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Rheumatol Int (1985) 5:61-67

Clinical and Experimental Investigations

© Springer-Verlag 1985

Antibodies to rheumatoid arthritis nuclear antigen (RANA) in Japanese patients with rheumatoid arthritis K. Nakabayashi 1, M. Saito 1, T. Nagasawa 1 ,, and M. Takada 2 a First Department of Internal Medicine, Kyorin University School of Medicine, Mitaka, Tokyo, Japan 2 Nissui Seiyaku Institute, Ibaraki, Japan Received March 2, 1984 / Accepted August 15, 1984

S u m m a r y . We studied antibodies to rheumatoid arthritis nuclear antigen (RANA) by the Ouchterlony method in 0.5% agarose plates, using soluble antigen extracted with 0.25 M sucrose solution from cultured Raji cells. AntiRANA antibody was found in sera from 24 to 40 (60%) patients with rheumatoid arthritis (RA), from 4 of 20 (20%) patients with systemic lupus erythematosus (SLE), and from 2 of 30 (7%) healthy controls. When sucrose extracts from BJAB, Ramos, and JM cells were used as the cellular antigens, no anti-RANA precipitin lines were detected. Indirect immunofluorescence study, using Raji cells or h u m a n B lymphocytes transformed by EB virus as substrate tissues, demonstrated anti-RANA antibody as fine speckled nuclear staining. Although RA patients with positive anti-RANA antibody usually had high titers of anti-Epstein-Barr nuclear antigen (EBNA) and anti-viral capsid antigen (VCA) I g G antibodies, the Wilcoxon ranks sum test showed no close statistical correlation between the presence of anti-RANA antibodies and the titers of anti-EBNA or anti-VCA (IgG) antibodies. These data showed that the incidence of positivity of anti-RANA antibodies in Japanese RA patients is almost the same as that in American and European RA patients. K e y words: Anti-RANA antibody - Rheumatoid arthritis EB virus infection

(RAP) or anti-rheumatoid arthritis nuclear antigen (RANA) antibody; it reacts only with extracts from EB virus-infected B cells. However, the role of EB virus in the pathogenesis of RA remains a matter of controversy [6-10]. We investigated the presence of anti-RANA antibodies in the sera of Japanese patients with RA and systemic lupus erythematosus (SLE) using extracts from Raji cells, and compared the anti-RANA antibody and different well-known anti-EB virus antibodies with respect to their biological characteristics and incidence of positivity. We also considered the clinical implications of anti-RANA antibodies in the RA pathogenesis. M a t e r i a l s and m e t h o d s

Sera

Sera were obtained from 40 patients with RA, 20 patients with SLE, and 30 healthy subjects. The ages of the study population ranged from 22 to 60 years; the mean age of RA and SLE patients and healthy subjects was 44, 29, and 40 years, respectively. The controls were age- and sex-matched with the RA patients. All patients satisfied the ARA diagnostic criteria for RA and SLE. Serum samples were inactivated at 56 °C for 30 min and stored at -20 °C until use. Detection of anti-RANA antibodies

We used the immunodiffusion and the indirect immunofluorescent methods. Introduction

In 1975, Alspaugh and Tan used immunodiffusion methods to demonstrate three different antibodies in the sera of SjOgren syndrome patients [1]. They referred to these antibodies as precipitin A, B, and C. Based on subsequent studies, they suggested that precipitin C was closely associated with patients who had sicca syndrome (SS) accompanied by symptoms of rheumatoid arthritis (RA) [2-5]. Subsequently, precipitin C, mainly found in the sera of RA patients, was called rheumatoid arthritis precipitin * To whom correspondence should be sent

1. Immunodiffusion studies using the Ouchterlony method. Using a slight modification of the method of Alspaugh and Tan [1] antigen was obtained from Raft cells. These cells were cultured in a CO2 incubator at 37 °C in RPMI-1640 medium containing 10% heatinactivated fetal calf serum (Gibco). The culture medium was replaced with fresh medium every 3 days to ensure continuous cell growth. Then the cells were adjusted to 1 × 106/ml of culture medium. A one-liter volume of Raji cell suspension was centrifuged for 20 rain at 350 g and then the Raji cell pellet was washed three times with phosphate buffered saline (PBS, pH 7.2). To obtain soluble cellular antigen from the Raji cells, extraction by either 0.25 M sucrose solution or PBS was performed. The final cell pellet was resuspended in an equal volume of 0.25 M sucrose solution containing 0.004 M CaC12 that had been adjusted to pH6.2 with 0.01 M phosphate buffer, or in an equal volume of PBS (pH 7.2). The Raji cells in these solutions were sonicated five

62 times with 30 s bursts at 30 s intervals using a 150 W sonicator (Tomy Model UR-150, Japan). The supernatant for soluble cellular antigens was separated from the cell debris by centrifugation for 30 min at 105 000g and 4°C. For comparative studies with cells devoid of the EB virus genome, BJAB [11] and Ramos cells [11] (B cell line cells) and JM cells [11] (T cell line cells) were also cultured and sonicated by the same procedures. After measuring the protein content in these supernatants by the microliter method of the trichloracetic acid assay system with a Dupont Aca-II automatic clinical analyzer [12], the protein content was concentrated to approximately 20 mg/ml using a minicon concentrator (Amicon Corp., Danvers, USA). The supernatants containing soluble antigens were stored at -70 °C until use. For the immunodiffusion studies by the Ouchterlony method, 0.5% agarose (Hoechst) plates were used; 7 wells 5 mm in diameter and 2.5 mm apart were made. The antigen was placed in the center well; the reference serum, anti-RANA positive serum kindly donated by Dr. Tan, and experimental sera were placed in the other wells. Immunodiffusion was for 48-96 h at room temperature, and the plates were inspected for precipitin lines fused with the reference serum.

buffer at pH 8.1 in the presence of 0.0115 M calcium ions; RNase digestion in 0.01 M phosphate buffer (pH7.4) with 0.15 M NaC1 in the presence of 0.006 M magnesium ions; and DNase digestion in PBS (pH 7.4). The treated extracts were tested against reference sera.

2. Indirect immunofluorescent method For the immunofluores-

Incidence of positivity of anti-RANA antibody and biophysiological characterization

cence study of anti-RANA antibody, we prepared cell smears from Raji cells, B95-8 EB virus-infected B cells [13, 14], and normal human peripheral blood lymphocytes. Raji cells were washed three times with PBS, and one sample was spread on the slide. Then the slide was centrifuged at 1500 rpm for 15 min and dried at 37°C for 30 rain to fix the cells. For the preparation of slides with B95-8 EB virus-infected B cells, a minor modification of the method of Miller and Lipman [13] was used. Briefly, B cells from normal volunteers were suspended in culture medium containing the supernatant from 4-day cultures of a B95-8 cell line; eventually, the B cells became EB virus-induced transformed cells. These cells were harvested 20, 30, 40, and 50 days after exposure to the supernatant and cell smears were prepared as described above. Besides dry-heat fixation, cell fixation with acetone, methanol, 10% formalin, or carbon tetrachloride was carried out for comparative studies of antigen preservation. Smear slides of normal human peripheral blood lymphocytes were made using the same fixation method as described for Raji cells, or using acetone fixation. The cell smears were incubated (37°C, 30min) with sera diluted 10 times with PBS, and then washed three times with PBS. They were again reacted for 30min at 37°C with rabbit antihuman immunoglobulin (IgG, IgA, and/or IgM) labeled with fluorescein isothiocyanate (FITC) (Hoechst, FP ratio: 2.0). Subsequently, the slides were washed three times with PBS and mounted under an immunofluorescentmicroscope.

3. Detection of anti-EBNA and anti-VCA (IgG) antibodies. AntiEBNA antibodies were detected by the anti-complement immunofluorescence technique described by Reedman and Klein [15]. Anti-VCA (IgG) antibodies were assayed by the indirect immunofluorescence method of Henle and Henle [16]. In these immunofluorescence studies of anti-RANA, antiEBNA, and anti-VCA (IgG) antibody, control studies were carried out using JM cell slides to exclude the effects of anti-nuclear antibodies.

5. Statistics. Statistical analyses were based on the following tests. The relationship between the incidence of positivity of antiRANA antibodies in RA patients and healthy subjects or SLE patients was evaluated by the chi-square test. The correlation between the presence of serum anti-RANA antibodies and the EBNA or VCA (IgG) antibody titers was assessed by the Wilcoxon ranks sum test. The significance of differences in anti-EBNA or anti-VCA (IgG) antibody titers between anti-RANA antibodypositive and -negative RA patients or controls was calculated by Student's t-test. Differences in clinical manifestations and laboratory findings between anti-RANA antibody-positive and antibody-negative RA patients were also evaluated by Student's t-test.

Results

As shown in Table 1, sera from 24 of 40 (60%) RA patients were positive for a n t i - R A N A antibody; this was true for sera from 4 of 20 (20%) SLE patients, a n d 2 of 30 (7%) healthy subjects. The a n t i - R A N A antibody precipitin lines were usually not as strong as those of a n t i - D N A and antiRNP antibodies; however, they were clearly differentiated from those of anti-DNA, anti-RNP, a n d anti-Sin antibodies (Fig. 1). The SLE sera precipitin lines, which included various kinds of anti-nuclear antibodies, interfered with a n t i - R A N A antibody precipitin lines on several plates; this made it difficult to recognize the a n t i - R A N A antibodies in SLE sera. To compare the antigenic differences between soluble cellular antigen extracted by 0.25 M sucrose solution a n d by PBS, we used a n t i - R A N A positive sera from 4 RA patients, 2 SLE patients, and 2 controls a n d a n t i - R A N A negative sera from 4 RA patients, 2 SLE patients, 2 SLE patients, a n d 4 controls. PBS-extracted antigen demonstrated more precipitin lines than 0.25 M sucrose-extracted antigen, and irrespective of the extraction m e t h o d used, a n t i - R A N A antibodies were detected only in confirmed antibody-positive cases. In comparative studies in which the antigens were extracted with 0.25 M sucrose from BJAB, Ramos, a n d JM cells, no

Table 1. Positivity incidence of anti-RANA antibody (Ouchterlony method) No. tested

4. Biophysiological characterization of extracted soluble cellular antigens from Raji cells using 0.25 M sucrose solution. Extracted cellular antigen was heated at 56 °C for 30 rain and used in the immunodiffusion study. In addition, antigen solutions were digested (2 h, 37 °C) with enzymes such as trypsin, ribonuclease (RNase), or deoxyribonuclease (DNase) (Sigma), and the digested antigens were checked for their influence on antigenicity. The ratios by weight of the enzyme to substrate were as follows: trypsin to substrate, 1 : 20, RNase to substrate, 1 : 20; DNase to substrate, 1 : 40. Trypsin digestion was carried out in 0.04 M Tris

RA SLE Controls

40 20 30

* )¢ P values< 0.01 ** Z2 Pvalues<0.1

Anti-RANA Ab Positive cases

Positive %

24 4 2

60 * ** 20"* 7"

63

Fig. 1. Immunodiffusion study showed a line of identity between precipitins of anti-RANA antibody-positive reference serum and RA sera, but not between precipitins of anti-RANA antibodypositive reference serum and anti-DNA or anti-Sm/RNP antibody-positive sera. Raft cell extract was placed in the center well

anti-RANA antibody was demonstrated. RANA was heat stable at 56 °C for 30 rain, and resistant to RNase and DNase digestion but sensitive to trypsin digestion, which abolished its antigenicity completely. Thus, we postulate that the antigenic properties of RANA are part of a nuclear protein with a molecular weight of less than 0.5x 106 daltons, but that RANA does not constitute a part of DNA or RNA, because it exhibited the abovementioned biophysiological characteristics, was extracted by weakly acid solution, was present in the supernatant obtained after 30-40rain centrifugation at 105 00091, and was distributed mainly in the nuclei of EB virus genome-containing cells, as shown by the immunofluorescence studies discussed below.

Indirect immunofluorescence study of anti-RA NA antibody Immunofluorescent studies with Raji cells and B95-8 EB virus-infected B cells showed that anti-RANA antibody exhibited a fine speckled pattern, mainly in the nucleus but also to some extent in the cytoplasm (Fig. 2). AntiRANA antibody was stained brightly with anti-human IgG and weakly with anti-human IgM, but not at all with IgA and C3. B95-8 EB virus-infected B cells were stained at 20, 30, 40, and 50 days post-infection; those stained on day40 exhibited the brightest fine speckling (Fig. 2A), while the others were less intensely immunofluorescent and showed fewer fine speckled granules. A comparison of B95-8 EB virus-infected B cells at 40 days post-infection 1 Centrifugation at 105 000 # for 30-40 min sediments substances greater than 0.5 x 106 daltons in molecular weight [17]

showed that in those fixed by dry heat (Fig. 2A) the fine granules were stained more brightly than in those fixed with acetone, methanol, 10% formalin, or carbon tetrachloride (Fig. 2B). When B95-8 EB virus-infected B cells were dry heat-fixed at 40 days and reacted with antiRANA negative sera, no fine granules were visualized in the cell nuclei. These cells exhibited only peripheral immunofluorescence, probably ascribable to surface immunoglobulin and cytoplasmic immunoglobulin, and/or blocking of Fc receptors with FITC-labeled immunoglobulin (Fig. 2C). The staining of normal peripheral blood lymphocytes with anti-RANA antibody-positive sera from four RA patients did not result in positive immunofluorescence (Fig. 2 D). All cases of RA patients and controls examined by immunodiffusion for anti-RANA antibody were also evaluated by immunofluorescence for the presence of the antibody. In RA patients, all of 24 positive sera samples and 10 of 16 negative sera samples examined by immunodiffusion for the detection of anti-RANA antibody were also found to be positive for the antibody upon immunofluorescence staining. In samples from controls, 2 of 2 positive sera samples and 1 of 28 negative sera samples found to contain anti-RANA antibody after immunodiffusion were also stained positive for the antibody upon immunofluorescence.

Relationship between anti-RANA antibodies and anti-EBNA antibodies The geometrical mean values of anti-EBNA antibodies in patients with anti-RANA antibody-positive RA, antiRANA antibody-negative RA, and healthy subjects were 18.5, 11.7, and 7.73, respectively (Fig. 3); there were no statistically significant differences in the anti-EBNA antibody titers of these three groups. Furthermore, there was no statistical correlation between the anti-EBNA antibody titers and positivity for anti-RANA antibodies. In fact, there were four RA patients who were positive for antiRANA antibody but negative for anti-EBNA antibody (Fig. 3).

Relationship between anti-RANA antibodies and anti- VCA (IgG) antibodies The geometrical mean values of anti-VCA(IgG) antibodies in patients with anti-RANA antibody-positive RA, anti-RNA antibody-negative RA, and controls were 242.6, 339.2, and 175.6, respectively (Fig. 4); there was no statistically significant difference in the anti-VCA (IgG) antibody titers between the patients and the control group. Furthermore, there was no definite correlation between the anti-VCA (IgG) antibody titers and positivity for antiRANA antibody.

Relationship between anti-RA NA antibodies and clinical manifestations The differences in clinical manifestations between antiRANA antibody-positive and -negative RA patients were

Fig. 2A-D. Indirect immunofluorescence study of Raji cells, B95-8 EB virus-infected B cells inspected at 40 days post-infection, and normal human peripheral blood lymphocytes. The cells were fixed by dry heat (37 °C, 30 min) (Inset, A, C, D) or acetone (B) and reacted with anti-RANA antibody-positive (Inset, A, B, D) or -negative (C) serum. A remarkable finely speckled staining pattern was observed only in the nuclei of Raji cells (Inset) and EB virus-infected B ceils (A) which had been heat-fixed and reacted with anti-RANA antibody-positive serum; cells fixed with acetone (B) did not exhibit the bright, fine granules. Cells reacted with anti-RANA antibodynegative serum exhibited no fine granules in their nuclei, but showed only peripheral immunofluorescence (C) (the second antibody staining was with IgG). Peripheral blood lymphocytes reacted with anti-RANA antibody-positive serum manifested no immunofluorescence (D). (x 400)

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Fig. 3. Anti-EBNA titers in patients with anti-RANA-positive RA, anti-RANA-negative RA, and healthy subjects

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Fig. 4. Anti-VCA (IgG) titers in patients with anti-RANA-positive RA, anti-RANA-negative RA, and healthy subjects

65 examined. Although no major differences were noted, anti-RANA antibody-positive patients had a higher incidence of sicca syndrome (6 of 24, 25%) than did antiRANA antibody-negative patients. While the association with sicca syndrome was not statistically higher in antiRANA antibody-positive RA patients, 2 of the 6 RA patients with sicca syndrome were found to have anti-SS-A and anti-SS-B antibodies. While the laboratory findings on RA patients with or without anti-RANA antibodies were not significantly different, anti-RANA antibody-positive patients exhibited a higher incidence of hypergammaglobulinemia (P<0.01). There was also no statistically significant differences between anti-RANA antibody-positive and -negative patients with respect to the presence of RA factors and anti-nuclear antibodies.

Discussion Although the pathogenesis of RA is not yet well understood, immunologic, genetic, and environmental factors are thought to play important roles in this disease. Several investigators have implicated EB virus in the pathogenesis of RA based on the experimental data such as the demonstration of RANA antibody [4], and the findings that RA lymphocytes infected in vitro with EB virus can synthesize large amounts of IgM RA factor compared to lymphocytes

from healthy subjects [18, 19], RA lymphocytes exposed in vitro to EB virus are easily transformed into lymphoblastoid cell lines [20], and EB virus-specific suppressor T cells in RA are defective [21]. Nevertheless, questions still remain about whether EB virus is important in RA pathogenesis. Based on these observations, we investigated antiRANA antibodies as well as EB virus-associated antibodies in Japanese patients with RA. For the detection of anti-RANA antibody, we used Raji cells and performed an immunodiffusion study using the Ouchterlony method, and indirect immunoflnorescence staining. Soluble antigens for the immunodiffusion study were extracted from Raji cells by 0.25 M sucrose solution or PBS. Using sucrose-extracted antigen, we found anti-RANA antibody in 60% of sera from RA patients, in 7% of sera from controls, and in 20% ofsera from SLE patients. When using PBS-extracted antigen, although more precipitin lines were exhibited than with sucrose-extracted antigen, no anti-RANA antibodies were observed in anti-RANA antibody-negative sera with sucrose-extracted antigen from RA and SLE patients, nor in the samples from controls. When immunofluorescence staining for the detection of anti-RANA antibodies on all sera from RA patients and controls examined by immunodiffusion was performed, all sera positive for the antibody by immunodiffusion were

Table 2. Reported incidences of positivity of anti-RANA antibodies detected by the Ouchterlony method Investigator

1975 Alspaugh 1976 Alspaugh 1978 Alspaugh 1979 Catalano 1980 Venables 1981 Venables 1981 Alspaugh 1981 Ferrell 1982 Furuta 1983 Nakabayashi a ° This study

Antigen source (extraction method)

Wil-2 cells (sucrose) Wil-2 cells (sucrose) Wil-2 cells (sucrose) Wil-2 cells (PBS) Raji cells (PBS) Wil-2 cells (PBS) Wil-2 cells (sucrose) Wil-2 cells (sucrose) Raji cells (pBs) Raji cells (sucrose)

RA

Controls

No. Positive/ tested

Positive %

No. Positive/ tested

Positive %

24/37

65

1/22

4.5

59/90

66

6/71

8.5

19/29

66

6/71

8.5

44/47

94

12/48

25.0

51/80

64

10/90

11.1

86/100

86

49/93

52.7

43/48

90

1/16

6.3

63/89

71

3/53

5.7

59/60

98

40/44

90.9

24/40

60

2/30

6.7

66 also found to be positive for the antibody upon immunofluorescence. However, sera which were anti-RANA antibody-negative from 16 RA patients and 28 healthy subjects upon immunodiffusion were found to be positive in 10 of RA patients and 1 healthy subject in the immunofluorescence study. Our comparative studies using soluble antigens from cells that did not contain the EB virus genome (B JAB, Ramos, and JM) revealed no identity lines for anti-RANA antibody in all RA patients and healthy subjects. When correlations between the presence of anti-RANA antibodies and titers of various well-known EB virusassociated antibodies were evaluated, we could not find any significant statistical correlations between the presence of anti-RANA antibodies and titers of anti-EBNA antibodies or anti-VCA (IgG) antibodies. Previously published data on anti-RANA antibodies in RA patients and normal subjects are summarized in Table2. These studies documented a high incidence (65-90%) of anti-RANA antibodies in RA patients and a low incidence (less than 15%) of the antibodies in healthy subjects except for reports by Catalano et al. [6], Venables et al. [9], and Furuta [10]. They also concluded that antiRANA antibodies were present whether the patients were seropositive or not for IgM RA factors. Our experimental data agree with these previous reports, showing that our RA patients manifested a high incidence of anti-RA antibodies. On the other hand, Catalano et al. [22, 23], Venables and Maini [24], and Furuta [10] cast some doubts on these data with the following experimental results. Catalano et al. [23] used the "sensitive" indirect immunofluorescence method and stated that they could frequently find the anti-RANA antibodies in healthy subjects (50% of healthy subjects with previous EB virus infection). Venables and Maini [24] questioned the relationship between the presence of anti-RANA antibody and EB virus infection, because their experimental data showed that anti-RANA antibodies were observed even in cell extracts without EB virus genomes. Furuta's preliminary report [10] showed that in the incidence of anti-liANA antibodies was 98% in Japanese RA patients and 81% in healthy Japanese subjects. In contrast, our experimental data obtained through immunofluorescence and immunodiffusion studies with soluble antigens from non-EB virus genome-containing ceils did not agree with these data. First, even through the immunofluorescence study, which was more sensitive than our immunodiffusion, we found only 10% (3 cases out of 30 sera) positivity of anti-RANA antibody in normal subjects, while the incidence of anti-VCA (IgG) antibodies in healthy Japanese adults is reported to be 85-90% positively [25]. Second, soluble antigens from non-EB virus genome-containing cells did not reveal any identity lines for anti-RANA antibody. Accordingly, we conclude from our experimental studies that anti-RANA antibody is one of the nuclear antibodies, that it reacts with the extracted soluble cellular antigen from cells containing EB virus genome, that it is

present mainly in the sera from RA patients, and that it is not completely associated with any of the different wellknown EB virus antibodies. Our data also suggest that in RA patients the regulation of the immune response to EB virus infection might be altered and that this may be associated with the pathogenesis of RA.

Acknowledgement. We would like to thank Professor E. M. Tan for the donation of reference serum and for his helpful advice. References

1. Alspaugh MA, Tan EM (1975) Antibodies to cellular antigens in Sjtgren's syndrome. J Clin Invest 55:1067-1073 2. Alspaugh MA, Tan EM (1976) Serum antibody in rheumatoid arthritis reactive with a cell-associated antigen. Arthritis Rheum 19:711-719 3. Alspaugh MA, Buchanan WW, Whaley K (1978) Precipitating antibodies to cellular antigens in Sjtgren's syndrome, rheumatoid arthritis, and other organ and nonorganspecific autoimmune diseases. Ann Rheum Dis 37:244-246 4. Alspaugh MA, Jensen FC, Rabin H, Tan EM (1978) Lymphocytes transformed by Epstein-Barr virus. Induction of nuclear antigen reactive with antibody in rheumatoid arthritis. J Exp IVied 147:1018-1027 5. Alspaugh MA, Henle G, Lennette EL, Henle W (1981) Elevated levels of antibodies to Epstein-Barr virus antigens in sera and synovial fluids of patients with rheumatoid arthritis. J Clin Invest 67:1134-1140 6. Catalano MA, Carson DA, Slovin SF, Richman DD, Vaughan FH (1979) Antibodies to Epstein-Barr virus-determined antigens in normal subjects and in patients with seropositive rheumatoid arthritis. Proc Natl Acad Sci USA 76: 5825-5828 7. Ng KC, Brown KA, Perry JD, Holborow EJ (1980) AntiRANA antibody: A marker for seronegative and seropositive rheumatoid arthritis. Lancet I:447-449 8. Ferrell PB, Aitcheson CT, Pearson GR, Tan EM (1981) Seroepidemiological study of relationships between EpsteinBarr virus and rheumatoid arthritis. J Clin Invest 67:681-687 9. Venables PJW, Roffe LM, Erhard CC, Maini RN, Edwards JMB, Porter AD (1981) Titers of antibodies to RANA in rheumatoid arthritis and normal sera. Relationship to EpsteinBarr virus infection. Arthritis Rheum 24:1459-1464 10. Furuta Y (1982) Antibodies to rheumatoid arthritis nuclear antigen (RANA) in patients with collagen diseases, sicca complex and in normal subjects. Okayama Igakkai Zasshi 94: 519-529 11. Nilsson K (1979) The nature of lymphoid cell lines and their relationship to the virus. In: Epstein MA, Achong BG (eds) The Epstein-Barr virus. Springer, Berlin Heidelberg New York, pp 227-281 12. Westgard JO, Lahmeyer BL (1972) Comparison of results from the Dupont "ACA" and Technicon "SMA 12/60". Clin Chem 18:340-348 13. Miller G, Lipman M (1973) Release of infectious Epstein-Barr virus by transformed marmoset leucocytes. Proc Natl Acad Sci USA 70:190-194 14. Takada M, Murata M, Takei M, Nakagawa S, Okumura H, Kawamura A (1979) The rescue of Epstein-Barr virus from primary cultured cells of nasopharyngeal carcinoma. Comp Immunol MJcrobiol Infect Dis 2: 177-189 15. Reedman BM, Klein G (1973) Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 11:49%520 16. Henle G, Henle W (1966) Immunofluorescence in cells derived from Burkitt's lymphoma. J Bacteriol 91:1248-1256 17. Dahl JL, Hokin LE (1974) The sodium-potassium adenosinetriphosphatase. Annu Rev Biochem 43:327-356

67 18. Rosen A, Gergely P, Jondal M, Klein G (1977) Polyclonal Ig production after Epstein-Barr virus infection of human lymphocytes in vitro. Nature 267:52-54 19. Slaughter L, Carson DA, Jensen FC, Holbrook TL, Vaughan JH (1978) 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 20. Bardwick PA, Bluestein HG, Zvaifler NJ, Depper JM, Seegmiller JE (1980) Altered regulation of Epstein-Barr virus induced lymphoblast proliferation in rheumatoid arthritis lymphoid cells. Arthritis Rheum 23:626-632 21. Tosato G, Steinberg AD, Blease RM (1981) Defective EBVspecific suppressor T-cell function in rheumatoid arthritis. N Engl J Med 19:1238-1243

22. Catalano MA, Carson DA, Niederman JC, Feorino P, Vaughan JH (1980) Antibody to the rheumatoid arthritis nuclear antigen. Its relationship to in vivo Epstein-Barr virus infection. J Clin Invest 65:1238-1242 23. Catalano MA, Slovin SF, Freer SS (1980) Anti-RANA antibody and rheumatoid arthritis. Lancet 1: 824 24. Venables PJW, Maini RN (1980) Antibodies to RANA and other nuclear antigens in infectious mononucleosis (IM) and rheumatoid arthritis (RA). Ann Rheum Dis 39:191 25. Hinuma Y, Ohta-Hatano R, Suto T, Numazaki Y (1969) High incidence of Japanese infants with antibody to a herpes-type virus associated with cultured Burkitt lymphoma cells. Jpn J Microbiol 13:309-311