin. Three days after implantation, extensive cell degeneration is observed together with the appearance of stellate macrophages. After five days, chamber membranes show extensive mitotic activity with colony-like organization of lymphocytes and polyblasts as well as advancing sheets of fibroblasts. By two weeks, sheets of medium and smallsized lymphocytes are observed together with collections of plasma cells. General cellular organization is observed with little further evidence of cellular degeneration. The protective effect of host cell exclusion permits this technique to be adapted to the study of cell populations implicated in the production of rheumatoid factor, STRUCTURAL STUDIESOF RHEUMAT~IDFACTOR BY CHROMATOGRAPHIC AND ENZYMATICTECHNIQUES Wallace V. Epstein and Margaret Tan, San Francisco, California Macroglobulins of high rheumatoid factor activity as measured by the F 11 tanned cell heinagglutination technique were isolated by preliminary separation of euglobulin and non-euglobulin proteins using a polymerized dextran system (Sephadex C-25). The euglobulin proteins were then eluted by ionic strength gradient (pH8) from an anionic exchange resin (DEAE) and final purification achieved by precipitation in low ionic strength buffer. The product with a protein content of 0.1 mg./ml. showed a log, titer of 26 and had s , ~ of approximately 19s and 26s with 19s material as the major component. The macroglobulins were treated for 16 hours at 37 C. with papain derived from mercuripapain in the presence of ethylene- diamine tetracetic acid and cysteine. The reaction was stopped by the addition of the sodium salt of para-Chloromercuribenzoate. Dialysis against 0.01 ionic strength phosphate buffer pH6 produced a crystalline protein product and the supernate showed a single peak by ultracentrifugation of approximately S3.5. Fractionation on a Chl-cellulose cxchange column revealed three peaks; two were eluted with 0.01 M pH6 phosphate buffer and one following ionic strength gradient. The fractions were also separable by starch gel electrophoresis at pH5.5. All serological activity in the F I1 hemagglutination test was lost following enzymatic digestion. No inhibitory activity toward rheumatoid factor was exhibited by these fractions. THEHEATLABILITY OF RHEUMATOIDFACTOR AS MEASURED BY PRECIPITATION WITH HUMANF M ~ I OIIo N Wallace V. Epstein and Mnrgaret Ross, San Francisco, California The heat lability of rheumatoid factor doubling dilution technique to changes less was shown by heating the serum from pa- than 50 per cent of the total serum activity. tients with peripheral rheumatoid arthritis Further heating of the supernate of the to 56 C. for 30 minutes. The effect was precipitation system causes a further fall shown by decreased activity of the heated in hemagglutinating activity; since this sera in both precipitation reactions with change is proportional to the amount of human Fraction I1 and agglutination by the Fraction I1 added, the effect appears due F I1 tanned cell technique. For one serum to the activation of the adled Fraction 11. Although the Fraction I1 precipitation the difference mounted to 110 yg. N/ml. at the point of maximum precipitation, The system is insensitive to small amounts of associated decrease in hemagglutinating ac- rheumatoid factor in serum, it will permit tivity as measured by the F I1 hemagglu- a demonstration of changes in the serum tination test was evident, but not as dra- content of thermolabile and thermostabile matic, because of the insensitivity of the rheumatoid factors that cannot be made with any certainty by techniques involving *Read by title. doubling dilutions. 11.1 AMERICAN RHEUMATISM ASSOCIATIOIL' PERIPHERAL NCUROPATHY IN RHEVMATOID ARTHRITIS Richard H . Ferguson and Charles H. Slocumb, Rochester, Minnesota The occurrence of peripheral nenropathy of several varieties in patients with rheumatoid arthritis h.is been observed with increasing frequency in recent years. A study has been made at the hlayo Clinic of all cases of rheumatoid arthritis seen in the last 10 years in which an isolated nerve pdsy or peripheral polyneuropathy was found. The incidence of the various types of neuropathy was compared with that in an earlier era. Particular attention was focused on 61 patients who had peripheral polyneuropathy seen in the last decade, and data pertaining to clinical, pathologic, laboratory and electromyographic stud:es will be presented. Classification of this latter group into foiir categories on the basis of the type and exttnt of a neurologic involvement offers a clinical means of separating patients with nezrotizing arteritis from those in whom the prognosis is better. The possible role of adrenal-steroid therapy in the pathogenesis and treatment of these states will bc discussed. SERUM SUBSTANCES BEHAVING LIKERHEUMATOII) FACTOR AND LUPUSFACTOR IN CHRONIC PSYCHOSIS* W. J . Fessel, San Francisco, Calif. Psychiatric and psychosock1 factors have been thought by many to be important in the pathogenesis of certain connective tissue diseases. This report gives some preliminary results of an investigation into the possibility that connective tissue disease may be important in the pathogenesis of certain psychiatric disorders. FII latex and nucleoprotein (N.P.) latex agglutination tests were made on sera from about 1100 patients, in a State mental hospital, having various chronic psychoses. 8.2 per cent had positive FII tests and 1.3 per cent had positive N.P. tests; the corresponding figures for control sera, from nonpsychotic institutionalized persons, were 1.4 per cent and zero respectively. Ultracentrifuge analyses of 9 sera showing positive FII tests demonstrated elevated macroglobulin levels in 5. Physical examination of the patients with positive agi?Jutination tests showed the *Read by title. following diseases: rheumatoid arthritis 7; probable systemic lupus 4; Marfan's syndrome 3; Ehlers Ilanlos syndrome 2; retinitis pignientosa 4. In addition, there was a high incidence of congenital skeletal deforniities. A randomly selected group of 46 psychotic patients having negative FII and N.P. latex tests also had a high incidence of congenital skeletal deformities and produced another example of the Ehlers-Danlos syndrome. The above findings confirm the fact that rheumatoid arthritis is uncommon in a psychotic population and suggest that other connective tissue diseases might OCccisionally be important associations of a chronic psychosis. The explanation is not yet clear for the positive FII tests in the psychotic patients who do not have clinical evidence of il connective tissue disease. The possibility is being explored that the psychosis in the latter patients is a defense mechanism which is alternative to a connectivc tissue diskase. THETOXOPLASMA INTRAPERMAL TFST IN RHEUMATOIDARTHRITIS' Arnfn E. Good, Ann Arbor, Michigan Toxoplusmu gondtt, a protozoan infectious agent of virtually world-wide distribution, has shown in numerous population studies a high infectivity rate, varying geographically and increasing with age. Little is known of the course of acquired chronic, 'Read by title. subacute, or inapparent toxoplasmosis, but the following, selected, reported manifestations may also occur during the course of rheumatoid arthritis: fatigue, myalgia, arthralgia, lymphadenopathy, splenomegaly, chorioretinitis, pulmonary infiltration and macroglobulinemia. To investigate a possible- association be- 112 AMERICAN RHEUMATISM ASSOCIATION tween toxoplasmosis and rheumatoid arthritis, the intradermal test with toxoplasmin, prepared from peritoneal exudate of mice infected with toxoplasma, was carried out in 45 consecutive paticnts with definite or classical rheumatoid arthritis and in 45 agematched controls, selected at random from hospitalized patients free from rheumatic symptoms or stigmata of connective tissue disorders. Two of the rheumatoid group and one of the controls were receiving corticosteroids at the time of the skin testing. Twenty-three (51 per cent) of the rheumatoid group and 28 (62 per cent) of the controls showed positive intradermal tests. The hemagglutination test and SabinFeldman dye test, unavailable for this study, would have lent increased precision or specificity. The consensus of workers in this field is that a positive skin test with toxoplasmin is nearly always associated with dcmonstrable antibody. The cutaneous test is generally accepted as a useful aid in population surveys for late detection of toxoplasma infection. This pilot study utilizing the intradermal toxoplasn~intest showed no evidence for a positive correlation between rheumatoid arthritis and toxoplasmosis. OF THE E L B ~ W IN PATIENTS WITH 1~HEUU4TOlDARTHRITISARTHROPLASTY AN END RESULT STUDY' 1. Paul Harcer~,Jr., New York, N. Y. This is a report on the end result of 15 arthroplasties of the elbow done at the Hospital for Special Surgery during the past 20 years, the longest being 20 years postarthroplasty, the shortest 3 years, and the average 11 years. All the patients were Stage 3 or 4, according to the American Rheumatism Association Classification of the process. Using activities of daily living as criteria, close to 50 per cent of the patients have shown improvement. A few of the remain'Read by title. INHIBiTORS OF der have shown improved range of motion hut lack stability. The remainder have frank instability or refusion secondary to progression of the disease. The poor results seem to be secondary to definite progression of disease, although a few patients have shown instability immediately postoperatively. The best results are present in those who have had severe juvenile rheumatoid arthritis. This procedure is usually associated with procedures on other involved areas in the upper and lower extremities. COMPLEMENT FIXATION-AN APPAnEXT NEW CHARACTERISTIC OF SERUMOF CONNECT~VE TISSUE DISEASES Ralph Heimer, Josue M. Corcos and C u r b Nosenzo, New York, N. Y. and Jersey City, N. J. Although it is known that rabbit, guinea pig and pig sera in moderate dilutions exert inhibitory effects on complement fixation reactions, the behavior of human sera in such reactions has received scant attention. We have ex'amined the effect of added human serum on a system fixing complement, and have found that only sera from patients with certain connective tissue diseases interfered markedly with such reactions. Our experimental model for testing inhibitors of complement fixation (ICF) was as follows: Human thyroglobulin and rabbit anti-human thyroglobulin, fixing 10 to 12 50 per cent hemolytic units of guinea pig complement, were incubated in the pres- ence of dilute human serum. Guinea pig coinplement w.is then added, and after further incubation the residual complement was titrated with sensitized sheep cells. Of over 200 sera tested, only those of patients with rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis contained appreciable amounts of ICF. In a variety of other diseases, including osteoarthritis, spondylitis and gout, little or no ICF was found. Moreover, when present, such activity could be removed partially with zymosan. ICF in sera of patients with connective tissue diseases is heat-stable and not dialyzable. It is absmbed by antigen-antibody 113 AMERICAN RHEUMATISM ASSOCIATION complexes, but not by sheep cells or zymosan. ICF was purified by DEAE cellulose chromatography, appeared in a fraction containing mostly albumin and alpha-globulins, and lacking rheumatoid factor ( R F ) activity. Similar fractionation of control sera, failed to yield ICF. Zone electrophoresis on starch of purified ICF recovered from DEAE cellulose resulted in inactive subfractions. On reconstitution of the albumin and alpha-globulin subfractions ICF activity was recovered. Similar results were obtained on electrophoresis of whole serum. ICF is therefore a multicomponent system. Evidence will be presented that ICF, although reacting with antigen-antibody systems, is neither RF nor an incomplete RF. It is proposed that ICF together with RF comprises a unique immunological system characteristic of certain connective tissue diseases. Whether ICF levels reflect the clinical course of disease or are modifyable by treatment remains to be elucidated. ICF, however, promises to provide a new tool for diagnosis and for study of the etiology of connective tissue diseases. THE RELIEF OF INTRACTABLE ARTHRITICPAINBY EXTENDED SYMPATHETIC DENERVATION’ Robert A. Herfort, White Plains, N. Y. The principal source of disability presented by most patients with chronic arthritis is pain originating in the weight bearing joints. Over the course of the past five years, the author has employed a technique of so-called extended sympathetic denervation in 72 patients with advanced painful rheumatoid arthritis and osteoarthritis of the hips and/c;r knee joints. The surgery consists of a retroperitoneal resection of the lumbar sympathetic trunks from the superior margin of the common iliac vessels to the second lumbar vertebra encompassing accessory ganglia on the rami communicantes and also decussating fibers in the retro-aortic prevertebral plexus. The procedure is, at present, carried out bilaterally ‘Read by titlc. at one sitting. This surgery in middle aged and elderly arthritic invalids has had no mortality. In the immediate postoperative period, the patients exhibit, consistently, relief of arthritic pain with concomitent improvement in ioint mobility and general functional capacity. The patients have been followed for periods up to 5 % years after surgery; the relief of pain and improved range of motion have been maintained in 67 of the 72 patients. There have been no undesirable effects noted after extended sympathetic denervntion. Neuropathic disorganization of the affected joints has not occurred. It is suggested that the principal afferent pain pathways from the articular surfaces of the hip, knee and ankle joints traverse the lumbar sympathetic trunks and plexuses. “FALSE POSITIVE” L. E. CELLPREPARATIONS IN LEPROSY’ Conrad Herr, New Orleans, Louisiana Results are reported of 500 L.E. cell preparations done on blood from 325 l e p rosy patients hospitalized at Carville, Louisiana. A case of concomitant S.L.E. and lepromatous leprosy stimulated this survey. Since the significance of the L.E. cell, the L.E.-like cell, “the tart-cell,” and the presence of leukoagglutination is still in doubt, the preparations were classified as Negative; Class A (many typical L.E. cells); Class B (rare to occasional typical L.E. cells with or without leukoagglutination or leukophagocytosis); or Class C (leukoag‘Read by title. glutination or leukopbagocytosis without L.E. cells). The preparations, throughout the study, were done by the two-hour clot technique. Correlation with the type and activity of the patients’ disease; with types of drug therapy; with concomitant systemic diseases unrelated to leprcsy; and with the presence of secondary amyloidosis is attempted. A survey for cryoglobulins was made, and their effect on the L.E. cell preparation is demonstrated. Only the patient with clinical S.L.E. presented Class A preparations. All with Class B preparations ( 2 1 cases) 114 AMERlCAN RHEUMATISM ASSOCIATION had lepromatous leprosy. Nine had biologic false positive serologic tests for syphilis; ten, abnormal liver functions; five, "reactive episodes of leprosy"; several single cases, diseases unrelated to leprosy; and two, secondary amyloidosis. Those with Class C preparations (58 cases) formed a more homogeneous group which could not be distinguished from the average patient population. However, all but one had lepromatous leprosy, and one carries the only recorded diagnosis a t Carville of concomitant leprosy and rheumatoid arthritis. The implications of this work are discussed. Histochemical staining and studies to demonstrate autoimmune antibodies are in progress. TRANSFER OF AUTOIMMUNE NEPHROSIS IN RATSBY MEANSOF LYMPHNODECEL.LS E w l y n V. Hem, Charles T.Ashworth unrl -Vowis Zif, Dallas, Texas Recent studies have demonstrated that nephrosis may be induced in rats by the intraperitoneal injection of rat kidney homogenate with Freund's adjuvant. The disease is characterized by proteinuria, hypoproteinemia, hypercholesterolemia and hyperlipemia; histological studies have shown a membranous gloinerulonephritis. The method of induction suggests an autoimmune basis for this condition. Hemagglutination and precipitin tests have not demonstrated serum antibody. The possibility that cellular antibody was coiicerned in this phenomenon was accordingly investigated by attempting transfer of the disease to immunologically tolerant recipients by means of lymph node cells. A total of 28 rats were pretreated neonatally with spleen cells from the prospective donors. When lymph node cells from 10 nephrotic donors were injected intra- venously into 10 prepared recipients, chemical and histological evidence of nephrosis was observed in 9 of the latter. No abnormalities were noted in any of 18 prepared recipients who received cells from nonimmunized animals or animals injected with Freund's adjuvant alone. Successful transfer was accomplished in only 1 of 8 recipients who had been pretreated neonatally with spleen cells from rats other than those providing the donor lymph node cells. Nontolerant recipients of lymph node cells from nephrotic rats did not develop disease. Immunofluorescent and electron microscopic as well as histological studies have confirmed the transfer of the rat nephrosis. These experiments provide evidence for the role of cellular antibody in the renal lesion produced in rats by immunization with kidney tissue with Freund's adjuvant. GREATEREFFECTIVENESS OF SMALLDOSES OF CHLOROQUINE IN THE TREATMENT OF RHEUMATOLD ARTHRITIS* William K . lshmuel a d R. W. Pnyne, Oklahoma City, Oklahoma Chloroquine phosphate was administered to 40 patients with rheumatoid arthritis in doses of 500 mg. daily (Group .4)and 250 mg. daily (Group B ) for periods of from 3 to 36 months. Of the 18 patients receiving the larger doses of Arden (Group A ) , the following subjective responses were noted: Grade 1-0; Grade 2--5; Grade L 5 ; Grade 46. Toxicity requiring discontinuance of medication occurred in 5 of these patients. The average time of beginning response to - 'Read by title. -- nicdication was 6 weeks. The smaller dose of .4ralen was wed in the treatment of 24 patients (Croup B ) with the following subjective responses: Grade 2-18; Grade k 5 ; Grade 1-1; Grade 4-2. Toxicity necessitating discontinuance of medication occurred in only one of these patients. Beginning response to medication averaged three months. Effects of the two dosage schedules on body weight loss and on serum protein pnlysaccharide ratio further indicated reduced toxicity nnd greater therapeutic usefulness of the lower dose schedule of Aralen. 115 AMERICAN RHEUMATISM ASSOCIATION CHOLESTEROL-ASSOCIATED PROTEINS PRECIPITABLE FROM RHEUMATOIDPLASMA ‘WITH CHONDR~ITIN SULFATE Grace P. b b y , Durham, N. C. Levels of acid mucopolysacharides in the euglobulin fraction of plasma from patients with rheumatoid and nonrheumatoid inflammatory disease increase nonspecifically in parallel with the euglobulin fraction. (Kerby, 1958). The present study explored the proteins remaining in the supernatant plasma (after isoelectric precipitation of euglobulin) which were then precipitable by adding chondroitin sulfate. Salt concentration and pH were important in determining amount of protein additionally precipitated, as noted by Badin and Schubert ( 1955). In adidtion, plasma dialysis itself increased later precipitability of a protein fraction from the supernatant plasma with chondroitin sulfate. This effect was reversed by adding to dialyzed plasma (prior to euglobulin precipitation) a pressure dialysate of plasma. The factor involved resisted heating to 100 C but not incineration. The amount of biuret-positive material (related to mg. of standard albumin) precipitable by chondroitin sulfate from the supernatant fraction of plasma under rigidly controlled conditions was significantly ( p < .01) greater in rheumatoid plasma (371 f 175 (S.D.) mg. per 100 ml. plasma) than in either nonrheumatoid inflammatory (225 f 160) or noninflammatory 176 f 117) plasma. The material from rheumatoid plasma, diluted to serum equivalence, reacted with FII-coated latex particles in about o n e half of the instances. Its cholesterol content (6.94 -t 5.66 mg. per 100 of plasma) far exceeded that from inflammatory (1.82 +1.94) or nonidammatory (1.66 -I- 1.24) plasma. The ratic of cholesterol to biuretpositive material in the fraction showed a high correlation with plasma cholesterol in the two non-rheumatoid groups ( p < .01) but not so in tht rheumatoid group ( p > .1). The protein additionally precipitable with chondroitin sulfate after isoelectric removal of euglobulins offers opportunity for study of factors determining aggregation of globulins, of particular interest since the material precipitable from rheumatoid (as compared to nonrheumatoid inflammatory and nonidammatory) plasma was significantly increased and in some instances reacted with FII-coated latex particles. ONEPOSSIBLE MECHANISM IN THE UPTAKEOF SEROTONIN BY HUMANPLATELETS“ Grace P . Kerby and S. M. Taylm, Durham, N. C. Enhancement of dye reduction by human platelets in the presence of serotonin and a heat-stable, nondialyzable plasma factor or factors (“5HT redox effect”) has been shown to be correlated with increased uptake of serotonin by intact platelets. Investigations into the cause of the increased uptake revealed that serotonin was capable of solubilizing calcium phosphate in vitro, suggesting some type of complex formation. Prior mixing of serotonin with Mg++ did not affect serotonin uptake by platelets, nor did disodium calcium EDTA. Disodium EDTA enhanced both 5HT redox effect and uptake. However, prior mixing of serotonin with Mg-++ followed by chelation with di‘Read by title. sodiuni EDTA diminished both 5HT redox effect and uptake of serotonin by intact human platelets in the presence of plasma. The results suggested the possibility that, in the presence of plasma, serotonin which was free to combine as a complex with a divalent cation was taken up by human platelets as a complex with a divalent cation bound to a nondialyzable plasma factor. It is evident from studies previously reported by others (e.g., Weissbach and coworkers, 1958, 1960) that factors which appear important in the uptake of serotonin by platelets in vitro vary with the in vitro conditions imposed. Resultant data cannot at this time be assumed to relate to in vivo events. 116 AMERICAN RHEUMATISM ASSOCIATION OSTEOARTHROSIS: UNUSUAL,MANIFESTATION’ Harry F. Klinefeltez, Jz. and John I € . Y(irdIq, Baltimore, Maryland Degenerative changes in the inetacarpophalangeal joints of the hands is exceedingly rare. A 68-year-old carpenter showed siich changes as well as involvement of the left hip. The hand joints were treated by capsulotomy and resection of the osteophytes; pathological examination of the tissue showed only degenerative changes in the cnrtilage, with fibrous tissue reaction, but no inflammation. Marked improvement followed surgical treatment. Clinically, the patient was thought to have rheumatoid arthritis, hut there was no supporting evidencv for this diagnosis, either radiologically, serologically, or hematologically. The benign course of his disease is characteristic of osteroarthrosis. “Read by title. STUDIES OF A WALDENSTHOM-TYPE hIACROGLOBLI.IN WITH RHEUMATOIDFACTORPROPERTIES Julius Kritzmun, Henry G . Linkel, John McCarthy and Robert C . Mellms, Boston, Mass. and New York, N. Y. A macroglobulin obtained from the blood of a patient with typical Waldenstrom’s macroglobulinemia has been shown to have serologic properties similer to rhriunatoid factor. In other respects (behavior in the iiltracentrifuge, reaction with rabbit antimacroglobulin serum, cryoprecipitation, electrophoretic mobility and carbohydrate content), it was a typical macroglohiilin. An important difference between it and rheumatoid factor is its pH optimum of about 6.0, whereas tht. optimum for rheumatoid factor’s serologic activity is ahout 8.6. This difference in pH optimum affects both the precipitin reaction with aggregated gamma globulin and the agglutination of vcarious test particles. The macroglohiilin AN ATTEMPT TO CHARACTEHIZE THF was demonstrated in the cytoplasm of spleen ‘and lymph node cells by the use of fluorescent aggregated gamma globulin. Extracts of splenic tissue (obtainsd at autopsy) had the same serologic activity as the serum factor. The morphology of the cclls of origin of this protein is to be described in detail. The patient from whom this protein was obtained had no joint symptoms at any time. The resenihlance of his cellular proliferation to that seen in other diseases where proteins are elaborated that brhave like antibodies C t J gamma globulin suggest‘s d hroadcr significance of the rheumatoid factor phenomenon than a pathogenetic role in iirthritis. ANTIGENIC SPECIFICITS O r THE L. E. CELL FACTOR P. J. Lachmann, Cambridge, England The failure to produce the L.E. cell factor by immunization of experimental mimals has made it neccssary to rely solely on the analysis of the lupus Phenomenon as given by human lupus sera for information on its antigenic specificity. A two-stage indirect L.E. cell test has been devised for this purpose. This technique allows the initial, immunological stage of the lupus phenomenon to bc studied independently of the later changes, and can be u s e d for exprinients involving either variation in the substrate particle or inhibition of the reaction. It has been shown that liipus inclusion can be formed not only from a variety of cell nuclei but also from sperm heads. Inhibition experiments have shown that efficient inhibition of the lupus phenomenon is given by DNA-nucleoproteins only in their native state. Denaturation of the nucleoproteins or degradation with DNAase or papain destroys their inhibitory activity. The results of such experiments have lent support to the concept that it is the “backbone” configuration of DNA-nucleoproteins ( both DNA-histone and DNA-protamine) in their native state that carries the relevant antigenic determinants. 117 AMERICAN RHEUMATIShl ASSOCIATION A SPECIFIC ENZYMATIC SPECTnOPH01.0hiETRIC METHOD TO I>ETERMlNE HOMOGENTISIC ACID IN BIOLOGICAL MATERIAL B . N. La Du, V. G . Zunnmi, L. Laster and J . E . Seegmiuer, Bethesda, Maryland Methods currently available for the estimation of homogentisic acid depend upon its reducing properties and therefore lack both specificity and precision when applied to biological material. We have developed a specific enzymatic method utilizing purified homogentisic acid oxidase to convert homogentisic acid to maleylacetoacetic acid. The ultraviolet absorption of the latter acid is measured spectrophotometrically at 330 mp. The method is specific for homogentisic acid since no other compounds are known to be oxidized by homogentisic acid oxidase, and as little as 1 or 2 micrograms of homo- gentisic acid can be measured accurately. After suitable deproteinization and extraction, honiogentisic acid can also be determined in tissues, such as cartilage, s k i n and synovial fluid. Plasma levels of homogentisic acid and the variation during a single day have been determined for nlcaptonuric patients. .4ttempts to detect a partial defect in the ability to metabolize homogentisic acid in heterozygous carriers of alcaptonuria are being made and some results of these studies will be presented. IN V m o FORMATION OF 7s AND 19s GAMMA GLOBULINS BY TISSUES FROM NOFMALSUBJECTS AKD PATIENTS WITH RHEUMATOIDARTHIWITS Howard I. Levene, Edward C . Franklin and G. Jeunette Thorhecke, New York, N . Y. Recent studies using fluorescein labelled antibody techniques have demonstrated the presence of 7s and 1% gamina globulins in lymphoid tissues from normal and diseased human siibjects. These studies however could not distinguish between prodnction and storage of these proteins by the tissues. In order to answer this question, experiments have been perfonned to demonstrate the incorporation of C,, labelled amino acids into gamma globulins by human lymphoid tissues in tissue culture. Specimens of lymph node m d splenic tissue from 13 normal subjects and 3 patients with rheumatoid arthritis were incubated in a medium cop,taining ovalbumin and a mixture of amino acids, Some of which were labelled with C,,. After a 24 hour period of incubation, the supemates were twice absorbed with n diphtheria toxoid equine antitoxin precipitate, following which gamma globulins, were specifically precipitated with rabbit antisera against 7s or 19s gamma globulins. The radioactivity of the specific precipitate was compared to that nonspecifically absorbed from the same culture fluid by the second absorption precipitate. 7s gamma globiilin formation was d s tected in 13 of 28 culture tubes prepared from specimens from 13 normal subjects and in 3 of 9 tubes from 3 patients with rheumatoid arthritis. 19s gamma globulin fonnation was found in 7 of I0 tubes from three patients with rheumatoid arthritis but was never detected in 22 tubes prepared with tissues from normal subjects. Attempts to detect formation of rheumatoid factor by the F-I1 tanned cell agglutination technique and by measuring the specific absorption of radioactivity to heat precipitated human gamma globulin were unsuccessful. THEPRODUCTTON OF 19s ANTIBODIES IN ADULTSAND PREMATURE INFANTS Joseph LoSpalluto, William M i l k , Jr., Chester Fink and Barbara Dorward, Dallas, Texas Normal individuals, premature infants, and patients with a variety of diseases have been immunized with a mixed typhoidparatyphoid vaccine with a view toward studying duced in obtained course of the size of the antibodies response to these antigens. after either one injection three injections of vaccine proSera or a were 118 AMERICAN RHEUMATISM ASSOCIATION fractionated by anion exchange chromatography on DEAE cellulose. The 7s and 19s Containing fractions were examined for antibody activities with standard agglutination tests. In 23 individuals (group l ) , including patients and normal subjects, who had received three weekly injections of the mixed vaccine for the first time, the chief respcnse three weeks after the initial injection was the production of a preponderance of 19s antibodies against each of the antigens. Small amounts of 7s antibodies to three of the four antigens given were found. Antibodies to Typhoid 0, the fourth antigen administered, were consistently found in the 19s fraction only. In another group, consisting of 32 individuals who had undergone previous immunization (group 2), the major response observed was the formation of 7s antibodies. Comparison of the antibody levels in &e 7s and 19s fractions in this group has indicated that, although 19s antibody levels are present, the 7s antibodies predominate. Further studies on those individuals who had produced a preponderance of 19s antibodies (group 1 ) has indicated that after a three month period there is an increase in the production of 7s antibodies with little change in the titers of 19s fraction. In a group of premature infants, however, an almost complete conversion from the production of 19s to 7s antibodies occurred within a three month period. The results obtained indicate that the earliest response to the antigens administered in this study has been the production of 19s antibodies and that this is followed, in time, by production of 7s antibodies. Preliminary studies on a relatively small group of patients with rheumatoid arthritis suggest that conversion from 19s to 7s antibody production requires somewhat longer periods. Additional data, however, are required. DEPOLYMERIWTIONOF HYALURONIC Acm BY BACTERIAL HYALWRONIDASE: THE QEAV-4GE OF THE GLYCOSIDIC LINKAGE Julio Ludowteg, Sir@ Vennesland and Albert Dwfman, Chicago, Ill. and San Francisco, California Hyaluronic acid is a straight chain heteropolysaccharide containing alternating units of N-acetyl glucosamine and glucuronic acid linked by glycosidic bonds [J.A.C.S. 76, 1753 (1954)l. Bacterial hyaluronidase degrades hyaluronic acid to a disaccharide containing a double bond in its uronic acid moiety [J.B.C. 279, 13 (1956)l. The enzymic degradation of hyaluronic acid may take place by two different types of hydrolysis that can be distinguished by carrying out the reaction in a medium containing H,O*e. Thus, the bond may be broken initially between C-1 of N-acetylglucosamine and oxygen or between C-4 of glucuronic acid and oxygen. In the former case, the 1sOH from the medium will be taken up by the C-1 of the N-acetylglocosamine moiety of the hal product. In the latter case, the 18OH of the medium would not appear in the disaccharide product, The results of degrading hyaluronic acid with purified hyaluronidase from C1. Welchii in a medium containing H201*shows that the reaction does not involve any incorporation of label in the unsaturated disaccharide above the amount which occurred as the result of exchange reaction unrelated to the enzymatic degradation. Polarimetric studies were also made of the depolymerization of hyaluronic acid by bacterial hyaluronidase. These studies show that the reaction product first released must have the same 't3-configuration at the reducing group as hyaluronic acid itself. The above mentioned results provide strong support for Meyer's conclusion [J.B.C. 219, 13 (195e)I that the reaction catalyzed by bacterial hyaluronidase should be formulated as a simple elimination reaction. SPONTANEOUS A c m SEPTIC BURS^* Andre March-, Roberte S h i m , David S. Howell and Haroey E. Brown. Jr., Miami. Florida 119 AMERICAN HHEUMATISM ASSOCIATION Attention is called to this entity because of its apparent rarity. In this small series (7 patients) all were young-to-middle aged males without evidence of chronic internal medical diseases. Involvement was confined to the prepatellar or olecranon bursae. Organisms responsible for the infection were identified as staphylococcus aureus in 6 patients and diplococcus pneumoniae in one. Two patients had been under treatment with adrenocortical steroid derivatives by outside physicians who had labelled the condition “gouty arthritis.” Six patients were cured by antibiotics alone, administered according to, in vitro, sensitivity studies. One patient required an incision and drainage in addition to antibiotics. Possible causes of acute septic bursitis and the relationship of the present data to the rising frequency of staphylococcal infections in the community will be discussed. Failure to recognize this illness may result in its mismanagement. ‘Read by title. IN RHEUMATOID ARTHRI-IIS PELVO-SPONDSLITIS William Martel and Icun F. D u f , Ann Arbor, Michigan Involvement of the pelvic bones in ankylosing spondylitis is well known but it is not widely recognized that erosions frequently occur at the ischial tuberosities, symphysis pubis and femoral trochanters in otherwise classic rheumatoid arthritis. The sacroiliac joints and cervical spine iue frequently abnormal in these individuals, but the radiographic RS well as clinical features STUDIESos differ from ankylosing spondylitis. There is a tendency among rheumatologists to underscore the clinical differences between rheumatoid arthritis and ankylosing spondylitis. Roentgen features of the pelvospondylitis in rheumatoid arthritis often seems to support this differentiation. However, in some patients both conditions seem to coexist. URINARY HYDROXYPROLINE E . Meilman and M. Lrriuetsky, New Hyde Park, New York Comparative studies of “bound” (or peptide) hydroxyproline excreted in urine have been undertaken as part of a study of in vivo collagen metabolism to compare excretion patterns in various connective tissue disorders. Urine specimens (24 hour) were collected from the following subjects after 24 to 72 hours of a hydroxyproline-free diet: a normal adult male, an adult female with sclerodenna, an adult male with scleroderma, an adult male with Marfan’s syndrome, three postpartum females, and an adult female with lupus erythematosus. In addition, a urine from the normal adult was collected after a normal diet, Amino acids and peptides present in the urine were isolated by batchwise adsorption onto and subsequent elution from Dowex 50 ( 2 b 5 0 mesh). The eluates were concentrated by flash evaporation and aliquots of the concentrates were used to charge 1 x 1-50 cm. Dowex 50 (200-400 mesh) columns which were then eluted using a p H and K+ ion gradient. Effluent fractions were separated in a fraction collector; ninhydrin and hydroxyproline assays were run on alternate tubes in order to 13rate hydroxyproline peptides. T h e elution pattern showed 4 to 5 welldefined hydroxyproline peaks. The pstterns obtained from the patients studied showed remarkable qualitative similarity, with some quantitative differences being noted in the relative amounts of certain hydroxyproline fractions. Free hydroxyproline was low, varying from 0 to 0.3 mg. per 24 hour smple. The normai individual on a regular diet showed 1.5 mg./24 hr. of free hydroxyproline; the postpartum women on a hydroxyproline-free diet showed 1.5-2.5 mg./ 24 hr. on the third postpartum day, gradually diminishing over the fourth and fifth postpartum days. Bound hydroxyproline was present in a range of 30 to 80 mg. per day, the largest amounts being present in the Marfan’s patient. A detailed investigation into the nature and composition of the isolated peptides is in progress. 120 AMERICAN RHEUMATISM ASSOCJATION DETECTION OF CELLULAR RHEUMATOIDFACTOR WITH FLUORESCENT IMMUNE COMPLEX Robert C. MeUors, Adam Nowoslawski, Leonhard Korngold and Beth L. Sengson, New York, New York In previous work (J. Exp. Med., 110, 875, 1959) fluorescein-labeled aggregated human gamma globulin was demonstrated to be a sensitive reactant for the detection of rheumatoid factor in frozen sections of synovial membranes and lymph nodes obtained from patients with active rheumatoid arthritis. Similar observations have now been made with fluorescent immune complex and are reported herewith. The starting preparatory material was rabbit antiserum against bovine serum albumin. An immune precipitate was formed at antigen equivalence with fluorescein-labeled bovine serum albumin and, after thorough washing, was converted to a soluble complex by addition of labeled antigen at two times equivalence. With this fluorescent immune complex, cellular rheumatoid factor was detected in synovial and lymph node biopsies obtained from adults and children with active rheumatoid arthritis. Plasma cells in the synovial membranes and germinal-center cells, and rarely plasma cells, in the lymph nodes contained cellular rheumatoid factor demonstrable with the fluorescent complex. In the identical tissues, cells of similar kinds but in more abundant number also contained rheumatoid factor detectable with fluorescein-labeled aggregated human gamma globulin. Comparable sections of normal and pathological tissues obtained in diseases other than rheumatoid arthritis did not stain with the fluorescent immune complex. EXCRETION AND STORAGE OF MUCOPOLYSACCHARIDFS IN HURLER’S SYNDROME Karl Meyer and Philip Hoffman, New York, N.Y. In Hurler’s syndrome, two mucopolysaccharides, chondroitin sulfate B ( ChS-B) and heparitin sulfate (hep. S.), are excreted in the urine and stored in various organs. In the majority of the cases studied, between 60 and 80 per cent of the urinary mucopolysaccharide is ChS-B and 40 to 20 per cent hep. S. The mucopolysaccharides of the organs vary greatly in type and quantity. Liver shows a great predominance of hep. S. over ChS-B in five out of six cases, while in spleen the ratio is reversed. In kidney and brain, the two polysaccharides are present in approximately equal concentration. In two cases, the polysaccharides of rib cartilage and bone were isolated and characterized. In both cases, ChS-B was isdlated from the cartilage and bone, while the presence of hep. S. was questionable. The major component was a mixture of STUD^ OF TKE ChS-A and C (60 to 80 per cent) with C predominating, and keratosulfate (12 to 30 per cent). Sections of these tissues showed broad intensely stained collagen bands. It is suspected that the abnormal presence of ChS-B induces, in these organs, abnormal fibrous tissues. Another striking feature in the disease is the generalized involvement of large and small arteries. This is of special interest since ChS-B and heparitin sulfate are normally major constituents of aorta and probably of other blood vessels and both mucopolysaccharides increase with age andlor arteriosclerosis. The primary lesion of Hurler’s syndrome probably lies in an abnonnal synthesis of ChS-B, which in turn may cause a secondary release of the chemically unrelated hep. S. presumably from mast cells. MECHANISM OF AWORPTXON AND SENSITIZATION OF TANNED SHEEP CELLS WITH H W GAMMA GLOBULIN Irwin Oreskes, Jacques M. Singer and Bernard Liebennan, Brooklyn, N. Tanned sheep cells adsorb human gamma globulin in amounts proportional to the concentration of the added HGG. Logarithmic plots of amounts adsorbed verses amounts Y. added are linear, corresponding to a Freundlich type of adsorption isotherm. Maximum adsorption is about 1,500,000 HGG mole cules per red cell. Under conditions of the 121 AMERICAN RHEUMATISM ASSOCIATION FII S.C. procedure, and at a sensitizing amount of 1,000 PgN, one red cell adsorbs about 500,000 HGG molecules. When the volume of the system is doubled, the number of molecules adsorbed per cell at 1,000 pgN added HGG decreases to 350,000. At saturation each HGG molecule occupies 4,000 A2 of red cell surface. This is the same area occupied by an HGG molecule adsorbed in a monolayer to a 0.8 p diameter latex particle. Both 7s HGG and aggregated THEEFFECTOF NUTRITION ON HGG are adsorbed to tanned sheep cells, and the corresponding isotherms are very similar. However, only aggregated HGG is capable of sensitizing tanned sheep cells for reaction with rheumatoid factor. Maximum and constant titers were obtained with tanned cells sensitized with 37.5 to 6,000 PgN HCG, even though the amount of protein adsorbed increased continilally in this range. EXPERIMENTAL “ADJUVANT DISEASE” Carl M . Pearson, Shennan A4eUinkoff, Fae D . Wood, and Om01 Eshelman, Los Angeles, California Periodically within the past several years evidence has appeared from various laboratories for the nutritional dependence of ( a ) increased bacterial resistance or SIISceptibility of a test animal, ( b ) the caliber of antibody production, or ( c ) the susceptibility of an experimental disease such as allergic encephalomyelitis. Experimental “adjuvant disease” has been produced in about 90 per cent of our laboratory rats by the administration of Freund‘s adjuvant and has been the subject of previous reports. Its most constant feature is a polyarthritis, but often associated also are a balanitis, an intis, a chronic dermatitis and mild diarrhea. The disease has been postulated to be the result of a delayed hypersensitivity reaction to a component of the acid-fast bacillus in the adjuvant. In the present study, utilizing over 200 animals, the disease was found to be quite susceptible to dietary influences. Hence, when weanling rats were maintained for 6 weeks prior to inoculation with adjuvant on a diet which contained 50 per cent protein (lactalbumin) as well as all dietary supplements, they were mildly resistant to “adjuvant disease”; on 80 per cent protein they were significantly resistant; m d on 98 per cent protein (and 2 per cent fat) they were resistant at the highly significant level, Animals on high carbohydrate diet showed no variation from control groups but those on a high fat diet (corn oil) showed a greater susceptibility, both in incidence and severity of various lesions. The animals on the highest protein diet gained less weight than did the other groups but appeared otherwise healthy. The inoculation sites healed promptly and in none of the 40 animals in this group was the arthritis more than mild in degree. It is postulated that dietary factors, notably protein or amino acid composition in the present seric-s, may modify enzymatic mechanisms within the body to the extent that the normal mechanics of the delayed hypersensitivity xesponse are grossly altered. PREPARATION AND PROPERTIES OF HYALURONIC Ac~D” Ward Pigmun, S. Rizoi and Howard L. Holley, Birmingham, Ala. and New York, N. Y. Roseman, Watson, Duff and Robinson ( 1955) have used an electrodeposition method for the isolation of hyaluronic acid from a number of sources. The method has been described more recently (1959) by Balazs and Sundblad. In the present work, the method has been *Read by title. applied to the preparation of hyaluronic acid from bovine synovial fluid. After five repetitions of the process, products were obtained in yields of about 20 per cent which appeared to be free of proteins and only slilrhtly degraded relative to the original synovial fluids. The intrinsic viscosity in 0.2 M pho.rphate buffer was about 60. The use of 5 to 10 per cent ethanol or a small amount 122 AMERICAN RHEUMATISM ASSOCIATION of tolue-ie in the solutions decreased the degradation and offered other advantages. Purifiid hyaluronic acid products could be stored as lyophilized powders or gels at -10” if free of salts. With 20 per cent ethanol present, solutions could be kept at room temperature. THEDEGRADATION OF HYALUHONIC ACIDBY SmuM PROTEINS* Ward Pigmun, S. Rizci and Howard L. Hollcy, Birmingham, Ala. and New York, N. Y. A hyaluronidxse-like action of blood serum, previously reported in the literature, has been found to result from the depolymerizing action of certain blood serum fractions. Whole blood serum, serum proteinfractions, and other selected proteins have been studied for their degrad:itive effect on highly polymerized hyaluronic acid at p H 7.3 and 4.2. Degradations were assessed by irreversible decreases of viscosity. It was found that at pH 7.3 Cohn serum-fractions IV-4 (-globulins) and V (albumin) and other selected proteins caused a slow degradation. hole serum and fraction II-1,2 were found to have no effect at pH 7.3, but at pH 4.2 they produced appreciable degrdation. The degradation by most serum proteins had a pH optimum at about 3.0, but was extensive at p H 4.2. The so-called serum hyaluronidase, active at weak acidities, seems, thus, to be a nonspecific action of several serum fractions and of other proteins. Wead by title. STUDIESON SERUMHAPTQGLOB~N I N EXPERIMENTAL DISORDERS OF CONNECTIVE TISSUE Leslie Roberts, Paolo S . Mombelloni and Piwfranco Crosti, Chicago, Illinois Serum haptoglobin ( H p ) was determined by a colorimetric modification of Jayle’s “activation’’ method in guinea pigs and rabbits before and after the induction of several different types of connective tissue alteration. In guinea pigs, the experimental lesions and the results obtained were as follows: ( 1 ) Guinea pigs with carrageenin granulomas had average Hp values about four times higher than those of normal controls; ( 2 ) acutely scorbutic guinea pigs had average Hp values eight times higher than those of normal controls; (3) scorbutic animals bearing carrageenin granulomas had soniewhat lower values than the preceding group, at five times the normal level; ( 4 ) pair fed controls to the scorbutic animals, receiving a 2.5 mg./day supplement of ascorbic acid, had a significantly higher Hp value than the normal controls. In rabbits, the normal serum Hp is significantly higher than in guinea pigs. ExperiSTUDIESOF TEE mental procedures and results were as follows: ( 1) Biweekly subcutaneous injection of turpentine leading to the production of amyloidosis induced high titers of Hp. After three months of treatment, values levelled off, but in some cases, a second rise occurred after 4-S months. ( 2 ) I.V. injection of papain in young rabbits produced the well known cartilage changes and also a very marked rise of Hp reaching a maximum between 18 and 50 hours after treatment. The average level then was four times the initial value but may reach 7.5 times. ( 3 ) I.V. hyaluronidase injection produced a moderate rise in Hp while injections of saline, ovalbumin, trypsin and chymotrypsin induced no signilkant changes. The possible relation of serum Hp to the different types of connective tissue changes obtained in the present experiments will he discussed. RABBITANTIBODIESWHICH SENSITIZERm BLOODCELLSFOR AGGLUTINATION BY RHEUMATOID FACTORS John H . Rockey and Hennj G . Kunkel, New York, New York Observations from a number of laboratories have indicated that the sheep red cell agglutinating and hemolytic antibodies in rabbit antisera represent a mixture of the high and low molecular weight types. However, little specific information is available 123 AMERICAN RHEUMATISM ASSOCIATION about the characteristics of the rabbit antibody responsible for the sensitization of red cells for agglutination by rheumatoid factors. Separation of these two classes of antibodies was accomplished by the use of zone electrophoresis, sucrose density gradient preparative ultracentrifugation and DEAE column chromatography. Clear differences in their capacity to sensitize cells in subagglutinating titer for agglutination by rheumatoid factors was demonstrated. The low niolecular weight antibodis possessed the capacity in high degree. Thr liigh molecular weight antibodies completely lacked this capacity. Observations on the use of antibody from individual rabbits of differing globulin allotypes and on rabbit incomplete antibodies will be described. The rabbit incomplete antibody was shown also to be of the low molecular weight type. DECREASED RENAL EXCRETION OF URIC ACID AS A CAUSEOF HYPERURICEMIA IN GOUT J. E . SeegmiUer, Arthur I . Grayzel, Lois V . Liddle and Candace Pluto, Bethesda, Maryland The cause of the hyperuricemia of gout has been under investigation in recent years with the use of isotopically labeled uric acid and its precursors. An excessive production of uric acid was found in a substantial portion but not in all of the gouty patients studied. Seven of 16 gouty subjects showed an incorporation of isotopically labeled glycine into urimry uric acid that was indistinguishable from normal. Correction for an increased extrarenal disposal of injected uric acid-2-ClJ present in two subjects left 5 patients in whom no evidence of increased uric acid production could be found by either glycine incorporation or urate turnover values. Renal function studies showed that the group of gouty subjects who did not overproduce uric acid had urate/inulin clearance ratios significantly lower than those found in the group of “overproducers” of uric acid or in a group of normal control subject. in whom an increased uric acid production and hyperuricemia was produced by the administration of ribose nucleic acid. It seems unlikely that a single metabolic defect common to all gouty subjects could cause both an excessive production of uric acid in some patients and a decreased excretion of uric acid in others. We are left then with the view that the hyperuricemia of gout is the result of a variety of metabolic or physiological disturbances. Although excessive production of uric acid may be the major factor contributing to hyperuricemia in the majority of gouty subjects, a decreased renal excretion D f uric acid can also be the primary factor in producing a hyperuricemia in the absence of excessive uric acid production. TI~E PRODUCTION OF RENALDISEASE EXPERIMENTAL STRF.PTOCOCCALINFECTIONS: IN THE WHITEMOUSE John T.Sharp, Detroit, Michigan It has been reported that about 60 per cent of male, Webster, white mice inoculated with more than 108 chains of live, group A streptococci (strain D58, type 3) developed albuminuria, hypoalbuminemia, generalized edema, and histological changes in the kidneys. These studies were extended to examine daerences in host responses. Thirty-seven female Webster mice infected with more than 108 chains of D58 coccus remained edema free. Only 1 of 24 male CSI/BL mice and 5 of 46 male Hau-M mice similarly infected developed edema. Studies of dose response of male Webster mice revealed that only 3 of 45 animals inoculated with between 107 and 10s chains of streptococci developed edema, whereas 121 of 232 animals challenged with more than 108 chains became edematous. Albumin was observed in pooled urine samples as early as the 3rd day of infection and was excreted in largest amounts in those groups in which a high proportion of animals later developed edema (2.89 to 23.7 mg.!mouse/ day). Animals that did not develop edema either excreted no albumin in their urine or only small amounts !1.31 mg./mouse/day). Serum albumin of 13 edematous animals averaged 13.2 per cent of serum proteins. In animals without edema, serum albumin was 29.2 per cent of serum proteins for 32 streptococcus infected animals, 29.8 per cent 124 AMERICAN RHEUMATISM ASSOCIATION for 14 E. coli infected animals, 24.2 per cent for 4 staphylococcal infected animals and 39.2 per cent for 9 animals inoculated with heat-killed organisms or sterile susppnsions. It is concluded that male mice of the Webster strain are more likely to develop kidney disease when inoculated with large numbers of group A streptococci (strain PRESENCE OF D58) than female Webster inicr or inalv mice of other strains studied. This renal disease is manifested by the development of albuminuria, hypoalbuminemia, and edema in many instanccs. It is believed that these manifestations reflect a glomendar lesion induced by the streptococcal infection. “SERDLOGICALLY ACTIVE MACROGLOHULINS” IN SERA OF SOME WITH ACTIVE PULMONARY TUBERCULOSIS Jucyues 31. Singer, Francisco A.! PATIENTS Perdta, Harold U.Lyom and Charles Id.Plotz, Brmklyn, N.Y. Sera from 220 cases of active pulmonary tuberculosis were examined with the FII latex particle procedure. It was found that 38 of these sera were positive, although none of these cases e-xhibited clinical evidence of rheumatoid arthritis. In addition, 12 sera out of the 38 gave :a positive serological test for syphillis. Presence of macroglobulins in FII latex particle positive sera was deniocstratc.d by analytical ultracentrifugation, DEAE cel- lulose column chromatography, and density gradient ultracentrifugation. Mercaptoethan01 treatment resulted in loss of FII laiex particle activity. FII latex particle positive sera and an equal number of matched negative sera were examined by paprr electrophoresis and livm function tests. No correlation was found between clinical activity and serological activity, protein variations and liver function tests in tuberculosis. THEACTIONOF GRISEOFULVIN IN ACUTEGOUTY ARTHRITIS Roberte Slonim, David S . Howell and Hnrliey E . Brown Jr., Miami, Florida A fungistatic antibiotic, griseofulvin, was observed to have a resemblance to colchicine in regard to structural formulae, interference with purine nietabolism and cell division. The agent was used to treat 22 acute attacks of gouty arthritis in 20 patients. Four to 12 Gm. of griseofulvin were administored over a 48 hour period. Complete remission of symptoms was obtained in 12 to 24 hours without any definite toxic reactions, and stiffness, pain, crythema, swelling and heat decreased greatly or disappeared during this period in 14 patients. Of the 6 patients who had a slight or moderate response to griseofulvin, 5 had previously received colchicine in adequate dosage without improvement. Serum levels of griseofulvin in 5 patients indicated peaks of 6 to 9 pg. per cent at 18 hours. If the antiarthritic response is produced by colchicine and griseofulvin at the same biochemical sites, certain similarity of their chemical structure may provide a clue to the active groups. However, a less specific antiphlogistic action of the drug remains to be excluded. SERUM GLYCOPROTEIN ABNORMALITIES IN WHIPPI~E’S DISEASE,INCLUDING A FAMILY STUDY Mary Betty Steum, Baltimore, Maryland There are conflicting data regarding the character and significance of the increased serum glywproteins reported in Whipple’s disease, and family investigations have been limited to siblings with this disorder. T h e glycoprotein abnormalities in a proven case of Whipple’s disease were recorded serially over a 15-month period, during which the ‘Read by title. clinical activity varied and corticosteroids were not administered. Chemical and electrophoretic data were also obtained on 12 family members. In the index patient, seromucoid, total protein-bound hexosamine, and the hexosamine:protein ratio were all strikingly increased. Most of the glycoprotein elevation was accounted for by increased seromucoid. Electrophoretically, the elevation in the al- 1% AMERICAN RHEUMATISM ASSOCIATION pha-1 carbohydrate-rich globulin persisted throughout the observation period; the increase in the alpha-2 glycoproteins was more variable; and transient hypergammaglobulinemia was not associated with a concomitant increase in gammaglobulin glycoprotein. Although the elevations of the glycoproteins were maximal when the disease was clinically active, abnormally high levels continued during spontaneous remission of symptoms. Serum protein and glycoprotein determinations were normal in 10 (2 male, 8 female) of the 12 relatives studied. One brother, suspected clinically of having Whipple’s disease, had persistent mild elevation of the alpha-1 globulin and a transient increase in the carbohydrate-rich components. The patient’s only son had an “alpha3 globulin” electrophoretic pattern as the only abnormality; the four daughters had normal findings. DISSOCIATION AND REASSOCIATION STUDIES OF A 19s hlAC:ROGLOBUI.IN RHEUMATO FACTOH ~ YHOPEH~TES WITH Thomus B. T o m s i and Henry G . Kiinkt.1, New York, N. Y. Human 19s macroglobulin of both normal and pathological sera can be dissociated by means of sulfhydryl compounds into 7s subunits. Recently certain well d d n e d 19s antibodies including Rh saline agglutinins, isohemagglutinins, and cold agglutinins have been found to dissociate in a similar manner. In all well defined instances dissociation results in a complete loss of biological activity. Rheumatoid factors which resemble this class of antibodies loses on dissociation their ability to precipitate aggregated gamma globulin as well as their activity in the latex and sensitized sheep cell tests. Despite partial reaggregation to 19s material following removal of the reducing agent, in none of the reported cases has biological activity been regained. A 19s macroglobulin has been encountered recently in the serum of a patient with the diagnosis of Waldenstom’s macroglobulinemia which behaved like a rheumatoid factor in a variety of ways, including its ability to give a precipitin reaction with ag- gregated gamma globulin. This macroglobd i n was isolated and dissociated to 7s subunits in .1 Methylmercaptan. Renioval of the sulfhydryl reagent resulted in reassociation into a heterogeneous group of higher polymers, of which the major constituent was 19s. Dialysis against iodoacetamide completely prevented reassociation. Quantitative precipitin analyses performed on the native, dissociated, iodoscetamide treated, and reaggregated products indicated a complete loss of precipitin activity in the dissociated and iodoacetamide treated samples. However, the reaggregated product showed a return of about 90 per cent of the activity of the original preparation. This return of activity could be blocked in large part by reassociation in the presence of dissociated heterologous inacroglobulins. The results are interpreted in terms of the structure of the macromolecule. They suggest that the high molecular weight configuration is of primary importance in determining the activity of this molecule. OF THE NUCLE~PR~TEIN-REACTIVE GAMMAGLOBULINS IN QUANTITATIVE ESTIMATES SYSTEMICLUPUSEHYTHEMATOSUS Ahan& S. Tounes, C . R. Steuart, Jr. a d dbrahnin G. Osler, Baltimore, Mary!and As a basis for immunological studies in systemic lupus erythematosus ( SLE ) and related disorders, effort has been directed toward developiilg a method for quantitative estimations of gamma globulins which r.=act with aucleoprotein. Particulate suspensions of calf thymus niicleoprotein are reacted with dilutions of test serum. Particles are recovered by centrifugation and thoroughly washed in isotonic saline. The amount of gamma globulin bound to these particles is then estimated by means of a quantitative complement fixation reaction with a constant quantity of rabbit anti-human gamma globulin serum. Results are expressed as equivalents of gamma globulin nitrogen by reference to a calibration curve established under identical conditions with known amoiints of human gamma globulin. In patients with SLE, whose sera induce AMERICAN RHEUMATISM ASSOCIATION L.E. cell formation> the quantity of equivalent gamma globulin ranges from approximately 10 to 40 micrograms nitrogen per ml. (reproducible within 10 per cent). In these sera, uptake of gamma globulin by nucleoprotein is associated with loss of antinuclear antibody as defined by a fluorescent anti-globulin technique. Values for normal sera range from 0.5 to 3 micrograms nitrogen per ml. Some rheumatoid and hyperglobulinemic sera give EVALUATIONOF THE AGGLUTINATIDNS IN values above the normal range, occasionally as high as those for SLE sera. In a preliminary series these reactive sera also give a positive FII latex test in high titer. In contrast, SLE sera have shown negative FII latex tests. Further studies are underway to expend these observations in order to clarify the nature of interaction between gamma globulin and nucleoprotein in SLE as compared to other disease states. SENSITIZED HUMANCELL, SENSITIZED SHEEP CELL AND THE LATEX INDIVIDUALS, IN PATIENTS WITH ~ U M A T O I DARTHRITIS A N D PATIENTSWITH OTHER DISEASES NORMAL M. V. Wuller, B. Decker, E . C. Toone, W. R . 1rby und N . E l . Cur?/, Richmond, Virginia This study was designed to ( 1) evaluate individuals with “false positive” reactions for rheumatoid factor, ( 2 ) compare the SHC with the better known latex and SSC agglutinations and (3) correlate the three types of rheumatoid serological reactions with various clinical features of rheumatoid arthritis. The SHC tests were done by the method of Waller and Vaughan, the SSC tests by the method of Heller, and the FII latex agglutination was done with commercial reagents ( Hyland Laboratories ) . The study was carried out on a total of 500 patients which included 340 normal individuals, 87 patients with rheumatoid arthritis and 73 patients with other diseases. There were 14 (49;) positive reactors detected among the 340 seemingly normal individuals chosen from among blood bank donors and a prenatal clinic. The SHC agglutination was positive in 3 per cent, the latex in 3 per cent and the SSC in 0.3 per cent. Nine of the 14 reactors were studied in greater detail and 5 had either suggestive family histories, mild rheumatic symptoms or hypergammaglobulinemia. No abnormalities were found in the remaining 4. A detailed serological and clinical study of the normal individual with the highest titre of rhewnatoid factor (SHC-2048,SSC-256), followed bi-monthly for two years, will be presented in detail. In 87 cases of rheumatoid arthritis, the rheumatoid factor was found to be present in 89 per cent; in 82 per cent by the SHC; in 86 per cent by the latex; and in 86 per cent by the SSC agglutinations. Positive agglutinations for rheumatoid factor were significantly more common in stage 111-IV, classic-definite rheumatoid arthritis but could not be correlated with age, duration of disease, nodules, sex or race. These studies indicated that ( 1) the rheumatoid factor may be present in normal persons, (2) the SHC method, as used here, parallels the latex agglutinations, both mc-thods being more sensitive and less specific than the SSC agglutination, ( 3 ) positive agglutinations for rheumatoid factor were more common in classic-definite, stage 111-IV rheumatoid arthritis. OF DEEP SOMATIC PAIN* A QUANTITATIVE MEASURE B. Berthold Wolfl and Murray E . Jumik, New York, N. Y. Pain threshold as well as tolerance and reaction to pain may form good bases for the prediction of success in the rehabilitation of the chronic arthritic patient, but an adequate objective and quantitative e.xperimenta1 method is required for this. A modified ver‘Read by title. sion of Lewis’s and Kellgren’s technique was followed by injecting hypertonic saline intramuscularly to produce deep somatic pain, which is subjectively described as a poorly localized, long-lasting and unpleasant dull ache, starting several seconds after injection, increasing in intensity during 10 to 45 seconds, and persisting at least 30 seconds. In 127 AMERICAN RHEUMATISM ASSOCIATION this investigation, using the verbal responses of 14 normal subjects, it was found that ( a ) the best technique consisted of using 25 gauge 1%inch hypodermic needles, inserted intramuscularly at an angle of 45" through the anesthetized skin in rosettes of 4 each, so that in the muscle the points of the needles were about 1 inch apart; ( b ) intradermal injection of procaine satisfactorily abolished the perception of cutaneous pain; and that ( c ) volume of saline and rate of injection were important variables in the production of deep somatic pain. However, when tested with isotonic saline, a quantity of 0.1 or 0.2 cc. injected slowly satisfactorily minimized error due to volume or pressure. In 10 subjects the mean deep somatic pain threshold of the gastrocnemius on injection of 0.1 cc. of hypertonic saline at &minute intervals was found to be 2.34 NaCl, with a median of 2.5% NaCl and a standard deviation of -C 0.69 per cent NaCl. In another experiment with 7 subjwts the mean deep somatic pain threshold of the gluteus maximus on injection of 0.2 cc. of hypertonic saline at 2-minute intervals was found to be 4.5 per cent NaCI, with a median of 5.0% NaCl and a standard deviation of 1.61% NaC1. In addition 2 chronic arthritic patients in a pilot study described the experimentally produced pain as similar to their arthritic pain. The subjective deep somatic pain responses, measured as percent concentration of saline, are also meaningful psychophysically, because they range from zero at an isotonic level to 100 per cent at 4 per cent NaCl for the gastrocnemius and at 8 per cent NaCI for the gluteus maximus. Therefore, these results suggest that the hypertonic saline technique is a satisfactory objective and quantitative method for the measurement of deep somatic pain. ADMINISTRATION OF TRIAMCINOLOXE ACETORIDE AND TRIAMCKNOLONE DIACETATE IN THE TREATMENT OF RHEUMATOID ARrHIuTlS: h E L I M I N A R Y &PORT* INTRAMUSCULAR Jack Zuckner and Robert H. Ramey, St. Louis, Missouri The acetonide and diacetate derivatives of triamcinolone were injected intramuscularly into patients with rheumatoid arthritis. There were 73 injections, 44 with triamcinolone acetonide and 29 with triamcinolone diacetate. The dose per injection was 100 mg. The maximum duration of benefit was 49 days after an acetonide injection and 50 days after the diacetate form, averaging 18.1 and 15.4 days, respectively. Eighteen of 21 patients receiving triamcinolone acetonide and 15 of 19 receiving triamcinolone diacetate had satisfactory responses. (The latter was defined as greater than 50 per cent improvement for a period of seven or more days. ) Such improvement followed approximately two-thirds of injections with either derivative. When compared to hydrocortisone acetate injected intramuscularly in 100 and 500 mg. doses, the triamcinolone prepsrations gave greatly superior results, particularly in reference to duration of effect. Fifteen patients were receiving other steroids orally at the time the intramuscular injections were administered. In about half these individuals the close of the oral steroid could then b e lowered, and in several patients, the total of the oral plus the parenteral dose was less than the total oral dose given previously. Toxic reactions were as follows: euphoria, 2 patients; leg cramps, 2; nausea, vomiting, anorexia, and weakness, 3. The duration of the study did not allow long term evaluation of toxicity. *Read by title. -- - - A.R. A. N E W S ABSTRACT DEADLINE FOR 1961 ANNUAL MEETING Is Those wishing to present papers at the 1961 Annual Meeting (June 22 and 23, Hotel Roosevelt, New York City) should send ten copies of abstracts (maximum length: 250 words) to the Program Chairman, Dr. R. W. Lamont-Havers, Room 1700, 10 Columbus Circle, New York 19, N. Y. 128 The Program Committee will make special efforts to select for the meeting’s plenary sessions a relatively large number of clinical papers, if their quality merits acceptance. Abstracts of all papers on the program and a selective list of those to be read by title will be published in the journal, ARTHRITIS A N D RHEUMATISM. Ten minutes are allowed to each speaker, followed by a five-minute discussion period. There may be a series of three specialized concurrent sessions. The Chairman of each of these sessions will report a summary of the proceedings to the plenary sessions so that everyone in the audience will have an opportunity to inform himself of the highlights of all sessions. Among other plans is a panel discussion, jointly sponsored by the A.R.A. and the American Academy of Orthopedic Surgeons, on “Reconstructive Surgery of the Shoulder, Elbow and Knee.” For the first time, the Program Committee will select a Westhoff Memorial Lecturer. This lecture, to be given for a series of annual meetings, is named in memory of the late Mrs. Anna H. Westhoff of St. Petersburg, Florida, who bequeathed a substantial part of her fortune to the American Rheumatism Association. Dr. John Talbott, editor of the J o u m l of the American Medical Association, will give a luncheon address on the subject of “Trials, Triumphs and Thwartways of a Medical Editor.” The annual business meeting has been scheduled for the afternoon of June 23. To bridge the gap between the A.R.A. meeting and the annual convention of the American bledical Association (June 26-30), the New York Rheumatism Association is planning a postgraduate seminar on arthritis and rheumatism at the Roosevelt Hotel, Saturday, June 24 and Sunday morning, June 25. The New York Rheumatism Association will also be host to A.R.A. members and their wives at a reception Thursday evening, June 22, at the Hotel Roosevelt’s Terrace Room. Members will receive room reservation forms several weeks before the meeting, Printed programs will be distributed to the membership early in June. A limited number of rooms will ;dso be A.R.A. NEWS available at Olin Hall, residence hall for the Cornell Medical School, 69th Street and York Avenue. Accommodations ($3.00 per day per person) are available for both men and women. A few double rooms are also available. Olin Hall is within convenient distance from the Roosevelt Hotel. Room rates at the Roosevelt Hotel range from $8.50 to $1850 for singles, and $13.50 to $23.50 for doubles. For the entertainment of members and their wives, the Local Arrangements Committee and the Ladies’ Sub-committee have set aside a hospitality room, immediately adjoining the Roosevelt’s Grand Ballroom, site of the plenary sessions. Information on shopping, sightseeing and entertainment will be available at the Hospitality Room. A block of 50 tickets for the Loew-Lerner Broadway musical, “Camelot,” has been secured. Out-of-town members who wish to obtain tickets for Friday, June 24, will be given preference. To reserve tickets, send your reservation and check to the American Rheumatism Association, 10 Columbus Circle, New York 19, N. Y. All tickets are priced at $8.05. DALLAS INTERIM MEETING A total of 36 papers was presented at the Seventh Interim Scientific Session of the A.R.A. in Dallas, Texas, December 9 and 10, 1960. The meeting was attended by nearly 300 physicians, more than half of these A.R.A. members. A panel discussion on lupus nephritis was moderated hy Dr. Halsted R. Holman. Panel members were Drs. Robert M. Kark, Lawrence E. Shulman, Conrad L. Pirani and Howard Worthen. At the business meeting, 115 new applicants were elected to membership. This brings the total of new members gained during the last twelve months to 226. Among distinguished visitors from abroad were Professor Franpis Coste, of Paris, France, President, International League against Rheumatism, and Dr. Oswald Savage, of London, England, Secretary of the Empire Rheumbtism Council. Professor Coste, as guest of President Dr. Edward F. Hartung, attended not only all scientific sessions but also the all-day meeting of the A.R.A.’s Executive Committee. The Dallas educational television station, A.R.A. NEWS KEFU, devoted a half-hour program to the subject of arthritis. Participating in this round-table discussion were Drs. Edward F. Hartung, Morris Ziff, Howard C. Coggeshdl. R. W. Lamont-Havers and MI. Robert Straws, President, North Texas Chapter, Arthritis and Rheumatism Foundation. The Foundation’s Dallas Chapter helped to underwrite part of the cost of thtb scientific sessions and presented President Hartnng with a ten-gallon hat. POSTGRADUATE COURSES FROM MARCH-MAY 1961 Among postgraduate courses on rheumatic disorders scheduled for the next few months are two at New York University Medical Center and one at the IJniversity of Michigan, Ann Arbor. A special coiirse for the experienced clinician and research worker, under the direction of Drs. Cuirier McEwen and Edward F. Hartung, will be given March 13 through March 17, 1961 at New York University Medical Center. Tuition will be $100. For application, write to the Office of the Dean, New York University Post-Graduate kledical School, 550 First Avenue, New York 16, N. Y. The course is designed for those physicians who are already familiar with the fundamental data essential to an understanding of arthritis and related disorders. In general, applicants should have had five or more years of experience in an arthritis clinic or its equivalent. Standard differential diagnosis and treatment will not be covered. Particularly stressed will be newer concepts of etiology, newer research technics and recent advances in our !,asic knowledge of what underlies these disorders. Among the guest lecturers will be Drs. Ward Pigman, New York; Jerome Gross, Boston; C. William Castor, Ann Arbor; 129 Philip H. Henneman, Jersey City; Maciyn McCarty and Alexander Gutman, both of New York. The same Center will hold another course for general practitioners May 15 through 19, 1961. Clinic and bedside teaching will be stressed. Tuition is $85. A postgraduate course in rheumatology is being offered April 24, 25 and 26, 1961 by the University of Michigan, Department of Postgraduate Medicine, at the University Hospital, Ann Arbor. The course consists of three days of instruction on the newer coiicepts of diagnosis and management of rheumatic disease, including lectures, case demonstrations, as well as consideration of laboratory aids and physical therapy. Dr. Richard H. Freyberg, Professor of Clinical Medicine, Cornell Univeriity, will participate as guest lecturer in the program. The prograni and application forms may be obtained from Dr. John hl. Sheldon, Director, Department of Postgraduate Medicine, University Hospital, Ann Arbor, Michigan. NEW BIBLIOGRAPHY AVAILABLE The first three issues of The National Foundation’s new bibliography on Arthritis and Related Diseases contains abstracts of 192 articles from 91 journals. Of these, 124 articles were from 46 American journals, and 68 from 45 foreign journals. .4.R.A.’s official journal, ARTHXUTIS AND R H E m ~ A T I s M , contributed the largest number ( 3 2 ) from any one journal. The monthly biblography is distributed to 3,500 professional individuals and institutions, including medical and associate professional schools, voluntary and governmental agencies. In addition, it i5 sent to 651 libraries in this country and abroad, and to 370 medical journals.