Seroepidemiological study of relationships between Epstein-Barr virus and rheumatoid arthritis.

To elucidate the relationship between Epstein-Barr virus (EBV) and rheumatoid arthritis (RA), we measured antibodies to RA-associated nuclear antigen (anti-RANA) and three other EBV-related antigens in the sera of RA patients and controls. Our study groups consisted of 89 patients with definite or classical RA, mean age 56, male/female ratio 47:42; and 53 normal and osteoarthritis controls, mean age 51, male/female ratio 25:28. In addition to anti-RANA, we measured antibodies to viral capsid antigen (anti-VCA), early antigen (anti-EA) and EBV-associated nuclear antigen (anti-EBNA). Anti-RANA was detected in 71% of RA patients but in only 6% of controls. Elevated anti-VCA titers (greater than 1:160) were more common in RA patients than controls, 31% compared with 15%. The geometric mean titer of anti-VCA was significantly higher iun the RA group, 133 compared with 58. Anti-EA was present in 53% of RA patients but only 19% of controls. Anti-EA in elevated titers (greater than 1:20) was present in 26% of RA patients but only 7% of controls. Characterization of the anti-EA antibodies revealed that the RA patients reacted primarily with the diffuse component, whereas the majority of the controls reacted with the restricted component of the EA complex. In contrast, the frequencies, distributions, and geometric mean titers of anti-EBNA were not significantly different between the two groups. Correlative analysis of these antibodies showed highly significant relationships between anti-VCA and anti-EA, and anti-RANA and anti-EBNA in the RA group. These data are compatible with the interpretation that RA patients have either more active EBV infections than controls or an altered regulation of their immune response to this infectious agent.     

Antibody to the Rheumatoid Arthritis Nuclear Antigen: ITS RELATIONSHIP TO IN VIVO EPSTEIN-BARR VIRUS INFECTION

Most patients with seropositive rheumatoid arthritis, and a variable but lesser percentage of normal subjects, have precipitating antibodies to a nuclear antigen, rheumatoid arthritis nuclear antigen, present in Epstein-Barr virus-infected human B lymphoblastoid cells. We have used a sensitive indirect immunofluorescence assay for antibody to rheumatoid arthritis nuclear antigen in a study of patients with infectious mononucleosis and healthy control subjects. Of 110 sera from normal, college-age cadets, 58 were from individuals without prior Epstein-Barr virus infection, as indicated by the lack of antibody to viral capsid antigen. All of these also lacked activity to rheumatoid arthritis nuclear antigen. 52 sera were positive for antibody to viral capsid antigen, and antibody to rheumatoid arthritis nuclear antigen was present in 26 (50%) of these. In 67 sequential sera from 11 college-age students with infectious mononucleosis who became positive for antibody to rheumatoid arthritis nuclear antigen, only 2 were positive during the 1 mo. Thereafter the incidence and titers increased progressively through the 1st yr after infection. This time-course resembled that for the development of antibody to Epstein-Barr nuclear antigen, another transformation antigen in Epstein-Barr virus-infected B lymphocytes. The development of positivity for both was much later than that of antibody to the structural viral capsid antigen, which in the current study was always positive by 1 wk. Thus, antibody to rheumatoid arthritis nuclear antigen is present in a large proportion of normal individuals and can now be clearly ascribed, from both in vivo and in vitro studies, to prior infection with Epstein-Barr virus.     

Detection of antibody to a new antigen induced by Epstein-Barr virus in rheumatoid arthritis.

A new antigen which is different from Epstein-Barr virion antigen was detected in NC-37 cells infected with Epstein-Barr virus (EBV). A significant elevation of the titer of antibody to this new antigen was observed in the sera of the patients with rheumatoid arthritis (RA), but not in the sera of controls. From the standpoint of the etiologic role of EBV in RA, it is interesting that the antibody to the new antigen is not detected in the healthy persons in contrast to other viral antibodies. Therefore, it differs from any other viral antibodies so far reported.     

Comparison of the classical immunofluorescent antibody tests for Epstein-Barr virus with commercially available test kits

Twenty reference sera with previously reported titers plus 10 normal seropositive and 10 seronegative sera were used to determine which assay for the detection of antibodies to Epstein-Barr virus should be utilized in our laboratory. By three commercially available test systems and by slides prepared in our laboratory, all techniques detected the same number of samples positive for IgG antibodies to viral capsid antigen (VCA) and Epstein-Barr virus-determined nuclear antigen (EBNA), but the geometric mean titers (GMTs) varied. Major discrepancies arose in the sensitivity of detection of early antigen-diffuse (EA-D) and -diffuse and restricted (EA-DR). Slides prepared in our laboratory gave the best results, detecting 12 of 12 (100%). Slides prepared according to Gull and Hillcrest detected 11 of 12 (92%), and those prepared according to Zeus detected 9 of 12 (75%). From these data, we conclude that all of these methods for detection of VCA and EBNA are adequate. However, in determining antibody levels to EA-D and EA-DR, great care should be taken as to the source of the slide and specificity and sensitivity.     

Polynucleotide antibodies in connective tissue disease: viral markers or disease mediators?

Antibodies to polynucleotides are seen primarily in systemic lupus erythematosus (SLE), but also occur in a variety of other connective tissue diseases. We looked at the prevalence of antinucleotide antibodies (double- and single-stranded RNA and DNA [dRNA, sRNA, dDNA, and sDNA]) in the sera of patients with SLE (70), rheumatoid arthritis (RA) (31), juvenile rheumatoid arthritis (JRA) (68), osteoarthritis (12), and of 22 patients with a preceding viral illness. In comparison with sera from a control population, elevated mean antibody levels to sRNA were found in the sera of all the patients with connective tissue disease, as well as in the sera of patients with preceding RNA, but not DNA, viral infections. Elevated mean levels of antibodies to dRNA were seen in all groups with the exception of RA. Elevated mean antibody titers to sDNA were not seen in patients with JRA nor were they present in the sera of patients with preceding RNA viral infections. Elevated mean anti-dDNA titers were seen only in sera from patients with SLE. High correlation coefficients between the levels of antibodies to sRNA and dRNA in sera from SLE and RA, and between sDNA and dDNA in sera from SLE suggest cross-reactivity of the antibodies in these diseases. Immunization of an elderly population with influenza (RNA) viral vaccine induced antibodies to sRNA only. These studies further document the prevalence of antipolynucleotide antibodies in the sera of patients with connective tissue diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epstein Barr virus-specific immune defects in patients with persistent symptoms following infectious mononucleosis

In a number of patients recovery from infectious mononucleosis (IM) following primary Epstein Barr virus (EBV) infection, is complicated by the persistence of symptoms for months or years. Normally recovery from infectious mononucleosis is associated with the development of EBV-specific antibodies and memory cytotoxic T-cells, which are present in the peripheral blood of all normal seropositive individuals. We studied four patients who had persistent symptoms for more than two years after infectious mononucleosis to determine if this abnormality was associated with a defect in EBV-specific or non-specific immune responses. All four patients had normal immunoglobulin concentrations, T- and B-cell numbers, T-cell proliferative responses and natural killer cell activity. However three of the four had reduced or absent antibodies to the EBV nuclear antigen (EBNA) although other EBV-specific antibody titres were normal. All four also had reduced EBV-specific cytotoxic T-cell activity as measured by the EBV regression assay. This defect was probably EBV-specific as alloreactive cytotoxic T-cell responses were normal. In addition, three of three patients tested had reduced in vitro antibody synthesis following pokeweed mitogen stimulation. These studies indicate that the syndrome of persistent symptoms following EBV mononucleosis may be associated with a defect in EBV-specific immunity, and thus suggest a possible immunological basis for the syndrome.     

Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis

Antibody responses to candidal polypeptides and mannans were studied in patients with systemic candidiasis, candiduria, and other fungal and bacterial infections, and in healthy laboratory personnel to determine the diagnostic value of these immunologic responses. When tested by immunoblot analysis, sera from 15 patients with systemic candidiasis frequently contained antibodies to three antigens: 15 of 15 sera from patients with invasive disease reacted to a molecular species having a molecular weight (Mr of 90-200 kd, 13 of 15 reacted with a 45-kd polypeptide, and 12 of 15 reacted with a 17-kd polypeptide. Lesser reactivity was observed in 11 of 15 sera with a 28-kd candidal antigen and in 9 of 15 to a 57-kd candidal antigen. Quantitation of antibody titers against the 45-kd candidal polypeptide demonstrated much higher immunoreactivity in patients with systemic candidiasis than in patients with superficial candidal infections, bacterial infections, other systemic mycoses, and healthy individuals. Antimannan antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA) and these titers were also higher in patients with systemic candidiasis than in patients in the other categories. These differences, however, were less than those observed with the anti-45-kd polypeptide antibody. Therefore, the ability to detect systemic candidiasis is improved by testing sera for immunoreactivity to polypeptide and to mannan antigens from Candida albicans. Detection of polypeptide antibodies improves the serodiagnosis of systemic candidiasis.     

Evaluation of antibodies to the Epstein-Barr virus immediate early gene product ZEBRA by a new enzyme-linked immunosorbent assay.

For the serodiagnosis of Epstein-Barr virus (EBV) infections, we have developed a new enzyme-linked immunosorbent assay (ELISA) for antibodies to the ZEBRA product of EBV immediate early gene BZLF1. ZEBRA protein fused with glutathione-S-transferase (GST) was expressed in Escherichia coli and purified by affinity chromatography with glutathione-Sepharose 4B. An ELISA sandwich capture system was constructed with the GST-ZEBRA immobilized on plastic microtiter plates which had been coated with a mouse monoclonal antibody to GST. ZEBRA-IgG antibodies in patients' sera with chronic active EBV infection (CAEBV) and infectious mononucleosis (IM) had, respectively, very high and high titers. Anti-ZEBRA antibodies were also detected at low titers in sera of some healthy controls. ZEBRA-IgM antibodies were detected in sera of patients with IM and CAEBV but not in sera of healthy controls. In sera of patients with CAEBV, the titers of IgG antibodies to ZEBRA correlated with the antibody titers to early antigens obtained with an immunofluorescence assay, but not to EBV nuclear antigens. This ELISA is a useful diagnostic and prognostic test for EBV infection.     

Enzyme-linked immunosorbent assay for detection of virus-specific IgM antibodies to varicella-zoster virus

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of varicella-zoster virus (VZV) IgM antibodies. The antigen consisted of a sonically disrupted extract of VZV-infected human embryo cells. The tested sera were absorbed with Staphylococcus aureus (strain Cowan I) before analysis. Rabbit anti-human IgM peroxidase conjugate was used to detect human IgM bound to viral antigen. The results were compared with those obtained by the indirect fluorescent antibody to membrane antigen (IFAMA) technique. Comparison of titers obtained by ELISA with those obtained by IFAMA for sera of chickenpox patients showed agreement between the results in 8 of 9 patients. In 1 chickenpox patient, no VZV IgM antibodies could be detected by IFAMA, while a titer of 3,200 was obtained by ELISA. The ELISA technique described gave titers more than 100 times higher than those obtained by IFAMA. VZV IgM antibody was detected by ELISA and IFAMA in only 1 of 5 zoster patients. No VZV IgM antibodies were found by ELISA in 45 control sera (healthy adults and hospitalized patients with various other diseases). Neither were they found in paired sera of 6 patients with acute herpes simplex infections, 2 patients with Epstein-Barr virus infections, and 3 patients with human cytomegalovirus infections.     

Seroepidemiologic evidence of Epstein-Barr virus reactivation in a veterans' nursing home

Endogenous reactivation of latent Epstein-Barr virus (EBV) infection is thought to occur primarily in immunocompromised hosts, although little is known about the prevalence of this condition among institutionalized older adults. We tested serum specimens from 145 residents of a veterans' nursing home for the presence of EBV-specific antibodies to viral capsid antigen (VCA), early antigen (EA) and nuclear antigen (EBNA) by indirect immunofluorescence. Assays were similarly performed on a convenience sample of 153 sera submitted to the VA reference laboratory from other sources. Serial specimens were obtained on two or more occasions in a subset of 24 nursing home residents. All but two (99%) of the 145 nursing home residents were seropositive for EBNA. VCA IgG titres were ≥1 : 320 in 131 (90%) and ≥1 : 2560 in 39 (27%), with an overall geometric mean of 1 : 1832. EA antibody titres were ≥1 : 80 in 45 (31%) subjects, all of whom had VCA IgG titres ≥1 : 320. The mean titres and seroprevalence of EBV antibodies was similar in the comparison cohort. Among nursing home residents who had serial measurements, a majority (15 of 24) had significant changes in the levels of VCA IgG and/or EA antibody between two or more different specimens. These findings suggest a high prevalence of EBV reactivation among debilitated older adults living in a nursing home setting, and possibly within the veterans' population as a whole. Studies are needed to assess more accurately the clinical impact of persistent EBV infection in this population.     

Inhibition of Epstein-Barr virus production in P3HR-1 cells by Epstein-Barr virus-seropositive human serum.

The effect of human serum with or without Epstein-Barr virus (EBV) antibodies was characterized on virus production in P3HR-1 cells. Cell culturing with EBV-seropositive sera reduced both production of infectious virus and amounts of virion DNA in the supernatants. EBV DNA was also reduced in the cells. Such reductions in cell-associated EBV DNA depended upon the concentration of seropositive serum and incubation time. Decreased frequencies of productive EBV DNA-replicating cells were observed in cell populations which had reduced levels of cell-associated EBV DNA. The inhibitory effect of seropositive serum was reversed upon switching the cells to medium with seronegative serum. In serial sera of an acute infectious mononucleosis patient the EBV DNA-reducing activity arose in parallel to antibodies against EBV membrane antigen and nuclear antigen. Possible mechanisms were discussed for antibody-mediated inhibition of EBV production.     

Antibody pattern to human cytomegalovirus in patients with adenocarcinoma of the colon

The role of human cytomegalovirus (CMV) was examined according to serological patterns in 37 patients with adenocarcinoma of the colon (ACC). The sera were examined for the presence of IgG antibodies by the immunoperoxidase antibody to membrane antigens (IPAMA) method and by the complement-fixation (CF) test. Antibody determinations were also performed by the IPAMA method for three other members of the herpesvirus group: Epstein-Barr virus (EBV), herpes simplex virus (HSV) and varicella-zoster virus (VZV). Comparison groups included normal subjects, ACC patients treated with chemotherapy, and patients operated on for benign diseases. No significant difference was found in the geometric mean titers (GMTs) for CMV and the other herpesviruses in the sera of nontreated ACC patients when compared with the control groups. However, a significantly elevated antibody titer to CMV was found in chemotherapy-treated ACC patients by both the IPAMA and CF methods. In this group, elevated titers were found by the IPAMA method for EBV and HSV, but not for VZV. The significance of serological studies in elucidating the role of CMV in ACC patients is discussed.     

Analysis of antinuclear antibody titers and patterns by using HEp-2 and primate liver tissue substrate indirect immunofluorescence assay in patients with systemic autoimmune rheumatic diseases

BackgroundIndirect immunofluorescence assay (IIFA) is viewed as a preliminary standard to assess antinuclear antibodies (ANAs). Our aim was to explore ANA positivity rate, titers, and patterns in patients with systemic autoimmune rheumatic diseases (SARD), including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjögren''s syndrome (pSS), systemic sclerosis (SSc), and mixed connective tissue disease (MCTD), compared with healthy controls (HC).MethodsAssess antinuclear antibody titers and patterns were retrospectively identified and compared by IIFA using human epithelial cells (HEp‐2) and primate liver tissue substrate according to international consensus in SARD. Serum complement 3 (C3), C4, and immunoglobulin G were compared among subgroups with different ANA titers. The positive predictive values (PPV) for different ANA titers were calculated.ResultsThere were a total of 3510 samples, including 2034 SLE, 973 RA, 155 SSc, 309 pSS, and 39 MCTD cases. There was no difference in age between HC and SARD, excluding RA. ANA positivity rate in SARD and HC was 78.7% and 12.2%, respectively. A titer of ≥1:320 revealed a PPV of 84.0% in SARD. SLE patients with ANA titers ≥1:320 had significantly lower levels of C3 and C4. AC‐4 (31.2%) was the major pattern in patients with SARD, followed by AC‐5 (23.9%) and AC‐1 (18.8%). SLE mostly presented with AC‐4 (30.3%). Several mixed patterns provided a significant hint for SSc and SLE. The major pattern in HC was AC‐2 (12.2%).ConclusionsAssess antinuclear antibody positivity, titers, and patterns display differences in various SARD, contributing to the classification of SARD.     

Immunoblotting reactivity of sera from patients with autoimmune connective tissue diseases against Epstein-Barr nuclear antigen (EBNA) polypeptides

The antibody responses to Epstein-Barr nuclear antigen (EBNA) polypeptides were analyzed by immunoblotting in 93 patients with autoimmune connective tissue diseases (ACTD) in comparison with 50 clinically healthy control subjects. Antibody frequencies to EBNA-2, -4 and -6 were signficantly higher in patients than in controls. Among the patients with ACTD, those with systemic lupus erythematosus (SLE) showed a significant increase in the frequency of anti-EBNA-3 antibodies. These results confirm the particularity of the antibody responses against Epstein-Barr virus (EBV) polypeptides in patients with ACTD; they could either reflect basic immune disturbances or suggest a participation of EBV in the pathogenesis of the disease.     

Post-perfusion syndrome due to Epstein-Barr virus. Report of two cases and review of the literature

The post-perfusion syndrome is rarely due to Epstein-Barr virus (EBV), because most adult patients already carry protective neutralizing antibodies in their sera. Data are presented from two patients in whom heterophil-antibody-positive infectious mononucleosis developed 35 and 48 days after open-heart surgery. The diagnoses of their predominantly febrile illnesses were delayed because of late appearance of atypical lymphocytes and heterophil antibodies. EBV-specific antibody responses were also delayed, with peak titers appearing several weeks or months after onset. The differences in evolution of the present cases, compared to those of classic infectious mononucleosis, were presumably due to different portals of entry (blood stream versus oropharynx).     

Serial estimation of anti-RNP antibody titers in systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis

Anti-nuclear ribonucleoprotein (RNP) antibody titers were estimated serially in 19 anti-RNP antibody-positive patients, which included 10 patients with systemic lupus erythematosus (SLE), 6 patients with mixed connective tissue disease (MCTD) and 3 patients with rheumatoid arthritis (RA). Counterimmunoelectrophoresis was used for antibody detection and a calf thymus extract was utilized as an antigen. Anti-RNP antibody titers in most patients with MCTD and RA did not fluctuate significantly, whereas those in patients with SLE fluctuated by more than a four-fold serum dilution. The anti-RNP antibody titers in SLE seemed to correlate with disease activity. Previous reports have stated that anti-RNP antibody titers do not fluctuate easily throughout the disease course. The present report shows that they do fluctuate in patients with SLE. The use of high dose of steroids in SLE might be responsible for the fall in antibody titer.     

Real-time Epstein-Barr virus PCR for the diagnosis of primary EBV infections and EBV reactivation.

BACKGROUND: The serological diagnosis of primary Epstein-Barr virus (EBV) infections is often difficult, whereas the relevance of elevated immunoglobulin G (IgG) antibodies against early antigen (EA) for the diagnosis of EBV reactivation has increasingly become a matter of dispute. Recently, EBV PCR has been added as a diagnostic tool. Positive EBV PCR has been demonstrated in the serum of patients with primary EBV infections and EBV reactivation. OBJECTIVES: To compare classical serological diagnosis of primary EBV infection and EBV reactivation with real-time EBV PCR. STUDY DESIGN: Sera from 45 patients were selected with detectable immunoglobulin M (IgM) antibodies against EBV viral capsid antigen (VCA), and 62 sera were selected with a reactivation profile. A real-time EBV PCR was performed with DNA extracted from these sera. RESULTS: Based on serological data, the diagnosis of primary EBV infection was established for 24 of the 45 IgM VCA-positive patients. By performing PCR, seven extra cases of primary infection were diagnosed for which no heterophilic antibodies could be detected. In five cases of primary infection, no EBV DNA could be detected by PCR. Only in two of the 62 sera with a reactivation seroprofile could EBV DNA be detected. CONCLUSIONS: Based on these data, we suggest that for the diagnosis of primary infections, EBV PCR could lead to an increase of >16% in the number of positive diagnoses by confirming a positive IgM VCA in the absence of heterophilic antibodies. Furthermore, EBV PCR is positive in only 3% of sera with elevated antibodies against EA, raising doubt as to the utility of EA titers for diagnosing EBV reactivation.     

Acid pH-induced changes in the immunoreactivity of specific antigen and antibody in circulating immune complexes from tuberculosis sera.

The effect of acid pH treatment of circulating immune complexes (CIC) derived from tuberculosis sera was studied on the relative titers of specific antibody (CIC Ab) and mycobacterial antigen (CIC Ag) in the complexes. While the specific antibody titers increased, the titers of CIC Ag declined as a result of acid pH treatment of CIC, both changes being highly significant statistically. Direct exposure of TB sera to pH 2.8 also resulted in significant enhancements in the titers of antibodies directed against Mycobacterium tuberculosis (M.tb.). Competition experiments indicated that the acid pH-treated antibodies retained their antigen specificity. TB sera treated with pH 2.8 for 30 min and then neutralized back to pH 7.4 retained the enhanced antibody reactivity even after 7 days of storage at 4 degrees C. Our results indicate that acid pH treatment induces higher antigen binding ability in anti-M.tb. antibodies, present in free or complexed form in TB sera. These changes appear to be irreversible. Dissociation of CIC and analyzing the titers of released antibody and antigen components offered no advantage with respect to the immunodiagnosis of tuberculosis, as compared to the levels of undissociated CIC components or the serum antibody.     

Reactivity of agalactosyl IgG with rheumatoid factor

BACKGROUND: The difference in the reactivity of rheumatoid factor (RF) with agalactosyl IgG and galactosyl or intact IgG is controversial. METHODS: ELISAs for IgM-RF, using agalactosyl IgG and galactosyl IgG as a coated antigen, were developed. The sera of patients with RA, systemic lupus erythematosus (SLE), chronic liver diseases (CLD) and healthy subjects were measured for IgM-RF. An absorption test was performed using these IgGs with or without heat-pretreatment to determine any differences in the antigenicity between agalactosyl IgG and intact IgG. RESULTS: IgM-RF titers were higher against agalactosyl IgG than those against both heated-intact and intact IgG in three of nine RA patients' sera. Three other serum samples showed similar titers against all three forms of IgG and the remaining three serum samples showed varied results. IgM-RF titers were higher in sera of RA patients than those of SLE, CLD and healthy subjects. Absorptions of RA sera showed that RF was completely absorbed by 5x of absorptions with agalactosyl IgG as well as intact IgG. CONCLUSIONS: The reactivity of IgM-RF is higher with agalactosyl IgG than with heated-intact or intact IgG. The antigenic epitopes of agalactosyl and intact IgG were shown to be closely similar by the absorption test.     

Evaluation of four commercially available ELISA assays for the serologic diagnosis of Lyme disease

We evaluated four commercially available ELISAs for detection of antibody to Borrelia burgdorferi with 21 sera from patients with clinically diagnosed Lyme disease and 89 patient control sera. Patient control sera included 28 sera from patients with rheumatoid arthritis (RA), 17 sera from patients with systemic lupus erythematosus (SLE), and 44 sera containing antibodies reported to cross-react in some Lyme disease tests. The ELISAs tested (Cambridge Bioscience, Diamedix, 3M, and Zeus) detect antibodies (IgM and/or IgG) that bind Borrelia burgdorferi antigen attached to microtiter wells. Antibody reactivity in the sera from patients with clinically diagnosed Lyme disease was characterized by using Zeus immunoglobulin class-specific assays (IgM and IgG). Sensitivities in early and late Lyme disease were as follows: Cambridge and Diamedix, 57% and 100%; 3M, 57% and 93%; and Zeus, 71% and 86%. Reactivities within a patient control population were: Cambridge and Diamedix, 3%; 3M, 7%; and Zeus, 10%.