Proceedings of the 41st annual scientific session of the american rheumatism association a section of the arthritis foundation december 911 1976 miami beach florida.код для вставкиСкачать
105 Proceedings of the 41st Annual Scientific Session of the AMERICAN RHEUMATISM ASSOCIATION A Section of The Arthritis Foundation December 9-11, 1976 Miami Beach, Florida Abstracts of Papers Presented Chondromalacia Patellae as a Separate Entity Peter J . A bernethy. Paul R . Townsend, Robert M . Rose, and Eric L. Radin, Edinburgh, Scotland and Boston, Massachusetts Chondromalacia can be defined as cartilage fibrillation that does not progress to exposure and eventual eburnation of the underlying bone. It is often asymptomatic. Most authors feel it is a precursor of osteoarthrosis. It has been suggested that the progression of cartilage lesions is related to the state of its underlying subchondral bone. In order to investigate the relationship between cartilage fibrillation, its progression, and the relative state of the underlying subchondral bone, 24 patellas obtained at autopsy were examined. Meachim’s India ink technique was used to grade their cartilage damage by the Collins scale. The quantitative density of the underlying subchondral bone of various regions of the patella was measured. This parameter has been shown to be directly related to the relative stiffness of the bone specimens. Previous studies of the contact surface area of the patello-femoral joint at various angles of flexion had shown that the periphery is basically non-weight-bearing, the “odd (extreme medial) facet” is in contact above about 135” of flexion, and the central medial and lateral facets are in contact through much of knee motion. It was found that the earliest cartilage fibrillation occurred along the unloaded periphery and habitually loaded central medial areas. These changes were age-related and tended to be nonprogressive. The bone underlying these areas was relatively osteopenic. The cartilage changes on the habitually loaded lateral facet tended to occur in older individuals and tended to be progressive. The bone in this area was denser and stiffer than the bone of the medial facet. An “odd facet” was observed at 30% of specimens. There was no correlation between the changes on this facet and those of the rest of the patella. When the “odd facet” was present it was crossed by a synovial fringe 40% of the time. I t was concluded that nonprogressive chondromalacic changes occur in both loaded and unloaded areas of the patella. Erosion of cartilage to bear bone (Collins grade IV) was unlikely over relatively osteopenic bone and was more likely over stiffened bone. The distribution of stiffness in the underlying bone related well to both the degree and rate of cartilage damage. The observations were consistent with the hypothesis that the symptoms of chondromalacia patellae could well be due to synovial irritation rather than cartilage fibrillation. This series clearly needs to be enlarged. Antibodies to an Acidic Nuclear Antigen in Connective Tissue Diseases M . Akizuki, M . J . Boehm-Truitt, S. S. Kassan. A . D. Steinberg. and T . M.Chused, Bethesda. Maryland The soluble extract of mammalian cell nuclei contains several distinct antigens against which patients with connective tissue diseases produce antibodies. Determination of the specificity of such antibodies is important because of increasing evidence of correlations between antibody specificity and the clinical characteristics of patients with autoimmune disease. At present the major problem in the identification of antibodies to soluble nuclear antigens is the lack of purified preparations of the individual antigens, which would allow specific and quantitative titration of antibody. In this study an acidic nuclear protein, distinct from the Sm and RNP antigens and tentatively named the Ha antigen, was purified approximately I ,000-fold and a specific, sensitive assay for detecting antibodies was developed. The antigen was purified from calf thymus nuclear extract by ammonium sulfate fractionation (60-8070 saturation), DEAE Sephadex chromatography (eluted between 0.25 and 0.38 M NaCl in 0.05 M Tris buffer, pH 7.4), and affinity chromatography utilizing DEAE purified IgG from a patient with a high titer of antibody to the antigen covalently bound to Sepharose. This preparation was iodinated by the Arthritis and Rheumatism, Vol. 20, No. 1 (January-February 1977) method of Bolton and Hunter and finally purified by gel filtration through a Sephadex GI00 column. On polyacrylamide gels the antigen is homogeneous, has an apparent molecular weight of approximately 53,000, and is composed of two components of approximately 33,000 and 20.000 daltons. It is heat labile, resistant to nucleases, but destroyed by trypsin treatment. Antibody to Ha antigen in 20 pl serum was assayed by the ammonium sulfate method, by using 5 ng of I-Ha. Binding greater than 24%, two standard deviations above normal, was observed in 73% (32/44) of patients with Sjogren’s syndrome (SS) without rheumatoid arthritis (RA). and in 85% (17/20) of patients with SS and systemic lupus erythematosus (SLE). Only 6% (1/18) of patients with the combination of SS + RA, and only 3% (2/58) of patients with SLE had anti-Ha, results suggesting that they represent distinct populations. Patients with other connective tissue diseases and healthy subjects did not have anti-Ha antibody. This rapid and accurate method of measuring antibody to Ha may be useful in the diagnosis of SS and in the recognition of subpopulations of patients with the sicca complex. ARA ABSTRACTS 106 Circulating Immune Complexes in Acute Uveitis Brian S. Andrews, Valerie Petts. Jill Mclntosh, and Ronald Penny, La Jolla, California and Sydney, Australia The aim of the study was to determine if circulating immune complexes (IC) were present in 39 consecutive referred patients presenting with acute uveitis. Of 15 patients (38%) who were HLA-B27 positive (827 (+), 12 were males (80%). 12 (80%) had radiologic evidence of sacroiliitis, but systemic disease was uncommon. In the 24 HLA-B27 (B27 (-) negative patients (62%), 16 were females (67%). sacroiliitis was present in I (4%). and systemic disease, including polyarthritis, was common. IC were sought in serum by the following techniques: a ) complement (C) activation (plasma C3, C4 levels, C 3 fragments in plasma, Clq precipitins, and anticomplementary activity in serum); b) rheumatoid factor ( R F ) in serum; c ) monoclonal R F precipitins; d ) presence of cryoglobulins; e ) inhibition of EA (IgG) rosette formation by blockage of Fc receptors on human peripheral blood lymphocytes (PBL); f) increased numbers of PBL with surface membrane bound Ig (Smlg); g ) spontaneous neutrophil chemotactic activity in plasma. The mean f 2 SD for normals in all of the above tests was determined and values outside this range were regarded a s abnormal. Thirty-six percent of uveitis patients had evidence of C activation, in particular anticomplementary activity in serum (P < 0.005); 14% had cryoglobulins; 33% inhibited EA rosette formation (P < 0.05); 32% had increased numbers of Smlg positive PBL; and 47% showed neutrophil chemotactic plasma activity (P < O.Ol), while R F precipitins were uniformly absent. Anticomplementary activity was more common in the 827 (-)than in the 827 (+)group (30% vs loo/,), as was neutrophil chemotactic activity (63% vs 21%). One o r more tests were positive in 60% of B27 (+) and 96% of B27 (-) patients, while 2 or more were positive in 27% of B27 (+) and 67% of 827 (-) patients. Thus we have demonstrated that when a variety of techniques is employed, circulating IC are frequently detected in patients with acute uveitis. In addition, acute uveitis in the B27 (-) group is more frequently associated with circulating IC, a fact suggesting that B27 ( - ) patients differ from the 827 (+) patients either in their immune response to antigen o r in their handling of circulating IC. HLA-A28 in Patients with B27-Associated Rheumatic Diseases Frank C. A rnett. Jr., Bernice Z . Schacter, Marc C. Hochberg, and Wilma B. Bias, Baltimore, Maryland Tissue typing for HLA-B27 has become a widely applied test in various rheumatic disorders. However the association of B27 with the spondylitic group of diseases remains an unexplained biologic phenomenon, and other factors determined by the HLA region may play a role in disease susceptibility. HLA-A2 has been reported to be increased in B27-positive patients with disease; however the high prevalence of this antigen in control populations (50%) makes statistical confirmation difficult. HLA-A28 is a cross-reactive antigen with A2 and may be technically difficult to define if A2 is also present. Our studies indicate that A28 may be increased in white B27-positive patients with rheumatic disease, but not in white B27-positive normal individuals. Thirty-eight unrelated, white 627-positive patients were studied with careful attention directed toward A locus antigens. Thirteen had ankylosing spondylitis (AS), 21 Reiter’s syndrome (RS), and 4 juvenile chronic polyarthritis (JCP). The A28/B27 phenotype was found in 9 (23.7%). Of 38 age, sex, and race matched controls, 4 (10.5%) had 827, 2 (5.3%) had A28, but none had A28/B27. In 210 normal, healthy, unrelated white controls, 29 (13.6%) had B27, 11(5.2%) had A28, but only I (0.5%) possessed the A28/B27 phenotype. The difference between the incidence of A28/B27 in the B27positive disease (9/38 o r 23.7%) and B27-positive control (1/29 o r 3.4%) groups is significant (P 5 0.05). In addition, calculations based on actual gene frequencies would predict an A28/B27 phenotypic frequency of 3.8%, irrespective of whether their alleles were on the same or different haplotypes. The A28/B27 phenotype was found in 31% of AS, 51% of JCP. and only 10% of RS patients. The A2 antigen was present in 53% of B27-positive disease patients, 41% of matched controls, 37% of B27-positive normal controls, and 45% of the 210 white population controls (nonsignificant differences). In addition to these Caucasian patients, studies of 11 blacks (RS 7, AS 3, and JCP 1) revealed 827 in 10 (90%). None had A28. These results suggest that A28 may be an additional marker for 827 disease or may be a contributing factor in pathogenesis. Further determinations of multiple gene products determined by the HLA region in disease states are needed. HLA-B7 Cross-ReactiveAntigens in B27-Negative Reiter’s Syndrome Frank C. Arnett, Jr.. Bernice Z . Schacter, Marc C . Hochberg. and Wilma B. Bias, Baltimore, Maryland Reiter’s syndrome (RS) is strongly associated with HLA-B27, yet there is good evidence for infectious agents in pathogenesis. The “molecular mimicry” and “cellular surface receptor site” theories of histocompatibility antigen and infectious agent interaction to produce disease have been demonstrated in animals and proposed as possible explanations for HLA and disease associations in man. The finding of HLA antigens cross-reactive t o B27 could lend indirect support for these concepts when the HLA antigen is itself involved and make the “linked immune response gene” hypothesis less likely. Thirty-one patients with RS have been studied with respect to clinical features and HLA phenotypes. A clinical diagnosis of RS was made prior to tissue typing in all. The complete triad of nonspecific urethritis (NSU), conjunctivitis, and typical arthritis was present in 13 (42%). Eighteen (58%) had incomplete Reiter’s syndrome (IRS). Of these, 8 (26%) had NSU and arthritis (IRS-I) and the remaining 10 (32%) had arthritis alone (IRS-11). Twenty-four were white, 6 black, and I tri-racial. Only 2 were female, and both had the complete triad. HLA-B27 was present in 25 (80%), including all 10 with IRS11. Six patients did not have 827; 3 had complete RS and 3 IRS-I (including the I black patient without B27). Of these 6 B27-negative patients, 5 had HLA-B locus antigens cross-reactive with B27 (HLAB7 cross-reactive group). In the complete RS group, 1 had B7, I had BW22, and 1 had no 8 7 cross-reactive antigens. In the IRS-I group, 2 had BW22 and the 1 black patient had an unidentified B7 crossreactive antigen. There were no significant clinical differences between the 827 positive and negative groups, except for the absence of uveitis and radiographic sacroiliitis in the latter. Two B27 negative families have been studied, and a 10-year-old daughter of the black patient has unilateral sacroiliac pain, heel pain, and an elevated sedimentation rate, but as yet normal X rays. She does possess the same B7 crossreactive antigen. Therefore further studies of HLA phenotypes and genotypes in R S should direct special attention t o B27-negative patients. ARA ABSTRACTS 107 A Recently Recognized Familial Arthropathy in Children Balu H . A threya, H . Ralph Schumacher. and Peter Vanace, A tlantic City, New Jersey, and Philadelphia, Pennsylvania Three siblings with a recently recognized form of familial a r t ~ o p a t h yhave been studied clinically and by light and electron microscopy of the synovium. T w o of these children were girls and I was a boy. All of them had symmetric swelling, effusion, and limited motion of large joints starting within the first 6 months of life. All of them also had flexion deformity of the PIP joint of one of the fingers from early infancy and resembled the patients reported briefly by Jacobs ef al (Pediatrics 57:696, 1976). One of these children was studied extensively at age 3. CBC, urinalysis, sedimentation rate, rheumatoid factor, and antinuclear factor were all normal or negative. The synovial fluid from one knee was straw-colored, had good viscosity, formed a good m u c h clot, and showed 300 WBC/m13 with predominantly mononuclear cells. Only soft tissue swelling was seen o n roentgenograms of the knee and wrist joints. Needle synovial biopsy was done o n this girl. Synovium was available from synovectomy in the 2 siblings. All of them showed synovial hyperplasia, necrotic synovial villi alternating with normal villi, deposition of eosinophilic and PAS-positive material in the subsynovial layer, and large numbers of multinucleated giant cells with pink cytoplasm. There was n o vasculitis and there were only occasional lymphocytes o r polymorphonuclear leukocytes. Alcian blue and amyloid stains were negative Electron microscopic study of synovium in I patients revealed many connective tissue cells with dilated rough endoplasmic reticulum. Some phagocytic cells contained degenerated collagen in vacuoles. Acid phosphatase was normally distributed and was prominent in the giant cell lysosomes. Giant cells also had profuse rough endoplasmic reticulum. Ruthenium red stains identified normal amounts of mucopolysaccharide. There was granular and fibrin-like interstitial material but no amyloid. This symmetric noninflammatory arthropathy now reported in 2 families appears to have a characteristic histologic appearance of synovium that allows its identification as distinct from other joint diseases. Kinetics of Amyloid Protein SAA in Casein-Induced Amyloidosis Merrill D. Benson. Morton A . Scheinberg, Tsuranobu Shirahama, Edgar S . Cathcart, and Martha Skinner, Boston. Massachusetts and Indianapolis, Indiana Secondary amyloid protein (AA) is derived from a circulating alpha globin (SAA) which is elevated in chronic diseases such as rheumatoid arthritis. SAA is also elevated in acute diseases, including bacterial and viral infections, and in cancer, in which n o association with secondary amyloid is known. T o study the relationship between SAA production and amyloid deposition and to determine factors involved in the cause of secondary amyloid in chronic inflammatory diseases, the murine model of casein-induced amyloidosis was used. Amyloid was induced in CBA/J mice given daily casein injections for 28 days. SAA levels were measured throughout the course by radioimmunoassay (RIA), by using antiserum to protein AA. SAA levels were also monitored in casein-treated A/J mice, bovine serum albumin (BSA) treated CBA/J and A/J mice, and casein-treated nude (athymic) mice. SAA levels were reported in microliter equivalents of a serum standard. SAA levels rose within 3 hours after a single casein injection and peaked by I2 hours (I350 & 350 units vs normal 7 f 2 units). SAA remained elevated throughout the preamyloid and amyloid phases, but dropped rapidly after cessation of casein injections. N o significant difference was found between the SAA response of CBA/J mice t o injections of casein and A/J mice, a strain relatively resistant t o the induction of amyloid. Also, treatment with BSA, which has a low potential for inducing amyloid, gave high levels of SAA. Athymic mice were shown to produce levels of SAA as high as those produced by CBA/J mice (1860 & 401 units 18 hours after a single casein injection). These data show that T lymphocytes are not necessary for the production of SAA. Although the production of SAA may be necessary for the formation of secondary amyloid. other factors must be involved in the deposition of the fibril protein. These factors may be important in the degradation of SAA by the reticuloendothelial system, and certain substances, such as casein, may interfere with this process so as to accelerate the formation of amyloid. Similar mechanisms may be involved in the production of secondary amyloid in chronic inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, and granulomatous colitis. In Vitro Culture of Articular Cartilage Stimulates Collagen Synthesis and Maintains the Differentiated Collagen Phenotype Paul D. Benya and Marcel E. Nimni. Los Angeles. California Thin slices of articular cartilage from rabbit hip and shoulder were incubated in a 10% CO,, 90% air environment in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% (v/v) penicillin-streptomycin. Duplicate dishes were cultured for 0, 1, 2, 3, and 5 weeks and fed every third day. Radioactive collagen was synthesized during the last 24 hours of culture by incubating the slices in media supplemented with tritiated proline (50 pCi/ml), B-aminoproprionitrile (25 mM), and ascorbate (100 pg/ml). The medium and slices were lyophilized together and extracted at 4" with 1 M NaCI, 50 mM Tris, pH 7.5. The extracted collagen was treated with pepsin and purified. The collagen types were fractionated by SDS-polyacrylamide gel electrophoresis on 5% gels t o give discrete peaks identified by cyanogen bromide peptide analysis as: Type Ill. X (a new collagen chain), a1 (11). and a2. In the first 2 weeks of culture, collagen synthesis increased approximately 5-fold; by the third week, 20-fold. N o significant changes were detected in the number of viable cells per milligram of cartilage. In the first 24 hours of culture the cartilage slices synthesized 1.0% Type 111, 3.2% X. 95% Type I I , and 0.6% a2. At 5 weeks cells synthesized 1.7% Type 111,0.6% X,87.8 Type 11, and 2%a2. Thus the differentiated collagen phenotype of these cells remained relatively unchanged during long-term culture, in contrast t o what we have previously observed for subcultured chondrocytes. This finding emphasizes the importance of some as yet undefined part of the cell environment for maintaining the proper phenotype. The behavior of these chondrocytes t o organ culture should provide a phyisiologic model t o elucidate further the factors that control the synthesis and turnover of macromolecules in normal and diseased cartilage. ARA ABSTRACTS 108 Auranofin: An Oral Chrysotherapeutic Agent for the Treatment of Rheumatoid Arthritis Frans-Erik Bergliif: Kerstin Bergliif: and Donald T. Waltz, Ostersund, Sweden and Philadelphia, Pennsylvania This study was undertaken to evaluate the safety and clinical efficacy of auranofin (SK&F D-39162) (2,1,4,6-tetra-O-acetyl-l-thioP-D-glUCOpyranOSat0-S) (triethylphosphine) gold, in the treatment of rheumatoid arthritis (RA). Eight patients with established R A by the ARA criteria were treated with auranofin at an oral dose of 3.0 mg, b.i.d. for 3 months. Oral absorption of auranofin was evidenced by the blood and urine gold levels. The mean blood gold level of the 8 patients the first week of treatment was 0.26pg/ml, which increased to 0.70 pg/ml at week I2 of treatment. Improvement in the biochemical, immunologic, and objective/subjective clinical parameters were seen in the 5th-7th week and continued throughout the study. Single episodes of auranofin-related diarrhea (1- o r 2-day duration) were seen in 3 patients. A fourth patient withdrew from auranofin treatment (week 10)after four episodes of drug-related diarrhea (1 t o 4 days duration). The efficacy of auranofin treatment was evident by alterations of the biochemical parameters toward normal levels as evidenced by the following observations: increases in albumin and albumin/globulin ratios; decreases in alpha-2-globulin, rheumatoid factor titers, sedimentation rate, and in the level of immunoglobulin G. Clinically, effects were evident with auranofin treatment by the reduction of the number of painful and swollen joints, knee and finger joint size, and morning stiffness, by a decrease in walking time, and by an increase in grip strength. The data are summarized in the table. ~~ Parameters (units) Painful joints (no.) Swollen joints (no.) Knee joint size (cm) Finger joint size (mm) Morning stiffness (Hrs) Walking time (Sec) Grip strength (kg/cm*) Clinical Mean Values Pre-Drug Week 12 32 39 38 74 4 52 0.2 I 8 14 36 68 1 33 0.29 No. RA Patients Responding/Treated 7/7 8/8 8/8 7/8 8/8 6/8 7/8 Reflex Neurovascular Dystrophy in Childhood Bram Bernstein. James Kent. Berne Singsen, Karen Koster King, Helen Kornreich, and Virgil Hanson. Los Angeles, California Reflex neurovascular dystrophy ( R N D ) has been reported only in adults, usually as a complication of trauma o r surgery. During the past 7 years we have observed 20 instances of R N D in 19 children who ranged in age from 9 to 15 years (median: 12 years). There were 15 girls and 4 boys. A recognizable traumatic event preceded the onset of symptoms by 2 to 24 months in 9 of 20 cases. The duration of illness before diagnosis ranged from 1 week to 24 months (median: I month), and previous diagnoses included collagen-vascular diseases, gout, tendinitis, spinal cord tumor, and other neurologic diseases. Previous treatments included casting, corticosteroids, other antiinflammatory agents, antibiotics, acupuncture, and narcotic analgesics, but symptoms persisted in all cases. Twelve children had ankle and foot involvement, 4 had knee involvement, and 4 had the hand-shoulder syndrome. All complained of severe pain, and there was exquisite tenderness of the involved area in 18 of 20 cases. Changes at the involved site included swelling in 16 of 20, decreased temperature in 15, skin mottling o r discoloration in 8, decrease in pulses in 5 , and asymmetry of perspiration in 2 cases. The CBC, creatine phosphokinase, antinuclear antibody. and rheumatoid factor tests were negative. In 19 of 20 cases the erythrocyte sedimentation rate was normal. Radiographs showed diffuse demineralization of the involved site in 8 of 19. Seventeen cases were hospitalized (mean duration: 22 days) and 3 were treated as outpatients. All children received physical therapy. Emphasis was placed o n mobilization and weight bearing, in spite of pain. It was strongly suggested that improvement would follow participation in this program. Fifteen children underwent psychologic evaluation and treatment. They were found t o be accepting of responsibility beyond their years, to have difficulty in expressing anger, a n d to show little anxiety about their illness or in its outcome. There was a high frequency of long-standing family conflict; the illness appeared t o allow the child to avoid this. At the time of discharge 5 of 19 were well, 1 I showed marked improvement, and 3 showed moderate improvement. R N D occurs in children; it should be considered in the child with an exquisitely tender and painful extremity. Vigorous physical therapy, in spite of pain, appears effective. The high frequency of family conflict suggests that psychologic intervention is also important. Formation of Single-Strand Regions in Native DNA by Lupus Sera Ronald N . Berzofsky. Carole A . Dorsch, and Mary Betty Stevens. Baltimore, Maryland Although antibodies t o multiple nuclear antigens have been described in several connective tissue diseases, antibodies specific for native DNA have become the hallmark of SLE. In an attempt to determine the nature of the association of native DNA and antinative DNA antibodies, we observed that sera from patients with SLE were capable of generating single-strand regions in highly purified preparations of native T 7 DNA under conditions normally employed for binding assays. That this observation might be due to the perturbation of the double helix by the binding of antibody was further investigated. The formation of single-strand regions was observed in the following manner. Native T7 DNA, after incubation with the appropriate serum or serum factor, was subsequently incubated with the single-strand specific endonuclease from Pseudornonas Bal 31, and the ARA ABSTRACTS 109 reaction products were analyzed by velocity sedimentation through alkaline sucrose gradients. Although native T7 D N A is resistant to the Bal endonuclease, the combined action of SLE serum and the Bal enzyme results in a dramatic decrease in the sedimentation of the DNA. Surprisingly, the incubation of T7 DNA and serum alone also caused a decrease in the sedimentation of the DNA, but never as dramatic an effect as seen with a combined digest. This latter observation suggests the presence of intrinsic serum nucleases capable of degrading native D N A . To determine the role of antibody to DNA in the synergistic effect of lupus serum and Bal enzyme, an IgG fraction of lupus serum still possessing DNA-binding activity was tested in the above assay. N o shift in the sedimentation of DNA was observed with this fraction either alone o r with the Bal enzyme. Furthermore, normal serum possessing no detectable D N A binding activity, but with significant levels o f serum nucleases, was capable of further decreasing the sedimentation of D N A in conjunction with the Bal enzyme. These observations suggest that intrinsic serum nucleases are responsible for most of the single-strand regions produced by serum and not antibody binding. In support is the finding that a lupus serum with high DNA binding activity but with n o detectable serum nuclease, as well as normal sera lacking nuclease, was unable t o shift the sedimentation of DNA. The role and effect of serum nucleases on the measurement of anti-DNA antibodies are under investigation. The mechanism of binding of antibody to D N A remains t o be determined. Antineuronal Activity in Systemic Lupus Erythematosus (SLE) Serum Harry G. Bluestein, San Diego, California SLE patients with central nervous system (CNS) disease have increased titers of lymphocytotoxic antibodies that bind to human brain. This fact suggests that antineuronal antibodies may be responsible for some C N S manifestations in SLE. SLE sera were tested on a neuronal cell line, SK-N-SH, established from a human neuroblastoma, for complement-dependent neurocytotoxicity and for their effect on Veratridine-induced sodium fluxes across the cell membrane. At a 1 . 2 dilution 25/32 S L E sera were cytotoxic (> 10% 5'Cr-release) t o SK-N-SH cells. The mean 6*Cr-releasefor the entire group was 31.3%. Only 5/16 seropositive rheumatoid arthritis (RA) sera were cytotoxic, mean cytotoxicity 8.2% (P < 0.001). Almost all of the cytotoxic activity in SLE sera was recovered in the exclusion peaks from Sephadex G-200. Anti-IgM (p-chain specific) completely inhibited the killing. Absorption of S L E sera with SK-N-SH cells removed all neurocytotoxicity; lymphocytotoxicity was reduced but not eliminated. After absorption with a human lymphoblast cell line (WIL-2). most of the neurocytoxicity remained. When compared in I8 SLE sera. antineuronal and antilymphocyte cytotoxicity titers were weakly correlated ( r = 0.5; P < 0.05). Veratridine stimulates z2 N a incorporation in cells with elec- trically excitable membranes. Cell line SK-N-SH responds to lo-' M Veratridine with 4-fold increased uptake of 22 Na. Addition of 26 SLE sera to the cultures during the period of Veratridine stimulation significantly reduced 22 Na. Addition of 26 SLE sera to the cultures during the period of Veratridine stimulation significantly reduced 22 N a uptake to 56% of the amount of sodium incorporated with a standard normal human serum. In contrast, the Veratridine response with sera from 21 RA patients and 8 healthy volunteers was 92 and 99% of the standard. In 13/26 of the SLE sera, the Veratridine response was less than 50% of the standard. None of the RA or control sera were less than 50%. Veratridine stimulation is not reduced by the addition of a rabbit anti-human brain antiserum to the cultures. The suppressive factor(s) in SLE sera are nondialyzable, precipitate with 50% (NH,)Z SO,, and their activity is not blocked with antiimmunoglobulin serum. SLE sera contain at least 2 factors that can interfere with neuronal function: a ) IgM antibody cytotoxic t o neurons in the presence of complement, and b ) a nonantibody factor that blocks the opening of sodium channels in the neuronal membrane. These observations support the hypothesis that attributes C N S manifestations of S L E to circulating antineuronal factors. Acute Monocytic Arthritis Arthur D . Brawer and Edgar S . Carhcart, Boston, Massachusetts Five patients presented with a triad of fever, skin rash. and acute polyarthritis. The fever was low grade, continuous, and without spikes: the skin rash was papular in 4 and urticaria1 in I ; the arthritis involved small and large joints with knees and ankles affected in all cases. One patient had transient renal involvement, but none received medications prior to the illness. Acute rheumatic fever, viral hepatitis, infectious mononucleosis, and bacteremia were excluded as possible diagnoses. Synovial membrane biopsy was performed in Case No. 5 and skin biopsies in Cases 2, 3, and 5. All showed vasculitis with a predominant mononuclear infiltrate in small blood vessel walls. Multiple joint aspirations were performed in all cases. The initial synovial fluid analyses revealed inflammatory effusions with marked monocytosis and low percentages of polymorphonuclear cells and lymphocytes (Table). Other synovial fluid findings included normal C3 (complement) levels and negative aerobic and anerobic cul- tures. Mononuclear cells from the synovial fluid of Case No. 1 were purified on a Ficoll-Hypaque gradient and demonstrated 20% E (erythrocyte) rosettes, less than 5% E A C (erythrocyte, amboceptor, and complement) rosettes, and 80% EA (erythrocytes coated with 1gG amhoceptor) rosettes. This last finding, plus the observation that most of the mononuclear cells were phagocytic for latex particles, confirmed the monocytic origin. In summary. we have attempted t o describe a new and distinct syndrome characterized hy synovial fluid monocytosis. I t appears t o be caused by more than one etiologic agent and bears striking similarities to certain viral arthritides. Although synovial fluid analyses have not been previously reported in systemic vasculitis. it also deserves consideration as a form of acute hypersensitivity angiitis. Synovial Fluid Analyses in Acute Monocytic Arthritis Vol. Aspirated Case No. (ml) WBC/mms % Poly 1 25 15 15 4,400 12,600 I 13,000 2,300 3 4 18 5 0 2 3 4 5 10 1 ,om % Lymphs % Monocytes Mucin Test 14 4 0 15 35 83 92 82 80 65 Good Fair Fair Fair Fair ARA ABSTRACTS 110 Antibody-Forming Cells in the Peripheral Blood of Patients with Active SLE D. R . Budman, E. B. Merchant, B. Doft, M . E. Gershwin, E. Lizzio, J . P. Reeves, and A . D . Steinberg. Bethesda, Maryland Thirty-seven patients with systemic lupus erythematosus (SLE) and 18 normal controls were studied for spontaneous background IgM plaque forming cell number per 10' cells (PFC) to 5 specific chemical haptens (Dansyl, T N P , DNP, NIP, Sulfonate) in 2 separate series of experiments (Table). Active SLE patients had significantly more PFC in their peripheral blood t o these chemical determinants than did patients with inactive disease or controls (P< 0.005). The greatest increase was in PFC, with specificity for the Dansyl and T N P haptens. The increased PFC number correlated with depressed serum C3, but not with other laboratory measures by Spearman rank order analysis. The finding of elevated numbers of spontaneous PFC to chemical haptens supports the concept that in active SLE there is a widespread increase in B-cell activity toward a variety of antigens consistent with a generalized loss of immune regulation. Group 2 Group 1 Patients Active SLE Inactive SLE Controls No. PFC (log,, f SE) No. PFC (log,, f SE) 9 13 10 838 (2.92 f 0.11) 174 (2.24 f 0.23) 146 (2.16 f 0.13) 8 7 8 6140 (3.79 f 0.28) 216 (2.33 f 0.38) 184 (2.26 f 0.32) Connective Tissue Activation: Stimulationof DN A and Glycosaminoglycan Synthesis by a Platelet Factor C . W . Castor, M . E. Scott, J . C . Ritchie, a n d S . L. Whitney, Ann Arbor, Michigan Earlier studies identified a platelet factor or factors that stimulated glycosaminoglycan (GAG) synthesis and glycolysis in human synovial cell cultures. The connective tissue activating peptide (CTAP111) clearly differed from CTAP-I (of lymphocyte origin) in its lability to thiols and in the added capacity t o stimulate G A G synthesis in human dermal fibroblast cultures. Current data indicate that CTAP-I11 is an acid-ethanol extractable basic protein with a molecular weight of 9,300 daltons containing one or more disulfide groups essential for its biologic activity. CTAP-111 is mitogenic in that it stimulates incorporation of ['HI methyl thymidine into DNA in cell cultures. CTAP-111 stimulated DNA synthesis in human synovial cells, cartilage cells, skin and thyroid fibroblasts, and N R K (rat) cells, but not in continuous cultures of human epithelial or lymphoid cells. Mitogenic activity was retarded by concurrent addition of M dithiothreitol, cysteine, and glutathione. but not by cystine, ascorbic acid, or cortisol. CTAP-I11 stimulated increased hyaluronate synthesis and glycolysis not only in human synovial cells and dermal fibroblasts, but also in thyroid fibroblasts and cartilage cells. G A G stimulating capacity of CTAP-Ill was not blocked by cytosine arabinoside, but it was inhibited by cycloheximide and actinomycin D. Both PGE, and dibutyryl cyclic A M P potentiated the G A G stimulating action of C T A P- I I I. Platelets aggregated by bovine thrombin extruded CTAP-111 into the aqueous suspending medium. Much of the fibroblast mitogenic activity of serum appeared t o be derived from the clotting process, because plasma supported less [9H]methyl thymidine incorporation into synovial cell DNA than did serum. The diverse stimulatory properties of CTAP-III are appropriate for a mediator designed to initiate the connective tissue component of the inflammatory response. Systemic Lupus Erythematosus: Failure to Detect Endogenous Baboon Leukemia Virus or Mammalian Type C Virus Interspecies Antigens in Tissue Extracts Howard P. Charman and Paul E. Phillips, Frederick, Maryland and New York. New York Recent studies suggest enhanced type C virus expression in systemic lupus erythematosus (SLE). We have examined human SLE tissues for evidence of mammalian type C virus interspecies antigens using sensitive radioimmunoassays (RIAs). Eight patients with definite, and 1 each with probable or possible SLE (ages 25-58, duration 0.5-15 years, 3 active, 5 on prednisolone and/or lmmuran) provided 14 tissues including 5 placentas, 5 spleens, and 4 kidneys. Thirty percent (w/v) extracts were homogenized, solubilized with Triton X100, clarified, and examined for mammalian type C virus p30 and an antiserum prepared by sequentially immunizing with several purified p30s. This RIA detects currently known isolated type C viruses with equivalent sensitivity. These tissues were also tested for BaLV p30 in a heterologous interspecies RIA employing 12s1-labeled BaLV p30 and an antiserum to the highly related endogenous cat virus, RD-114. All extracts but one were unreactive in both assays at levels of negativity of less than 0.1 ng RD-I 14 p30 per mg of soluble protein. Despite the incorporation of phenylemethanesulfonylfluoride in all buffers and assay conditions restricting incubation at 37°C to 1-1.5 hours, one kidney extract degraded labeled BaLV p30 to produce false positive results in both assays. This is a well-known complication of RIAs. Interestingly, although 3 of the tissues contained structures resembling type C viruses, type C virus isolation studies on 6 tissues were negative as previously reported. These results strongly suggest that the structures observed in SLE are unrelated either to the endogenous BaLV or to other known type C viruses. Thus we are unable to confirm previous reports of type C virus p30 expression in human SLE. ARA ABSTRACTS 111 Serum Complement Values During Pregnancy in Patients with Systemic Lupus Erythematosus Sherwood M . Chetlin, Thomas A . Medsger. Jr., and Steleanos N . Caritas. Pittsburgh. Pennsylvania, and Anthony G. DiBartolomeo. Morgantown, West Virginia It is well recognized that clinical manifestations of S L E may exacerbate o r first appear during pregnancy or the early postpartum period. Because immunologic alterations frequently antedate the expression of SLE, serum complement values in pregnant patients with lupus may be an important guide to disease activity. Serum complement determinations (C, and C, by radial immunodiffusion) were performed o n over 400 women in various stages of uncomplicated pregnancies and during the postpartum period. The mean C, level rose progressively from a baseline of 145 mg% in nonpregnant women t o 194 mg% a t 39-40 weeks gestation (Table). At 6-8 weeks postpartum, the mean level had dropped to 172 mg%. Eight patients with SLE were followed serologically and clinically through ten pregnancies. The mean pre-pregnancy serum c, level was 121 mg%, which is significantly lower than for normals. C, increased during pregnancy but, in contrast to normals, peaked a t 32-36 weeks and declined thereafter. Four patients developed exacerbation of SLE, and in all cases the symptoms were preceded by a fall in serum complement. In 2 instances these changes occurred in the third trimester and in the remaining 2 during the postpartum period. Three of these 4 patients had serum C, values less than 100 mg%. N o individuals developed renal involvement associated with pregnancy. Increased doses of corticosteroid therapy were employed only as clinically indicated and not as therapy for hypocomplementemia alone. c, levels generally paralleled the C, levels. It is concluded that mean serum complement levels are significantly reduced during the entire course of pregnancy in SLE patients compared with normal women. A declining serum complement level is often the harbinger of increased disease activity. As in nonpregnant SLE patients, values of less than 100 mg% most commonly antedated new symptoms. Weeks of Gestation Normals C, (mg%) SLE C, (mg%) Nonpregnant 8-12 16-20 24-28 32-36 39-40 6-8 Weeks Postpartum 145 121 162 108 I62 I24 I68 135 I82 I58 194 I39 I72 I37 Plasma Renin Activity (PRA) in Progressive Systemic Sclerosis (Scleroderma) with Malignant Hypertension Sherwood M . Chetlin, Alvin P. Shapiro, Gerald P. Rodnan. Thomas A . Medsger, Jr.. Sanford F. Tolchin. Daniel E. Leb, and Peter Cohen, Pittsburgh, Pennsylvania The onset of malignant hypertension in progressive systemic sclerosis is most often associated with rapidly advancing renal failure. In 8 recently observed patients with renal involvement by scleroderma, the peak levels of PRA by immunoassay ranged from 81.4 to 1000 ng/ml (angiotensin released in 3 hours incubation). These values are 16-200 times the upper limit of normal for this laboratory (5.0 ng/ml in supine subjects). In 2 of these patients, PRA had been measured as recently as 6 months before the onset of this complication and was found to be normal. In 32 normotensive scleroderma patients, the mean was 5.6 ng/ml (range: 0.8-16); in this group patients with the CREST variant had significantly lower values. Six of the 8 patients died despite hemodialysis in all and bilateral nephrectomy in 3. The remaining 2 have survived 6-8 months on dialysis after nephrectomy. T w o additional scleroderma patients with hypertension and azotemia had P R A values elevated to only 31.7 and 54.1 ng/ml. Both have survived with intensive antihypertensive therapy, and P R A has subsequently fallen t o normal levels. All 8 patients were admitted with the onset of renal failure between the months of October and March. It is possible that this vascular complication of scleroderma may begin with a Raynaud’slike phenomenon in the kidney vessels followed by marked renin release and resulting in accelerated arteriolar necrosis. Musculoskeletal Manifestations of Bacterial Endocarditis (BE) Melvin A . Churchill, Jr., Joseph E. Geraci, and Gene G. Hunder, Rochester, Minnesota Although musculoskeletal (MS) symptoms are recognized in BE, they have been poorly studied. To determine the frequency, character, and relationship of these manifestations t o the onset of disease, we reviewed the histories of 192 patients with BE seen between 1965 and 1975. Eighty-four of the 192 patients (44%) exhibited MS findings. Arthralgias were most common (34 cases) and tended t o involve larger joints asymmetrically, but were occasionally monoarticular or migratory. Symmetric arthralgias were less common (8 cases), but in 2 instances the combination of shoulder and hip pain and stiffness, anemia, and elevated ESR led to a diagnosis of polymyalgia rheumatica until blood cultures were obtained. Synovitis was present in 26 patients and was monoarticular in 16 and polyarticular in 10 (usually 2 or 3 asymmetrically involved joints). The ankle and knee joints were affected most often, but others-including the sternoclavicular and acromioclavicular joints-were sometimes involved. One patient had migratory polyarthritis for 3 months before the onset of fever. The two synovial fluids aspirated were sterile. Low buck pain occurred in 28 cases and was the presenting complaint in 15. The pain was frequently unilateral, mimicking sacroiliitis or ruptured disk. A disk space infection was found in 5. Diffuse myalgias (16 cases) were usually related to the onset of high fever. In 10 other cases myalgias were limited to the thigh o r calf and at times were severe and incapacitating. In 1 right thigh myalgia antedated fever and the diagnosis by 3 months. Less common findings included nail clubbing, Achilles tendinitis, avascular necrosis (hip), and hypertrophic osteoarthropathy. In 52 of the 84 patients MS complaints were the first o r among the first manifestations of BE. In 20, M S complaints were the only presenting symptom and preceded fever in 16 of these 20. Fortyeight of the 84 were tested for rheumatoid factor and 16 (33%) were ARA ABSTRACTS 112 positive. Antinuclear antibodies were found in the serum of 5 of 25 patients tested. Only 1 of 35 patients tested had low serum complement (migratory polyarthritis and focal glomerulonephritis). The relatively high incidence, frequent distinctive character, and tendency to occur early in the course of the disease emphasize the importance of MS manifestations in BE. An awareness of these various symptoms may aid in early diagnosis. Sjogren's Syndrome Associated with the Lymphocyte-Defined Antigen HLA-DW3 Thomas M . Chused, Stuart S . Kassan, Haralampos M . Moutsopoulos, Gerhard Opelz, and Paul Terasaki, Bethesda, Maryland and Los Angeles, California An increased frequency of the HLA-BB antigen has been observed in Sjogren's syndrome (SS) as well as in other organ-specific autoimmune diseases. Regulation of the ability of the immune system to respond t o antigens in the salivary and lacrimal glands might play a role in triggering the immunologically mediated destruction of these secretory tissues in SS. In mice it has been shown that the Ia antigens are probably the product of immune response genes and that they determine the intensity of the mixed lymphocyte reaction (MLR), i.e., they are lymphocyte-defined histocompatibility antigens. Although immune response genes have not yet been described in man, by analogy with the mouse they might be associated with the human lymphocyte-defined antigens of the HLA-D locus. For this reason we asked whether SS is more closely associated with HLA-B8 or with HLA-DW3. the lymphocyte-defined antigen known to be in strong linkage disequilibrium with HLA-B8. Twenty-five unrelated patients with SS were typed for HLAA and HLA-B antigens by the microcytotoxicity method. HLA-DW3 typing was done by determining the M L R responsiveness of the subject's peripheral lymphocytes t o homozygous HLA-AI, HLA-B8, and HLA-DW3 stimulator cells. Lymphocytes bearing the HLA-DW3 antigen gave a low response. The Haldane method was used for statistical analysis. Of 25 SS patients, 14 were B8+ (corrected P = (0.003) and 17 were DW3+ (P = 0.00009). Of 19 patients with SS in the absence of another connective tissue disease, 14 were B8+ (corrected P = 0.00009) and 16 were DW3+ (P = 0.00001). Of 6 patients with SS and rheumatoid arthritis, none was B8+ (P = 0.25) and 1 was DW3+ (P = 0.84). By dividing the patients with SS alone into subgroups positive and negative for the HLA-B8 o r HLA-D3 antigens and then testing for association of SS with the other antigen, the interference from the linkage disequilibrium can be removed. This technique showed that the primary association of SS is with DW3 (P = 0.002) rather than with B8 (P = 0.17) These results suggest a) that there are two subpopulations of SS patients, those with SS alone, 84% DW3+, and those with SS and RA, 17% D W 3 f (compared to 24% in normals); and b ) that in the first subpopulation the primary association of SS with DW3 is consistent with the involvement of an immune response gene in the pathogenesis of their disease. A Prospective Clinical Analysis Comparing Polycentric and Geometric Knee Replacement with 2 to 4 Years Follow-Up Andrea Cracchiolo. 111, Harlan C . Amstutz, and Gerald A . M . Finerman, Los Angeles, California A prospective comprehensive analysis of 100 polycentric and 75 geometric replacements with a follow-up of 2 to 4 years has demonstrated the effectiveness and pitfalls of surface replacement arthroplasty in rheumatoid and osteoarthritic knees. Standard treatment regimens and operative techniques were used in both groups. Both the polycentric and geometric groups were similar, except that the polycentric patients were an average of 10 years younger and 58% had rheumatoid arthritis while 59% of the geometrics had osteoarthritis. Pain was the prime indication for surgery. Data were collected by using six standardized proformas with 480 entries, which were transferred to a specially designed computer program for in-depth analysis. A successful result was maintained in 187 knees, and pain relief showed the most improvement-being comparable in both groups-as was the improvement in stability, alignment, and motion. Functional motion increased because the preoperative flexion contracture was significantly reduced in most knees. The improvement in walking and overall function was related to the specific disease and involvement of other joints. Results appear to correlate best with the type of disease, the degree of preoperative knee pathology, and the presence of complications. Twenty-one knees (20 patients) failed. Of this group 80% were osteoarthritics, more were men, and knees previously operated upon failed more frequently. The most common causes of failure were component loosening, ligamentous instability, and postoperative varus alignment; sepsis occurred in only four knees. Thus, following this intermediate evaluation period, we can determine the results of treating various types of osteoarthritic and rheumatoid knee deformities and establish more specific indications for knee arthroplasty. Specific patient management procedures that have improved our results are the use of antibiotics, anticoagulation, proper wound drainage, and early physical therapy. Prostaglandin Production by Synovial Cells: Stimulation by a Human Lymphocyte Factor Jean-Michel Dayer, Dwight R. Robinson, and Stephen M . Krane, Boston, Massachusetts Prostaglandins (PG ) have been implicated in the bone resorption of rheumatoid arthritis (RA) and some neoplasms. Culture media from R A synovial explants as well as isolated adherent synovial cells (ASC) produce large quantities of PGE, and collagenase. Dexamethasone (< IOnM) inhibits production of PGE, and collagenase >90%, whereas indomethacin (IOpM), while inhibiting PGE,, stimulates collagenase production (PNAS 73:945, 1976). We reasoned that products of lymphocytes, which we have previously shown to increase produc- tion of collagenase, would also influence P G production. Normal and R A peripheral blood lymphocytes separated by Ficoll-Hypaque gradients were further cultured (after removing adherent monocytes) at 37°C in Dulbecco's medium with 10% fetal calf serum at 2X l(P cells/ml for 3 or 4 days. The supernatants at dilutions 1.5 to 1:60 were incubated with ASC and PGE, and assayed by radioimmunoassay (using specific antisera); the identity of PGE, was confirmed by thin-layer chromatography. After 3-7 passages of ASC, ARA ABSTRACTS when levels of PGE, and collagenase in culture media had decreased markedly (normalized to cell number), PGE, release was increased 5-10-fold by stimulating factor (SF) from lymphocytes (e.g.. from (-)SF, -50 ng to (+)SF -500 ng/lCP cells/d). In the same cultures collagenase (assayed by using labeled reconstituted collagen fibrils) was increased 10-100-fold. In some cultures the levels of PGE, and collagenase produced by ASC in the presence of SF approached those found in early primary cultures of ASC. Indomethacin blocked the effect of SF and PGE, production by ASC but potentiated the effect of 113 SF on collagenase production. A similar although not identical dose response for both PGE, and collagenase was observed; enhancement of production of SF by phytohemagglutinin was found and was evident at all dilutions tested. Similar apparent MW by gel filtration (-8000-12,000) was obtained for the factor that stimulated both PGE, and collagenase. PG and collagenase production by connective tissue cells stimulated in vivo by lymphocytes could play a role in connective tissue resorption of RA, as well as in other pathologic processes such as neoplastic invasion. Sympathetic or Reflex Footpad Swelling Due to Crystal-Induced Inflammation in the Opposite Foot Charles W . Denko. Cleveland, Ohio Although patterns of inflammatory joint swelling in rheumatic disorders are well documented, their immediate causes remain obscure. Symmetric joint swelling is characteristic in patients with rheumatoid arthritis, whereas asymmetric joint swelling is characteristic in patients with gout. Joint swelling due to deposition of various calcium salts can mimic that of rheumatoid arthritis or that of gout. Joints previously involved are sites of subsequent attacks. Techniques were devised to investigate patterns of footpad swelling in rats due to inflammation induced by several crystals known to be human pathogens. Swelling (inflammation) was quantified by measuring the foot diameter by a mechanical spring dial. Initial or primary inflammation was induced in adult rats by injecting the footpad of a rear foot with fixed amounts of crystals in suspension. Monosodium urate (MSU), xanthine (X).calcium oxalate, and hydroxyapatite were used to induce primary or initial bouts of inflammation as well as subsequent episodes. MSU and X induced swelling in the opposite uninjected footpad as well as in the footpad for which they were primary irritants. MSU injected initially into one foot induced marked swelling in the opposite foot that had previously been inflamed by MSU, calcium oxalate, or hydroxyapatite as the primary inflammogen. These effects appear dose-related. Larger inocula produce larger swelling. When the initial inflammation had not completely subsided, the injection of a second inflammogen in the opposite foot was accompanied by a pronounced swelling of the initially inflammed foot. The foot receiving the injection always had more swelling than the opposite uninjected foot. The sympathetic swelling is thought to be due to elaboration of a humoral factor locally following reflex or overlapping nervous stimuli initiated by the crystal injection to the opposite foot. This work is an experimental model for reflex dystrophy. Role of Calcium in the Phenotypic Expression of Rabbit Articular Chondrocytes in Culture Kalindi Deshmukh, Indianapolis, Indiana Rabbit articular cartilage synthesizes Type I I collagen, comprised ofa,(II) chains, in vivo or in vitro. Chondrocytes from thesame tissue have the ability to produce Type I collagen [Za,(I).a,],which is normally present in skin, bone, tendon, etc., or Type II collagen, depending upon the culture system. The cells synthesize Type I collagen in monolayer cultures. Upon transfer from monolayer to suspension cultures, they synthesize Type I1 collagen in the medium with no CaCI, and Type I collagen in the complete medium. Incubation of the cells with ionophore A23 I87 increases the transmembranous flux of Ca+, into the cells, intracellular levels of c-AMP, and causes the cells to synthesize Type I collagen in the medium containing no CaCI,. Similar results were obtained by addition of dibutyryl c-AMP to the medium. Each type ofcollagen was identified by incubation of the cells with the medium containing aH-proline, ascorbic acid and @-aminopropionitrile; by pepsinization and salt-precipitation of labeled collagen; and by study of the composition of the chains and their CNBr peptides with CM-cellulose chromatography. The change in the synthesis of collagen by normal chondrocytes from Type I1 to Type I collagen in the presence of extracellular calcium is of great significance in light of the fact that osteoarthritic joints show signs of remodeling of subchondral bone and ectopic calcification. Increase in the pyrophosphate concentration and deposition of calcium phosphate in the synovial fluid and the extracellular matrix of the cartilage of osteoarthritic joints have been recently reported in the literature. Crystal-Enhanced Membrane Permeability in Liposomes: Dissolved Urate Reverses the Protective Effect of Albumin Charles A . DiSabatino. Mary G. Breitenstein, and Stephen E. Malawista. New Haven. Connecticut Anionic liposomes have been used successfully to show the disruptive effects of silica and of monosodium urate crystals on membranes that are not protected by protein in the ambient media. Thus urate crystals within digestive vacuoles, when plasma-derived proteinaceous material has been enzymatically degraded, are thought to cause perforation of the vacuolar membrane and rapid cell death, which may in turn contribute to the violence of gouty inflammation. We have now found that urate in solution can reverse the protective effect of protein on liposomes challenged by crystals. We used anionic liposomes composed of lecithin, cholesterol, and dicetyl phosphate (7:1:2), with I T glucose trapped in their aqueous phase. In the representative experiment shown, quadruplicate aliquots of liposomes were incubated in a standard salt solution at 37°C for 30, 60. and 90 minutes, with or without additives. The mean percent release of their total radioactivity, f 1 SEM. is recorded in the table on the next page. Silica crystals, 15 mg/ml (used rather than urate crystals because the former do not dissolve) enhanced membrane permeability. Human serum albumin, 0.5 mg/ml, significantly protected the liposomes from silica-induced leakage. Urate in solution, 8.4 mg%, reversed the protective effect of albumin. Soluble urate alone had no effect on the permeability of liposomes (not shown in the table). We conclude that urate in solution enhances membrane permeability by reversing the protective effect of albumin. Thus the increased concentrations of dissolved urate characteristic of gout may hasten crystal-associated disruption of the phagolysosomal membrane, and thereby contribute to gouty inflammation. A R A ABSTRACTS 114 Mean Percent Release of Total Liposome Radioactivity * 1 SEM Time of Incubation (minutes) No silica Silica Silica + albumin Silica + albumin + urate 30 P 60 P 90 6.5% f 0.9 38.8% f 1.5 20.3% f 0.4 34.0% f I . I <.001 <.01 <.001 9.4% f 0.6 41.2% f 1.3 22.0% f 0.6 36.1% f 1.2 <.001 <.Ol < .01 43.4% f 1.4 P 12.2% f 0.8 24.6% f 0.4 38.4% f 1.0 <.001 <.01 < .oo I Clinical Significance of Antibodies to Extractable Nuclear Antigen (ENA) in Systemic Lupus Erythematosus (SLE) Carole A . Dorsch, Edward J . Feinglass, and Mary Betty Stevens, Baltimore, Maryland Although antibodies to ENA, especially antiribonucleoprotein (RNP), have been described in other connective tissue disorders, they have been associated primarily with the mixed connective tissue disease (MCTD) syndrome. The significance of these antibodies in patients with SLE, selected only on the basis of clinical diagnosis, has not been fully established. Antibodies to ENA were determined in 81 patients with SLE by means of a combination of Ouchterlony immunodiffusion and hemagglutination of tanned, ENA-sensitized, sheep red blood cells before and after ribonuclease digestion. Both techniques were required to detect optimally the incidence and specificity of antibodies present. Patients' histories were independently reviewed for clinical and laboratory features of SLE. Forty-three of 81 sera (53%) were positive for anti-ENA: 6 had anti-RNP alone, 13 anti-Sm alone, and 4 both anti-RNP and anti- Sm. Raynaud's phenomenon, myositis, and sicca symptoms were significantly more frequent in the 30 patients having anti-RNP antibodies. These features were absent in the 13 patients having anti-Sin alone, with the exception of 1 patient who had Raynaud's phenomenon. Vasculitis and leukopenia occurred more often in anti-ENA positive patients and were significantly associated with the presence of anti-Sm. There was no difference between anti-ENA positive or negative groups with respect to renal disease, hypocomplementemia, and the presence of antibody to denatured DNA. It thus appears that antibodies to components of ENA are associated with specific disease features in SLE. For anti-RNP these represent features common to MCTD. However, unlike MCTD, renal involvement remains a prominent feature in anti-RNP positive SLE. Periosteocytic Demineralization : A Phase of Bone Erosion in Rheumatoid Arthritis Howard Duncan, C . H . E. Mathews, Donna Witzgall. Detroit, Michigan At the level of cellular activity, bone erosion, juxtaarticular demineralization, and joint destruction in rheumatoid arthritis have not been wholly understood. In many rheumatoid patients, a paucity of multinucleated osteoclasts suggests that these bone-resorbing cells are not exclusively the cause of bone erosion. Most rheumatoid inflammatory cells lining bone surfaces are mononuclear and do not share the staining features of osteoclasts. Some bone damage is thought to depend upon an initial demineralization that exposes the collagen matrix to the numerous enzymes synthesized by rheumatoid synovitis. The means by which demineralization could occur have not been clear. In bone removed at surgery, we observed a periosteocytic demineralization that exposes the underlying matrix. Fully mineralized serial sections, 5 p thick and appropriately stained, were used. We observed areas close to the invading rheumatoid inflammation where individual osteocyte domains demineralize beyond the matrix of their osteocytic lacuna to a distance of 3 to 4 p . Coalescence with adjacent demineralizing osteocytes occurs, and the closer to the inflammatory tissue, the more frequent is the confluence of adjacent cell domains. Occasionally the osteocyte lacuna itself is enlarged, but the initial process appears to be a demineralization rather than a simultaneous collagen disruption. There is subsequent invasion by inflammatory tissue, which replaces the collagen with a typical rheumatoid vascular inflammatory reaction. A mechanism for bone erosion independent of osteoclasts is identified in some areas of active rheumatoid joints removed at surgery. This occurrence is due to the amalgamation of the rheumatoid inflammatory tissue. (Supported by a grant from the Michigan Chapter of The Arthritis Foundation) Nonconstrained Metal to Plastic Total Elbow Arthroplasty in Rheumatoid Arthritis Frederick C. Ewald. William H . Thomas, Richard D. Scott, Clement B. Sledge, and Robert Poss. Boston, Massachusetts A nonconstrained metal to plastic total elbow replacement fixed with methyl methacrylate has been developed, and clinical trials are in progress. This paper is a report of the design concept and a review of the first 50 replacements in patients with rheumatoid arthritis. The maximum follow-up is 2 years 6 months, and minimum follow-up 6 months. Results have been encouraging, with a significant increase in postoperative flexion (X = 135" P <0.005) and pronation (X = 71" P<0.0025) but no significant increase in postoperative extension or supination. Pain-relief and functional improvement have been uniformaly excellent. Preoperative evaluation based on a predetermined rating system was 37% of normal compared to 97% of normal postoperatively. There has been no loosening of components or avascular necrosis of the olecranon process, but two procedures have failed. The first failure was for peptococcus sepsis and the prosthesis was removed. The second failure was in a patient with a previous fascia1 arthroplasty with no ligaments or capsule, and this elbow had to be converted to a hinge type of replacement. The two other serious complications were caused by previously unrecognized severe cubitus valgus in the rheumatoid elbow. Two elbows with cubitus valgus of 32" and 27' dislocated at time of surgery and stable reduction could not be achieved. One elbow was salvaged by revision with a 15" ARA ABSTRACTS 115 cubitus valgus fixation stem in the humeral component instead of the standard 5" stem. Other complications have been two ulnar nerve palsies (one transient, one permanent), two single dislocations with stable reductions and no further problem, two small posterior skin flap losses secondary to splint pressure, and one new bone formation. Contraindications to the procedure include previous fascia1 arthroplasty or loss of collateral ligaments or capsule, previous sepsis, or excessive loss of bony stock as in giant rheumatoid bone cysts. There has been no experience with post-traumatic arthritis, and at this writing the prosthesis is not recommended as salvage for these problems. Early Clinical Features That Predict Outcome in Rheumatoid Arthritis (RA) :Results of a Prospective Study Using Multivariate Analytic Techniques Seth L. Feigenbaum, Stanley B. Kaplan, Alfonse T. Masi and Robert W. Chandler, Memphis, Tennessee In an ongoing follow-up study of newly diagnosed RA in younger adults, entry variables were analyzed to identify predictors of the subsequent course in 52 patients closely observed for a mean interval of 5 years. All satisfied ARA criteria for at least probable RA. These cases included 42 females and 10 males, with 28 blacks and 24 whites. Outcome was classified initially by the presence (N=26) or absence (N=26) of joint swelling during the last 2 years of follow-up. I n addition, patients without swelling were subdivided into outcome groups with (N=19) or without (N=7) pain or tenderness (P/T). Similarly, patients with joint swelling were subdivided into those who did (N=9) or did not (N= 17) develop bone erosions on hand films during the course of follow-up. Stepwise discriminant function analysis identified entry varia- Entry Variables Predicting Outcome Total number ofjoints involved Weight loss around onset (&3 mos) Serum complement (C3, mg/dl) Sex (male) Rheumatoid factor positive Lymphadenopathy on examination Raynaud's phenomenon bles which correlated significantly with outcome. Seven entry variables correctly predicted the outcome into swelling and no swelling groups in 48 (92%) of the 52 cases. These variables are ranked in the table according to their predictive value, with mean results of percentage of frequency at entry. With few substitutions these variables correctly predicted classification into the four subgroups with 80% accuracy. R F was raised to second rank in that analysis because 8 of the 9 patients who developed erosions had R F at entry (P<O.OOl). Thus the number of initially involved joints is the best predictor of continued joint swelling in RA, with R F predicting erosive disease. Multivariate analytic techniques are valuable in sorting out complex interactions and can offer new insights into disease relationships. Entry Results in Outcome Groups Rank Order N o Swelling Swelling I 2 3 4 5 6 7 7.7 19% 183.1 30% 15% 4% 0% 18.1 58% 166.2 8% 50% 27% 15% Univariate Significance P P P P P P < < 0.001 0.05 NS < 0.05 < 0.01 < 0.05 < 0.05 Cellular Infiltrates in Scleroderma Skin Raul Neischmajer and Jerome S . Perlish, Philadelphia, Pennsylvania . The purpose of this study was to estimate the incidence and nature of cellular infiltrates i n scleroderma skin. Skin biopsies, including the subcutaneous tissue, were taken from 65 patients with systemic scleroderma (SS) and 43 with localized scleroderma (LS). Ten SS and 4 LS skin specimens were processed for electron microscopy (EM). 'H-thymidine labeling of vascular and extravascular cells was estimated by in vitro autoradiography in 14 SS and 5 LS patients. Most cell infiltrates were perivascular, although they were also noted around nerves and skin appendages. By EM we identified blast cells (T-blasts and plasmablasts), plasma cells, mature lymphocytes, macrophages, and fibroblasts with marked RER. T-blast cells were noted within the wall of some capillaries. Cell contact between macrophages and lymphocytes or plasma cells was frequently noted. Capillaries showed marked alterations of endothelial cells (vacuolization. nuclear degeneration, release of organelles to the lumen), whereas other endothelial cells showed a well-developed RER and numerous microfilaments. There was a marked increase in the 'H-thymidine labeling index of endothelial cells i n SS (5.50+0.92; normal: 0. I6-+0.12; P<0.0005), whereas increased labeling of extravascular cells was noted in both SS and LS. This study suggests that B and T cells participate in the tissue immune response in scleroderma skin and probably play a role in the mechanism of the fibrovascular alterations. The Actions of IgM Rheumatoid Factor (RF) on an IgG-Mediated Reverse Passive Cutaneous Arthus Reaction in the Rat Michael Floyd and Joseph T. Tesar, Chicago, Illinois Vasculitis and extraarticular complications of rheumatoid arthritis appear more frequently in patients with high titers of RF. We have directly investigated the actions of an RF on an experimental immune vasculitis in rats. This rheumatoid factor was obtained from the serum cryoglobulin of a patient with symptoms of purpura, arthralgia, and hepatospenomegaly. The cryoglobulin was IgM-IgG, and after purification by multiple cold precipitations and washing at 4°C. the IgM antiglobulin component was obtained by acid dissociation and preparative ultracentrifugation in a 10-25% sucrose dentity gradient in pH 2.8, 0.2 M tris HCL buffer, using an IEC ultracentrifuge with an SBI 10 rotor. The pure 19s fraction was obtained after extensive dialysis with sterile phosphate-buffered saline, pH 7.4. I t was concentrated to 4 mg/ml for use. Intravenous (IV) injection of the antiglobulin induced a prompt hypocomplementemia in the rat only ARA ABSTRACTS 116 after the administration of noncomplement fixing reduced and alkylated heat-aggregated human IgG. A complement-dependent vasculitis was induced by the intradermal injection of 20 pg of rabbit IgG antibody to BSA (AB-BSA) followed 40 minutes later by IV BSA 10 mg/100 g. This lesion was characterized by a striking local dermal leakage of simultaneously IV administered '*'Iodinated rat serum albumin marker, and histologically by infiltration of dermal vessels with neutrophil granulocytes. Decomplementation with cobra venom factor totally inhibited the appearance of this vascular injury. After incubation with R F the injected AB-BSA induced a significantly greater leakage of isotope marker than that generated by AB-BSA preincubated with normal human IgM (P<O.001). Intradermal antibody alone or after incubation with R F and normal human IgM did not induce a measurable vasculitis in the absence of parenteral BSA. Further decisive evidence of a role for R F in this experimental inflammation was obtained with IgG antibody rendered noncomplement fixing by carbamylation. Thus treated, the antibody alone or with normal human IgM failed to induce vasculitis following intravenous BSA. In contrast, carbamylated antibody preincubated with R F consistently gave a marked lesion characterized by extensive leakage of isotope marker and perivascular infiltration with neutrophil granulocytes. Automated Quantitative Particle Aggregate Assay (QPAA) for Rheumatoid Factor ( R F ) with the Coulter Counter Eric Gall, David Templin. and William Dito. Tucson, Arizona In an attempt t o automate the R F assay, a method utilizing the Coulter Channelyzer (H-4) with attached Coulter CountercR'has been devised. These instruments electronically determine particle size. R F causes agglutination of IgG-coated latex beads. As the concentration of R F rises, aggregate particle volume increases. Histograms of populations of particle size are displayed, and an arbitrary unit-the MPRF-is derived from the median size of a dilution that produces a histogram meeting predetermined criteria. The M P R F is curvilinearly related to the concentration of the RF. Previous work in this laboratory has confirmed the precision of this method. One hundred and twenty-eight tests were run on patients with a variety of rheumatic DiseaseGroup RA-poss, prob RA-Def RA-class JRA Vasculitis Gout-chondrocal DJD Nonartic rheum Lung fibrosis Rheum variants diseases. IgG-coated latex particles obtained from Behring (Beh) and Hyland (Hyl) laboratories were utilized to determine R F by using standard latex tube dilutions and the QPAA. Correlation coefficients comparing the standard tube assay to the QPAA were 0.89 with both Hyl and Beh particles, P < 0.00001. Mean f SE results of the R F assay for each disease group tested are disulayed in the table. classic RA chronic fibrotic lung Patients with definite disease are statistically distinct from the other disease groups by the QPAA (Duncan's multiple range test). The QPAA provides a rapid, reliable R F assay, comparing favorably with techniques in use at this time but with the added advantage of automation. + + No. of Cases Beh Tube Titer Beh QPAA M P R F Hyl Tube Titer HylQPAA M P R F 15 0 f.0 795 f 356 I254 f 701 20 f 20 10f 10 23 f 23 OkO Of0 1892 f 780 1214 282 20 f 20 80 f 80 II fII Of0 Of0 448 f 234 Of0 3fl 676 f 188 703 f 190 89 f 89 33 f 32 13 f 12 1 f .5 Of0 193 f 62 5 216 f 120 2 f 1 663 f 157 646 f 161 4 f 3 55 f 54 I f 1 I f .6 6 f 4 766 f 693 13 Of0 Of0 29 29 4 8 7 13 9 Of0 * Of0 Histidine Inhibition of the Oxidation of Serum Protein Sulfhydryl Groups: A Possible Explanation for the Low Serum Sulfhydryl Concentration in Rheumatoid Arthritis Donald A . Gerber and Kee-Yee Shum, Brooklyn, New York The concentration of serum protein sulfhydryl groups is low in patients with rheumatoid arthritis and this abnormality, like the low free serum histidine concentration in patients with rheumatoid arthritis, is proportional to the activity of the arthritis. Cystine reacts with serum protein sulfhydryl groups by means of a copper-catalyzed sulfhydryl-disulfide interchange reaction. Because histidine is an excellent copper ligand, and because patients with rheumatoid arthritis have a low serum histidine concentration, we have studied the effect of Lhistidine on the interaction between L-cystine, copper, and serum protein sulfhydryl groups. Serum sulfhydryl groups were measured by using 5,5'-dithiobis (Znitrobenzoic acid) in the presence of EDTA. Reactions were carried out in 0.1 M phosphate buffer, p H 7.4, NaCl = 1 M. Serum was diluted 1:37. In the presence of 2 pM cystine and 25 pM cupric ion, 16.2% of the serum protein sulfhydryl groups disappeared after incubation at 37°C for 1 hour. When, in addition to these reagents, 2.8 mM L-histidine was also present, only 3.4% of the protein sulfhydryl groups disappeared after incubation for 1 hour. Other amino acids did not have this effect. In 85 sera from patients with rheumatoid arthritis, there was a correlation ( r = 0.4, P = 0.0005) between the free serum histidine concentration and the free sulfhydryl group concentration. These results suggest that the low serum histidine concentration of rheumatoid arthritis may contribute t o the low serum protein sulfhydryl concentration found in this disease by allowing a coppercatalyzed interaction between serum sulfhydryl groups and some disulfide, perhaps cystine. ARA ABSTRACTS I17 HLA-D and Ia-Type Alloantigens in Juvenile Rheumatoid Arthritis (JRA) M.Eric Gershwin. Gerhard Opelz, Paul I. Terasaki, James J . Castles, and Theresa Gorman, Davis and Los Angeles. California Study of the murine histocompatibility system (H-2) led to the realization that the H-2 complex has broad implications for the genetic control of immune responsiveness and disease susceptibility. Analogous human studies identified a comparable complex, the HLA system and its four major series: HLA-A, B, C, and D. Subsequently numerous studies of the HLA supergene identified several significant associations between HLA-B alleles and seronegative arthropathies and autoimmune diseases. The explanation for these associations is unclear but appears due to the close linkage of HLA-B with Ir genes (immune response genes) and/or Ir gene products. Although identification of Ir associated (Ia) antigens in inbred mice has been relatively simple, their definition in humans is considerably more complex. Nevertheless, several la-type alloantigens have been described by means of platelet-absorbed sera from multiparous women in cytotoxic reactions against purified B cells. Thus, in parallel with the use of homozygous stimulator cells in MLC reactions to define HLA-D loci of responders, it is possible to genetically characterize B-cell markers. Because of the association of an MLC-defined locus and adult RA, and a questionable association of HLA-B27 with JRA, we have prospectively examined 46 consecutive unrelated children, 28 female and 18 male, for HLA-A, B, D, and la-type antigens. The data were analyzed according to mode and age of onset, as well as extraarticular features. Patients with JRA had an elevated frequency of HLA-B27 (23% versus 8% of controls, P < 0.01). However the most striking associations were apparent when patients were divided into either a monoarticular or polyarticular (including Still's) group. Patients with polyarticular disease had an HLA-DW3 frequency of 64% compared to 22% in normals and 21% in monoarticular disease (P < 0.001). Furthermore, of 12 polyarticular patients thus far la typed, I I possessed the la allele B5. This latter association and its significance will require further typing and more refined definition of the genetic region involved before definitive conclusions are possible. However the associations of the loci HLA-DW3 and B5 (Ia) underscore the relationship between genetic markers and susceptibility to JRA. Further definition may aid both in understanding the mechanism of pathogenesis and in the classification and prognosis of childhood onset arthritis. Age-Dependent Loss of Morphologic and Functional Maturation of New Zealand Mouse Thymic Epithelium M . Eric Gershwin. Floyd Wilson,and Moshe Shifrine. Davis, California Numerous immunologic abnormalities have been catalogued in New Zealand mice, including development of autoantibodies. immune complexes, and progressive loss of T-cell function with age. Although several plausible theories have been advanced to explain the spontaneous occurrence of immunopathology, the precise mechanism remains to be elucidated. Nonetheless, present evidence indicates that one of the most critical features in the development of autoimmunity is a progressive imbalance of thymic function. Several previous studies of thymic histology from both New Zealand mice as well as patients with SLE have disclosed premature thymic involution, with a predominant degeneration and vacuolization of epithelial cells. Because of recent evidence that thymic epithelial cells are critical for both development of the thymus in utero as well as the continued maintenance of thymic dependent function, an attempt was made to study the growth and immunologic characteristics in vitro of isolated cultures of murine thymic epithelial cells. Thymic explants were cultured in vitro and serially observed in 2-week to I-year-old NZB, BALB/c, DBA/2, and N:GP(S) mice. Within 48 hours of culture, control mice demonstrated a proliferation of large PAS positive epithelial cells in a mosiac-like arrangement. Such cells contained prominent nucleoli and intense granular material and were both relatively radioresistant and extremely adherent. They could be readily separated from fibroblast contamination by selective treatment with trypsin-EDTA. In contrast, proliferation of epithelial cells was noted only in young NZB mice. Indeed, clones of epithelial cells derived from these young NZB mice underwent vacuolization and degeneration with age, an occurrence suggesting a preprogrammed defect. Furthermore, isolated thymic epithelial cells from control strains, but not NZB mice, induced T-cell surface markers on spleen cells derived from congenitally athymic (nude) mice. These abnormalities of thymic epithelial cells in NZB mice occur before the loss of tolerance to foregn proteins (BGG) or the appearance of thymocytotoxic antibody. Although a considerable amount of immunocytotology remains to be performed, we suggest that functional and morphologic alterations of thymic epithelial cells, perhaps genetically or virally induced, may be critical in the primary development of autoimmunity in New Zealand mice. DNA Binding to Normal Skin Connective Tissue as a Localizing Factor for DNA: Anti-DNA Complexes James N . Gilliam. Dallas, Texas There is a considerable body of evidence indicating that Clq and basement membrane collagen have similar chemical, structural, and binding characteristics. It is also well known that Clq efficiently binds DNA. In systemic lupus erythematosus (SLE) DNA.anti-DNA complexes often accumulate in the connective tissue (CT) structures of the upper dermis, glomerulus, choroid plexus, and vasculature. This study was undertaken to examine the possible role of DNA binding to skin CT as an explanation for the localization of immune complexes to the subepidermal region in SLE. Cryostat sections of normal human skin were incubated for 60 minutes in moist chambers at room temperature, with either calf thymus DNA in 0.15 M Tris buffer or buffer alone, and then washed extensively in 0.01 M PBS. Sections on glass slides were left untreated or were treated with DNAase, RNAase, or sodium metaperiodate. The enzyme-treated and nontreated tissues were then incubated with normal human serum or sera containing DNA or RNP antibodies in high titer. After washing, fluorescein-conjugated goat-antihuman-yglobulin was used to detect tissue bound antibody by fluorescence microscopy. Strong fluorescence of dermal CT was observed in the specimens that were preincubated with DNA and reacted with serum containing DNA antibodies. The fluorescence was most intense in the subepidermal zone. Preincubation with normal serum and RNP-positive serum gave negative results. DNAase digestion of the tissue or removal of DNA antibodies from the serum abolished the CT staining. Digestion of tissues with RNAase and sodium metaperiodate failed to interfere with the CT staining. This in vitro demonstration of DNA binding to skin CT in a location that corresponds to the usual area of immunoglobulin deposi- ARA ABSTRACTS 118 tion in SLE is strong evidence for a localizing effect o n DNA:antiDNA complexes by DNA-CT binding in vivo. C T binding of DNA may also account for the tendency in SLE for DNA-containing im- mune complexes to accumulate along basement membranes at other sites. Increased Sensitivity of Platelets and Neutrophils to Aggregated IgC in the Presence of the Lipid Moiety of Bacterial Endotoxin Mark H. Ginsberg. Peter M . Henson. and David C. Morrison, La Jolla. Cali$ornia In the course of study of the interaction of human platelets with highly purified bacterial endotoxins (Epopolysaccharides, LPS) we noted that mixtures of y-globulin preparations and LPS of S minnesofa Re595 were potent stimuli for platelet serotonin release. LPS or y-globulins alone were orders of magnitude less potent platelet activators. It seemed likely that the Re595 activity resided in the lipid moiety (lipid A ) of this LPS, because lipid A is the major constituent of Re595, is part of all bacterial LPS, and has been shown t o account for much of the biologic activity of LPS. This hypothesis was confirmed by acid hydrolysis of several different LPS preparations to produce isolated lipid A fractions, all of which were active in this system. Sephadex (3-200 gel filtration chromatography of the y-globulin preparations demonstrated that the active constituent migrated in the void volume, a finding suggesting that it was not monomeric IgG. Gentle heating of previously inactive unaggregated 1gG produced platelet serotonin releasing activity in the presence of LPS Re595. Velocity sedimentation analysis of heated IgG o n sucrose density gradients indicated that the newly acquired activity was found in material of sedimentation coefficient greater than 7s. Material sedimenting at 9 s (IgG dimer) and above was active. In the presence of 25pg/ml LPS Re595, significant platelet secretion was induced by as little as 0.5 pg aggregated IgG/ml. Platelets were at least 25-fold more sensitive to aggregated IgG-induced serotonin release in the presence of LPS Re595 than in the absence of LPS. Induction of neutrophil lysozyme secretion by IgG aggregates was also enhanced by LPS Re595. Because IgG aggregates share many of the properties of immune complexes, these data indicate a possible mechanism of amplification of immune complex activation of mediator cells by a product of gram-negative bacteria. Further, these data suggest that platelet serotonin secretion in the presence of the lipid moiety of LPS may be used to detect minute quantities of IgG aggregates or immune complexes. Altered Numbers of Circulating and Pokeweed-Mitogen-Induced Immunoglobulin Secreting Cells in SLE William W . Ginsburg. Peter E. Lipsky, Fred D. Finkelman, and Morris Z i x Dallas, Texas The number of immunoglobulin secretingcells (ISC) found in peripheral blood and the number generated by in vitro stimulation with pokeweed mitogen (PWM) were determined in 5 normals and 6 patients with active, untreated SLE. Peripheral blood mononuclear cells (PBM) prepared by Ficoll-Hypaque centrifugation were assayed for 1SC immediately or after culture for 7 days with or without PWM. 1SC were identified by a reverse hemolytic plaque assay that allowed quantitation of total ISC or cells secreting lgG, IgM, or IgA. Sheep erythrocytes coated with rabbit anti-human immunoglobulin (RAHI) were mixed with PBM in agarose and incubated for 1 hour at 37°C. After development with RAHl or class-specific anti-p, anti-y, or anti(Y and complement, ISC were detected as hemolytic plaques. Such plaques represented active ISC and not passive carryover of Ig into the assay because they were a ) abolished by cyclohexamide, b ) unaffected by 37OC preincubation and washing of the cells, and c) not found in PBM from patients with X-linked agammaglobulinemia. In normals, 945 ISC/IV PBM (815-1,IOO)were found,60.4% secreting lgG, 14.6% IgM, and 38.5% 1gA. Five of 6 SLE patients had markedly increased numbers of circulating ISC, with 11,166/KP (5.250-20.424) found. 74.5% lgG, 7.2% IgM, and 22.1% IgA. Stimulation of normal PBM with PWM for 7 days generated 9,894 ISC/IV (5.900-16.484). 61.8% IgG. 45.3% IgM, and 13.3% IgA. Similar incubation without PWM yielded only 534f141 ISC/lW. Culture of SLE PBM for 7 days yielded strikingly different results in that incubation without PWM resulted in a 7-fold decrease in ISC (1,683 352/1V) compared to the number found in fresh SLE PBM. Moreover, incubation with PWM failed to increase significantly the number of 1SC (2,095*731/1V) in 5 of 6 SLE patients. These data indicate that SLE is characterized by both a marked increase in the number of circulating ISC in vivo and a significantly diminished capacity of PBM t o generate ISC when stimulated with PWM in vitro. * Computer Analysis of Factors Influencing Frequency of Infection in SLE Ellen Ginzler. Herbert Diamond, David Kaplan. Max Weiner. Michael Schlesinger, and Mitchel Seleznick. Brooklyn, New York Infection has become an increasing cause of both morbidity and mortality in patients with SLE. Among 182 patients followed for an average of 3.2 years (0.1-lo), 334 infections occurred in 129 patients. Bacterial or fungal infections occurred 168 times; 166 viral syndromes were diagnosed. O f 4 8 total deaths, infection was the cause or a major contributing factor in 26 (54%). Data from a prospective study of clinical course in SLE was analyzed by computer t o determine the influence on frequency of infection of a ) dose of adrenocorticosteroid, b) treatment with an immunosuppressive agent (azathioprine), and c) three separate parameters of disease activity: new disease exacerbations, low serum C3, and elevated ESR. Results were as follows. a ) The frequency of all infections increased progressively with increasing steroid dose. The increase was even more striking when only bacterial and fungal infections were considered (Table). b) Patients both receiving and not receiving azathioprine had similarly increased rates of infection with increasing steroid dose, azathioprine itself not accounting for an increased risk of bacterial, fungal, or nonspecific viral syndromes (183 observed vs 191 expected). Azathioprine was, however, independently associated with a greater than 3-fold increase in the frequency of herpes zoster; steroids were not. c) New disease flares, low C3, and high ESR were associated with a 20-100% increase in infection rate. However few patients with inactive disease received high-dose steroids, and when patients taking comparable steroid doses were compared, the difference in frequency of infections was no longer observed. Disease activity itself did not predispose to infection. ARA ABSTRACTS 119 We conclude that the risk of bacterial and fungal infection in patients with SLE is directly related to the dose of steroids they receive. In view of the associated high morbidity and mortality, studies designed to evaluate the possible steroid-sparing action, as well as efficacy of drugs used in combination with steroids appear to be warranted. Steroid dose (mg/day) 0 1-10 1 1-20 21-40 41-60 61-100 All infectiondpt-yr Bacterial and fungal in fections/pt-yr 0.32 0.44 0.61 0.95 1.06 2.28 0.10 0.13 0.35 0.57 0.68 I .64 Accelerated Intravascular Coagulation in Scleroderma Geoflrey Gratwick, Ronald S. Klein. John S . Sergent, and Charles L. Christian, New York, New York Fibrin has been demonstrated by electron microscopy and specific fibrin staining in the intima of renal arterioles and along the glomerular basement membrane in scleroderma. The current study was designed to determine whether the coagulation system is activated and fibrin consumed more rapidly in scleroderma patients than in normals. Eleven patients (9 women and 2 men) with a clinical diagnosis of scleroderma have been studied with either autologous or heteroOur 6 normal controls had a logous fibrinogen labeled with fibrinogen half-life of 88.3 hours SD 6.5 hours, while our 11 patients had a fibrinogen turnover of 59.5 hours SD 12.4 hours. Using identical techniques to study patients with systemic lupus erythematosus, Sergent et al (Arthritis Rheum 19:195, 1975) found a fibrinogen half life of 60.5 SD I2 hours, with their normal controls running 90.1 SD 1 I hours. In the present study the usual parameters of intravascular coagulation including platelet count, staphylococcal clumping, partial thromboplastin, and prothrombin times were normal. There was no correlation between the fibrinogen half-life and the ESR, complement, creatinine clearance, pulmonary function tests, or upper GI series. Six patients with severe disease (3 have died) had a half-life of 55.8 SD 10.9 hours, while 6 patients with less severe disease had a fibrinogen half life of 63.0 f 13.8 hours. These observations support the hypothesis that there is ongoing disseminated intravascular coagulation in scleroderma contributing to vascular injury. Preliminary results suggest that heparin may prolong the survival of autologous fibrinogen in patients with scleroderma. Relative Roles of Receptors for Complement Components, Clq and C3 in Binding of Immune Complexes to Raji Lymphocytes Ramesh C . Gupta, Frederic C. McDufie, Gerhard Tappeiner, and Robert E. Jordon, Rochester, Minnesota Theofilopoulos, Wilson, and Dixon (J Clin Invest 57:169, 1976) have developed a radioimmunoassay for measuring soluble immune complexes (IC) which depends on binding of IC via receptors for complement (C) on Raji cells, a B lymphocyte culture cell line derived from Burkitt’s lymphoma. Though these authors have cited evidence that the binding of such complexes occurs via receptors for C3b and C3d, we found that Raji cells were capable of binding IC and aggregated human gammaglobulin (AHG) after incubation in normal human serum (NHS) to the same extent in the presence or absence of EDTA, so that a role for C3b or C3d in binding seemed unlikely. AHG incubated in Clq-deficient NHS at 37°C for 30 minutes failed to bind to Raji cells but did if purified Clq was added. Raji cells were found to possess receptor sites for Clq capable of binding 5.4 X I @ molecules of lallClq to Raji cells that were specifically inhibited by unlabeled Clq. Another cell culture, MOLT,a T-lymphocyte cell line derived from acute lymphoblastic leukemia and lacking C3b receptors, possessed a comparable number of Clq receptors. MOLT cells were capable of binding IC to the same extent as Raji cells, and when substituted for Raji cells in a radioimmunoassay, gave results comparable to those of Raji cells on the sera of 20 patients with immune complex diseases. Results with both assays were similar to those found with the 1311 Clq precipitin test. To further assess the possible role of C3b receptors in the binding of IC by Raji cells, these receptors were blocked by incubating Raji cells with inulin-converted C3 in Clq-deficient NHS, and then further reacted with erythrocyte antibody complement complex (EAC) and AHG. There was a marked inhibition of EAC-Raji cell rosettes, but no significant change in uptake of AHG. We conclude that Clq receptors on Raji cells play a major role in binding to soluble immune complexes. C3b receptors on lymphocytes probably play a significant role in the binding of particulate complexes, such as EAC. Nonvasomotor Postinjection Reactions Associated with Chrysotherapy James T.Halla, Joe G. Hardin, and Julius E. Linn. Birmingham, Alabama Transiently increased rheumatic symptomatology after individual gold injections is an acknowledged, but uncharacterized, reaction. This study was designed to determine the frequency of nonvasomotor postinjection reactions (PIR) and to define their nature and significance. In addition to record review, a partially structured interview was used to elicit a history of all untoward gold responses from 100 patients who had received regular gold within a year prior to evaluation, but who were otherwise unselected. Care was taken not to suggest to the subject the reaction being investigated. N o postinjection symptoms or typical vasomotor reactions occurred in 85 subjects. Thirteen described a PIR predominately of new or increased rheumatic symptoms, all initially occurring after gold sodium thiomalate (GST)injections and beginning within 1 hour postinjection in I , and within 6-24 hours postinjection in 12 subjects. Reactions persisted from 1/2 to 5 days (mean: 2) and consisted of new or increased stiffness (l3), arthralgia (12). myalgia (8), constitutional symptoms (5) and joint swelling (3). Initial reaction occurred during the first month of therapy in 5 subjects, during the second month in 4, ARA ABSTRACTS 120 and after the third month in 4; and the PIR had recurred > 5 times in 10 and >I0 times in 4 subjects. Therapeutic manipulation had been felt to be warranted in 6 patients because of the severity of the reaction. For these 6 gold thioglucose (GTG) was substituted for GST with no subsequent PIR in I and with decremental decreases in reaction severity in the remaining 5, so that the reaction had disappeared by the fourth GTG injection in 4 patients. The milder PIR continued predictably in the remaining 7. A predictable nonrheumatic PIR-consisting only of constitutional symptoms beginning 12-24 hours after GST injection and lasting 3-5 days-was described by 2 additional subjects. Other untoward gold reactions, besides the PIR, occurred in 43% of the 85 subjects without, and in 41% of the 15 with PIRs, a result suggesting no tendency for the PIR to occur in a reaction-prone group. Fifteen percent of 100 unselected subjects experienced new or increased symptoms, primarily rheumatic, within hours of GST injection. Contrary to prior beliefs, these reactions may begin late as well as early in the course of therapy, tend to persist, and may be severe enough to require intervention, in which case GTG substitution may result in waning of the PIR. It is suggested that these PIRs are significant and deserve further study. Application of the Radioimmunoassay for Fibrinopeptide A to Serial Evaluation of Patients with SLE John A. Hardin, Malcolm Cronlund, Edgar Haber, and Kurt J . Bloch, New Haven, Connecticut and Boston, Massachusetts We have previously reported the development of a radioimmunoassay for fibrinopeptide A (FPA) which is useful for detecting activation of the clotting mechanism. Application of this assay to the study of patients with systemic lupus erythematosus (SLE) demonstrated higher FPA levels in patients with active disease than in patients with inactive disease or in normal donors. Elevated levels correlated directly with the presence of antibodies to native DNA and inversely with depression of serum C3 concentrations. These findings suggested that measurements of FPA might be useful for assessing disease activity in patients with SLE. To test this hypothesis, serial determinations of FPA levels were carried out in 21 patients who were followed for a mean of 18 months. In individual patients it was found that FPA levels fluctuated in parallel with disease activity; exacerbations were accompanied by a rise in the FPA level and improvement was accompanied by a fall. FPA levels were always elevated when clinical evidence of inflammation was present, and the extent of elevation correlated with the extent of inflammation. Some patients with inactive disease also had elevated levels of FPA, a finding suggesting the possibility that subclinical inflammation was present (Table). These data indicate that activation of the clotting mechanism occurs in association with the inflammatory response in SLE. Measurements of FPA appear to provide a sensitive method for evaluating these patients. Correlation of FPA Values with Number of Organ Systems Involved Clinically with SLE Determinations FPA ng/ml (mean f 1 SD) Number of Systems Involved Normal Donors Clinically Inactive SLE One Two Three or More 20 0.9 f . 3 36 2.1 f 1 . 1 25 5.2 f 4. I 20 7.5 f 2.5 12 12.1 f 2.6 Costs of Monitoring Chrysotherapy Benjamin K . Harris, De Witt W . Englund, H . Arlene Ross, and Sanford H . Roth, Phoenix, Arizona Cost and potential toxicity are two factors that may deter both physician and patient from considering gold therapy for rheumatoid arthritis. In 1975 Liang and Fries presented a cost analysis of alternative monitoring strategies in administering gold. Their data, based on a survey of 10 rheumatologists and 3 hospitals in northern California, indicated that the cost of an initial course of gold therapy was approximately $800. A number of cost-reducing measures were suggested, including the use of a physician-assistant and a decrease in the frequency of certain laboratory tests. Since 1970 our approach to monitoring chrysotherapy has included many features advocated by Liang and Fries. We have re viewed our experience with gold therapy administered in 1975 to find the average cost to initiate therapy and to determine whether our costreducing measures in any way increased toxic reactions to gold. Gold therapy was begun in 64 patients in 1975. Forty-nine patients (77%) completed the initial course with a favorable response; in 8 patients (13%) gold was discontinued because of toxicity. Five patients were lost to follow-up, and 2 patients were considered treatment failures. Our monitoring plan called for an average of 5 office visits, 21 gold injections, 22 urinalyses, and 9 complete blood counts with platelet estimates. Average cost to the patient was $315. Major savings resulted from utilizing a physician-assistant to screen for toxicity and administer gold, and thus to reduce physician visits from weekly to monthly. If, as indicated by our data, alternative approaches do not increase risk to the patient or detract from gold effectiveness, further exploration for an optimal monitoring regimen appears warranted. HLA-B27-Associated Arthritis in 6 Families: Homozygosity, Patterns, and Severity of Disease Marc C. Hochberg, Frank C . Arnett. Jr., Bernice Z . Schacter. and Wilma B. Bias, Baltimore, Maryland HLA-B27 is strongly associated with ankylosing spondylitis (AS), Reiter’s syndrome (RS), and juvenile chronic polyarthritis (JCP). Although these disorders share certain clinical similarities, there are differences between them, most notably the predominance of ARA ABSTRACTS 121 peripheral arthropathy in RS and J C P and axial skeletal involvement in AS. Similarly, there exists a broad spectrum of severity within each disorder. T h e families of 6 patients with B27-associated arthritis were studied clinically and radiographically. H L A genotypes were determined independently and then correlated with clinical data. Two of the 6 probands (families I and 2) are homozygous for 827 and heterozygous a t the A, C , and D loci. Both have extraordinarily severe disease. The homozygous female proband of family 1 has a I 5-year history of J C P with severe peripheral and axial disease and recurrent uveitis. The mother and a sister with the maternal B27 haplotype have only mild, nondeforming, seronegative peripheral arthritis, whereas the father has only sacroiliitis (SI). The homozygous male proband of family 2 is crippled by a progressive, deforming peripheral and axial arthritis beginning at age 18 (JCP-AS), aortic regurgitation, and uveitis. A heterozygous brother has RS without crippling despite similar duration of disease (30 years). One heteroeygous child has mild JCP. Six B27-positive relatives are well. The male proband of family 3 is possibly homozygous and has RS with severe. deforming arthropathy and mucocutaneous disease. Two HLA identical brothers are well. Family 4 contains a mother, daughter, and son with RS. all heterozygous for 827, and none has axial disease. Family 5 consists of a mother, 2 daughters, and 1 son, all with SI, and 1 son with AS: all are B27 heterozygotes and none has peripheral disease. Family 6 contains 4 B27 heterozygous siblings, I female with severe peripheral and axial arthritis (JCP-AS), I male with mild AS, and 1 male with SI; 1 female is well. These data suggest that a ) homozygosity for B27 may result in more severe rheumatic disease; b ) there may be exclusivity to the type and pattern of rheumatic disorder associated with a given B27 haplotype within families; and c ) the presence of normal B27 positive individuals within these families implies that other environmental and/or genetic factors a r e necessary for disease expression. CRP in SLE Patients: An Aid in Diagnosing Superimposed Infections Stephen Honig, Peter Gorevic, and Gerald Weissmann, New York. New York The presence of C-reactive protein ( C R P ) in the sera of patients with SLE is not well documented. C R P positivity is, however, well established in sera from patients with various other acute and chronic inflammatory processes. In order t o determine the incidence of C R P in sera of patients with SLE, 70 patients with SLE were studied. Results of C R P positivity were correlated with disease activity and superimposed infections. Seventeen hospitalized patients with documented infections were also studied. Strong C R P positivity (2 2 + ) was found in the sera of 13 of 61 patients with active SLE (21%). Of these 13 patients, 11 had superimposed infections at the time that C R P was found in their sera. Only 2 patients with active SLE had strong C R P positivity in their sera in the absence of a superimposed infection. Excluding the SLE patients with infections, the incidence of CRP-positive sera in patients with active SLE was 4%. Only 1 patient continued t o have C R P in her sera after treatment of the superimposed infection. In the absence of infection, there is a lack of association between C R P positivity and active SLE ( P < 0.002). The activity of the uninfected SLE group is shown by the negative correlation between ESR and C, ( r = -0.16) and C, ( r = -0.41), results reflecting patients with elevated ESR and hypocomplementemia. The finding of C R P positivity in sera of patients with SLE is therefore strongly associated with the presence of infection (P < 0.002). There were n o instances of infection documented in the active SLE patients without strong C R P positivity in their sera. The 17 patients with infections, including 1 patient with a viral infection, all had C R P in their sera at the time of initial diagnosis. Appropriate treatment resulted in the subsequent loss of C R P from their sera and reaffirmed its utility as an indicator of acute infections. The data suggest that patients with active SLE rarely have strongly positive C R P in their sera and, when positive, this acute phase reactant indicates a superimposed infection and not disease activity. Early Diagnosis and Treatment of Ischemic Necrosis of the Femoral Head David S. Hungerford, Baltimore, Maryland The hemodynamic status of the proximal femur has been evaluated in 90 hips with ischemic necrosis of the femoral head ( I N F H ) in 60 patients and 18 normal controls. The methods constitute a simple, rapid system for establishing the diagnosis of I N F H in the patient with suspicious clinical findings. Treatment of 70 hips in 44 patients has consisted of core decompression and removal of an 11 mm diameter plug of bone from the central axis of the femoral head and neck. Baseline intraosseous pressure (IOP) and the pressure response t o a 5 cc saline load injected intraosseously were measured by an electromanometer via a rigid, percutaneously inserted 3 mm cannula. In selected patients an intraosseous phlebogram, using Renografin, was carried out. All patients with I N F H had their diagnosis histologically confirmed. Thirty-eight of 90 hips with I N F H showed an IOP of greater than 30 mmHg and/or a sustained hypertensive response of more than 10 mmHg to the injected saline load. All controls demonstrated IOP less than 28 mmHg and none had a hypertensive response to the saline. lntraosseous venograms in hips with I N F H showed diaphyseal reflux. poor filling of extraosseous veins, and delayed intraosseous evacuation of dye, in marked contrast to the normal controls. Hips having had core decompression have been followed from 6 t o 42 months. All hips were painful preoperatively. Of 18 hips in the preradiologic stage, 17 are asymptomatic and show no radiologic changes at last follow-up. Successful relief of symptoms and prevention of progression decrease with advancing stages. Forty-four hips in this series were in patients with connective tissue disease who were on or had been on large doses of steroids. Ten of these patients and 18 in the whole series had painful hips but normal or near normal x-rays. The methods presented provide the opportunity of establishing the diagnosis of I N F H in the preradiologic stage of the disease, at a time when core decompression appears to be an effective means of interrupting the natural history of the disease. ARA ABSTRACTS 122 Influence of Interstitial Infiltration on Tubule Cells in Kidneys of NZB X NZW FI (B/W) Hybrid Mice and the EfFect of Cyclophosphamide (CY) E. R . Hurd and M . Zif, Dallas, Texas We have reported that kidney glomerular cell proliferation can be quantitated by 3HTdr injection, autoradiographic examination of kidney sections, and enumeration of labeled glomerular cells. In the present studies we have examined renal tubule cells (TC) by the same technique to assess the effect of interstitial infiltration on renal T C proliferation. Three, 5, 7, 9, and Il-month B/W mice were injected with SHTdr daily for 8 days prior to sacrifice. Autoradiographically labeled sections of kidney were prepared and the number of labeled TC per lo00 TC was counted. At the same time interstitial mononuclear cell infiltration, which is commonly present in these mice, was scored on a 0-4+ basis. Percentages of labeled T C adjacent to interstitial infiltrates and distant from them were tabulated. In other mice the effect of CY (15 mg/kg/day), injected over a 2-month period, o n both the interstitial infiltrate and amount of T C proliferation, was examined. NZB, CBA/J, and C57B1/6J mice were used as controls. In B/W mice interstitial infiltration began at 5 months and reached a peak at 9 months. Proliferation of T C began somewhat later, i.e. at 7 months, but also reached a peak (45 labeled cells per lo00 T C ) at 9 months. Control mice (15 labeled T C ) showed no increase with age. In the areas adjacent to infiltrates at 7 and 9 months, an average of 72 TC/1000 were labeled, as opposed to 19 cells at a distance, an increase of about 4-fold. CY administered 2 months before sacrifice virtually obliterated interstitial infiltration and significantly decreased TC proliferation at 3, 5, 7, and 9 months of age. These studies suggest that mononuclear cells in the interstitial infiltrate may produce a lymphokine or other factor that may be injurious to kidney T C and lead to regeneration after injury or death. CY reverses or markedly inhibits these phenomena. The present findings suggest that in diseases in which interstitial nephritis is present, such as Sjogren’s syndrome, the interstitial infiltrate may contribute to the observed T C dysfunction by immunologically induced injury. Characterization of the Proteins Synthesized by Sclerodermatous Skin in Organ Cultures Sergio A . Jimenez. Philadelphia, Pennsylvania Full-thickness skin biopsies from the leading edge of actively involved sclerodermatous lesions were incubated in an organ culture system in the presence of “C-proline. At the end of the incubation, the labeled proteins from the media and the tissues were separated and characterized. The organ cultures studied showed marked increase in the incorporation of “C-proline and in the synthesis of “C-hydroxyproline when compared to normal skin. The distribution of radioactivity between media and tissue proteins showed that 77-93% of the total 14C-prolineincorporated and 6 1-9 I % of the IT-hydroxyproline synthesized, were present in the media. Determination of “C-hydroxyproline showed that only 1-3’70 of the total “C-proline incorporated was hydroxylated to “C-hydroxyproline, indicating that collagen represented only a minor fraction of the labeled proteins synthesized during the incubation. When the media proteins were examined by gel filtration under denaturing conditons, it was found that most of the labeled proteins were of small molecular weight, and that only 10-12% of the radioactivity eluted in a region corresponding t o collagenous molecules. The elution of “C-hydroxyproline-containing proteins in- dicated that essentially all the labeled collagen in the media was present as procollagen. Determination of the hydroxyproline content of the purified procollagen indicated that the protein was markedly under-hydroxylated, because it contained only one-third of the hydroxyproline expected for a fully hydroxylated molecule. The small molecular weight proteins did not contain hydroxyproline and therefore they were noncollagenous. N o molecules with molecular weights greater than that of procollagen were present in the media. The tissues were sequentially extracted with 0.15 M NaCI, 0.5 M acetic acid, 100 jtg/ml pepsin, and I % sodium dodecyl sulfate. About 40% of the labeled collagen was extracted in the neutral salt and acid buffers. An additional 35% was solubilized after pepsin digestion of the tissues, and the remaining was solubilized under denaturing conditions. In contrast, approximately 75% of the total “C-proline incorporated was extracted in the neutral salt and acid buffers, indicating that these buffers extracted preferentially noncollagenous molecules or underhydroxylated collagen. Detection of Circulating Immune Complexes in Patients with Rheumatic Diseases and Vasculitis Gary M.Kammer, David N . Glass, Nicholas A . Soter, and Peter H . Schur, Boston, Massachuserts The sera of patients with rheumatoid arthritis or Sjogren’s syndrome complicated by vasculitis, and of patients with SLE were studied for circulating immune complexes. The IC assays performed were Clq-1z51 binding and inhibition of antibody-dependent lymphocyte-mediated cytotoxicity (ADLMC). Both Clq binding and A D L M C inhibition are expressed as the equivalent of micrograms of aggregated IgG (AgglgG) per ml. Mean Clq binding in 34 patients with vasculitis, histopathologically defined a s cutaneous necrotizing venulitis (CNV), was 315.5 pg AggIgG/ml (range: 10-1840), and in 34 normals it was 44 pg AgglgG/ml (range: 10-200) ( r = 4.16, P < 0.001). Of the 34 patients with CNV, hypocomplementemia and Clq binding were detected in 1 I , normocomplementemia and Clq binding in 8, and both normocomplementemia and n o detectable Clq binding in 15. The 19 CNV patients with Clq binding had significantly higher binding [572.6 pg AggIgG/ml f 474.1 (1 SD)] when compared with patients with active SLE [155.0 jtg AgglgG/ml f 125.7 ( I SD)] (f = 3.31, P < 0.001). Cryoproteins were detected in 5 of the CNV patients with Clq binding. Mean ADLMC inhibition in 13 of the 34 CNV patients studied was 1.97 pg AgglgG/ml (range: 0.1-9.0). and in 17 normals, 0.17 pg AgglgG/ml (range: 0.1-0.82) ( I = 2.40, P < 0.01). In the 13 patients assayed by ADLMC inhibition, there were 4 with hypocomplementemia and inhibition, 4 with normocomplementemia and inhibition, and 5 with normocomplementemia and without inhibition. There was n o significant difference between CNV and SLE patients with the A D L M C inhibition method. These data suggest a ) that there are circulating immune complexes in sera of some patients with CNV complicating rheumatic diseases; b ) that there is a relationship between hypocomplementemia. Clq binding, and ADLMC inhibition; c ) that sera from CNV patients contain appreciably more Clq binding material than do sera from patients with active SLE; and d ) that both the Clq binding and ADLMC inhibition assays are more sensitive than hypocomplementemia as criteria for the detection of immune complexes. ARA ABSTRACTS 123 Increased Incidence of Malignancy in Sjogren’s Syndrome Stuart S. Kassan. Robert Hoover, Robert P . Kimberly, Daniel R . Budman, John L. Decker, and Thomas M.Chused. Bethesda. Maryland The case records of 128 women and 6 men with Sjiigren’s syndrome (SS) treated at the National Institutes of Health (NIH) from 1955 through 1975 were analyzed to estimate the risk of malignancy. Only those cases of malignancy developing after the first admission were included as cases in calculating risk of cancer in this population. The number of cancer cases in the study group was compared to the expected incidence of various forms of cancer in the population derived from the Connecticut Cancer Registry (for the incidence of lymphoma) and the cancer registries of Dallas-Fort Worth and Iowa (for the incidence of skin cancer). The relative risk ( R R ) for each type of cancer observed, observed casedexpected cases, was then determined. Five patients developed non-Hodgkin’s lymphoma (RR = 100, P < 0.001) from I t o 9 years following their first NIH admission for SS. The R R for lymphoma in patients with sicca complex alone was similar to that for SS patients with rheumatoid arthritis. Six cases of Waldenstrom’s macroglobulinemia occurred in this study group, but control incidences for this entity are not available to allow an estimation of the RR. Four patients developed nonmelanotic skin cancer, a figure in excess of that expected from rates in either low(Iowa) o r high-risk (Dallas-Fort Worth) areas in the US. Other tumors occurred at their expected frequency. Low-dose therapeutic parotid irradiation may have predisposed to lymphoid malignancy in SS patients so treated. SS patients who received low-dose parotid irradiation had an incidence of lymphoma 33 times that of nonirradiated SS patients. This particular mode of therapy may, however, have selected a subpopulation of SS patients at greater risk of developing malignancy. Cytotoxic drug therapy had no evident effect on tumor development in this SS population. However the number of patients treated with cytotoxic drugs was small (13) and the duration of follow-up for these patients was short (longest = 6 years). This is the only quantitative consideration of this matter available. T h e nature or severity of SS upon referral to N I H may of course introduce selective biases, but none was apparent. HLA-D Locus Typing in Ankylosing Spondylitis Kip L . Kemple. Richard A . Gatti, Rodney Bluestone, and James Klinenberg. Los Angeles. CaliJornia The high association of the HLA antigen B27 with ankylosing spondylitis (AS) and other spondyloarthropathies has led to the speculation that a disease susceptibility gene within the major histocompatibility complex is responsible for disease production. The incriminated gene is presumed to be in marked linkage disquilibrium with the 827 antigen and possibly with other histocompatibility determinants. Analogous Ir genes in the mouse H-2 complex are known to be located near or at the main MLR locus. We have therefore evaluated the patterns of D locus typing in humans suffering from AS to determine whether there is a n altered distribution of L D alleles comparable to that seen with the 827 antigen. T o date 44 patients with definite or probable AS have been evaluated. Both B27-positive (35) and 927-negative (9) patients a r e included. Typing was carried out with a panel of 24-40 stimulator lymphoblastoid cell lines which represent the Sixth International Workshop specificities DW 1-6 plus 107-108. These lines a r e derived from individuals known to be homozygous at the D locus. They are mitomycin-treated and frozen in typing plates for storage at -80”. When they are thawed for use in standard one-way M L R testing, they give patterns of stimulation comparable to typing with fresh cells. Results have been compiled by separating weak (typing) responses from strong MLR responses pending more sophisticated computer analysis of the data. These studies indicate that there are no significant differences between AS patients (either 927-positive or negative) and controls in the distribution of DW alleles or in responses to 40 individual cell lines. Specific gene frequencies will be discussed. In addition, there is no significant difference in the overall level of M L R responsiveness between AS patients and controls. It appears on the basis of these results that disease susceptibility in AS is not determined by factors associated with the D locus, as appears t o be the case with multiple sclerosis and possibly other diseases. Effects of Aspirin on Renal Blood Flow, Glomerular Filtration Rate, and Renal Prostaglandins in SLE Robert P . Kimberly, John R . Gill Jr., Robert E. Bowden and Paul H . Plotz, Bethesda. Maryland Antiinflammatory doses of aspirin have been shown to affect serum creatinine (SCr), blood urea nitrogen (BUN), and creatinine clearance (CCr) in some patients, but the nature of the changes in renal function remains controversial. Five patients with systemic lupus erythematosus (SLE) have been studied under controlled metabolic conditions in order to examine the effects of approximately 7 days of oral aspirin (3.3-6.0 g/day) on SCr, BUN, CCr, and urinary excretion of PGE-like material determined by radioimmunoassay. Inulin and PAH clearances were performed before, during, and after aspirin therapy. The BUN rose an average of 5.8 f 3.1 mg/dl (mean f SEM); SCr increased 0.3 f 0.06 mg/dl (P < 0.01); and CCr fell 15.6 f 1.9 ml/min ( P < 0.01). PAH clearance (CPAH) dropped 29% or 128 f 22.5 ml/min (P < 0.01). while inulin clearance (CIn) fell 15% o r 12.2 f 2.4 ml/min (P < 0.01). With the cessation of aspirin, all parameters showed a significant return toward baseline (P < 0.05) within 5 days, except for CIn which demonstrated some variability. Two additional patients without complete CIn and CPAH studies also showed increases in SCr and BUN with decreases in CCr. However in I the change in CCr had regained the control level by the end of the treatment period. Measurement of urinary PGE-like material showed a mean decrease of 47% (P < 0.001) during aspirin therapy. These data demonstrate that aspirin given in antiinflammatory doses to patients with SLE for several days causes a significant decrease in renal blood Row and glomerular filtration rate, which may be reflected in serum parameters. These changes are reversible with the cessation of aspirin therapy. The decrease in renal blood Row concomitant with the measured decrease in renal prostaglandins suggests that the mechanism for the aspirin-induced change in renal physiology may be the inhibition of renal prostaglandin synthesis. This hypothesis predicts that this effect will not be limited t o aspirin in the class of nonsteroidal antiinflammatory drugs. ARA ABSTRACTS I24 Efficacy of Antiviral Agents in the Treatment of Lupus Nephritis in NZB/NZW F, Mice L. W . Klassen. D . R. Budman, G. W . Williams,A . D . Steinberg. and N . L. Gerber, Bethesda. Maryland Four antiviral agents were evaluated in the treatment of lupus nephritis in NZB/W mice. One hundred fifty-two NZB/W female mice aged 20 weeks were randomly selected to receive I P injections of ribavirin (250 mg/kg) twice weekly; phosphonoacetic acid (750 mg/kg) five times weekly; levamisole (10 mg/kg) twice weekly; ara-A (600 mg/kg) twice weekly or saline twice weekly. All mice were followed for development of proteinuria, anti-DNA antibodies, and survival. Control mice had a median survival of 10 months with a 15% survival at 14 months. Ribavirin therapy was associated with 76% survival at 14 months. Levamisole and phosphonoacetic acid prolonged median survival to I I .4 and I 1.6 months respectively, and were associated with a 2% survival at 14 months for both groups. Ara-A treated mice had a median survival of 9 months (Table). This study was designed t o determine the efficacy of selected antiviral agents in prolonging life in NZB/W female mice. The results clearly demonstrate the efficacy of ribavirin in prolonging survival, reducing proteinuria, and decreasing the titer of antibodies against native DNA. Elfects of Antiviral Agents on Proteinuria and DNA Antibodies in NZB/W Mice Therapy Group Evaluation Proteinuria* Age (mo) Ara-A Levamisol Phos Acid 7 0/25t 0/7t 64 f 3 62 f 7 4/22 11/17 55 f 3 65 f 74 13/28 11/17 52 f 2 69 f 6f II Anti-DNA$ 7 II * Animals with urinary protein concentration greater than I00 mg/day/number t P < 0.05 vs saline control by x 2 analysis. Ribavirin 10/29t 0/26t 44 f 24 42 f 2 Saline 21/31 8/13 59 f 2 47 f 4 surviving. $ Expressed as percent binding f SEM. < 0.05 vs saline control by student's I test. tj P Effects of Soluble Immune Response Suppressor (SIRS) on the Course of Autoimmune Disease in NZB/W Mice Randall S . Krakauer, Warren Strober, and Thomas A . Waldmann,Boston. Massachusetts, and Bethesda, Maryland Female NZB/W mice develop autoimmune abnormalities similar to those seen in systemic lupus erythematosus (SLE). As they age, these mice lose precursors of suppressor T cells, and their ability to make soluble immune response suppressor (SIRS) in response to concanavallin A exposure for 36 hours. Yet even the adult mice respond to the suppressor signals of SIRS produced from other sources. These observations suggested the possibility that such mice could be treated with the suppressor T-cell product, SIRS. Therefore we felt SIRS may provide a means of adoptive immunotherapy for these mice, and by extension, for SLE. We treated female NZB/W mice, beginning at age 4 weeks, with thrice weekly injections of SIRS, the supernatant of splenocytes incubated with concanavallin A (2 rg/cc) for 36 hours, from BALB/c and young (4-week-old) NZBiW mice spleen cells. Treated mice over the 28-week observation period had decreased immunoglobulin levels (75% reduction in IgG and IgA, 50% reduction in IgM) and strikingly less antinuclear antibody (absent or present in very low titer in all treated animals, present in higher titer in all untreated animals) compared to animals receiving a control preparation. They also had less proteinuria (22 f 24 mg/100 ml urine in treated animals. 105 f 28 mg/100 ml in untreated animals) and renal pathology (by light microscopy, severe glomerulonephritis in untreated animals, near normal glomeruli in treated animals) compared to NZB/W mice receiving a control preparation or no treatment at all. At the end of a full year of study, 93% of the treated animals and 7% of the control animals survived. Thus SIRS administration appears to be an effective therapy of the autoimmune disease found in this animal model of SLE and holds promise for the successful treatment of human SLE. Liver Involvement in Essential Mixed Cryoglobulinemia (EMC) Yoram Levo. Peter Gorevic, Hannah Kassab. Hillel Tobias, and Edward C. Franklin, New York, New York Essential mixed cryoglobulinemia is characterized by purpura arthralgia and weakness. Although an immune complex glomerulonephritis is the major cause of death. the frequency of clinically apparent liver involvement has not been emphasized. Between 1960 and 1976, 104 patients with cryoglobulins were seen, 30 of whom were considered to have EMC. Purpura was present in 90%. arthralgia in 70%. and renal disease in 43%. Twenty-six cryoglobulins were composed of IgG and IgM; 4 also contained IgA. The IgM was polyclonal in 22 and monoclonal (IgMk') in 8. Evidence of hepatic involvement was present in 25 (83%). Hepatomegaly was noted by physical examination in 18 and by liver scan in only 3 additional ones. Seventeen patients had abnormal liver function tests, with elevation of alkaline phosphatase the most sensitive parameter. Four patients had severe clinical manifestations of liver involvement such as ascites or hepatic encephalopathy. Liver tissue was obtained from I I patients and showed pathologic changes in 10. Two patterns of liver pathology were observed-one similar to that in chronic active hepatitis characterized by periportal infiltration with lymphocytes, histiocytes, and plasmacytes, piecemeal necrosis, and fibrosis; the other resembling portal cirrhosis. All 4 patients with IgA in their cryoprecipitate had liver involvement documented by biopsy. Because of the high incidence of hepatic dysfunction, sera and in several instances cryoprecipitates from 21 subjects were tested for H&Ag only in their cryoprecipitate and one had it in his serum (14%). Twelve ARA ABSTRACTS 125 patients (57%) had significant titers of anti-H$, antibodies. Thus 62% of the patients had evidence of exposure to the HBV. These findings emphasize the frequency of hepatic involvement in EMC and raise the possibility that this syndrome may be another example of extrahepatic manifestations as a secondary immunologic response to HBV. Presence of y-Carboxyglutamic Acid in the Proteins Associated with Ectopic Calcification Jane B. Lian. Martha Skinner, Melvin J. Climcher, and Paul M . Gallop, Boston, Massachusetts A specific calcium-binding amino acid, y-carboxyglutamic acid (Gla), discovered in the blood clotting factor prothrombin, is synthesized in a vitamin K-dependent and bicarbonate ion requiring post-translational enzymatic reaction. Recent studies have demonstrated the presence of Gla in an EDTA-soluble, noncollagenous protein in both chicken and bovine bone. Because a Gla-containing protein is normally present in bone and absent in unmineralized tissues, it was of interest to examine several kinds of ectopic calcifications. Studies were done on a 21-year-old female with dermatomyositis and a 52-year-old woman with a 30-year history of scleroderma. In the case of dermatomyositis, the patient had many large nodules of soft tissue calcification throughout her body. Due to the acid lability of Gla, hydrolysis in 2 N KOH for 22 hours is required to detect it by amino acid analysis. Alkaline hydrolysis of biopsy material from both the normal and calcified skin and subcutaneous tissue from the patient with dermatomyositis revealed the presence of Gla only in the plaque specimens. The chalky paste-like exudate from subcutaneous nodules and from an infrapatellar knee bursa of the patient with scleroderma showed a higher concentration of Gla (6 res/IOOO amino acids) than normal whole bone (0.67 res/1000). The EDTA-soluble and nondialyzable proteins extracted from this exudate were enriched in Gla (20 res/IOOO amino acids). Because X-ray diffraction analysis showed hydroxyapatite to be the major mineral component of the plaque in these two cases, the question arises as to whether the Gla-containing protein(s) in the plaque is similar to the Gla-containing protein in normal bone. I t is also relevant to inquire if the Gla in the plaque is derived from the vitamin K-dependent circulating blood clotting factors or if perhaps it is synthesized de novo by cells at the site of calcification. In view of the known calcium and phospholipid binding properties of y-carboxyglutamic acid, the presence of Gla in pathologically mineralized tissues is a potentially significant finding. Effect of Prostaglandins on Metabolism of Isolated Chondroycytes Louis Lippiello, Dragoslav Mitrouic. and Henry J . Mankin, Boston, Massachusetts Prostaglandins (PG)are found at elevated levels in synovial fluid from patients with inflammatory arthritis and have been implicated as a possible agent in erosion of the articular cartilage of such joints. Experiments were designed to examine the role of PGs in articular cartilage metabolism and to define the nature and specificity of their effect on proteoglycan metabolism in vitro. Chondrocytes isolated from calf metatarsal cartilage were counted and assayed for viability with the tryphan blue dye exclusion test. Cells (1V) were aliquoted to tubes containing I ml media, and the cells “rested” for 24 hours. The media was changed and PGs at varying dosages were added for an additional 24 hours. At this time labeled substrate (ssSO,+ SH-glucosamine) was added for 30 minutes, and incorporation of isotope was terminated by heating cells media at 100” for 5 minutes. Radioactivity of the TCA insoluble (lo%), I N NaOH solubilized material was assayed by liquid scintillation counting. The results showed that there was considerable variation in the response of the cells to different members of the PGs. With the exception of PGA,, only PG of the 2 series (PGA,, PGE,, PGF-) + inhibited uptake of substrate. The observed inhibitions were statistically significant and the most potent inhibitor was PGA, (>SO% at a dose of0.5 pg/ml; <50% at a dose between 0.06pg/ml and 0.5 pg/ml). Concentrations of PGA, of less than 0.03 pg/ml had little effect. Inhibition by PGA, at 25 pg/ml was irreversible up to 60 hours after exposure, and treatment of cells with indomethacin (5 pg/ml) for 6 hours before exposure of the cells to PGA, at 25 pg/ml did not alter the inhibitory pattern. The results indicate that calf chondrocytes in suspension culture are very sensitive to and selectively inhibited by PG PGA, and all members of the 2 series (PGA, > PGA, > PGE, > PGF,). The absence of a reduction of the inhibition by indomethacin confines the action of the drug at the PG synthesis level, rather than after the active material is formed. Previous demonstrations of an inhibitory effect of PG on cartilage in vivo and in vitro in organ culture, and now using isolated cells, supports the thesis that these fatty acids may play a major role in the development of cartilage destruction in inflammatory arthritis and conceivably in osteoarthritis. Joint Allotransplantation: A Metabolic and Biomaterial Study Mitchel Lipton, Joseph M . Lane, Carl T. Brighton, John Irwin, and Chester Shadle, Philadelphia, Pennsylvania and New York. New York Joint replacement with allotransplants has received renewed clinical interest. A successful transplant necessitates the continued viability of the articular chondrocytes and structural stability of the cartilage matrix. Utilizing a mature rabbit osteochondral joint allotransplant model, we studied the metabolic, biochemical, and biomateria\ properties of the transplanted cartilage for up to 1.5 years, and the cartilage remained metabolically and materially intact in the majority of long-term rabbit transplants. Forty mature New Zealand rabbits underwent osteochondral shell joint allotransplantation of the left femoral condyle. The major- ity of long-term transplants remained grossly and histologically intact for as long as I .5 years. The subchondral bone of the shell graft was replaced by creeping substitution by 2 months. Twenty-eight percent of the transplants failed because of the surgical defects (pseudarthrosis, malalignment, incongruous fit) and manifest secondary osteoarthritic morphologic changes with no evidence of rejection. At 1.5 years the transplanted cartilage was comparable statistically to control cartilage in proteoglycan hexosamine, collagen hydroxyproline, and protein content. The transplanted cartilage was indistinguishable biomate- ARA ABSTRACTS 126 rially from control in terms of relaxed sheer modulus (0.9 vs 0.9) and instantaneous sheer modulus (5.1 vs 5.0) (dynes/cmP X 1V). Successful long-term joint allotransplantation with viable chondrocytes and intact cartilage matrix is possible. The limited failures appear to be directly related to surgical malalignment and incongruities resulting in secondary osteoarthritis. N o rejection was evident morphologically. These metabolic and biomaterial studies give strong support for continued clinical trials ofjoint allotransplantation. Hexosarnine Control Transplant (ag/mg dry wt) Hydroxyproline Olg/mg dry w t ) 52.6 f 0.5 43.7f 11.3 75.6 f 8.5 85.2f 10.6 Protein dry w t ) (mg/mg 0.48 f 0.09 0.58f 0.07 In Vivo Gold Kinetics-Lymphocyte Binding and Effect on Lymphocyte Responsiveness Arthur Lorber, Timothy Simon, and Stuart Wilcox. Long Beach, California tionation. The lymphocyte fraction, >95% lymphocytes by Geirnsa stain, was removed and washed until the effluent was devoid of detectable gold. Aliquots of 0.25-0.5 ml containing 3 X l(P cells of pre- and post-gold specimens were sonicated, and then 5-pl volumes were quantitated for gold content. This procedure included the application of an internal standard. Aliquots taken before sonication were used to assess the effect of superficial (membrane) bound gold on lymphocyte PHA and concanavallin A response and t o compare these values with the differences in lymphocyte gold content of the I-hour post-gold administration. We observed in all patients a decreased lymphocyte responsiveness and increased gold binding 1 hour post-gold administration, when compared to responsiveness and binding of lymphocytes obtained immediately before gold administration. A consistent suppression of the post-gold injection specimen is a remarkable finding, not attributable to usual sources of variability in the mitogen procedures. Carbon rod atomic absorption analysis (CRA) enables the quantification of gold in soluble and cellular materials of small sample volume ( 1 5 p l ) containing <I0 pg of gold at a precision of 6% RSD. The application of this technique was developed to quantitate the gold content of circulating lymphocytes in RA subjects receiving chrysotherapy. Hence we are able t o monitor blood gold kinetics and binding to lymphocytes and their subpopulations under in vivo conditions during intervals following gold administration. Prior methods for in vivo cellular quantification of gold, i.e., atomic absorption flame analysis, neutron activation analysis. or isotopic applications, have not been practical or feasible for long-term gold administration, because of limited sample size, detection limits, or amount of isotope required. Circulating lymphocyte gold content was quantitated by C R A analysis after timed periods of gold administration. Blood specimens were collected in heparinized tubes and subjected to Ficoll-Hypaque frac- Patient Serum gold fig% Pg Au/ lo* lymphocytes Pre Post Pre Post 1 2 3 4 5 I32 441 1310 1510 99 381 360 490 93 313 210 320 238 495 380 720 205 644 650 3470 PHA c p m X lo3 6 7 n 9 10 94 214 360 234 306 144 45 34 229 666 295 I57 209 745 887 764 214 61 I 339 I32 - Osteonecrosis of the Knee Without Bone Collapse Paul A . Lotke and Malcolm L. Ecker. Philadelphia. Pennsylvania Osteonecrosis of the knee presents with severe pain in the knee, x-ray evidence of subchondral radiolucent lesion in the femoral condyle, and a positive radionuclide bone scan. This report reviews osteonecrosis of the knee and broadens its spectrum to demonstrate that radiographic changes d o not have to be present. Eight patients, aged 58 to 75 years old, presented with the sudden onset of pain over the medial (7) or lateral ( I ) aspect of the knee. The pain was worse at night and unrelieved with antiinflammatory medication. Examination revealed trace synovitis, trace effusion, and marked tenderness just above the joint line. Synovial fluid analysis did not reveal any abnormalities. X-rays were normal in 5 patients, and 3 patients showed minimal degenerative changes. Laboratory studies did not reveal apparent cause for knee pain. In all patients the technetium diphosphonate radionuclide bone scans were markedly positive with definite localization of radionuclide uptake over the affected condyle. Technetium sulfur colloid scans of 2 patients did not reveal abnormalities in the marrow. Craig needle bone biopsies in 2 patients revealed small spicules of dead bone without new bone reaction. An autopsy performed on 1 patient showed an osteonecrotic focus within the subchondral bone plate, localized within 3 mm of the subchondral plate. Histologic section showed a well-localized necrotic area within the subchondral plate. There was a fibrocartilage surface and fibrous tissue reaction within the adjacent marrow space. In summary, we have shown that osteonecrosis of the femoral condyle can present without x-ray changes and it must be considered in patients without other obvious causes for knee pain. Because this problem may resemble diseases treated surgically, i.t. torn meniscus, recognition of this diagnosis can help avoid unnecessary surgery. Association of Autoantibodies to Certain Cytoplasmic Antigens with Systemic Lupus Erythematosus and Other Rheumatic Diseases Peter J . Maddison, Morris Reichlin. and Thomas T. Provost, Buflalo, New York Antibodies t o a soluble cytoplasmic antigen (Ro) have been found in approximately 25% of patients with systemic lupus erythema- tosus. In order t o investigate their clinical significance we have studied 60 patients in whom these antibodies have been identified by double ARA ABSTRACTS immunodiffusion. In 35% of cases they were accompanied by precipitating antibodies to another soluble cytoplasmic antigen, a ribonucleoprotein (La). Complement-fixing antibodies to ssDNA were present in 40%. and in 7 instances anti-Ro was associated with antibodies to nuclear-RNP. Sixty percent of the patients fulfilled the preliminary criteria of the ARA for SLE. The clinical course of these patients resembled that of other patients with lupus, but noteworthy were the presence of arthritis in almost 100%.and a higher incidence of photosensitive skin rashes. Sjogren’s syndrome occurred in 20%. All the patients were remarkable for a high degree of hyperganimaglobulinemia and for positive tests for rheumatoid factor, which occurred in 60% of those with antibodies to Ro alone and in 100% of those who also had antibodies to La. A further 20% had features typical of lupus but failed to fulfill the ARA criteria. A group of 10 patients (17%) had 127 a connective tissue syndrome consistent with lupus, although 5 did not meet the criteria, but their sera lacked demonstrable antinuclear antibodies. Of the remaining 20%. half had evidence of other connective tissue diseases, while the other half had apparently unrelated disorders. Although antibodies to Ro are not exclusively found in patients with SLE, they are most commonly found in this context, with a significant overlap with Sjogren’s syndrome. It is interesting that certain patients respond predominantly to cytoplasmic antigens, with a low incidence of antibodies to the soluble nuclear antigens, nuclearR N P and Sm. Furthermore, we have identified a population of patients with lupus in whom no antinuclear antibodies can be detected at all. ProliferativeSynovitis in Hemophilia: Morphologic and Biochemical Observations Carlo L. Mainardi, Zena Werb, and Edward D. Harris, Jr., Hanooer, New Hampshire Recurrent hemarthroses in hemophiliac patients lead to synovial proliferation, deposition of iron pigment in articular structures, and accelerated joint destruction. Most previous studies have examined the effects of hemarthrosis on articular cartilage. We report here light and electron microscopic studies of the proliferative synovitis along with measurements of neutral proteinase and collagenase release by primary cultures of the synoviuni and by monolayer cultures of enzymatically dissociated synovial cells. Fresh tissue was obtained from synovectomy of the knee of a 10-year-old boy with hemophilia A. The synovium was deep brown in color, quite distinct from the color of rheumatoid synovium. Microscopic studies showed synovial lining cell proliferation without inflammatory cell infiltration and iron pigment in the superficial cells. This pigment was also seen in the dissociated cells and was found t o be membrane-bound by electron microscopy. Many of these cells had Fc receptors on surface membranes. After passage, Fc receptors were found rarely, intracellular pigment decreased, and fibroblast-like cells began to predominate the culture. Medium from primary cultures of synovial fragments was activated with 10 pg TPCK trypsin at 22°C for 30 minutes followed by a 4-fold excess of soybean trypsin inhibitor, and was subsequently assayed for enzyme activity. Large amounts of latent collagenase (73.5 14.2 p g collagen degraded at 37’C/min/mg DNA/48 h) and neutral proteinase (gelatin and azocasein substrates) were found. Lysozyme was detectable in the medium from primary cultures but not from the monolayer cultures. Collagenase secretion (60.25 & 13.5 pg collagen degraded/min/mg cell protein) and neutral proteinase secretion by the monolayers were significant until they were passaged, then the enzyme activity fell precipitously. We conclude that recurrent hemarthrosis in hemophilia stimulates synovial proliferation and an increase in collagenase and neutral proteinase release by synovial cells. The uniform, dense iron deposits are greater than those seen in rheumatoid arthritis, but the proteolytic enzyme release is similar in quality and quantity t o that released by rheumatoid cells in previously reported studies. This proliferative lesion has the potential to destroy articular structures. These observations support the rationale for early synovectomy in selected joints of certain patients with hemophilia. + Abnormal Collagen Synthesis by Cultured Dermal Fibroblasts in Osteogenesis lmperfecta Ralph E. Marcus and Peter G. Bullough. New York. New York Osteogenesis imperfecta (01)is a primary connective tissue disorder that is broadly divided into two clinical types-a severe, spontaneously occurring “congenita” type, and a milder, inherited “tarda” type. Both are characterized by a diminished amount of matrix in tissues normally rich in type I collagen, and to a lesser extent type I l l collagen, its fetal analog. To determine if the normal amounts of types I and 111 collagen are produced by 01 dermal fibroblasts, cells were obtained from the skin of 6 0 1 (4 congenita, 2 tarda) and 4 normal patients and grown in secondary monolayer culture in the presence of ascorbic acid and 6-APN. Procollagen in the medium, labeled with C-14 glycine and proline, was converted to collagen by pepsin digestion, purified on DEAE-cellulose. and finally separated into component a chains o n a CM-cellulose column. In control cultures the ratio of type 111 to total (types I + 111) collagen in the medium was 0.085-0.143 (av: 0.1 16). inversely dependent on patient age. In 3 of the 4 01 congenita cultures, however, the ratios were 0.160-0.259 (av: 0.220). The ratios for the 01 tarda specimens were only slightly elevated above controls. Electron micrographs of the cultured 01 fibroblasts and fibroblasts from intact 0 1 skin revealed the cytoplasm to be packed with increased amounts of microtibrillar material as compared to normal controls. Previous investigators have found that the total amount of skin collagen is reduced in 01 patients and that urinary excretion of cysteine, found in type 111 but not type I collagen. is increased in 01 patients. With this new data we postulate that there is a quantitative defect in the synthesis (or transport) of type 1 collagen in 0 1 congenita, and that in many instances there is a relative increase in the synthesis of fetal type 111 collagen, altering the normal ratio of the two collagen types. Responses of Whole and T-Lymphocyte Populations to Mitogens in Patients with SLE, Rheumatoid Arthritis, and in Normal Volunteers Joseph A . Markenson, John W . Morgan, Catherine L. Joachim, and Michael D. Lockshin, New York, New York To define more precisely the reason for poor lymphocyte response to mitogens in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), mononuclear cells from SLE and RA patients and from normal volunteers were fractionated and tested for ARA ABSTRACTS 128 their responses to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and passed over rabbit anti-human Fab columns. In some experiments half of the initial sample was incubated at 37OC overnight in a glass petri dish t o remove the adherent cell population. Whole mononuclear cell preparations or the separated or reconstituted fractions were cultured in RPMI with 10% AB serum and varying concentrations of mitogen, and were assayed for response at 3 days by H, thymidine uptake. In normals and RA patients the response of the columnpurified T-cell population was less than that of the unfractionated population for PHA, Con A, and PWM. SLE patients demonstrated an increase in the response of isolated T cells compared to unfractionated cells for PHA and Con A, but not for PWM. Removal of the adherent cell population and subsequent fractionation also improved the low response of unfractionated SLE cells but did not alter the response of normal fractionated or unfractionated cells. The data suggest a suppressor cell population among the adherent cells in patients with SLE, which may account for poor lymphocyte response to mitogen. The poor response of lymphocytes from RA patients is not explained by this suppressor cell population alone. Mechanism of Probenecid-Induced Uricosuria: Inhibition of Reabsorption of Secreted Urate Allen D. Meisel and Herbert S.Diamond, Brooklyn, New York , Probenecid-induced uricosuria may be attributed t o diminished reabsorption of either filtered or secreted urate. To study the mechanisms of probenecid uricosuria, 72 clearance studies were performed in 18 normal subjects. Probenecid was adminstered as a I-g intravenous dose. PAH administration as a constant intravenous infusion resulted in a decrease in probenecid excretion (UPbV) (control UPbV. 20.1 f 5.0 pglmin; UPbV during PAH infusion, 9.2 f 3.2 pg/min), and probenecid administration resulted in a decrease in tubular secretion of PAH (TmPAH) (control TmPAH, 103 f I2 mg/min; TmPAH post-probenecid, 55 f 1 1 mg/min, P < 0.01). This result suggests that probenecid and PAH compete for secretion a t a common secretory site, and that urinary probenecid is derived largely from secretion. PAH administration attenuated the uricosuric response to probenecid 13 f 1.7 ml/min, P < 0.005 (dCur) (dCur during PAH infusion, compared t o dCur with probenecid alone), a fact suggesting that probenecid must be secreted in order t o exert its uricosuric effect. Thus the intraluminal concentration of probenecid is likely to be low in the early proximal tubule, where reabsorption of filtered urate occurs, and + higher in a more distal segment of the tubule, where secreted urate is r eabsorbed. Probenecid and uric acid d o not appear to compete for secretion, because urate loading by feeding yeast RNA (16 g/day) did not alter probenecid excretion or affect the uricosuric response to probenecid (control dCur, 19.8 f 2.5 ml/min; dCur after RNA, 24.1 f 7.8 ml/min). Pyrazinamide, an inhibitor of urate secretion, did not alter probenecid excretion (UPbV, 14.3 f 3.6 pg/min; UPbV post-PZA, 16.6 & 3.6 pg/min). However pyrazinamide resulted in a marked decrease in the uricosuric response to probenecid (dCur after probenecid and PZA, -0.5 ml/min). Thus intact urate secretion was required for probenecid t o be uricosuric. It is concluded that a ) probenecid secretion is required for its uricosuric effect; b) probenecid and PAH are secreted a t a common site independent of the urate secretory site; and c) probenecid uricosuria results predominantly from inhibition of reabsorption of secreted urate. Spontaneous Activation of B Lymphocytes and Macrophages in Young New Zealand Black Mice H. M . Moutsopoulos, M . S . Meltzer, J . J . Oppenheim and T.M . Chused, Bethesda, Maryland New Zealand black (NZB) mice spontaneously develop autoimmune disease at about 6 months of age. Because the pathogenesis of their disorder is not understood, we have investigated the status of their immune system very early in life. The activity of B lymphocytes was assessed by measuring the IgM in spleen cell extracts, in supernatants of 4-hour cultures of spleen cells, and in serum with an ultrasensitive two-site immunoradiometric assay. From 2 to 5 weeks of age, 10' NZB spleen cells produced between 21 and 32 ng IgM in 4 hours of culture, compared to I to 2 ng by the same number of NZW or C57BI /6J spleen cells. This increased to 340 ng at.6 weeks and 2100 ng at 10 weeks. Spleen cells from NZW or C57B1/6J mice produced only 5-7 ng a t 8-10 weeks of age. NZB spleen contained 52 ng IgM/IO' cells at 2 weeks, 76 ng at 4 weeks, and 250 ng at 8 weeks. NZW and C57B1/6J spleen contained 5. 6, and 7 ng/lO' cells at 2, 4, and 6 weeks, respectively. Because IgM release requires active metabolism, because the amount released into the medium during4 hours culture is a large fraction of that present in the unincubated cells, and because there is no difference in viability of NZB and control spleen cells, the elevated level of IgM produced by the NZB lymphocytes is not due to cell lysis. NZB serum contains 0.017 mg IgM/ml at 2 weeks, 0.17 at 4 weeks, and 1.8 at 6 weeks. Normal mice have 10-fold less at each of these ages. The level of activation of peritoneal macrophages was assessed by the degree of monocyte chemotactic responsiveness and by the amount of lymphocyte activating factor (LAF, assayed by the stimulation of thymidine incorporation by mouse thymocytes) produced by supernatants of 72-hour cultures of adherent peritoneal cells. Both chemotactic responsiveness and L A F production of peritoneal macrophages from 6-week old NZB mice were elevated 6- t o 8-fold above controls. These findings indicate that both splenic B lymphocytes and peritoneal macrophages are spontaneously activated in NZB mice by 6 weeks of age, long before they develop symptoms of autoimmune disease. Whether this phenomenon is part of an immune response (perhaps to an unusually presented self-antigen) or a primary abnormality that may initiate the autoimmune sequence remains to be determined. ARA ABSTRACTS 129 Neutral Protease from Bovine Nasal Cartilage that Digests Proteoglycan Hideaki Nagase and J . Frederick Woessner, Jr.. Miami, Florida The observation that highly-purified cathepsin D is unable to digest proteoglycan at pHs greater than 5.5 prompted a search for proteases in cartilage that could act at physiologic pH. Such a protease has been detected and purified from bovine nasal cartilage, based o n an assay employing proteoglycan subunit (PGS) of bovine nasal cartilage as substrate. Following a suggestion of A. J . Barrett, the PGS is incorporated in beads of cross-linked polyacrylamide gel. Uronicacid-containing fragments are released from the beads following protease action on the protein backbone of PGS. The bovine enzyme was purified about 4000-fold from crude cartilage extracts by cetylpyridinium chloride precipitation of polysaccharide material, 40-7570 ammonium sulfate fractionation of the supernatant, chromatography on Bio-Gel P- 100, DEAE BioGel-A Sephadex G-50, and C M Bio-Gel-A. The protease has an apparent molecular weight of approximately 15,000daltons based on Sephadex G-50 filtration. The p H optimum is 7.5-8.0, and split products of PGS are shown t o arise by proteolysis, not by splitting of carbohydrate chains. T h e protease is inhibited by EDTA and o-phenanthroline, and activity is restored by several divalent cations. It is concluded that the enzyme is a metalloprotease. N o effect is produced by soybean trypsin inhibitor, TPCK, TLCK, AcAla,CK, Trasylol, iodoacetate, cysteine, EACA, or pepstatin. These properties distinguish the cartilage protease from proteases found in leukocytes and synovial membrane. Such an enzyme may play a role in normal matrix turnover and in pathologic destruction of the matrix. (Supported by NIH Grant AM-16940.) Impaired in Vitro Immunoglobulin Synthesis by Peripheral Blood Lymphocytes in SLE Kenneth M . Nies and James S . Louie, Torrance, California Despite increased in vivo synthesis of immunoglobulin (Ig) in systemic lupus erythematosus (SLE), lymphocytes stimulated in vitro with pokeweed mitogen (PWM) show a depressed Ig synthetic response. Peripheral blood lymphocytes (PBL) (2 X IP) isolated from 20 patients with SLE and 20 normal individuals were incubated at 37°C for 7 days in RPMl 1640 with 10% A B serum with and without PWM. The cultures were washed and pulsed with 'H-leucine for 6 hours in Medium 199 without leucine. Secreted Ig was isolated by immune precipitation and compared to total TCA precipitable radioactivity. Classes of Ig secreted were determined by polyarcylamide gel electrophoresis (PAGE). The total amount of Ig secreted, expressed in cpm, was decreased in SLE compared to normal lymphocytes (6.6*6.4 X I @ vs 24.2f I I .6X IDS), P<O.001. A smaller difference resulted when secreted Ig was expressed as a percentage of secreted protein (15.5f13.7% vs 24.7f I I % ) P=0.05. Unstimulated PBL cultures in both groups secreted little or no Ig. On PAGE, the major Ig class synthesized by PBL in SLE was IgG>lgA>lgM, whereas normal lymphocytes synthesized IgM>lgA>IgG. Co-culture of SLE lymphocytes with normal PBL and PWM produced a slight but not significant increase in Ig synthesis. Co-culture of SLE PBL with nylon wool separated normal lymphocytes (<4% membrane Ig bearing cells), and PWM led to a further depression of Ig synthesis. All abnormalities were most marked in patients with clinically active disease and those taking > lOmg/d prednisone. These findings suggest that nonspecific stimulation of PBL is impaired in SLE and cannot be normalized by co-culture with normal PBL. IgG rather than IgM synthesis suggests a fundamental functional difference between SLE and normal PBL. Modulation of Human Lymphocyte Response by Cartilage Proteoglycans Masaru Nozoe, Marie V . Dennis, and Jerome H . Herman, Cincinnati, Ohio Highly negatively charged proteoglycans (PG), in part sharing antigenicity with those contained in cartilage matrix, have been detected in pathologic synovial fluids and a cell-mediated immune response (CMI) to disruptive P G preparations identified in patients having diverse cartilagenous destructive disorders. Because immunologic events potentially play a significant role in the pathogenesis of articular inflammation, we determined the effect of a broad range of doses of more highly purified PG upon modulation of spontaneous DNA synthesis and phytomitogen (PHA-M) responsiveness of normal and rheumatoid (RA) peripheral blood lymphocytes (PBL) derived by Hypaque-Ficoll gradient sedimentation. PG were isolated by dissociative measures from normal human cartilage in conjunction with cesium gradient ultracentrifugation under aggregative and dissociative conditions to provide aggregate, link protein, and subunit fractions. PBL were cultured for 3, 5, and 7 day periods in microtiter wells containing cells alone, cells + P H A + PG fractions ranging from 200 t o 0.001y,and cells + P H A + P G in varying doses. D N A synthesis was assessed following (25-1OOy), cells 3H-thymidine pulsing. Without affecting PBL viability, PG significantly modified spontaneous D N A synthesis in both a stirnulatory and/or suppressive manner, depending upon the fraction employed, its concentration, and the duration of cell exposure. PG were able to'modulate mitogeninduced D N A synthesis in a similar manner, and total CPM was potentially suppressed and/or enhanced, depending on the quality and quantity of fraction employed. A dichotomy in mitogen response was observed wherein P G suppression of spontaneous D N A synthesis was associated with enhanced PHA responsiveness, spontaneous activation of synthesis by PG, and a suppressed PHA effect. RA response could be clearly differentiated from that of normal subjects. It thus appears that PG in synovial fluid, o n a concentration-dependent basis, may potentially modulate CMI responses. ARA ABSTRACTS 130 Cytotoxic Activity of Rheumatoid Arthritis Sera on Rheumatoid Synovial Cell Cultures Stephen A . Paget. Karen Anderson, and Paul E. Phillips, New York, New York The immunopathology of rheumatoid arthritis (RA) is consistent with a response to a chronic antigenic stimulus. Cell cultures from RA synovial membranes have persistent biochemical and immunologic abnormalities that suggest the in vivo abnormalities may persist in vitro. We have tried t o determine whether R A sera contain antibodies specific for persisting antigens in RA synovial fibroblast cultures by using a complement-mediated cytotoxicity assay. Synovial membranes from 16 patients with definite RA for 0.1-30 years and 10 non-RA control patients were trypsinized and cultured in medium with 20% heat-inactivated fetal calf serum for 1-30 months and 1-9 subcultures before testing as confluent monolayers in Linbro 24-well multiplates. Sera obtained aseptically from 2 I patients with definite RA for 0.3-30 years ( 8 of whom had synovial cultures as well) and non-RA controls were stored at 4'C and heat-inactivated before testing. The RA and control synovial cultures were 61 Crlabeled (Na Chromate, 100pCi/ml in medium at 37OC, 90 min) and washed 6 times. RA and control sera were diluted at 1:2 and 1:8 in medium containing 1:32 freshly thawed rabbit serum (stored at -70°C) as a source of complement and added to the cultures. After incubation (37°C. 60 min), serum dilutions were removed and Cr cpm counted; the cells were then removed by trypsinization and 61 Cr cpm counted. Percent cytotoxicity was calculated as serum cpm X 100/cpm in cells cpm in serum. The positive control was rabbit antinon-RA synovial fibroblast antiserum; negative controls were included. R A sera were tested on both R A and non-RA cells. Preliminary control experiments with herpes simplex virus (HSV)-infected non-RA synovial fibroblasts and HSV-antibody containing sera from both normal and RA patients showed that their HSV cytotoxic antibody titers were similar to their virus-neutralizing antibody titers, and that other constituents of RA serum did not interfere with this known antigen-antibody system. One hundred and twenty experiments using both autologous and heterologous RA culture-serum combinations were then done. No increased cytotoxicity over controls was found. Some RA sera showed anticomplementary activity, which was overcome by using rabbit complement at 1:8; repeat testing of 12 RA sera against 7 RA cultures showed similar negative results. Thus, in this system, R A sera did not contain detectable antibody specific for a persisting antigen in cultured R A synovial fibroblasts. + Circulating Complement-Fixing Immune Complexes in Mixed Connective Tissue Disease Michael D . Parker and Tony Marion, Winston-Salem, North Carolina The presence of high titers of antinuclear antibodies in mixed connective tissue disease (MCTD) reactive with nuclear ribonucleoprotein (RNP) suggests that immunologic factors might be important in the pathogenesis of this illness. T o determine whether circulating immune complexes (IC) might contribute to tissue injury, we have assayed to date sera from 8 patients with MCTD for presence of IC. Results were compared to those from 63 patients with other rheumatic diseases, including 8 with SLE, 46 with rheumatoid arthritis (RA), 3 with scleroderma, 1 with eosinophilic fasciitis, 2 with R A vasculitis, 2 with Reiter's disease, and 1 with psoriatic arthritis. The recently described Raji cell assay was employed to detect IC. Complement-fixing IC bind t o these lymphoblasts via surface complement receptors. Surface-bound IC are then detected by immunofluorescence. The sensitivity of the assay was 15 pg IC/ml Serum (Table). Results demonstrate that circulating immune complexes are found consistently in M C T D and thus establish MCTD as an illness in which vascular deposition of IC may play an important pathologic role. The frequency with which IC were detected was comparable t o that in SLE, wherein IC have been previously found in a similarly high percentage of patients. IC were infrequently found in uncomplicated RA. Preliminary studies indicate that titers of IC were equivalent in SLE and MCTD. Despite the presence in M C T D of circulating IC with the capacity to fix complement, serum concentrations of C3 were normal by radial immunodiffusion. In SLE, IC-especially those involving DNA-have been implicated in the pathogenesis of nephritis. The absence of nephritis among patients with M C T D despite the presence of circulating IC suggests that there is a fundamental difference in the pathologic potential of 1C involving D N A and those involving RNP. Disease No. of patients Percent positive MCTD SLE RA RA-Vasculitis Other 8 8 46 2 7 100 88 4 loo 30 . Characterization of an IgG, Rheumatoid Factor David N. Podell, Martin Isenman, John J . Condemi, and George N. Abraham, Rochester, New York Understanding of the unique properties of rheumatoid factors has been clarified by structural studies of these anti-IgG autoantibodies. These investigations have been primarily confined t o monoclonal IgG-K or IgM-K rheumatoid factors, because automated amino acid sequence analysis of most antibodies with lambda chains is difficult due to blocked amino termini. Protein LON is an IgG,-X cryogel rheumatoid factor isolated from a patient with Atrophie Blanche, a vasculitis that affects only lower extremities and occurs primarily in middle-aged women. It is the first reported human monoclonal IgG-X rheumatoid factor with a defined antigenic specificity and an unblocked amino terminus. Upon analytical ultracentrifugation of se- rum, this protein sediments as a very prominent 7.5 spike, which decreases t o normal concentration after serum is depleted of protein by cryogelation. Immunodiffusion, immunoelectrophoresis, and liquid and polyacrylamide gel electrofocussing were employed to identify and elucidate the nature of the isolated cryogel. LON consisted of at least two serum components: an IgG rheumatoid factor of restricted heterogeneity, and the third component of complement, with isoelectric points at pH 6.75 and 4.83 respectively. Reduction and alkylation of the intact immunoglobulin molecule yielded heavy and light chains which were monoclonal by isoelectric focussing. Automated amino acid sequence analysis revealed the light chains to belong to the V A-iii ARA ABSTRACTS variable region subgroup. The antigenic specificity was determined by hemagglutination-inhibition assay and was nearly equivalent for intact IgG and isolated heavy chains of subclasses 1,2, and 3, but not IgG,. Reactivity was with an antigenic determinant present on the Fc region of IgG. The specificity pattern noted has been reported for some 131 monoclonal IgM-K rheumatoid factors. This protein will be useful in determining the structural similarities or differences between autoantibodies with the same antigenic specificity, but with different light chain variable region subgroups. Chemotactic Attraction of Fibroblasts to a Fragment from the Fifth Component of Complement Arnold E. Postlethwaite. Ralph Snyderman. and Andrew H . Kang. Memphis, Tennessee and Durham, North Carolina Fibroblasts accumulate at sites of tissue damage and inflammation and synthesize new connective tissue elments that constitute the scar. The mechanism(s) by which these effector cells are attracted to such areas is poorly understood. The complement system participates in inflammatory reactions resulting from immune or nonimmune etiologies. We have examined the complement system for a possible source of a chemotactic stimulus for fibroblasts, and have found that activation of the complement sequence by either the classic or alternate pathways generates a factor that is chemotactic for human dermal fibroblasts. Aliquots of normal human serum were incubated with heataggregated y-globulin (HAGG), zymosan (Z), lipopolysaccharide (LPS), or saline (S) fractionated by gel filtration, and effluent column fractions were assayed for fibroblast and monocyte chemotactic activity (CTX). Fibroblast CTX was measured by a sensitive in vitro assay that employs gelatin-treated polycarbonate filters and modified Boyden chambers. Separate peaks of fibroblast CTX (MW: 48,000daltons) and monocyte CTX ( M W 14,000 daltons) were found when HAGG, Z, or LPS activated serum samples were fractionated, but such activity was not found when S- treated serum was fractionated. When fractions from the 48,000and 14,000 dalton peaks weyreacted with monospecific antibody to C5, fibroblast and monocyte CTX in the respective samples were destroyed. The 14,000dalton factor has been previously described as being C5a, a potent anaphylatoxin. Limited proteolysis of the 48,000 dalton factor by trypsin conjugated to polyacrylamide beads was performed, and the reaction products were fractionated by gel filtration. A factor with a MW of 14,000daltons with chemotactic activity for monocytes was recovered, suggesting that the fibroblast chemotactic factor contains a subunit similar to or identical with C5a. Activation of the complement cascade generates a fragment with a MW of 48,000daltons, presumably from the fifth component of complement, that is chemotactic for fibroblasts but not for monocytes. This factor might act as a chemoattractant for fibroblasts in inflammatory reactions mediated by the complement system. Substantial Purification of the S m Antigen and Association of High Titer Antibody to Sm with a Clinical Subset of Systemic Lupus Erythematosus (SLE) Runas Powers, Masashi Akizuki, Marilyn J . Boehm-Tmitt, Virginia Daly, and Halsted R. Holman, Stanford, California and Bethesda, Maryland Specific autoantibodies are associated with certain subsets of SLE, i.e., antibody to DNA associated with nephritis and antibody to nuclear ribonucleoprotein (RNP) associated with mixed connective tissue disease (MCTD). We describe another subset associated with high titer antibody to the Sm antigen and present data on the purification of the Sm antigen. The usual source of the Sm antigen is in the soluble nuclear extract from mammalian cells, where the antigen is thought to be inseparably associated with RNP. However RNP-free Sm antigen can be recovered by a digestion of isolated, insoluble chromatin with DNAse. Ammonium sulfate (3040%) fractionation of the supernatant from DNAse-digested chromatin yields the RNP-free Sm antigen, as determined by the appropriate pattern of enzyme sensitivity using agar diffusion, passage hemagglutination and complement fixation. Chromatin Sm antigen is further purified by affinity chromatography, using Sepharose 4B activated by cyanogen bromide coupled with IgG that has been separated from selected sera of patients with SLE and higher titer antibody to the Sm antigen. The resultant preparation migrates as an apparent homogenous and immunologically reactive band on polyacrylamide gel electrophoresis. Chromatin Sm is stable between pH 5 and 10, but its antigenic activity is lost after heating at 60°C for 45 minutes. Sm antigen is stable in lyophilized form, has certain characteristics of an acidic glycoprotein, is resistant to enzymatic digestion with RNAse and DNAse, but is gradually digested by tryspin. Sixteen consecutive patients with high titer antibody to the Sm antigen without demonstrable RNP antibody were characterized by major skin lesions, clinically mild and apparently nonprogressive nephritis, substantial hypocomplementemia, significant antibody to DNA (demonstrated by binding assay but not by passive hemagglutination), and positive LE cell tests. On the basis of these observations, these patients can be distinguished by at least some characteristics from classic SLE with antibody to DNA, from MCTD, and from patients with antibody to the Ha antigen. Factors Affecting the Wear of Articular Cartilage in Vitro Eric L. Radin, Nicholas J . Myttas, David A . Swann, and Igor L. Paul, Boston, Massachusetts I t is usually assumed that the degradation of articular cartilage by enzymes is enhanced by mechanical factors. The relative contribution of these two mechanisms and the relationship between the wear rate and the chemical structure of the cartilage, however, are not known. In an attempt to answer some of these questions, bovine metatarsal-phalangeal joints were worn under various conditions in a mechanical system and the degree of wear of articular cartilage was determined histologically. The treatments used to alter the structure of the articular cartilage included extraction with 4 M guanidinium chloride, fixation with glutaraldehyde, scarification (A la Meachim) to disrupt the surface, and replacement of subchondral bone under the contact area with methacrylate. The results showed that wear was enhanced when sub- I32 ARA ABSTRACTS chondral area was stiffened with methacrylate, when the cartilage was extracted with 4 M guanidinium chloride and then the wear test run with veronal buffer as the lubricant, and also when the wear test was performed with 4 M guanidinium chloride as a lubricant without prior extraction of the cartilage. Scarification alone had n o effect, whereas glutaraldehyde fixation of the cartilage retarded wear. These data suggest that the proteoglycans contribute significantly to the wear characteristics of articular cartilage, in that under conditions in which these constituents were removed from the cartilage. the wear was increased, whereas following crosslinking reactions that prevent loss of proteoglycan, wear .was decreased. These conclusions, based on the depth of cartilage remaining after the wear test, were supported by histochemical analyses. More detailed chemical studies are now in progress to confirm these observations. (Supported by N I H Grant No. A M 15216.) Sex Differences in the Formation of Anti-T-cell Antibodies E. S. Ravecht, L . Kfassen. and A . D. Steinberg, Bethesda, Maryland Hybrids of NZB and DBA mice were studied at I year of age for the presence of naturally occurring thymocytotoxic antibodies (NTA). All NZB parents had NTA, whereas none of the DBAs did. Half of the F, mice of both crosses were castrated at 5 weeks of age in order to determine the contribution of sex hormones to the mice. The titer was the last dilution giving greater than 50% I’ Cr release. A positive animal had a titer of a t least 1:4 (Table). Castration of the females did not affect the development of NTA, whereas manipulation of the males did. Castrated males reached the level of their normal female littermates. The data suggest that the lack of NTA in intact male NZB hybrids may be due t o the presence of male sex hormones that suppress the development of autoimmunity. Naturally Occurring Thymoeytotoxic Antibodies ( N T A ) Positive NTA Test F, Hybrid NZBQ X D B A d NZBQ X D B A d NZBQ X D B A d NZBQ X D B A d DBAO X N Z B d DBAQ X N Z B d DBAQ X N Z B d DBAQ X N Z B d Sex Q 9 castrated 6 castrated No./Total Percent 11/20 9/16 0/15 10/18 55 53 0 55 2/8 2/7 0/6 2/7 25 29 0 29 0 9 castrated d 6 castrated Synovitis and Periostitis in Secondary Syphilis A.J. Reginato. H.R. Schumacher, F. Valenzuela. S. Jimenez, and K . Maurer. Philadelphia, Pennsylvania and Santiago, Chile Arthritis is frequently described in secondary syphilis, and there have been n o previous studies t o support an infectious basis for the arthritis or periostitis. This report describes clinical, dark-field, serologic, and pathologic studies in 7 cases presenting synovitis or periostitis. There were 6 women and 1 man. Ages were 18-44. The diagnosis of a secondary syphilis was documented by maculopapular generalized rash in 7, oral or perigenital mucus plaques in 5 , positive STS and FTA absorption in 7, and positive dark-field examination of the perigenital mucus plaques in 5 . N o patients had antinuclear antibodies. Two patients presented with elastic, round, mildly painful, localized swelling of the scalp. X rays showed localized bone resorption of the outer table of the skull. Bone biopsy in I case revealed subperiosteal inflammation with gelatinous exudate. There were abundant spirochetes on dark-field study. Four patients had symmetric nonmigratory polyarthritis involving MCPs, PIPS, wrists, knees, and ankles. One of these and one additional patient without arthritis had symmetric tenosynovitis at wrists and ankles. Musculoskeletal manifestations appeared at the same time as rash and other systemic aspects of secondary lues. Synovial fluid in the 4 patients with arthritis was 5-20 cc, cloudy, and yellow with 4,100-13,OOO WBC. PMN predominated in 2 effusions, while PMN and mononuclear cell numbers were similar in 2. Dark-field, silver stain, EM, and fluorescent antibody study on I fluid were negative for treponemes; routine cultures were negative. Synovial needle biopsy in 3 cases showed synovitis with infiltration by lymphocytes, plasma cells and PMN, vascular congestion, and increased synovial lining cells. Electron microscopy in these 3 synovial membranes in addition showed many necrotic PMN, much interstitial debris and fibrin, and in I patient bodies characteristic of treponema pallidum in the interstitium and vessel lumens. There were no electron-dense deposits in vessel walls. In all cases the periostitis and tenosynovitis rapidly subsided with penicillin. Two patients had Herxheimer reactions. Periostitis of the skull, tenosynovitis, and symmetric polyarthritis can occur in secondary syphilis along with the mucocutaneous manifestations. Spirochetes can occasionally be identified in subperiosteal and synoival lesions. Recurrent Myoglobinuria Due to Muscle Carnitine Palmityl Transferase Deficiency Michael J . Reza. Nirmal C . Kar, Pieter Kark. and Carl M . Pearson, Los Angeles, California Recurrent myoglobinuria ( R M ) is a dramatic syndrome commonly thought to be “idiopathic.” The identification of specific defects in muscle carbohydrate metabolism, e.g. myophosphorylase deficiency (McArdle’s syndrome), has led to improved diagnostic ability. Yet there remain patients with RM in whom no defect in carbohydrate metabolism is apparent. Bank et a / have recently described 2 brothers in whom defec- tive muscle lipid metabolism due to absence of muscle carnitine palmityl transferase (CPT) was associated with RM. CPT is an enzyme that facilitates the transfer of cytoplasmic long-chain fatty acids into mitochondria, where oxidation takes place. We have since assayed CPT activity in frozen muscle tissue in all available cases of “idiopathic” R M a t the UCLA Medical Center during the past 10 years. Of I I patients studied, 3 were found to have absent or markedly reduced ARA ABSTRACTS levels of skeletal muscle CPT, compared to 9 normal controls and 5 patients with intermittent muscle cramps without myoglobinuria. Three patients had myophosphorylase deficiency. The 3 CPT-deficient patients had distinctive histories of life-long episodes of muscle pain, weakness, and cramps associated with myoglobinuria. The epidodes were usually exercise- induced, especially with prolonged exercise after fasting, but sometimes occurred spontaneously and could be induced by fasting alone. Patients were entirely normal between attacks. As opposed to patients with McArdle’s syndrome, these patients could sometimes engage in vigorous muscle activity without ill effects. In the I patient so studied, fasting-induced ketosis was absent, and excess 133 lipid was demonstrated on muscle biopsy. Administration of the ketone body, beta-hydroxybutyrate, to this patient resulted in a prompt increase in muscle strength. Our studies show that CPT is probably absent in the liver as well, and emphasize the importance of lipids as a fuel for muscle energy metabolism. In summary, of I I patients studied with RM, we have uncovered 3 additional cases of CPT deficiency, hitherto described in only 2 patients in the world’s literature. In the future, many of the cases of what we now label as “idiopathic” RM may well be traced to specific metabolic abnormalities. Eosinophilia and Serologic Abnormalities in Linear Localized Scleroderma Gerald P. Rodnan. Esther Lipinski. Bruce S . Rabin, and Morris Reichlin, Pittsburgh, Pennsylvania and Bufalo, New York The presence of both rheumatoid factor and antinuclear antibodies, including anti-DNA antibody, has been described in patients with localized (or focal) scleroderma (Hanson et al: Pediat Res 8:806-809, 1974). In an effort to obtain more information concerning the nature of the antinuclear antibodies present in these individuals, we have studied 17 patients with linear localized scleroderma (1 3 F, 4 M), I 1 patients with morphea (single or multiple plaques) (6 F, 5 M), and 2 patients with scleroderma en coup de sabre (2 F). Linear localized scleroderma: Rheumatoid factor (RF) was present in titers of 1:80-1:2560 in 4/16 patients. Antinuclear antibody (ANA) was detected (immunoflourescent technique utilizing rat liver slices) in 7/15 individuals in titers of 1:64-1:256, with a speckled pattern in all. As determined by a radioimmunoassay-double antibody technique, high titers of antibody to single- stranded deoxyribonucleic acid (denatured E coli and calf thymus DNA) were found in the sera of 6/6 patients with strong ANA reactions and in 1 young girl in whom this latter test had been only weakly positive. Four of these seven sera fixed complement with heat-denatured calf thymus DNA, while only one yielded a weakly positive complement fixation reaction with native calf thymus DNA. Levels of C3 and C4 components of serum complement were normal in 14/14 cases. Anti-ribonucleoprotein antibody (anti- RNP) was not found (I4 cases). Peripheral eosinophilia. ranging from 7 to 14% of the total white blood cell count, was noted in 9/14 patients and exceeded 600 eosinophiles per mms in 6. Both the eosinophilia and the serologic abnormalities were limited to patients with evidence of clinically active disease. These observations, together with the presence of heavy infiltrations of lymphocytes in the affected skin, subcutaneous tissue, and muscle suggest that immunologic mechanisms may play an important role in the pathogenesis of this form of localized scleroder ma. Morphea and scleroderma en coup de sabre: Serum immunoglobulin and complement levels (C3, C4) were normal in all patients, and serum RF, ANA, anti-I)NA, and anti-RNP reactions were. uniformly negative, with the exception of a low titer of anti-RNP (l:2560) in 1 case. Augmentation of Immunoglobulin (Ig) Production in Connective Tissue Disorders (CTD) I . Jon Russell, Doyt L . Conn, Charles H . McKenna. and John D. Stobo. Rochester, Minnesota Compelling evidence indicates the importance of soluble products of lymphocyte metabolism in the regulation of Ig synthesis by B lymphocytes. We have investigated the possible relationship of such factors to polyclonal elevations of Ig noted in 26 patients with systemic lupus erythematosus (mean total serum Ig = 20.1 mg/ml), and 6 patients with mixed connective tissue disease (mean total serum Ig = 18.3 mg/ml, normal = 14.2 mg/ml). Peripheral blood mononuclear cells (PBMC) from these patients and 16 normal individuals were suspended in 5% fetal calf serum (FCS) and incubated in a humidified atmosphere of 95% air, 5% CO,, at 37OC. At 2, 4, and 7 days, aliquots of culture fluid were removed and added, in a final concentration of 25%. to allogeneic normal PBMC in 5% FCS. These admixtures were then cultured for 7 days, without any allogeneic normal PBMC in 5% FCS. These admixtures were then cultured for 7 days, without any mitogens. The net total Ig synthesized and released during this time was measured by a competitive binding radioimmunoassay. After 48 hours of culture, PBMC from 18 of the patients (mean total serum Ig = 20.7 mg/ml) released a soluble material (AF) capable of augmenting Ig production (mean 6.0-fold). In contrast, culture fluids from 14 patients (mean total serum lg = 19.6 mg/ml) and the 16 normals failed to do so (mean augmentation = 1.1-fold). Comparable augmentation of lg synthesis by A F occurred despite variable histocompatibility differences between patients and the target PBMC. Culture fluids from patients’ T-cell enriched (92 4% T ) or Bcell and monocyte enriched (50 f 4% lg bearing, 40 f 8% esterase positive cells) preparations were relatively depleted of AF. This result indicates that interactions between these two populations may be required for its production. The ability of A F to augment Ig synthesis among target populations depleted of T cells was markedly reduced, a fact suggesting that augmentation of Ig synthesis by A F was T dependent. However AF was not blastogenic for T cells. Fractionation of culture fluids by column chromatography and density gradient centrifugation indicated that A F has a molecular weight of approximately 190,000 daltons. Most importantly, AF could be specifically depleted by solid support media to which anti-lg or monoclonal rheumatoid factor was covalently linked. These studies suggest that in some patients with CTD and hypergammaglobulinemia, an immune complex may augment Ig synthesis by B cells, perhaps through its effect on regulatory T-cells. + 134 ARA ABSTRACTS Multiple Forms of Neutral Metalloprotease Activity from Human Cartilage Asher I . Sapolsky, Harold D . Keiser. David S. Howell, and J . Frederick Woessner, Jr, Miami. Florida and Bronx, New York Human articular cartilage contains a novel metal-dependent neutral protease that can digest the proteoglycan of the cartilage matrix. Because this protease also penetrates cartilage slices and depletes them of proteoglycan, it is postulated that it may play a role in the pathogenesis of osteoarthritis and in normal matrix turnover. The neutral protease can be resolved into four distinct electrophoretic forms distributed between two weight groups of I1,OOO and 22,000 daltons. The forms are cationic, with isoelectric points up to pH 8.4. The protease forms are all inhibited by o-phenanthroline and D-penicillamine; this inhibition is reversed by zinc, cobalt, and ferrous ions. Three of the four protease forms are inactive against casein and histone, a fact indicating a certain degree of specificity for the proteoglycan substrate. This protease is believed to belong to a previously undescribed group of mammalian metalloproteases. A 400-fold purified preparation of this neutral activity that includes all the forms digests proteoglycan subunit optimally at pH 7.25, and produces fragments containing 5-12 chondroitin sulfate chains. However it does not digest the link proteins of proteoglycan complex nor destroy the antigenic determinants of the complex or link proteins. No single or fragmented chondroitin sulfate chains are produced. Thus the human cartilage neutral protease degrades proteoglycan by a limited attack on its protein core. Clq Metabolism in a Clq-Deficient Lupus Syndrome Frank R . Schmid, Adoracion F. Suarer, Chester R . Zeiss, Adrian M . Jaffer,and Henry Gewurz, Chicago, Illinois An unusual lupus-like syndrome (urticaria, erythema multiforme, vasculitis) has been described, in which Clq levels are disproportionately reduced compared to other complement proteins. We studied the metabolism and the assembly of radiolabeled Clq from normal donors in 2 such patients. The Clq tracers gave a single sharp 1 IS peak on ultracentrifugation and a single preciptin line by autoradiography in gel diffusion against whole human antiserum. They were >80% precipitable by immune complexes, and <0.67 pg restored hemolytic activity to 0.1 ml RClq reagent. In contrast to normals and classic lupus subjects, both patients showed an accelerated catabolism Patient Clq1-D Clql-B SLE-L SLE-R Normal-S Normal-Z of Clq without marked change in synthesis (Table ). Addition of the Clq preparation to one Clq-deficient serum doubled the number of functional CI molecules. In both patients 1 IS Clq rapidly became incorporated into a series of heavier (> 11-19s) units after intravenous injection or after in vitro addition to serum. Such units were dissociable by EDTA into lighter (<15-llS) units. Thus Clq appears to be formed and assembled normally into larger (CI?) units in these patients, but it is more rapidly catabolized by mechanism(s) still to be determined. Clq level Olg/ml) TM (hrs) Hourly Fractional Plasma Pool Catabolism Hourly Plasma Pool Turnover Rate (mg/kg) 32 14 .065 .064 45 8 28 28 32-48 39 .02 ,015 .015 ,096 .092 .072 160 180 180 250 - - Decreased Cytotoxic Potential in Synovial Fluid in Rheumatoid Arthritis Stuart L. Silverman, Donna M . Yonkosky, and Edgar S. Cathcart, Boston, Massachusetts Cytotoxicity in synovial fluid of patients with RA was found to be markedly decreased (7%) as opposed to synovial fluids of patients with gout (73%) and septic arthritis (50%). In contrast, cytotoxicity in peripheral blood of patients with RA (31 f 12%) was found not to be significantly different (31 & 12%) from that ofcontrols (24 f 12%). The amount of slCr released depended dosage of PHA and was directly related to the number of effector cells in suspension. There is evidence that both polymorphonuclear leukocytes and monocytes are effector cells in this assay (J Clin Invest 57:826, 1976). However, despite high percentages of both these cell lines in synovial fluid of patients with RA, the cytotoxicity was found to be markedly decreased. This abnormality in synovial fluid as opposed to normal findings in peripheral blood suggests a local phenomenon in synovial fluid. These observations have relevance in the clearer under- standing of the total cytotoxic potential of synovial fluid in RA and other disease states. Cellular cytotoxicity of synovial fluid and peripheral blood cells using the mitogen-induced cellular cytotoxicity (MICC) assay was studied in patients with erosive, seropositive rheumatoid arthritis (RA), and in controls. Hyaluronidase-pretreated synovial fluid cells and Ficoll-Hypaque-purified peripheral blood cells were cultured with chromated Y r chicken red blood cells in the presence of phytohemagglutinin (PHA). Percentage of cytotoxicity was determined by the release of W r from chicken red blood cell targets. Monocytes were quantified by the nonspecific serine esterase stain (NSE). Polymorphonuclear (PMN) and mononuclear (MN) cells were determined by a Wright Giemsa stain. Synovial Fluid Cytotoxicity (Mean f Standard Deviation) Diagnosis RA Gout Septic arthritis WBC/m ma % PMN % MN % NSE % MICC 13,000f6.000 9,700f700 37.000f 17,000 6 0 f I4 86f13 91f 6 40f I4 15f10 IOf 6 17f3 7f 2 73fll 50f20 10 2fI ARA ABSTRACTS 135 Total Hip Replacement (THR) in Children with Arthritis Bernhard H . Singsen. Alvin S . Isaacson, Michael J . Patzakis, Bram H . Bernstein, Helen K. Kornreich, Karen K . King, and Virgil Hanson, Los Angeles and Downey. California THR for rheumatoid hip involvement in children under 18 years old has received little attention in the literature. Between 1972 and 1975, 8 girls and 6 boys with severe, polyarticular juvenile rheumatoid arthritis, and 2 boys with ankylosing spondylitis, received 29 THRs. The median age at disease onset was 6.8 years (range: 1-13), the median duration of hip involvement was 7 years (range: 2-12), and the median age at THR was 15.2 years (range:12-18). All 29 hips had radiographic evidence of severe destructive changes, and none showed evidence of further growth potential. Clinically, 5 of 29 THRs were for pain alone, 16 for pain and severe flexion contractures (FC) and 8 because of severe FC alone. Among the 16 children, 7 were complete nonambulators, 6 household ambulators with support, and 3 limited community ambulators without support. At the time of THR 12 children had severe, bilateral knee involvement; 4 had normal knees. Previous hip surgery, done in 5 patients, included 8 synovectomies, 6 soft tissue releases, and 1 cup arthroplasty. The median preoperative hip FC was 35" (range: 10-85'). median hip flexion range of motion (ROM) was 25" (range: 0-80"), and the median abduction was 8' (range: - 15-35"). All prostheses were the Charnley type, and many were custom made because of small patient size. The greater trochanter was transplanted in 26 of 29 cases. Postoperative complications were limited to low-grade fever (<38.5") in 20 cases All 29 cases had 1 day of preoperative and 3 days of postoperative intravenous antibiotics. The median follow-up duration was 2.2 years (range:l-4), at which time hip ranges were: median FC 20" (range: 0-45'), median hip flexion ROM 61" (range: 35-105'), median abduction 20" (range: -5-35"). All children had complete pain relief after the initial postoperative period. The ambulatory status included 4 full community ambulators, 7 limited community ambulators, 4 household ambulators with support, and I non-ambulator. Late postoperative complications were 2 femoral shaft stress fractures, 1 acetabular component subluxation, 1 acetabular cement stress fracture, and 1 painful trochanteric wire. At follow-up, 15 of 16 children felt they were improved. THR can be successful in the severely afflicted rheumatoid child. Motivation and cooperation are limiting factors, but the late results are encouraging. Ferric Hydroxide Macroaggregates as Particles for Radiation Synovectomy Clement B. Sledge, Jonathan Noble, Sonya ShortkroJ, and Donald J . Hnatowich, Boston and Cambridge, Massachirsetrs Radiation synovectomy has been used in Europe, but possible side effects have precluded its acceptance in the United States. These include lymphocyte aberrations (10% of patients), and leakage of OOYfrom treated knees (52-91%). If leakage could be prevented, the advantages of this simple procedure would be enormous, especially since the results of radiation synovectomy compare favorably with those of surgical synovectomy. Our approach is based on the principle of having a short-halflife, beta-emitting particle, which could be readily produced and whose tissue penetration was effectively confined to the rheumatoid synovium. Retention of such an isotope in the joint is made possible and leakage minimized by incorporating it with biodegradable particles of a given size. The pure beta emitter "'Dysprosium, whose halflife is 137 minutes and which has a Mev of I .3, fulfilled these criteria. Ferric hydroxide macroaggregate and laDy can be associated with 99% efficiency. The 137 minute half-life of Dy prevents its use for tracer experiments. Therefore we used another rare earth, I%d, whose half-life is 240 days, but whose chemistry is similar to Dysprosium. The ferric hydroxide-lS3Gd macroaggregate was injected into the knees of rabbits. Eleven normal rabbits were injected and sacrificed at 5 hours, and a further 10 at 24 hours. The amount ofactivity in tissue specimens was expressed as a percentage of the dose injected into the knee. Greatest leakage was <0.33% in the liver. Identical experiments were then repeated in 7 rabbits with antigen-induced arthritis in which leakage rates were nearly identical to those found in normal knees. Finally, a further 7 normal rabbits were studied by means of a preparation of Fe(OH), and lsaDy. Leakage in this group given a therapeutic dose of lesDy was extremely low, a result suggesting improvement in particle preparation. Our conclusions are a) In all cases the leakage was <0.3390 and was greatest in the liver. b) Leakage in the arthritis model is similar to that in normals. c) A rabbit model has been produced in which the leakage of a short half-life, beta emitting-isotope, bound to ferric hydroxide macroaggregates never exceeded 0.33%, 5 and 24 hours after injection. This rate of leakage would be clinically acceptable. Xanthine and Uric Acid as Xanthine Oxidase Inhibitors Hugh A . Smythe, Toronto, Ontario, Canada A dramatic feature of primate evolution has been the rise in plasma urate concentrations, so that human values are about 10 times higher than those of monkeys or nonprimates. This elevation has been achieved in steps, by the deletion of uricase and by complex alterations in renal transport mechanisms. No biologic function served by this change has been identified. Uric acid (UA) is not re-utilized, and its immediate precursor, xanthine (X), is re-utilized only slightly. Hypoxanthine (HX) is however, extensively and rapidly re-utilized, and absence of its re-entry in the Lesch-Nyhan syndrome has destructive effects on human brain function. Flexible inhibition of xanthine oxidase (XO) by X and UA might protect HX levels at critical periods. The kinetics (KI) of XO inhibition by X and UA were therefore studied, and the results are shown in the table on the next page. The KI of X is close to its normal plasma concentration, so that it would be a very effective inhibitor of HX oxidation, especially in the postabsorptive period when purine supply must switch from exogenous to endogenous sources. The relatively high KIs for UA make a biologic role for hyperuricemia less obvious. However, at 7 mg%, inhibition of xanthine oxidation is sufficient to raise by 64% the concentration at which xanthine formation and oxidation are in equilibrium. For technical reasons, little is known about the diurnal variations for metabolism of Hx and X, but the results of these in vitro studies indicate the need for in vivo studies of the inhibitory effects of X and UA on xanthine oxidase functions. ARA ABSTRACTS 136 Source of XO Substrate KI ( r M ) Inhibitor KI ( r M ) KI (mg%) Human liver HX HX X 16 16 II X UA UA 9.3 4600 I600 0. I4 77.0 27.0 Induction of Cell-Derived Chemotactic Factor (CF) and of Arthritis by Amorphous Diamond Crystals Isaias Spilberg, David Rosenberg, and Jagdish Mehta. St. Louis, Missouri The phagocytosis of Na urate or Ca pyrophosphate crystals by neutrophils leads to the generation of a chemotactic factor (MW 8400). Current evidence suggests that the above CFs are identical. Therefore experiments were designed to test the ability of diamond dust to induce generation of chemotactic activity from neutrophils and to produce arthritis in rabbits. Peripheral blood neutrophils (6 X 10’) were incubated with 6 mg of diamond dust (2-12 microns) for 45 minutes at 37OC. Cells were disrupted and the lysosomal fraction was obtained by differential centrifugation. The fraction was passed over a calibrated Sephadex G-50 column, and the fractions were tested for chemotactic activity by radioassay. Chemotactic activity was found localized at 0.1 I of eluate (MW 8400). When the chemotactic eluate was placed on polyacrylamide gel electrophoresis, a line representing the chemotactic material appeared at the same distance from the cathod as the chemo- tactic factor induced by Na urate or Ca pyrophosphate crystals. Control fractions showed no activity. In a separate set of experiments, 10 mg of diamond dust in 0.3 ml saline was injected into the left knee joints of 6 rabbits. Contralateral joints were injected with saline. Animals were killed at 4 hours, synovial membrane was fixed, and cell counts were made in synovial fluid. Marked leukocytosis was observed in the joints injected with diamond dust, and only minimal leukocytosis in control joints. In conclusion, amorphous diamond dust is capable of inducing generation of chemotactic factor from neutrophils following phagocytosis and also of producing a violent arthritis in vivo. Although evidence is incomplete, it is conceivable that the generation of CF may represent a uniform response of the neutrophils to phagocytosis of different crystals and perhaps other materials. Lyme Arthritis: Serum Cryoprecipitates Associated with Skin and Joint Lesions Allen C. Steere. John A . Hardin. and Stephen E. Malawista. New Haven, Connecticut We have reported a cluster of 51 patients who developed a similar type of inflammatory, oligoarticular arthritis, “Lyme arthritis,” the epidemiology of which suggests transmission of a causative agent by an arthropod vector (Arthritis Rheum 19:824, 1976). Onequarter of the 51 patients described a preceding skin lesion thought to be an arthropod bite and consistent with the entity “erythema chronicum migrans.” Because circulating immune complexes might occur in this disease, we tested sera for cryoprecipitates in 14 new patients who developed the skin lesion, with or without subsequent arthritis, in June or July 1976. The initial lesion, usually on a proximal extremity or the trunk, was a red papule that developed into an expanding, red circle, as large as 50 cm in diameter, often with central clearing. The patients had a median of 5 such lesions (range: 1-20) that lasted from 0.5 to 8 weeks. All 14 patients had profound fatigue, 13 fever, 9 stiff neck and headache, 7 backache, 5 myalgia, 3 nausea and vomiting, 2 malar rash and periorbital edema, and 1 Bell’s palsy and sensory neuropathy of the TI0 dermatome. In the table below the patients have been grouped according to whether the skin lesion was acute (group A ) or fading (group B), or gone recently but followed by a first attack of arthritis (group C). Three additional patients had recurrent attacks of arthritis during this period (group D). As the table shows, there was a strong tendency for cryoprecipitates to be associated with active skin lesions-in all of 6 patientsand with attacks of arthritis-in 5 of 7, plus the 2 in group A, who also had active skin lesions. We are currently examining the presence and composition of cryoprecipitates in individual patients serially. Correlation Between Clinical Activity of Skin or Joints and Presence of Cryoprecipitates Skin Arthritis Total Patients Total Group A: Active Group B: Fading Group C: Gone, 2-4 wks Group D: Gone, 1 year (2 with 1st attack) None 1st attack Recurrent attack 6 4 4 3 6 0 3 2 Delinition of GrouDs Patients with Cryoprecipitates IBM IgG Both 0 0 2 0 1 0 2 1 4 0 I 0 Circulating DNA in SLE Patients with Active Vasculitis and/or CNS Disease Charles R. Steinman, New York. New York DNA, in association with antibody, has long been thought to play a central role in the pathogenesis of SLE. Until recently, however, only conflicting information was available regarding the prevalence of circulating DNA in normals or in patients with SLE. With improve- ARA ABSTRACTS 137 ments in technique and the recognition by Davis and Davis that most serum samples contain D N A released in vitro, it has been recently shown that normal plasma contains n o detectable DNA. Surprisingly, the prevalence of plasma D N A in SLE, determined by a technique capable of detecting as little as 1.5 pg/ml of plasma, was reported not t o differ significantly from that in normals. In the present study, a modified counterimmunoelectrophoresis technique capable of detecting as little as 20 t o 50 ng/ml of DNA in plasma (and which shows no DNA to be present in normals) was used to examine, blindly, plasma from patients with active SLE. Persistent circulating D N A was defined as detection of DNA in plasma samples collected o n at least 2 different days. Patients with a clinical condition other than SLE that had been previously known or suspected of being associated with circulating DNA were excluded. Because preliminary studies suggested possible association between circulating D N A and active vasculitis and/or C N S involvement, the presence of persistent D N A in patients with these manifestations was specifically examined. A total of 43 patients with suitable specimens were studied. Eighteen were found t o be positive for persistent circulating DNA. In patients with C N S involvement, 8 of 11 (73%) were positive. Of those with vasculitis with or without concomitant C N S disease, 8 of 13 (62%) were positive for circulating D N A . Of the combined group with C N S disease and/or vasculitis, 16 of 24 patients (67%) were positive. Of patients with active SLE but lacking vasculitis or C N S disease, 2 of 19 (1 1%) had circulating D N A . N o correlation with active nephritis was noted. Neither inflammatory disease per se, as judged either clinically or by elevation of the ESR, nor high dose corticosteroid therapy could account for these findings. It is therefore concluded that the presence of circulating DNA may be clinically useful as a diagnostic tool in some SLE patients. Further elucidation of the source, mode of release, and consequences of circulating D N A may shed light on pathogenetic mechanisms currently not understood. Reversible Renal Insufficiency in Systemic Lupus Erythematosus M . Sugarman. A . Kamdar, B . H . Barbour. F. P. Quisrnorio, S . Massry, and E. L. Dubois. Los Angeles, California Advanced renal failure in SLE has been associated with a universally poor prognosis. In the past 5 years we have seen 7 of 43 SLE patients with renal insufficiency (serum creatinine 4 mg/dL) whose renal function improved; 6 survived 2.5 to 4 years. Six presented with rapid renal deterioration extending over weeks, associated with SLE activity both clinically and immunologically. None had significant hypertension with the renal failure. Treatment included dialysis in 4. Three of these patients improved without recent treatment with corticosteroids o r other immunosuppressive drugs. Two of these 3 had been declared drug failures. The first underwent renal transplantation, graft rejection, and removal of the transplanted kidney 4 months prior t o improvement in her natural kidney’s function. Renal function of the other drug failure improved after 11 months of chronic hemodialysis. Of the 3 not requiring dialysis, all received high-dose corticosteroids, and in addition 1 was given intravenous nitrogen mustard. Renal histopathology in 5 showed active diffuse proliferative glomerulonephritis. Significant interglomerular variability in SLE was noted; some glomeruli were obviously lost, whereas others showed marked inflammatory reaction, which may be reversible. Inflammation involved the interstitium, which may have adversely affected renal function. Vascular changes were minimal and no fibrin thrombi were noted. These patients demonstrate that this disease is too variable to indicate withholding therapy because of poorly functioning kidneys. Even patients with apparent end stage kidney disease may improve. Renal biopsy evidence of intact glomeruli, profound interstitial and glomerular inflammation, and normal arteries in a patient with rapid renal deterioration and multisystem disease may be helpful in determining which patients have a more favorable prognosis. The role of hemodialysis in these patients is uncertain, but it has been shown to activate the alternative pathway of complement, which may dissociate immune complexes. Augmentation of Leukocyte Motility by a Neutrophil Cytoplasmic Factor Akiteru Takeuchi and Robert H . Persellin, San Antonio, Texas The chemotactic attraction of polymorphonuclear (PMN) leukocytes is one requisite of the inflammatory reaction. We have presented evidence that directional cell motility is influenced not only by the concentration of the chemotactic attractant, but also by cell density. The number of P M N leukocytes required for the augmentative response must be adequate to permit cell-to-cell contact. Further studies of this cytoplasmic factor were performed. Directional cell motility was studied in a modified Boyden chamber. Normal human neutrophils were layered on a 3-p Millipore filters by means of a cytocentrifuge. E coli lipopolysaccharide-activated serum served as the chemotactic stimulus. Granulocyte cytoplasm was found t o activate cellular response. Fractionation of isolated neutrophils in hypertonic sucrose was performed by differential centrifugation. Neither the nuclear (400 X g, 10 min), the lysosoma\ (8,200 X g, 15 min), nor the microsomal fractions (1 hour at 100,00 X g), would activate cell migration in response t o the chemotactic stimulus. However the post-microsomal supernatant fraction, when placed in the top chamber in direct contact with viable cells, increased chemotaxis, and this action was dose-dependent. Activation was not due to protein content alone, because equivalent amounts of protein from either other subcellular fractions or human serum albumin failed to cause a comparable migration. Heating at 56°C for 30 minutes did not alter the activity, but dithiothreitol at 5 mM inhibited its effect. Studies performed with the cytoplasmic fraction either alone or in combination with the chemotactic factor in the top and/or bottom chambers indicated that the cytoplasm did not function as a chemotaxin but acted t o potentiate the cellular response to C5a. To further investigate this phenomenon, cell electrophoresis was performed. Using a Rank Mark I1 cytophorometer, we measured the surface charge of cells suspended in 5% sorbitol, 0.13 mM phosphate buffer. Chemotactic factors decrease the negative surface charge and increase cellular motility. The lysosomal fraction, previously shown to contain a chemotaxin, prolonged cell motility 10.35 f 0.32 seconds (SEM) being required for travel of 48 p . However, the cytoplasmic fraction, at an identical protein concentration, did not alter motility, 7.45 f 0.26 seconds being the same as control. Thus the cytoplasmic activator of neutrophil migration does not induce directional migration but renders a cell more responsive to a chemotactic stimulus. ARA ABSTRACTS 138 Specific Extraction of DNA Antibodies by Extracorporeal Circulation over DNA Immobilized in Collodion Charcoal David S. Terman, Ronald I . Harbeck, and Ronald Carr, Houston, Texas and Denver, Colorado It is now well recognized that tissue inflammation in some cases of systemic lupus erythematosus (SLE) is mediated by antibodies to DNA which, when combined with circulating DNA, give rise to pathogenic immune complexes. The selective withdrawal of circulating DNA antibodies would therefore be a valuable therapeutic measure. A novel immunoadsorbent was prepared in which significant quantities of DNA were entrapped in collodion membranes adherent t o small particles of activated charcoal. These D N A collodion charcoal (DCC) particles were placed in an extracorporeal circulation system, and arterial blood was circulated through a continuous flow celltrifuge in which plasma was separated from formed elements. Rabbit anti-BSA and human anti-DNA antibodies were passively infused into mongrel dogs, and only plasma was pumped through the DCC at a flow rate of 40 ml/min. Complete removal of DNA binding activity in serum was observed over the ensuing 15 to 30 minutes, with only a slight change in BSA binding during the same time period. There were no significant changes in lZsI DNA on the charcoal before and after the perfusion experiments and no recovery of lZsl DNA in the serum, urine, or vital organs of dogs at the conclusion of the studies. Further, there were no demonstrable hematologic or biochemical alterations. These findings suggest that this extracorporeal immunoadsorbent may rapidly and specifically withdraw circulating DNA antibodies with minimal host toxicity and may offer a specific approach to therapy of SLE when it is desirable to remove D N A antibodies. The Arthritogenicity of Type I1 Collagen: A New Animal Model David E. Trentham, Alexander S. Townes, and Andrew H . Kang, Memphis, Tennessee Immunity t o collagen could play a role in inciting or perpetuating the inflammation that occurs in the connective tissue diseases. We have found that about 40% of Wistar rats injected with Type I I collagen from human or chick cartilage in incomplete Freund's adjuvent (ICFA) develop an inflammatory arthritis and both humoral and cell-mediated immune responses to the Type I1 immunogen. Outbred female Wistar rats were injected intradermally on the back with 0.5 mg of soluble native collagens extracted from human skin (Types I and HI), or human or chick cartilage (Type 11) with complete or ICFA. Booster doses consisting of the immunogen without adjuvant were given intraperitoneally on day 21. Forty-three of 106 rats injected with four separate Type I1 collagen preparations developed a chronic, erosive polyarthritis 15 to 60 days following primary immunization. Peak incidence occurred at 20 days. Type I or Ill collagen, Type II without adjuvant, MgCI, extractable cartilage proteoglycan, denatured a l ( I I ) chains, and complete or ICFA were not arthritogenic in a total of 206 rats. Booster doses of Type II collagen as long as 6 months after the initial immunization resulted in recurrence of inflammation only in previously arthritic animals. Pre- liminary results also indicate induction of arthritis following injection of homologous rat Type I I collagen. Passive hemagglutination using antigen fixed to glutaraldehyde-treated human 0 red cells indicates a significant (P < 0.001) correlation of mean antibody titers to Type I1 with arthritis (mean in 27 arthritic rats, 1 :5120; mean in 39 nonarthritic rats, I : 160). Peripheral blood lymphocyte cultures show type-specific transformation to Type I1 [mean stimulation index (SI) in 15 arthritic rats 10.8; in 14 nonarthritic rats 3.31 that also correlates significantly with arthritis (P < 0.05). Rats injected with Types I and 111 collagen yielded lower antibody titers (mean 1:40 with I 1:80 with 111) and lymphocyte transformation (mean SI 2.8 with I, 2.7 with Ill). N o antibody or transformation to collagen was observed in normals or rats immunized with cartilage proteoglycan. The demonstration of immunity to collagen in this new animal model of arthritis suggests a potential role for cartilage type of collagen in provoking or perpetuating joint inflammation in the rheumatic diseases. + Immunologic Study of Arthritis Associated with Adenovirus Infection Peter D. Utsinger, Chapel Hill, North Carolina Three unrelated patients who developed an acute arthritis associated with a febrile upper respiratory illness (URI) were studied. All were females and the age range was 18-35 years. The arthritis began 3 to 8 days after the onset of the URI and was symmetric involving large and small joints of the upper and lower extremities. On joint examination the involved joints were red, hot, and tender. Two patients had a macular erythematous rash. N o lymphadenopathy or hepatosplenomegaly was found. Synovitis decreased gradually and after 4 weeks the joint examination was normal. Lymphocytotoxic antibodies (LCTA) were determined by a microlymphocyte cytotoxicity assay. T lymphocytes were determined by spontaneous rosetting with sheep blood cells, B lymphocytes by surface immunoglobulin using F(ab'), antisera; the Raji cell test for immune complexes (IC) was done by the method of Dixon. C 3 and C4 were determined by a nephelometric technique. The presence of adenovirus infection was proved by throat culture and complement fixation. None of the patients had cold agglutinins, rheumatoid factor, Australia antigen, DNA antibodies, or antinuclear antibodies. Gonococcal and mycoplasma cultures were negative. All patients had serum and synovial fluid (SF)cryoglobulins and IC as determined by the Raji cell test. When Raji cells were incubated with these serum cryoglobulins and subsequently stained with a fluorescein-conjugated adenovirus antiserum (FCAA), immunofluorescent staining was found. Cryoprecipitable protein (CP) found in the S F of these patients also fixed to Raji cells and were also stained with FCAA. IgG and C 3 were also found in the cryoprecipitates. LCTA were found in 2 of the 3 patients. These antibodies had specificities for membrane antigen determinants on both T and B lymphocytes. Selective concentration of the LCTA in the cryoprecipitates was not found. The peripheral blood lymphocyte populations of these patients were decreased with proportionate reductions of both 7 and B lymphocytes. Serum C 3 and C 4 were decreased in 2 patients and normal in the third. Synovial fluid white blood cell counts were less than 4000/mma in 2 patients with predominance of mononuclear cells, and 8500/mma in the third with predominant PMNs. C D and IC were not detectable 3 to 4 weeks after the onset of the illness. These data suggest that serum and intraarticular IC are involved in the pathogenesis of adenovirus-associated arthritis. ARA ABSTRACTS 139 Immunologic and Therapeutic Study of Erosive Osteoarthritis Peter D. Utsinger, Donald Resnick, and Robert Shapiro, Chapel Hill, North Carolina, San Diego, and Sacramento, California The purpose of this study was to define immunologic abnormalities and the efficacy of intraarticular steroid treatment in erosive osteoarthritis (EOA). In 12 females and 3 males, originally seen because of a dip or pip arthritis, the diagnosis of erosive osteoarthritis was made on blind reading of hand radiographs. The patients, whose mean age was 62 (range: 48-70) were followed for 18-22 months. Constitutional symptoms consisted of morning stiffness less than 1 hour and rare afternoon fatigue. The initial joint examination was notable for erythema, warmth, and swelling of either pip or dip joints in all patients, with symmetry in 1 1. Four patients had knee effusions. Lymphocytotoxic antibodies (LCTA) and histocompatibility typing were performed by microlymphocyte cytotoxicity assays. T lymphocytes (L) were determined by spontaneous rosetting with sheep blood cells, and B-L by surface immunoglobulin using F(ab’), f r a g ment antisera. C 3 and C4 were determined by a nephelometric technique. Ficoll-Hypaque separated L were transformed by different PHA concentrations in 2-4 cultures. M L C were performed by the miniaturized microtechnique of Hartzman. Serum rheumatoid factor and A N A were absent. Serum C3 and C4 were normal. ESR ranged from 18 t o 43 with a mean 30. The percentage of T- and B-L was normal. L PHA and M L C responses were normal. There was no significant deviation of HL-A antigen frequencies as compared t o normals. Analysis of dip or pip synovial fluid revealed a WBC less than 2,000/mma in 10 patients and a WBC from 2,000 to 16,000/mm8 in 5 patients. LCTA were absent. Each patient had individual joints injected with 10 mg triamcinolone hexacetonide. Prompt decrease in objective synovitis was noted after 26/33 injections. Radiographs taken from 6 to 18 months after the injections revealed a n increase in erosions in 7/33 injected joints, no change in 18, and a decrease in 4. Similar changes were noted in noninjected joints. Marked pip subluxation developed in 2 injected joints and in none of the noninjected joints. These data d o not support the concept of significant immunologic abnormalities in EOA and suggest that little permanent benefit accrues from intraarticular steroid injection in EOA. Structural and Immunologic Changes of Synovium of Hypertrophic Osteoarthropathy (HPO) Angel F. Vidal. Roy D. AItman, Victoriano Pardo, and Duane Schultz, Miami, Florida Recent interest in immunology of malignancy and Schumacher’s findings of electron-dense deposits (Arthritis Rheum 19:629, 1976) in synovial blood vessels of patients with H P O have raised the question of whether the synovitis of H P O is due to an immunemediated mechanism. We attempted to confirm the presence of electron-dense particles in H P O synovitis and to investigate the possible immune origin of this disease. A study was made of 2 subjects with bronchogenic carcinoma and HPO-related synovitis. Synovial biopsies were examined by light microscopy (LM) and electron microscopy (EM), and cryostat sections were reacted with fluorescein-conjugated antisera t o the following human immunoglobulins: polyvalent IgG, IgA, IgM, C3, and C4. Functional hemolytic complement assays were assessed in synovial fluid utilizing control fluids from degenerative arthritis. The H P O synovial fluids were noninflammatory, and synovial LM demonstrated minimal infiltration with inflammatory cells. EM revealed lesions involving segments of the microcirculation underlying the synovial lining cells. Some capillaries and venules were dilated and contained masses of platelets, necrotic desquamated cells, occasional lipid droplets, and dense granular material in their lumens. Many endothelial cells exhibited swollen cytoplasm-poor in organelles-with endothelial cell gaps. Rare electron-dense deposits were observed in the subendothelial areas of thickened capillary walls. In this, the first investigation of its kind to our knowledge, there was no localization of fluorescein-conjugated immunoglobulins or complement components in the synovium, and functional synovial fluid complement levels were within the range of the matched controls. The etiology of HPO synovitis remains unknown. The electron-dense deposits of the inner portion of the vascular walls with the cytoplasmic abnormalities may represent a permeation of the vascular wall by plasma components as a result of increased vascular permeability. The preliminary results of our study, with normal complement assays and absence of immunoglobulin deposits in the synovia, d o not support an antigen-antibody immune deposit mechanism. Autoantibodies in Palmerston North (PN) Mice, a New Animal Model of SLE Sara E. Walker, Maureen Fulton. Robert H. Gray, and Richard D. Wigley,Ann Arbor, Michigan and Palmerston North, New Zealand In 1966 Wigley described polyarteritis-like lesions in outbred P N mice (Nature 21 1 :319). The current study investigated the natural history of spontaneous autoimmune abnormalities, vasculitis, and glomerulonephritis in these animals. Colonies of PN mice were established, consisting of mice selected for antinuclear antibodies (ANA) through 3 generations and inbred for 9 generations. Ninety mice were followed t o death to determine life spans, and 19 of these animals were bled a t 6-month intervals until death and autopsied. Ninety additional mice aged 1-19 months were bled to study age-dependent autoantibody formation. Serum was tested by indirect immunofluorescence o n guinea pig stomach, kidney, aorta, and liver. Anti-double-stranded DNA was assayed by modified Farr technique; binding greater than 20% was abnormal. Ten kidneys from mice aged 4-21 months were tested by direct immunofluorescence. Tissue from mice aged 14 and 18 months was exam- ined by electron microscopy. Results were compared with data from autoimmune B/W mice, recognized a s animal models of SLE. Mean life spans in PN mice were 1 I .4 months in females and 14.1 months in males (P < 0.01). Vasculitis was found in 10/19 old autopsied mice, and 11/19 mice had glomerulonephritis. Lymphomas arose in 10% of these animals. Sera from 35% of mice aged 6 months produced specific fluorescence of stomach smooth muscle and renal arteries. These positive tests persisted throughout life. ANA were positive in 55% of mice aged 6 months and 92% of mice I2 months of age. Anti-DNA were first detected in 6-week-old mice. Mean DNA binding increased from 22% in mice aged 4-5 months to 30% in mice aged 12 months. Granular deposits of IgG and C 3 were found in glomeruli of all kidneys tested. Irregular thickening of glomerular basement membranes and dense subendothelial deposits were identified in renal tissue examined by electron microscopy. Thymic tissue ARA ABSTRACTS I40 contained cytoplasmic viral particles 700 A in diameter that were not identical to C-type viruses. Like B/W mice, female PN mice died earlier than male mice. Although anti-DNA appeared earlier in PN mice, PN renal disease was less severe than B/W glomerulonephritis. Thymic ultrastructure was different in both strains of mice. PN mice are an anti-DNA producing model simulating human SLE more than polyarteritis. Endogenous Activation of Collagenase Secreted by Rheumatoid Synovial Cells: Evidence for the Role of Plasminogen Activator Zena Werb, Carlo L. Mainardi, Carol A . Vater. and Edward D. Harris, Jr., Hanover. New Hampshire Adherent cells obtained by dissociation of human rheumatoid synovium secrete collagenase in a latent form (E,,)into culture medium. We report that conversion of EL t o active collagenase (E') may be mediated by another enzyme produced by these cells, a plasminogen activator. Previous studies have shown that exposure of conditioned culture medium to trypsin resulted in activation of EL t o E'. Plasmin, a proteinase that could be found in rheumatoid joints, also activated EL; 100 pM of plasmin/ml (2OoC, 30 min) completely activated EL to E'. Both E' and E,, bound to reconstituted guinea pig collagen fibrils. Substrate-bound EL was resistant to inhibition by a,-macroglobulin but could be activated by adding plasmin to the system. Addition of plasminogen (Pg) alone did not activate EL. The capacity of synovial cells secreting EL t o activate Pg to plasmin was examined next. Pg activator was assayed by growing cells on radioactive fibrin layers in the presence of 5% acid-treated dog serum, a source of Pg. Time-dependent release of fibrin peptides occurred when Pg was present, but not when the serum used first had been depleted of Pg by passage through lysyl-Sepharose. Addition of purified Pg to the cultures reconstituted the system. The Pg-dependent fibrinolysis also could be measured in serum-free culture medium from these cells. In the presence of an excess of plasmin inhibitors such as a,-macroglobulin of a,-proteinase inhibitor in culture medium, no active plasmin was formed and collagenase was found only in latent form. In the absence of such inhibitors, addition of 10 pM of Pg/ml to the cultures for 48 hours resulted in conversion of the Pg t o plasmin and activation of El, to EE' (collagenolytic activity capable of degrading 8 pg of fibrillar collagen/min/ml a t 37°C). These data suggest that in rheumatoid arthritis, collagenase is secreted in a latent form, EL. that is trapped locally by binding to collagen fibrils, its natural substrate. The endogenous activation of EL to E' may be accomplished by plasminogen activator released from the synovial cells. Plasmin is generated which, after saturating proteinease inhibitors present locally, can activate EL to E', and collagen degradation can begin. Marrow-Dependent (M) Cell Function in Casein-Induced Experimental Amyloidosis Donna Yonkosky and Edgar S . Cathcart, Boston, Massachusetts We have previously reported that mitogen-induced cellular cytotoxicity is completely depleted in the amyloidotic CBA mouse. Recent results from our laboratory indicate that an effector cell in the MlCC assay is a marrow-dependent ( M ) cell. M cells are responsible for rejection of selected marrow allografts in lethally irradicated recipients, and there is evidence that the same cell population may control resistance to Friend virus and Listeria monocytogenes infections. In the present study normal WB and CBA. strontium 89, and caseintreated CBA mice were lethally irradiated and injected with 2-4 X 16' WB marrow cells. Four days later cellular growth was detected by uptake of iodinated deoxyuridine in spleen tissue. Results are reported with respect to 100%growth in syngeneic animals (Table). Marrow allograft rejection was completely abolished following multiple casein injections. Similar results were obtained in CBA mice receiving spa, a bone-seeking isotope that destroys marrow and selectively eliminates M cells. Increased rejection of marrow allografts was noted during the preamyloid phase, at a time when MlCC is also markedly enhanced. Thus MICC and marrow allograft rejection, two functions that detect M cell activity, are increased in the preamyloidotic phase and depleted in the amyloidotic phase. These new findings are additional evidence that M cells play a key role in pathogenesis of amyloid. Percent Growth WRCBA CBA/preamyloid CBA/amyloid CBA/S~~ 2 X I@ cells 4 X l(P cells 100 100 46 0 0 100 100 0 100 100 Prostaglandin E, (PGE,) Treatment of NZB/NZW Mice Robert B. Zurier, Donna M . Sayadoff, Ivan Damjanov, and Naomi F. Rothfield, Farmington. Connecticut Because experimental evidence suggests that prostaglandins might modulate immune/inflammatory responses, NZB/NZW F, mice were treated with PGE,. After 1 year of treatment (with 200 pg PGE, sc once or twice daily from 6 through 58 weeks of age), 18 of 19 female mice survived, whereas only 2 of 19 control (saline sc once or twice daily) female mice were alive. Male mice treated with 200 pg daily PGE, were also protected: 10 of 1 I alive vs 2 of 9 untreated control mice alive at 58 weeks. The 9 female mice treated with PGE, twice daily were sacrificed at 58 weeks. Immunofluorescence and light microscopy studies done on kidney tissue were compared to kidneys from 9 untreated female control mice that had died of nephritis (proteinuria > 2+, BUN > 25) between 6 and 10 months of age. All of the untreated NZB/NZW mice exhibited heavy (4+ fluorescence) diffuse and globular glomerular deposits of IgG, whereas there were only minimal (*-2+) mesangial or focal deposits of IgG in PGE,-treated mice.