Frequencies of epstein-barr virus В Эinducible IgM anti-IgG B lymphocytes in normal children and children with juvenile rheumatoid arthritis.код для вставкиСкачать
959 I FREQUENCIES OF EPSTEIN-BARR VIRUSINDUCIBLE IgM ANTI-IgG B LYMPHOCYTES IN NORMAL CHILDREN AND CHILDREN WITH JUVENILE RHEUMATOID ARTHRITIS SHERMAN FONG, JOHN J. MILLER Ill, TEKRY L. MOORE, CONSTANTINE D. TSOUKAS, JOHN H. VAUGHAN, and DENNIS A. CARSON The relative frequencies of IgM antiIgG autoantibody (rheumatoid factor) producing cells induced by the polyclonal B cell activator Epstein-Barr virus were measured in peripheral blood lymphocyte cultures of normal children and patients with juvenile rheumatoid arthritis. The frequencies of rheumatoid factor precursor B cells in normal children were lower than adults, but higher than neonates. The frequency increased with the age of the donor. In seronegative children with the systemic-onset or pauciarticular-onset types of juvenile rheumatoid arthritis, the number of IgM antiIgG inducible B cells was not significantly different ( D 0 . 0 5 ) from age-matched controls. Patients with seropositive juvenile rheumatoid arthritis or seropositive adult rheumatoid arthritis had significantly higher IgM antiIgG precursor cell frequencies than age-matched normal subjects (P<O.O1 and P<0.02, respectively). In contrast, the patients with seronegative polyarticular-onset juvenile rheumatoid arthritis had an average precursor frequency significantly lower than normal age-matched -_. - Supported by National Institutes of Health Grants AG 02267, AM 25443, AM 21 175, AM 07144, AM 00364. and KK 00833. Sherman Fong, PhD: Assistant Memher, Dept. of Clinical Research, Scripps Clinic and Research Foundation; John J. Miller, 111, MD: Director, Rheumatic Disease Service, Children’s Hospital at Stanford; Terry L. Moore, MD: Associate Professor, St. Louis University School of Medicine; Constantine D. Tsoukas, PhD: Fellow, Department of Clinical Research, Scripps Clinic and Research Foundation; John H. Vaughan, MD: Head. Division of Clinical Immunology, Scripps Clinic and Research Foundation; Dennis A. Carson, MD: Associate Member, Dept. of Clinical Research. Scripps Clinic and Research Foundation. Address reprint requests to Sherman Fong, PhD, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla. CA 92037. Submitted for publication September 4, 1981; accepted in revised form January 19, 1982. Arthritis and Rheumatism, Vol. 25, No. 8 (August 1982) controls (P<0.05), analogous to results previously noted in adult seronegative rheumatoid arthritis. Thus, both children and adults with seronegative polyarticular rheumatoid arthritis had a deficiency in B cells that produce IgM antiIgG and that are induced by EpsteinBarr virus. This distinguished them from seropositive juvenile rheumatoid arthritis and rheumatoid arthritis patients, normal subjects, and patients with the pauciarticular-onset and systemic-onset types of seronegative juvenile rheumatoid arthritis. In both normal adults and patients with rheumatoid arthritis (RA), Epstein-Barr virus (EBV) infection of peripheral blood B lymphocytes (PBL) induces the secretion of IgM antiIgG antibodies (I). The ability to produce some IgM antilgG is present at birth in most normal children, and the idiotype of the antilgG was inherited in I family ( 2 , 3 ) . However, as determined by limiting dilution analysis, the relative numbers of IgM antiIgG precursor B cells in the blood of normal subjects increase several-fold between birth and adulthood (2). The EBV-induced IgM antilgG response of PBL from adult patients with R A differs from control subjects in two important aspects. First, as expected, seropositive RA patients’ lymphocyte cultures produce significantly more IgM antilgG than normal lymphocyte cultures ( l , 4 ) . Second, PBL from adult patients with persistent seronegative RA produce notably less IgM antilgG than normal lymphocytes (4). The latter result suggests that I ) the peripheral bloods of adult seronegative patients are deficient in an EBVinducible subset of IgM antiIgG precursor B cells, without any generalized lack of response to the virus, and 2) the deficiency might be a marker characterizing FONG ET AL 960 seronegative RA as a disease entity distinguishable from seropositive RA and other forms of arthritis. It is now clear that juvenile rheumatoid arthritis (JRA) encompasses a spectrum of diseases with overlapping clinical manifestations (5). Depending on the assay used and the source of the patient population, 15-68% of JRA patients have detectable IgM antiIgG antibodies in serum or in the IgM-containing fraction isolated by acid-gel filtration (6). The purpose of the present investigations was to determine the frequency of EBV-inducible IgM antiIgG precursor B cells, both in a large series of normal children of increasing age and in patients with different onset-types of JRA. The results show that the frequency increases progressively during normal childhood. Children with seropositive JRA have significantly higher EBV-inducible IgM antiIgG frequencies in PBL than age-matched normal subjects. In contrast, children with the seronegative polyarticular-onset type of JRA, as defined by absence of both overt and “hidden” rheumatoid factor, have significantly lower frequencies of EBV-inducible cells that produce IgM antiIgG in PBL than do patients with seropositive polyarticular-onset JRA, patients with seronegative systemic-onset or pauciarticular-onset JRA, or normal children of the same age. MATERIALS AND METHODS Subjects. Blood samples were obtained from 21 children with polyarticular-onset JRA, 12 with pauciarticularonset JRA, 7 with systemic-onset JRA, 2 with systemic lupus erythematosus, 1 with dermatomyositis, 1 with linear scleroderma, 1 with progressive systemic sclerosis, 1 with sarcoidosis, 1 with subcutaneous nodules, 1 with Raynaud’s disease, 1 with thyroid carcinoma, and 1 with WiskottAldrich syndrome, as well as from 10 healthy children. Additional blood samples came from 4 patients with adultonset seropositive RA and from 15 normal adults. The diagnosis and classification of the JRA patients met the criteria established by the Committee on JRA of the American Rheumatism Association (7). All the children were patients at the Pediatric Arthritis Clinic of St. Louis University at Cardinal Glennon Memorial Hospital in St. Louis, MO or the Children’s Hospital of Stanford at Stanford, CA. Clinical laboratory tests at these two institutions included the latex fixation test (LFT) for rheumatoid factor (RF), a test for antinuclear antibody (ANA), and Clq binding radioimmunoassay for immune complexes (8,9). Complementfixing IgM R F was analyzed by Dr. Terry L. Moore, as previously described, both before and after acid-gel separation of serum on Sephadex G-200 (10,ll). Patients are referred to as “hidden” RF positive if their complement fixing IgM antiIgG titer was >]:I6 in the enriched IgM fraction, but not in whole serum. Cells and cell cultures. Two to 3 ml of heparinized blood specimens were added to 5-ml vials that contained 2 ml of Lebovitz L-15 medium (Flow Laboratories, Inc, Rockville, MD) with 0.01M Hepes buffer. On the day that they were drawn, all blood specimens were sent by air freight to La Jolla and were processed within 24 hours in each case. Several duplicate samples obtained at different times from the same subjects yielded comparable results. Peripheral blood mononuclear cells were isolated immediately on amval by Ficoll-Hypaque density sedimentation (12) and yielded an average cell viability of 80 2% (SEM), as measured by trypan blue exclusion. The cells were infected with a dose of EBV-containing supernatant fluid from B95-8 marmoset lymphoblastoid cells that had been previously titered to yield immunoglobulin secretion and cellular transformation in 100% of the cultures by 28 days (1,2). The infected cells were incubated at a density of 3 x 105 cells/ml in 96-well culture trays (Corning Glass Works, Corning, NY) with 0.3 ml FWMI-1640 medium per well, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ ml streptomycin (Flow Laboratories). The average number of cultures obtained was 42 2 (SEM) for 60 individuals. Ten cultures served as noninfected controls. The microcultures were incubated for 28 days, and the supernatant fluids were analyzed for IgM antiIgG autoantibodies and total IgM by solid-phase radioimmunoassay as previously reported (2). The relative frequencies of EBV-inducible cells that produced IgM antiIgG were based on the fraction of the total lymphocyte cultures producing IgM antiIgG, determined exactly as described previously (2). Positive cultures were scored as those secreting 220 ng/ml of IgM antiIgG. The relative frequencies were determined according to the equation for the Poisson distribution P(,,=e-@, where P(,) is the proportion of nonresponding wells, X is the number of cells cultured per well, and C$ is the relative responding cell frequency (13,14). In control experiments, monomeric IgG at concentrations up to 1 mg/ml did not affect the IgM-RF assay (15). This result is indicative of the enhanced avidity of interaction between multivalent antigen and IgM antibody in the solid-phase radioimmunoassay , as compared with the liquid phase. Hence, it is extremely unlikely that the small quantities of IgG in the culture supernatants (approximately 2001,OOO ng/ml) masked the detection of cultures that secreted IgM-RF. * * RESULTS Patient data. A summary of the clinical classification, sex, age, and laboratory data on all subjects is shown in Table 1. Only 2 patients in the polyarticular JRA classification had serum LFT titers >1:20. The same 2 patients and 2 other children had complementfixing rheumatoid factor in the partially purified IgM fraction. None of the pauciarticular- or systemic-onset JRA patients had LFT titers >1:20, although 6 had detectable complement-fixing rheumatoid factor in their separated IgM-containing fraction. All JRA patients had active disease at the time the tests were done. IgM ANTI-IgG B LYMPHOCYTES IN CHILDREN 96 1 Table 1. Patient clinical classification, sex. age. and laboratorv data No. patients with positive results/ total number tested RF Activity Clinical classification Females/ males Polyarticular-onset JRA Pauciarticular-onset JRA Systemic-onset JRA Other disease controls Normal children 1813 715 215 713 515 Age 13 9 8 12 12 2 SD 4 2 5 2 2 f5 c5 2 LFT* Hidden RFt ANA$ Clq binding§ 2/14 0112 017 013 0110 4/12 4/10 217 013 Oil0 519 6/12 015 1/3 0110 411 1 218 417 013 0/10 * Latex fixation test results positive (titer >1:20). 7 “Hidden” rheumatoid factor (RF), results positive (titer >1: 16). $ Antinuclear antibody test results positive (titer > 1:40)(mouse liver substrate). § Liquid Clq binding assay (8), results positive >9.5%. Frequencies of EBV-inducible cells that produce IgM antiIgG. Table 2 shows the frequencies of EBVinducible IgM antiIgG B cells in the children with JRA as compared with controls and children with other diseases. Two JRA patients whose PBL were seropositive in unfractionated serum had significantly higher frequencies of cells that produce IgM antiIgG autoantibody than did normal age-matched controls (RO.01). In contrast, seronegative polyarticular-onset JRA patients had IgM antiIgG precursor B cell frequencies significantly lower than normal controls (P<O.OS). Seronegative pauciarticular, systemic-onset, and “hidden” RF positive JRA patients showed insignificant differences from normal subjects or disease con- trols. For comparative purposes, the frequency of EBV-inducible, IgM antiIgG B cells in adult RA patients and normal adults is also listed in Table 2. Quantities of IgM antiIgG antibody and total IgM in cultures. Under the limiting dilution culture conditions described, IgM antiIgG autoantibody responses were confined to only a fraction of the individual culture wells. Shown in Table 3 are the average levels of IgM antiIgG and of IgM detected in the positive and negative wells. Similar levels of EBVinducible IgM antiIgG antibodies and of IgM were secreted in positive cultures in all the groups tested. Moreover, wells positive and negative for IgM antiIgG antibodies secreted similar levels of IgM. The “hid- Table 2. Frequencies of EBV-inducible cells that produce IgM antiIgG in the peripheral blood of normal healthy children and patients with juvenile rheumatoid arthritis* Groups and number of subjects tested 1. 2. 3. 4. 5. 6. 7. 8. 9. Normal children (n = 10) Seronegative$ pauciarticular-onset JRA (n = 8) Seronegative$ systemic-onset JRA (n = 6) Seronegative$ polyarticular-onset JRA (n = 17) “Hidden” seropositive JRA§ (n = 7) Seropositive polyarticular-onset (n = 2) Other disease controls (n = 10) Adult seropositive RA (n = 4) Normal adult (n = 15) Frequency of cells that produce IgM antiIgG in PBL 2 SEM 4.2 2 1.4 x 6.3 ? 3.8 x lo-‘ 8.4 f 6.4 X 1.6 2 0.5 X 12.2 2 5.1 X 28.5 2 17.5 x 5.7 ? 3.0 x 15.0 ? 3.0 X 7.6 2 1 . 1 X P valuest >O. 1 >O. 1 <0.05 >0.05 <0.01 >o. 1 <0.02 - * EBV = Epstein-Barr virus; PBL = peripheral blood lymphocytes; SEM = standard error of the mean; JRA = juvenile rheumatoid arthritis; RA = rheumatoid arthritis. t P values determined by Student’s t-test, compared with normal age-matched controls. $ Seronegative as determined by the latex fixation test or by the hemolytic assay for hidden rheumatoid factor testing. 5 Seropositive by the hemolytic assay for hidden rheumatoid factor testing. This group includes 4 pauciarticular, 1 systemic-onset, and 2 polyarticular JRA patients. FONG ET AL 962 tests than do children with earlier onset of disease. For this reason, it was necessary to compare the EBVinducible IgM antiIgG precursor frequencies in JRA patients with an age-matched control population. In comparison with the other 3 groups studied (normal children, patients with other chronic inflammatory diseases, and children with seronegative JRA of any onset type), the overtly seropositive JRA patients had significantly elevated EBV-inducible IgM antiIgG precursor B cell frequencies (28.5 t 17.5 per 106 PBL, P<O.Ol). Subjects with a positive complement-fixing antiIgG in the IgM-containing fraction partially depleted of IgG by acid-gel filtration had a somewhat higher frequency of EBV-inducible IgM antiIgG B cells from the normal age-matched controls, but this difference did not reach statistical significance. This group consisted of the so-called hidden rheumatoid factor patients among whom normal or aggregated IgG obscured the determination of accurate IgM antiIgG concentrations in whole serum (6). It probably includes patients like those found by Ziff et a1 (17,18) in early experiments to be positive for antiIgG only in euglobulin fractions of serum. In inhibition studies, the hidden IgM RF isolated from JRA patients has specificity similar to IgM RF-plaque-forming cells in adult subjects (19,20). In children, hidden RF titers correlate with the presence of active disease and immune complexes as detected by Clq solid-phase assay (6,21). The EBV-inducible IgM antiIgG precursor cell den” RF seropositive JRA patient group secreted slightly higher levels of IgM ( l Y 0 . 0 5 ) in noninfected control cultures than the other groups tested. Age-associated development of IgM antiIgG precursors. We have previously shown that the frequency of EBV-inducible IgM antiIgG cells was lower in neonates (1.5 t 0.5 per 106 PBL) than in young adults (20-40 years old, 8.0 k 1.8 per lo6 PBL) and aged adults (75-90 years old, 7.2 ? 1.3 per lo6 PBL) (2). Figure 1 extends this analysis to pre- and postpubertal normal children. As can be seen, children between the ages of 12 to 18 years exhibited higher frequencies than children between the ages of 3 to 11. The data are consistent with a gradual age-associated increase in the frequency of IgM antiIgG autoreactive B cells. DISCUSSION The frequency of EBV-inducible IgM antiIgG autoreactive B cells in the peripheral blood of normal humans increased gradually from birth to young adulthood (2). Thus, healthy children (average age 12 ? 4 years) had an estimated frequency of 4.2 ? 1.4 IgM antiIgG precursor B cells per lo6 PBL, a value 52% of that detected in young adults. Moreover, children 1218 years old had a higher frequency than children between the ages of 3 to 11. The percentage of latex fixation-positive JRA patients is related to the age of disease onset (16). Children between the ages of 12 and 16 years have a higher incidence of positive results on latex fixation Table 3. Average levels of IgM antiIgG and total IgM produced by EBV-stimulated peripheral blood lymphocytes* X IgM antilgG ngiml f + EBV Clinical classification I . Normal children (n 2. Seronegativet 3. 4. 5. 6. 7. IgM antiIgG positive wells = 10) pauciarticularonset JRA (n = 8) Seronegativet systemiconset JRA (n = 6) Seronegativet polyarticularonset JRA (n = 17) “Hidden” RF seropositive JRAt (n = 7) Seropositive polyarticularonset JRA (n = 2) Other disease controls (n = 10) 73 f 2.5 56 2 6 X IgM ng/ml t SEM SEM - IgM antiIgG negative wells 511 t 2 513 + EBV EBV - EBV Controls IgM antiIgG positive wells IgM antiIgG negative wells Controls <I0 758 t 174 799 t 178 18 f 6 2 <10 1111 f 202 1072 f 172 517 t 3 <I0 894 t 1.57 848 t 170 98 t 20 48 ? 63 * 11 48 f9 57 f 1 <I0 927 129 720 f 50 t 6 511 t 3 <10 994 t 179 99.5 * 107 511 1 < 10 983 f 17 932 ? 515 f 3 <lo 1240 2 239 216 2 174 63 * 13 f f 103 90 1000 t 105 20 f f 9 10 284 t 126 60 f 31 64t6 * EBV = Epstein-Barr virus; % = mean; SEM = standard error of mean; JRA =juvenile rheumatoid arthritis; R F = rheumatoid factor. t Seronegative as determined by the latex fixation test or by the hemolytic assay for hidden rheumatoid factor testing. t Seropositive by the hemolytic assay for hidden rheumatoid factor testing. This group includes 4 pauciarticular-onset, 1 systemic-onset, and 4 polyarticular-onset JRA patients. IgM ANTI-IgG B LYMPHOCYTES IN CHILDREN neonates n:ll 3-11 12-18 20-40 n=6 n:4 n:8 75-90 n=10 AGE in Y E A R S Figure 1. Relative frequencies of EBV-inducible autoantibody-secreting lymphocytes as a function of age. Data shown for the neonates, the young adults (20-40 years old), and the aged adults (75-90 years old) were obtained from previously published information (2). frequency in the polyarticular JRA patients seronegative for both overt and hidden RF had a mean of only 1.3 0.4 per lo6PBL, a value significantly lower than age-matched controls and equivalent to the IgM antiIgG precursor frequency assayed in cord blood. This deficiency in EBV-inducible cells that produce IgM antiIgG did not result from a lack of polyclonal B cell activation by EBV in the seronegative polyarticular JRA patients. In contrast, seronegative patients with the systemic-onset or pauciarticular-onset forms of JRA did not differ notably in precursor frequencies from normal children. The peripheral blood lymphocytes from all groups secreted equivalent amounts of IgM after in vitro EBV infection and developed into permanent B lymphoblastoid cell lines. Thus, among the subsets of JRA patients analyzed, only the overtly seropositive and seronegative polyarticular groups yielded EBV-inducible IgM antiIgG precursor cell frequencies significantly different from age-matched controls. Indeed, the results in the seropositive JRA and seronegative polyarticular JRA subgroups were remarkably similar to the findings previously reported in adult seropositive RA and seronegative RA, respectively (4). * 963 It is possible that many cases of RF-positive polyarthritis of childhood are the juvenile equivalent of classic adult seropositive RA. There are notable clinical similarities between the 2 syndromes (22). Besides the raised numbers of EBV-inducible IgM antiIgG precursor cells in each disease, both are associated with an increased frequency of HLA-DR4, compared with control populations (23,24). The relative deficiency in EBV-inducible B cells that produce IgM antiIgG in the peripheral blood of patients with both seronegative polyarticular-onset JRA and seronegative adult RA suggests an immunologic relationship between these 2 diseases. With modern radioimmunoassay techniques, only rarely have IgG antiIgG antibodies been detected in persistently seronegative patients (25 and DA Carson, unpublished). The incidence of HLA-DR4 is not increased in either population (23,26). The exact reasons for the deficiency in EBVinducible IgM antiIgG precursors in seronegative polyarticular RA patients of all ages remain unresolved. Genes related to the primary sequence of IgM antiIgG antibodies are inherited in some (and possibly many) individuals but are unlinked to the HLA locus (3). A few EBV-inducible IgM antiIgG precursor B cells are detectable at birth in nearly all normal subjects (2). The gradual increase in precursor cell frequency between infancy and young adulthood in normal subjects could be the consequence of immunizations, childhood infections, or an internally programmed maturation of the immune system. In any one or all of these aspects, seronegative RA patients could differ from their normal counterparts. In this regard Van Snick has recently shown that the ability to make antiIgG antibodies in mice is genetically controlled (27). However, even in genetically high responding strains, the elicitation of autoantibody depended on the particular environmental conditions under which the animals were maintained (28,29). Similarly complex interactions between genes and the environment probably govern EBV-inducible IgM antiIgG production in humans. To summarize, the frequencies of IgM antiIgG precursor B cells inducible by EBV increased with age in normal children. Patients with seropositive JRA had higher precursor frequencies than age-matched normal controls. Children with seronegative systemic-onset , pauciarticular-onset, and hidden rheumatoid factor seropositive JRA had IgM antiIgG precursor cell frequencies appropriate for their ages. However, children with polyarticular-onset seronegative JRA, like their adult counterparts, had EBV-inducible, IgM antiIgG FONG ET AL 964 precursor cell frequencies significantly lower than normal. Our conclusions are 1) that seropositive JRA is probably the childhood variant of adult seropositive RA, and likewise 2) that polyarticular-onset seronegative JRA and adult seronegative RA are closely related or identical diseases, different from seropositive RA and from the pauciarticular-onset and systemic-onset types of JRA. ACKNOWLEDGMENTS The authors wish to thank Ms Lee A. Frincke and Ms Lynne Olds for their technical assistance, and Ms Shari Brewster and Ms Anna Milne for their preparation of the manuscript. REFERENCES 1. Slaughter L, Carson DA, Jensen FC, Holbrook TL, Vaughan JH: In vitro effects of Epstein-Barr virus on peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal subjects. J Exp Med 148: 1429-1434, 1978 2. Fong S, Tsoukas CD, Frincke LA, Lawrance SK, Holbrook TL, Vaughan JH, Carson DA: Age-associated changes in Epstein-Barr virus-induced human lymphocyte autoantibody responses. J Immunol l26:9 10-9 14, 1981 3. Pasquali J-L, Fong S, Tsoukas CD, Vaughan JH, Carson DA: Inheritance of immunoglobulin M rheumatoid-factor idiotypes. J Clin Invest 66:863-866, 1980 4. Pasquali J-L, Fong S, Tsoukas CD, Hench PK, Vaughan JH, Carson DA: Selective lymphocyte deficiency in seronegative rheumatoid arthritis. Arthritis Rheum 241770-773, 1981 5. Hanson V, Kornreich HK, Bernstein B, King KK, Singsen BH: Three subtypes of juvenile rheumatoid arthritis: correlations of age at onset, sex, and serological factors. Arthritis Rheum (suppl) 20: 184-186, 1977 6. Moore T, Dorner RW, Weiss TD, Baldassare AR, Zuckner J: 19s IgM hidden rheumatoid factor in juvenile rheumatoid arthritis. Pediatr Res 14: 1135-1 138, 1980 7. Brewer EJ, Bass J, Baum J, Cassidy JT, Fink C, Jacobs J, Hanson V, Levinson JE, Schaller J, Stillman JS: Current proposed revision of JRA criteria. Arthritis Rheum (suppl) 20:195-199, 1977 8. Miller JJ 111, Osborne CL, Hsu Y: Clq binding in serum in juvenile rheumatoid arthritis. J Rheumatol7:665-670, 1980 9. Zubler RH, Lange G, Lambert PH, Miescher PA: Detection of immune complexes in unheated sera by a modified '2sI-Clq binding test. J Immunol 1 16:232-235, 1976 10. Moore TL, Zuckner J, Baldassare AR, Weiss TD, Dorner RW: Complement-fixing hidden rheumatoid factor in juvenile rheumatoid arthritis. Arthritis Rheum 21:935-941, 1978 11. Robbins D, Moore TL: Lack of hidden complement fixing IgM rheumatoid factor in adult seronegative rheumatoid arthritis. Ann Rheum Dis 39:64-67, 1980 12. Boyum A: Separaton of leukocytes from blood and bone marrow. Scand J Clin Lab Invest (suppl 97) 21:7789,1968 13. Lefkovits I: Induction of antibody-forming cell clones in microcultures. Eur J Immunol2:360-366, 1972 14. Halsall MK, Makinodan T: Analysis of the limitingdilution assay for estimating frequencies of immunocompetent units. Cell Immunol 11:456-465, 1974 15. Pasquali J-L, Fong S, Tsoukas CD, Slovin SF, Vaughan JH, Carson DA: Different populations of rheumatoid factor idiotypes induced by two polyclonal B cell activators, pokeweed mitogen and Epstein-Barr virus. Clin Immunol Immunopathol21:184-189, 1981 16. Hanson V, Rexler ED, Kornreich H: The relationship of rheumatoid factor to age of onset in juvenile rheumatoid arthritis. Arthritis Rheum 12:82-86, 1969 17. McEwen C, Ziff M, Carmel P, Di Tata D, Tanner M: The relationship to rheumatoid arthritis of its so-called variants. Arthritis Rheum 1:481-496, 1958 18. Ziff M, Brown P, Lospalluto J, Badin J, McEwen C: Agglutination and inhibition by serum globulin in the sensitized sheep cell agglutination reaction in rheumatoid arthritis. Am J Med 20500, 1956 19. Moore TL, Dorner RW, Weiss TD, Baldassare AR, Zuckner J: Specificity of hidden 19s IgM rheumatoid factor in patients with juvenile rheumatoid arthritis. Arthritis Rheum 24:1283-1290, 1981 20. Robbins DL, Moore TL, Carson DA, Vaughan JH: Relative reactivities of rheumatoid factors in serum and cells: evidence for a selective deficiency in serum rheumatoid factor. Arthritis Rheum 21 :820-826, 1978 21. Moore TL, Sheridan PW, Traycoff RB, Zuckner J, Dorner RW: Immune complexes in juvenile rheumatoid arthritis. Arthritis Rheum (suppl) 24: 101, 1981 22. Kredich DW: Polyarticular juvenile rheumatoid arthritis, Juvenile Rheumatoid Arthritis. Edited by JJ Miller 111. Littleton, MA, PSG Publishing Co, 1979, p 121 23. Hoyeraal HM, Forre 0, Dobloug JH, Thorsby E, Kass E: HLA-antigens in juvenile rheumatoid arthritis patients: association between immunoglobulin M rheumatoid factor production and the HLA-DR4 antigen (abstract). Paris, XVth International Congress of Rheumatology, 1981, p 673 24. Stastny P: Association of the B-cell alloantigen DRw4 with rheumatoid arthritis. N Engl J Med 298:869-871, 1978 25. Wernick R, Lospalluto J, Fink C, Ziff M: IgG and IgM IgM ANTI-IgG B LYMPHOCYTES IN CHILDREN rheumatoid factor levels in adult and juvenile rheumatoid arthritis by radioimmunoassay. Arthritis Rheum 23:761, 1980 26. Dobloug JH, Forre 0, Kass E, Thorsby E: HLA antigens and rheumatoid arthritis, association between HLA-DRw4 positivity and IgM rheumatoid factor production. Arthritis Rheum 23:309-313, 1980 27. Van Snick JL: A gene linked to the Igh-c locus controls 965 the production of rheumatoid factors in the mouse. J Exp Med 153:738-742, 1981 28. Van Snick JL, Masson PL: Age-dependent production of IgA and IgM autoantibodies against IgG2a in a colony of 129/Sv mice. J Exp Med 149:1519-1530, 1979 29. Van Snick JL, Masson PL: Incidence and specificities of IgA and IgM anti-IgG autoantibodies in various mouse strains and colonies. J Exp Med 151:45-55, 1980 Continuing Education Vlll Panhellenic Congress of Rheumatology. The Congress will be held in Athens, Greece, November 25-27, 1982 at the Caravel Hotel. The subject of the Congress is “Rheumatic Diseases of Childhood.” For further information contact Dr. Alex Andrianakos, Secretary, V l l l Panhellenic Congress of Rheumatology, 1 Pythias Street, Athens 809, Greece. Fifth Annual Colorado Pain Symposium. The Symposium will be held in Aspen, Colorado, January 8-15, 1983 at the Continental Inn. The subject of the Symposium, which is sponsored by the Pain Control Center at Boulder Memorial Hospital, is “The Surgical Management of Pain.” For further information contact Diana Buck, Public Relations, Boulder Memorial Hospital, 31 1 Mapleton Avenue, Boulder, CO 80302; telephone 303441-0464. Problems in Rheurnatology. This postgraduate course, sponsored by the Department of Internal Medicine, University of South Florida College of Medicine, will be held March 10-13, 1983, at the Don CeSar Beach Resort Hotel, St. Petersburg Beach, Florida. For further information, contact Bernard F. Germain, MD, Course Director, Division of Rheumatology, University of South Florida College of Medicine, Box 19, Tampa, FL 33612.