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We performed a pooled analysis of data on self-reported history of infections in relation to the risk of non-Hodgkin lymphoma (NHL) from 17 case-control studies that included 12,585 cases and 15,416 controls aged 16-96 years at recruitment. Pooled odds ratios (OR) and 95% confidence intervals (95% CI) were estimated in two-stage random-effect or joint fixed-effect models, adjusting for age, sex and study centre. Data from the 2 years before diagnosis (or date of interview for controls) were excluded. A self-reported history of infectious mononucleosis was associated with an excess risk of NHL (OR = 1.26, 95% CI = 1.01-1.57 based on data from 16 studies); study-specific results indicate significant (I(2) = 51%, p = 0.01) heterogeneity. A self-reported history of measles or whooping cough was associated with an approximate 15% reduction in risk. History of other infection was not associated with NHL. We find little clear evidence of an association between NHL risk and infection although the limitations of data based on self-reported medical history (particularly of childhood illness reported by older people) are well recognized.

A role for Epstein Barr virus (EBV) in Hodgkin lymphoma (HL) pathogenesis is supported by the detection of EBV genome in about one-third of HL cases, but is not well defined. We previously reported that an elevated prediagnosis antibody titer against EBV nuclear antigens (EBNA) was the strongest serologic predictor of subsequent HL. For the present analysis, we measured antibody levels against EBNA components EBNA1 and EBNA2 and computed their titer ratio (anti-EBNA1:2) in serum samples from HL cases and healthy siblings. We undertook this analysis to examine whether titer patterns atypical of well-resolved EBV infection, such as an anti-EBNA1:2 ratio ≤ 1.0, simply reflect history of infectious mononucleosis (IM), an HL risk factor, or independently predict HL risk. Participants were selected from a previous population-based case-control study according to their history of IM. We identified 55 EBV-seropositive persons with a history of IM (IM+; 33 HL cases, 22 siblings) and frequency-matched a comparison series of 173 IM history-negative, EBV-seropositive subjects on HL status, gender, age and year of blood draw (IM-; 105 cases, 58 siblings). In multivariate logistic regression models, an anti-EBNA1:2 ratio ≤ 1.0 was significantly more prevalent in HL cases than siblings (odds ratio, 95% confidence interval = 2.43, 1.05-5.65); similar associations were apparent within the IM+ and IM- groups. EBNA antibodies were not significantly associated with IM history in HL cases or siblings. These associations suggest that chronic or more severe EBV infection is a risk factor for HL, independent of IM history.

Although Epstein-Barr virus has been detected in prostate tissue, no associations have been observed with prostate cancer in the few studies conducted to date. One possible reason for these null findings may be use of cumulative exposure measures that do not inform the timing of infection, i.e., childhood versus adolescence/early adulthood when infection is more likely to manifest as infectious mononucleosis (IM). We sought to determine the influence of young adult-onset IM on the prostate by measuring prostate-specific antigen (PSA) as a marker of prostate inflammation/damage among U.S. military members. We defined IM cases as men diagnosed with IM from 1998 to 2003 (n = 55) and controls as men without an IM diagnosis (n = 255). We selected two archived serum specimens for each participant, the first collected after diagnosis for cases and one randomly selected from 1998 to 2003 for controls (index), as well as the preceding specimen (preindex). PSA was measured in each specimen. To explore the specificity of our findings for prostate as opposed to systemic inflammation, we performed a post hoc comparison of other infectious disease cases without genitourinary involvement (n = 90) and controls (n = 220). We found that IM cases were more likely to have a large PSA rise than controls (≥ 20 ng/mL: 19.7% versus 8.8%, p = 0.027; ≥ 40% rise: 25.7% versus 9.4%, p = 0.0021), as were other infectious disease cases (25.7% versus 14.0%, p = 0.020; 27.7% versus 18.0%, p = 0.092). These findings suggest that, in addition to rising because of prostate infection, PSA may also rise because of systemic inflammation, which could have implications for PSA interpretation in older men.

An epidemiologic study of index and family infectious mononucleosis and adult Hodgkin's disease (HD): Evidence for a specific association with EBV+ve HD in young adults. Infectious mononucleosis (IM) is an established risk factor for Hodgkin's disease (HD). A substantial minority (33%) of cases of HD have. Epstein-Barr virus (EBV) DNA within the malignant cells (are EBV+ve). It is unclear whether risk after IM applies specifically to EBV+ve HD. We report the results of a population-based case-control study of HD in adults (n = 408 cases of classical H D, 513 controls) aged 16-74 years; the case series included 113 EBV+ve and 243 EBV-ve HD. Analyses compared total HD, EBV+ve HD and EBV-ve HD with the controls and EBV+ve HD with EBV-ve HD cases using, mainly, logistic regression. Regression analyses were adjusted for gender, age-group and socioeconomic status, and were per formed for the whole age range and separately for young (< 35 years) and old adults ( greater than or equal to 35 years); formal tests of effect modification by age were included. For the young adults, reported 1M in index or relative was strongly and significantly associated with EBV+ve HD when compared to controls (odds ratio [OR] = 2.94, 95% confidence interval [Cl]: 1.08-7.98 and OR = 5.22, 95% Cl: 2.15-12.68, respectively). These results may be interpreted as indications that late first exposure to EBV increases risk of HD, especially in young adults; this applies primarily to EBV+ve HD. (C) 2003 Wiley-Liss, Inc.

Monoclonal antibodies (MAbs) were developed against immunodominant HHV-6 (GS isolate) late and early proteins. The major late protein was identified as a probable glycoprotein with a molecular weight of approximately 110 kDa (gp 110). Immunoblotting of the early antigen yielded proteins of 41 and 38 kDa (p41/38). The MAb to the early protein reacted with cells infected with 14 different HHV-6 isolates. In contrast, the MAb against the late protein reacted with only 10 of these isolates, indicating that there was strain variation in this glycoprotein. The percentage of antibody-positive sera reactive with gp110 in the ELISA ranged from 56% to 96% among the different serum donor categories. In contrast, only 10-30% of the sera were positive for antibodies to p41/38 with the exception of sera from patients with African Burkitt's lymphoma (ABL) and Hodgkin's disease (HD). These antibody patterns denote the presence of active HHV-6 replication in patients with ABL and HD.

We assessed 33 lymphoid tissues from 15 patients, including 7 with X-linked lymphoproliferative disease (XLP) and 8 pa tients with sporadic fatal infectious mononucleosis (IM), to determine whether the cellular infiltrate had the immu nophenotype and expressed Epstein-Barr virus (EBV)-encoded proteins characteristic of either EBV-immortalized lymphoblastoid cell lines (LCL) or EBV-carrying Burkitt lymphoma (BL) cells. The results of these studies revealed that in 13 cases the proliferating B cells were polyclonal, LCL-like, and in 2 cases they were monoclonal, malignant lymphomalike.

Selective protein kinase C inhibitors can either block or significantly reduce Epstein-Barr virus infectivity: inhibition of transformation and decreased 3H-thymidine (3H-TdR) incorporation in human B lymphocytes infected with B95-8 EBV, as well as a significant reduction in the induction of early antigens in Raji cells superinfected by P3HRI EBV was achieved by pre-treating the cells with the inhibitors. The inhibitors do not act by blocking binding of the virus to its cellular receptor CR 2, but rather are effective in the viral internalization process. Our results suggest that protein kinase C may be involved in the process of viral entry into cells.

DNA from malignant cells is present in the serum/plasma of cancer patients and DNA from this source is amenable to analysis by polymerase chain reaction (PCR). In the present study, we evaluated whether Epstein-Barr virus (EBV) DNA is present in the serum of patients with EBV-associated Hodgkin's disease (HD). Using conventional PCR, EBV DNA was detected in serum from 30/33 patients with EBV-associated HD but in only 6/26 patients with non-EBV-associated disease (p < 0.001). Samples from healthy individuals were negative and only 5/12 infectious mononucleosis samples were positive. Real-time quantitative PCR was subsequently employed to determine the concentration of EBV DNA present in serum; among positive samples the level ranged from 1 to 705 copies per 125 microliter of serum. Post-treatment samples from 5/14 cases with EBV-associated HD contained detectable EBV DNA; analysis of this small group of cases suggests that positivity in post-treatment samples correlates with risk factors indicative of a poor prognosis. Overall, our results are consistent with the notion that DNA from Reed-Sternberg cells is present in the serum of HD patients, and further suggest that serum EBV should be evaluated as a prognostic marker. Int. J. Cancer (Pred. Oncol.) 84:442-448, 1999.

Primary infection with the Epstein-Barr virus (EBV) results in fatal infectious mononucleosis in up to 70% of males affected by the X-linked lymphoproliferative syndrome (XLP). This rare disease is often associated with diverse natural killer (NK)-, B- and T-cell deficiencies. We describe experiments testing whether the B lymphocytes of affected males play a role in the pathogenesis of XLP due to a low susceptibility to T-cell-mediated immunity. Using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry we detected in these B cells the expression of viral proteins EBNA-I, EBNA-2, EBNA-3A, EBNA-3C, LMP-1 and LMP-2A, which provide targets for cytotoxic T cells. Major histocompatibility complex (MHC) class I, MHC class II and the B7 costimulatory molecule were present on the cell surface. Accordingly, the EBV-infected B cells were lysed in 51 Cr-release assays by T lymphocytes sharing MHC determinants with the targets. This MHC-restricted and specific lysis was confirmed in competition experiments using MHC-specific monoclonal antibodies (MAbs) and synthetic peptides. XLP-derived LCLs could also induce MHC class I-restricted memory and cytotoxic T lymphocytes. Thus, these XLP-derived B cells resembled normal LCls in vitro with respect to induction of EBV-specific cytotoxic T cells (CTL), the ability to present EB viral antigens and the susceptibility to EBV-specific and MHC-restricted CTL-mediated killing. The failure of the immune system to eliminate these virus-infected B cells in XLP is clearly not caused by a B-cell-specific defect.

We examined the patterns of viral gene expression in acute infectious mononucleosis (IM) patients and the clonality of the directly growing EBV-carrying cell lines. Both low- and high-density EBV-carrying B cells obtained from the patients' tonsils expressed EBNA1, EBNA2 and LMP1. Like LCLs and immunoblastic B-cell lymphomas, the in vivo EBV-carrying low-density cells used only the latency III program for viral gene expression. The in vivo EBV-carrying high-density B cells used both the latency I program, as indicated by the QUK-, and the latency III program, as indicated by the YUK-EBNA1. This suggests that the lymphoid tissues contained not only proliferating immunoblasts but also cells programmed for latent viral persistence in vivo. EBV-carrying cells that grew directly into permanent cell lines in the presence of virus-neutralizing antibody and a late viral inhibitor were polyclonal, as indicated by JH rearrangement. Two of the high-density-derived lines had identical JH and TR patterns, indicating a common parental origin. Our investigation indicates that EBV-carrying cells divide and survive in a fully competent immune system during the outbreak of acute IM.

Using a sandwich enzyme-linked immunosorbent assay (ELISA) we were able to detect a soluble form of the CD30 antigen (CD30s) in the supernatant of cell lines expressing membrane-bound CD30 and in T and B cells after transformation with human T-cell leukemia virus (HTLV-1) and Epstein-Barr-Virus (EBV). While CD30s was not found in 250 healthy controls, it was detected in the sera of patients with Hodgkin's disease (23/100), anaplastic large-cell (6/9), angioimmunoblastic (2/2) and one unclassified high-grade non-Hodgkin's lymphoma (NHL), as well as in 18/20 patients with acute adult T-cell leukemia (ATL, HTLV-1-positive). It was absent in a large number of patients with other high-grade NHL, all low-grade NHLs, acute or chronic leukemias and solid tumors. The only non-malignant disease with detectable levels of CD30s was infectious mononucleosis (9/10). The membrane-bound form of CD30 has a molecular weight of 120 kDa. Western blot analysis revealed that CD30s in the serum of patients has a molecular weight of 88 kDa, identical to the antigen released by cell lines in vitro. CD30s disappeared in all originally positive cases after successful treatment and reappeared in relapsing patients. Thus, CD30s may be useful as a specific marker for disease activity of certain types of lymphoma and ATL.

The EBV-determined nuclear antigen, EBNA, is the only known viral product to be regularly detected in all EBV-transformed cells. The anticomplement immunofluorescence (ACIF) staining detects an EBV-specific nuclear reaction that has recently been shown to be due to at least 2 different proteins, EBNA-1 and EBNA-2, encoded by different parts of the viral genome. We now report the existence of a third antigen of the EBNA complex, designated as EBNA-3. Serum from a patient with chronic infectious mononucleosis contained no detectable antibodies to EBNA-I and had only a low EBNA-2 antibody level. Nevertheless, it gave an EBV-specific nuclear reaction of normal intensity and stained EBNA-2-positive and EBNA-2-negative EBV-carrying lines equally well. Immunoblotting with the same serum identified a new EBV-specific nuclear protein of 143-157 kDa.

This report describes the first of 2 investigations studying mechanisms of Epstein-Barr virus (EBV) persistence in the infected host; specifically, we wish to determine the extent to which virus carriage within the B-cell system is dependent upon continued replication of the virus in permissive oropharyngeal epithelium. Levels of EBV infection at these 2 sites have been monitored in 21 acute infectious mononucleosis (IM) patients before, during and after treatment with high doses of acyclovir (ACV). Twelve patients received oral ACV for 10 days and 9 patients received i.v. ACV for 5 days before the 10-day oral course; all were followed prospectively for 28 days. Infectious EBV, detectable at high initial levels in the patients' throat washings, disappeared almost completely during ACV treatment, then returned again to high levels post treatment. In contrast, levels of virus-infected B cells in the blood showed no reduction linked to the period of ACV treatment nor any increase with resumption of EBV shedding. During IM, therefore, maintenance of high levels of virus carriage within the B-cell pool is not dependent upon the continual recruitment of newly infected B cells. This might reflect an inability of the immune T-cell response in acute IM patients to prevent continued expansion of the existing EBV-infected B-cell pool. Alternatively, it raises the possibility that EBV carriage in B cells in vivo is maintained through a virus: cell interaction which is not sensitive to virus-specific T-cell surveillance.

A protocol utilizing isokinetic gradients to isolate human lymphocytes is combined with another that purifies the C3 receptor-bearing B lymphocyte subpopulation, thus enriching the EB virus genome-carrying population. Also, rabbit antisera were prepared to the Epstein-Bar virus nuclear antigen (EBNA) and the early antigen (EA) and utilized in an indirect immunofluorescence test (IIT) to detect these antigens in human lymphocytes isolated from various disease states. Using these methods we demonstrated excellent correlation between standard methods previously employed to detect EB virus-coded antigens and our IIT employing xenogenic antisera. Such tests were done on lymphoblastoid cell lines as well as lymphocytes isolated directly from patients with EB virus lymphoproliferative diseases. Human palatine tonsil-derived lymphocytes from children with exudative tonsillitis and peripheral blood lymphocytes of infectious mononucleosis contained only EBNA in C3 receptor-bearing B lymphocytes. However, patients with lymphoproliferative disorders, including Hodgkin's disease, harbored in their spleens and lymph nodes lymphocytes producing both EBNA and EA.

Lymphocytes were explanted from EBV-seropositive donors including peripheral blood of infectious mononucleosis patients, healthy donors and EBV-genome-carrying cells from Burkitt lymphoma (BL) biopsies or nude mouse-passaged, BL-biopsy-derived lines. The explanted cells were mixed with fresh cord-blood lymphocytes from mice of the opposite sex. In all categories of derived lines, cord-blood cell progeny was predominant, as judged by the sex marker and other associated markers. Only one BL biopsy line, serially passaged in nude mice, gave rise to a monoclonal lymphoma line.

In attempting to relate immunoglobulin production to certain aspects of the neoplastic behavior of human lymphoid cells proliferating in vivo, we have analyzed the immunoglobulin cell products from 21 human tumor lines experimentally maintained in serial transplantation in neonatal hamsters. These lines were established in the neonatal hamster (usually facilitated by treatment with antilymphocyte serum) by direct implantation of cells from the patient, or after an intermediate stage in cell culture. Three lines derived from patients with acute lymphoblastic leukemia (conversion from lymphosarcoma) which also produced leukemic manifestations in the hamster, did not produce immunoglobulins. In all other lines, these being derived from patients with lymphosarcoma (without conversion to leukemia), infectious mononucleosis, Hodgkin's disease and Letterer-siwe's disease, human immunoglobulin components were detected in the serum of the hamster host by immunodiffusion methods. With rare exceptions H-chain was accompanied by L-chain, these components being specific for the tumor line under investigation and without variation as to the class of H-chain and type of L-chain detected throughout observation periods of as little as 3 months or as much as 2 years. One line showed a sequential deletion of both H- and L-chains. In another line an H-chain not usually present was detected for a short time midway in passage.

A concentration of disodium phosphonoacetate (PA) has been defined which will reduce the synthesis of infectious EB virus in a producer cell line to 1% of control values but which will not affect the growth of EB virus-transformed cells in a 12-week colony-forming assay. When total mononuclear cells or T-lymphocyte-depleted mononuclear cells from the blood of acute IM patients were cultured in the presence of PA at the above concentration, the regular establishment of EB virus genome-containing cell lines seen in control cultures was almost totally abolished. In further experiments, when T-lymphocyte-depleted IM mononuclear cells were co-cultivated with foetal cells of the opposite sex in the presence and absence of PA, cell lines of mixed or of exclusively foetal origin were obtained not only from control co-cultures but also on those rare occasions when transformed foci developed in PA-treated co-cultures. The results suggest that all cell lines derived from the blood of IM patients are initiated in culture by a two-step process of virus release and secondary infection, and argue against the occurrence of any direct outgrowth of IM cells transformed by the virus in vivo.

A micro-51chromium-release assay was used to examine the possible relationship between antibodies cytotoxic to lymphoid cells and EBV infections of the donors. No correlation was found between sera capable of killing lymphoid cells pre-labelled with51 chromium and the presence or absence of antibodies to the viral capsid antigen (anti-VCA). Sera obtained from patients with infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease known to have elevated titers to anti-VCA showed cytotoxicity similar to control values. There appeared to be both heat-stable and heat-labile complement-dependent cytotoxic antibodies.

The Epstein-Barr virus (EBV) has been consistently found to be associated with Hodgkin's disease (HD) in two ways: cases generally have elevated titer distributions of antibodies against the viral capsid antigen, and the occurrence of HD among persons with a history of EBV infectious mononucleosis is two or three times higher than expected. We evaluated this association by measuring the prevalence and level of antibodies against EBV and related viruses among 304 cases of HD interviewed in a population-based study in comparison to 285 of their siblings. The most significant finding was that antibody titers to the viral capsid antigen of EBV were elevated (greater than or equal to 1:320) in 39% of the cases and in only 14% of the sibling-controls; the relative risk adjusted for age and sex was 4.1. The geometric mean titer was three-fold higher among cases (175.6 vs. 58.1) Subjects who reported a history of IM had a higher distribution of titers than those who did not. Cases also had elevated titers against the early antigen of EBV - the D Component being most prominent. A significantly higher proportion of cases has elevated titers against CMV, relative risk = 3.4, but the prevalence of CMV antibody was relatively low and not consistently higher among cases. The findings support the hypothesis that EBV may play a role in the pathogenesis of HD among persons with elevated titers. The findings neither confirm nor deny a possible role of CMV.

Nineteen pediatric patients with Hodgkin's disease (HD) who had experienced primary Epstein-Barr virus (EBV) before, or in one case after, diagnosis, were studied longitudinally for changes in the titers and spectra of EBV-related antibodies, excretion of EBV into the oropharynx, the number of EBV-carrying lymphoid cells in the peripheral blood, and clinical signs and symptoms suggestive of reactivation of the latent virus. The incidence and geometric mean titers of IgG antibodies to viral capsid antigen (VCA) in the HD patients at the time of diagnosis and in the controls were similar. The anti-VCA titers of the patients rose above control levels during and after therapy and remained elevated for up to 7 years of observation. At no time were heterophil or VCA-specific IgM antibodies detected. Antibodies to EBV-induced early antigens were more common in patients (ultimately 80%) than in controls (9%). In contrast, antibody levels to EBV-associated nuclear antigen were disproportionally low in the patients. Excretion of EBV was noted at increased frequency in the patients but the number of circulating, EBV-carrying lymphoid cells was the same as in controls. No discrete clinical syndrome was associated with rising antibody titers or viral excretion. While these results are best explained by a presumed reactivation of the persistent EBV infection by immunosuppressive effects of HD or its therapy, they have not provided direct evidence for this suggestion.

The occurrence and incidence of a characteristic banding abnormality in a No. 14 chromosome has been studied in Burkitt-lymphoma-derived and certain other EB virus-associated lymphoblastoid cell lines. The abnormality was readily detected in 7 out of 7 Burkitt lines, and when present could be seen in 100% of cells with recognizable No. 14 chromosomes. In contrast, the abnormality was not observed in 775 cells from 31 infectious mononucleosis-derived lines nor in 450 cells from 18 lines obtained from cord blood lymphocytes experimentally transformed by EB virus in vitro. The significance of the abnormality as an indication of cells transformed by the virus in vivo is discussed, and the importance of this considered in relation to a possible oncogenic role for EB virus in man.

Monospecific conjugated (fluorescein isothiocynate and horseradish peroxidase) goat antisera, prepared against three human immunoglobulin classes, IM (mu), IgG (gamma) and IgA (alpha), were compared for their ability to detect human Ig classes possessing specificity for Epstein-Barr virus (EBV) viral capsid antigens (VCA) in a chronically infected human lymphoid cell line, P3J-HRIK. It was determined that the enzyme system was significantly more sensitive than immunofluorescence in detecting most of the immunoglobulins in sera from cancer patients. Some patients with nasopharyngeal carcinomas (NPC) had extremely high levels of EBV-specific IgA, whereas cancers other than NPC may have lower EBV-specific IgA titers.

Specific antibody responses against the 2 major subcomponents of EBNA, EBNA1 and EBNA2 were evaluated, in order to study whether this serological study was beneficial compared to classical EBV serology. During this investigation, 491 sera, obtained from blood donors and patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), infectious mononucleosis (IM), Hodgkin's disease, renal transplantation, rheumatoid arthritis and Human Immunodeficiency Virus (HIV) infection, were tested. While the anti-EBNA1 response followed the classical anti-EBNA/Raji response (99% of anti-EBNA/Raji-positive sera also recognize EBNA1), the anti-EBNA2 response was much less frequent and did not correlate with either anti-EBNA/Raji or anti-EA antibodies. In a control population, 8% of individuals had antiEBNA2 antibodies at titers greater than or equal to 10. The percentage was 45% in NPC and 38% in EBV-associated BL; thus, although not detected in all patients with EBV-associated tumors, anti-EBNA2 serology might be a useful marker in BL and NPC. No antibody was detected in the early course of IM, but in rheumatoid arthritis and in HIV-infected patients, the percentage of positive individuals reached 54 and 68, respectively. Seroconversion to EBNA2 was noted in a few cases, including renal transplant recipients, AIDS patients, and complicated IM. This suggests that in these situations, EBNA 2 serology might represent a useful marker related to modulation of the immune status or EBV reactivation.

Epstein-Barr virus (EBV) has long been implicated in nasopharyngeal carcinoma (NPC). Recent studies in our and other laboratories have shown a correlation between the disease and high antibody titers to EBV-specific DNase. These data led us to also examine serial sera from healthy adults and patients with infectious mononucleosis or NPC, for their capacity to neutralize the EBV-specific thymidine kinase (TK) activity from chemically induced EBV-carrying human lymphoblastoid cells. Our results were the following: (i) sera were found that efficiently blocked the EBV-specific TK activity of induced-Raji TK- cell extracts, but not the host-cell TK activity from EBV-negative BJAB cells; (ii) a relationship appeared between high levels of EBV-specific TK-neutralizing activity in sera and NPC pathology, even though in this preliminary study the degrees of EBV-induced TK-blocking activity detected in sera were not significantly correlated with EBV-specific antibody titers; (iii) the EBV-induced TK-neutralizing activity was found in the main IgG fraction derived from NPC sera. These data must be compared with other known antibody responses to EBV for their clinical interest in NPC control.

Throat washings from 26 nasopharyngeal carcinoma (NPC) patients from Hong Kong and Tunisia were studied for the presence of transforming EBV. Only six (23%) were found positive which led to the hypothesis of a neutralizing factor in such salivas. The search for EBV-specific antibodies showed that NPC saliva contained neutralizing VCA and EA IgA (54 and 27% respectively) and VCA and EA IgG (73 AND 54% respectively). Both transforming and non-transforming throat washings contained virus particles as visualized by electron microscopy, but in non-transforming salivas (containing IgA and IgG) the particles were found to be clumped. Comparative study of throat washings from patients with Burkitt's lymphoma (BL); infectious mononucleosis (IM), immunodeficiencies, other cancers, and healthy subjects showed that IgA were restricted to NPC cases.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.

Twenty-three patients with recent infectious mononucleosis were studied for cell-mediated immunity to Epstein-Barr virus (EBV)-associated antigens. By means of a virion-containing antigen preparation, (P3J), leukocyte migration inhibition was demonstrated in 6 of 13 patients with acute infectious mononucleosis (IM) and in 6 of 10 patients studied during the convalescent period. Inhibition with a soluble antigen (S) preparation was seen in only a few patients at all stages of the illness. Lymphocyte blast transformation on exposure to P3J occurred in 4 of 9 patients with acute IM; no other study group reacted. Control donors lacking anti-EBV antibodies did not demonstrate migration inhibition or blast transformation with either P3J or S. Control donors with evidence of previous EBV infection did demonstrate migration inhibition with P3J (8 of 12) and S (3 of 10). These studies indicated that cell-mediated immunity to EBV-associated antigens could be detected by either leukocyte migration inhibition or lymphocyte blast transformation, but neither assay is diagnostic of infectious mononucleosis.

Antibody to EB virus was assayed in 586 sera from several populations in Australia and New Guinea and from leukaemic and control groups. The prevalence of antibody to EB virus in a normal population in Brisbane increased gradually from about 31% in 1- to 5-year-olds to about 90% in adults. A similar age distribution occurred in a population from Griffith, N.S.W. In contrast, all 1- to 5-year-old Aboriginal children from the west coast of Cape York Peninsula and from New Guinea had antibody to EB virus, indicating a high rate of infection in early life. Aborigines from the east coast of Cape York Peninsula had a slightly lower prevalence. The geometric mean (GM) titres of 63 leukaemic cases from Sydney (1:72) and of 60 non-leukaemic hospital patients (1:80) were not significantly different, but each was significantly higher than that (1:44) of 57 normal controls from Griffith. No association was found between the degree of remission in 29 cases of acute myeloid leukaemia and either the presence of antibody to heat-labile EB viral complement-fixing antigen or the titre of antibody to viral capsid antigen, or to crude or soluble EB viral complement-fixing antigens. The significantly higher GM antibody titres in non-leukaemic patients and in Aboriginal children with non-malignant diseases suggest that some pathological process common to a variety of diseases results in moderately increased titres of antbody to EB virus.

These data describe the use of a passive hemagglutination test (PHAT) for the detection of antibodies to the Epstein-Barr virus (EBV) associated antigen, early antigen-D (EA-D). EA-D was obtained from EBV-superinfected RPMI 64-10 cell preparations which showed by immunofluorescent staining (IF) that 25-35% of the cells contained EA-D. Solubilization of EA-D was achieved by sonic oscillation, treatment of the pellet-associated material with triton X 100, ammonium sulfate precipitation, and chromatography on diethylaminoethyl celluiose. Successful use of the PHAT for detection of antibodies to EA-D was dependent on the degree of purity of the partially purified antigen. Throughout these studies preparations from uninfected 64-10 cells were used as control reagents. More than 100 human antisera from patients with EBV-associated diseases, which had been previously tested by standard IF procedures were assayed by PHAT. A good correlation was found between IF and PHAT titers for EA-D and, in addition, the PHAT was 50-100 times more sensitive. Most human sera gave no agglutination of red cells coated with control preparations. An inhibition (blocking) of PHAT was developed which was useful in the purification and characterization of EA-D. These data indicate that the PHAT may provide a rapid, reliable, objective and sensitive method for studying EA-D and possibly other virus-associated antigens.

A glycoprotein antigen (gp40) was previously identified as a major component of immune complexes isolated from sera of patients with Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC). In the present work gp40 was partially purified from a pool of BL/NPC sera. Sera of patients with BL and NPC as well as sera of patients with other malignant and non-malignant diseases were tested for antibodies against gp40, using a double antibody radioimmunoassay. Practically all normal sera and sera of patients with non-malignant diseases had antibodies capable of binding radioiodinated gp40. In contrast, sera of patients with BL or NPC had very low binding, while sera of patients with other malignant diseases had intermediate binding values. The low binding activity of BL/NPC sera was shown to be due to inhibition by excess gp40 present in such sera. Effusions of patients with breast or ovarian cancer also contained demonstrable amounts of gp40. The origin of gp40 is still unknown.

Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co-cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB-virus-transformed B-cell line. The fresh cells present in co-cultures from EB virus antibody-negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co-cultures from seropositive donors. Larger-scale co-cultures, set up at a ratio of 80-100 fresh UM cells to one autologous virus-transformed B cell, were harvested after 8 to 12 days and the T-cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co-cultures and were strongly active against the autologous virus-transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome-negative target cell lines chosen as sensitive indicators of non-specific cytotoxicity. Recognition of allogeneic EB-virus-transformed cells was restricted to those whose HLA-A and/or B and/or B and/or C antigen expression matched that of the effector cells themselves;; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB-virus-specific HLA-restricted memory T cells, present in the blood of previously-infected individuals, can be reactivated in vitro using the established autologous virus-transformed cell line as a stimulus. THe reactivated cytotoxic cells appear to recognize a virus-induced lymphocyte-detected membrane antigen, LYD-MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious monoucleosis.

Stimulated by a report on elevated IgA levels in nasopharyngeal carcinoma (NPC), we tested a total of 372 sera from patients with NPC, other carcinomas of head and neck or elsewhere, Burkitt's lymphoma (BL), infectious mononucleosis (IM) or healthy controls. The sera were titrated in indirect immunofluorescence tests for IgA antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and to the diffuse (D) or restricted (R) components of the EBV-induced early antigen (EA) complex. The results proved NPC to be outstanding in that prior to therapy 93% of the patients tested revealed IgA antibodies to VCA and 73% to D, often at high titers which occasionally matched the corresponding IgG antibody levels. The EBV-specific IgA titers increased from stages I or II to stages III or IV; i.e. with the total tumor burden. Conversely, many of the NPC patients examined 2-6 years after initial therapy had only low levels of EBV-specific IgA or none at all, and the majority of those with high titers were known to have residual or recurrent disease. In contrast to untreated NPC patients, less than 5% of 73 patients with other carcinomas or of 76 healthy donors revealed VCA-specific IgA and even fewer EA-specific IgA; only 28% and 4% of 54 BL patients tested at admission had IgA antibodies to VCA and R, respectively, and 38% and 3% of 37 IM patients showed transient VCA- or D-specific IgA responses, all at generally low titers. While sera from untreated NPC patients often contained IgA antibodies also to herpes simplex type 1 virus, their incidence and range of low titers were similar to those obtained with sera from patients with other carcinomas or from healthy donors. It thus appears that the elevated IgA levels in NPC might be due to EBV-specific antibodies. Possible reasons for this unique response in NPC have been discussed.

Critical evaluation of the anti-complement immunofluorescence (ACIF) test for Epstein-Barr virus-associated nuclear antigen (EBNA) in known EBV-associated cell lines was carried out. The ACIF procedure, in which the cell smears were thoroughly air-dried and fixed with carbon tetrachloride (for 15 min at room termperature), provided reproducible staining results with minimum variations. The fixed smears coulb be stored for at least 5 weeks at minus 80 degrees C without loss of reactivity. On the basis of the above evaluation, a total of 59 human cell lines, which had unknown or uncertain association with EBV, were examined for the presence or absence of EBNA by the ACIF procedure. All of the cell lines tested could be judged positive or negative for EBNA. All of 21 lines growing as monolayer or in a mixed state of monolayer and suspension, were negative. Of 38 lines grown in suspension, 36 lymphoblastoid lines were positive but the remaining 2 lines (seemingly not lymphoblastoid) were negative.

EBNA-positive lymphoblast cells were detected in 0.1 to 0.9% of the T-cell-depleted lymphocytes obtained from peripheral blood samples of five patients with infectious mononucleosis (IM). The same blood specimens from four of the five patients contained cells that formed EBNA-positive colonies in soft agar containing EBV antibodies. The ratio of the colony formers to EBNA-positive cells was higher in blood samples taken early in the disease than in those obtained in later stages of the disease. The present results strongly suggest that EBV-transformed cells are present in the peripheral circulation of IM patients and that such cells can directly give rise to immortalized cell lines in vitro.

Peripheral lymphocytes obtained from two individuals with a previous history of infectious mononucleosis were exposed to mitomycin-treated cells of the autologous lymphoblastoid cell line (LCL) established during the acute phase of the disease. This resulted in a stimulation of DNA synthesis, comparable to or even exceeding a one-way MLC with allogeneic lymphocytes. The cytotoxic effect of the stimulated lymphocytes was tested by colony inhibition or 51Cr release, against a large LCL panel, including the autologous line and allogeneic lines from patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), infectious mononucleosis (IM), leukemia, myeloma, or normal donors. While the majority of the lines were highly sensitive to the killing action, three were relatively resistant. The same pattern of sensitivity was obtained with effector cells stimulated by autologous LCL derived from IM or BL patients. The majority of the target LCLs had B-cell characteristics and carried the EBV genome, but three cell lines that were devoid of the EBV genome, were also sensitive. These lines included two lymphoid cell lines, one of them a T-cell line, and a myeloma line. Fresh peripheral lymphocytes from normal donors or acute IM patients, PHA-induced blasts and blast cells from a case of acute myeloid leukemia were resistant.

Chromosome analysis of three lymphoblast cell lines from New Guinea Burkitt lymphoma (QIMR-GOR, QIMR-AMB/1 and AMB/2), three lymphoblast cell lines from infectious mononucleosis (QIMR-DAR, QIMR-STE and QIMR-GOE) and two lymphoblast cell lines from leukaemia (QIMR-WIL and QIMR-FIN) showed abnormalities associated with Group A, C, and G chromosomes. Each cell line contained diploid and near diploid cells. Some of the cell lines also contained pseudodiploid cells. Extra Group C or Group G chromosomes were found in a proportion of cells from some of the cell lines. Abnormal Group A chromosomes were also found. However, no one abnormality could confidently be assigned to a specific source of origin of the cell line studied. Cellulaire comprenait des cellules diploides et quasi-diploides. Certaines lignees comprenaient egalement des cellules pseudo-diploides. On a trouve des chromosomes surnumeraires du Groupe C ou du Groupe G dans une fraction des cellules de certaines lignees. On a egalement trouve des chromosomes anormaux du Groupe A. Toutefois, aucune de ces anomalies n'a pu ětre attribuee de facon sǔre a une origine particuliare de la lignee etudiee.

Sera from infectious mononucleosis patients aged 2 to 35 years and from normal subjects aged 11 to 35 years were examined for the presence of antibody to the EB virus capsid antigen (VCA) in the indirect immunofluorescence test, and for the presence of antibody reactive with the soluble (S) antigen of lymphoblastoid cell lines in the complement-fixation test. Sera from all infectious mononucleosis patients taken either in the acute stage of the disease or up to 9 years after its onset contained VCA antibody. The sera taken in the acute stage of the disease were free of S antibody; out of 97 sera investigated only one was weakly reactive. Only one of the seven sera taken 4 to 5 months after the onset of the disease contained S antibody. After the 7th month following onset, the majority of the subjects possessed S antibody. These data indicate that the S antibody appears much later in the course of EB virus infection than the VCA antibody. The predominant majority of sera from subjects with no history of the disease possessed VCA antibody; most of these also possessed S antibody. The incidence of S antibody in these subjects and those who had been suffering from infectious mononucleosis 1 to 9 years previously were comparable.

An improved method for the detection of deoxythymidine kinase (TK) in human sera is reported. The method which utilizes 125I-iododeoxyuridine (IdUrd) as a substrate was used to measure TK in sera from patients with different diseases. Sera collected during the acute stage of infectious mononucleosis were found to contain elevated levels of TK, in most cases 10-40 times the normal value. The serum TK activity disappeared gradually and reached a normal level within 4 weeks. Sera from patients with other viral infections contained in most cases normal serum TK levels except in connection with measles, rubella, varicella, herpes simplex virus and cytomegalovirus infections. Additional studies revealed that sera from patients with different types of advanced lymphomas, acute leukemias, chronic granulocytic leukemia and lung cancer of the small-cell type with metastases, contained high TK levels which fluctuated in parallel with alterations in activity of the disease. The TK activity in sera from patients with both mononucleosis and tumor disease was characterized by electrophoresis and by its ability to utilize cytidine triphosphate as the phosphate donor. The results showed that the serum TK has the same properties as the human cytosolar TKI, except in connection with varicella.

A high incidence of oropharyngeal excretion of Epstein-Barr virus (EBV) has been observed in African children with Burkitt's lymphoma (BL) (48%) and matched controls (45%). This compares with an incidence of 77% in American patients with infectious mononucleosis (IM) and 13% in age-matched controls. Cross-neutralization tests between EBV strains derived from BL and IM patients and their sera failed to detect differences in the major neutralizing antigenic components. Cord-blood lymphocytes transformed by American EBV expressed only early viral functions (EBV nuclear and soluble complement-fixing antigens) and produced no detectable transforming activity. By contrast, cord-blood lymphocytes transformed by African EBV strains contained 0.2-0.3% of cells with EBV capsid and early antigen and produced EBV with transforming activity. These cells contained twice as many copies of EBV homologous DNA as the cells transformed by American EBV strains.

Antigens were solubilized and attached to glass slides. The coated glass slides were then used to detect relevant antibodies by a mixed hemadsorption technique. These studies focused primarily on solubilizing the Epstein-Barr virus-associated antigens VCA, EA and MA. Well-characterized, discordant sera from patients with Burkitt's lymphoma and nasopharyngeal carcinoma were then used to evaluate the antigenicity of slides coated with the solubilized antigenic extracts. Good correlations were observed between titers of these sera for VCA, EA and MA as determined by immunofluorescence methods and by mixed hemadsorption. Most antigenic extracts could be stored directly or as coated-glass slides for many months with only moderate decreases in antigenicity. Antibodies to Epstein-Barr-associated nuclear antigen (EBNA) were not detectable in these studies.

Infection of cells of the EBV-genome-negative human B-lymphoma Ramos line with viral isolates obtained from two EBV-transformed marmoset cell lines (B95/8; Nyevu) resulted in the induction of a nuclear antigen (RAM-ag) apparently different from other EBV-associated antigen complexes. This antigen is revealed by indirect immunofluorescence and shows no detectable cross-antigenicity with EBNA or any other known EBV-associated antigen. EBV-isolates from P3HR-1 cells fail to induce a similar antigen in Ramos cells although they induce EBNA. No RAM-ag was expressed, either after infection of cells of another EBV-genome-negative human B-lymphoma line BJAB with B95-8 EBV or in a series of EBV-harbouring cell lines. Thus the antigen appears to be cell-line-specific for Ramos cells. It is also induced upon infection of either B95-8 or P3HR-1 converted Ramos sublines with EBV from B95-8 cells. All human sera with RAM-ag-reactivity revealed antibodies against VCA. However, sera from patients with acute infectious mononucleosis containing high anti-VCA-antibodies did not react with RAM-ag. Seroconversion for this antigen apparently more closely coincides with the appearance of EBNA-directed antibodies.

Injection of concentrated EBV derived from cells of the Kaplan line of infectious mononucleosis (IM) origin resulted in malignant lymphoproliferation in one out of three cotton-top marmosets 6 weeks after inoculation. Two additional animals receiving the same isolate after incubation with an antibody-containing human serum did not develop tumors. Inoculation of concentrated virus derived from the P3HR-1 line of Burkitt origin did not lead to lymphoproliferations in five marmosets. Three of these received non-neutralized, and two received neutralized P3HR-1 virus. The tumor obtained with the Kaplan isolate revealed characteristics of a lymphosarcome. It contained EBV-specific DNA. In addition, EBV-synthesizing lymphoblastoid lines were established from a tumorous lymph-node, as well as from the spleen of the diseased marmoset. Virus recovered from these lines transformed lymphocytes derived from spleens of healthy marmosets. The tumor-bearing animal developed low levels of anti-VCA antibodies during the course of tumor growth. These data demonstrate the oncogenic potential of EBV directly derived from cells of IM origin.

After removal of SRBC rosette-forming T-cells from the peripheral blood, the residual, largely B-lymphocyte fractin of five infectious mononucleosis patients was found no contain 0.5-2% blast cells, positive for the EBV-determined nuclear antigen (EBNA). There was a rough parallelism between the presence of large lymphoblasts in the hematological smear, EBNA-positive large blasts in the B-cell fraction and the ability of the T-cell fraction to expert an EBV-specific lymphocytotoxicity on established cell lines in vitro. EBNA-positive B-cells and EBV-specific killer T-cells disappeared after the acute phase of the disease.

In a comparative study of populations at high and low risk of nasopharyngeal carcinoma (NPC), sera from 442 Eskimo and 770 Danish children and adolescents were tested for the presence of antibodies against Epstein-Barr virus (EBV). Eskimo children in Greenland were seropositive at an early age and showed significantly higher titers of IgG antibody to the viral capsid antigen (VCA) (p less than 0.0001) and soluble (S) antigen (p less than 0.005) than Danes matched for age and sex, but had similar levels of IgA antibody to VCA and IgG antibody to the early antigen (EA). The high geometric mean VCA (IgG) titers found in certain age groups of Eskimo children were as high as those previously reported from areas in Africa highly endemic for Burkitt's lymphoma. In Greenland, neither location nor household size was a determining factor for prevalence or titer of VCA (IgG). The high antibody titers among Eskimo children probably reflect exposure to a large inoculum of EBV at the time of primary infection, infection early in life and/or re-exposure due to the higher incidence of EBV infection in Greenland. In view of the high incidence of NPC in Greenland and the known association of this tumor with EBV, we speculate that the time and quantitative aspects of the primary infection are also factors of relevance in the etiology of NPC.

Transformation to continuous cell lines has been studied in cultures of peripheral leukocytes from infectious mononucleosis (IM) patients and in co-cultures of IM leukocytes and foetal cord blood leukocytes of opposite sex. The transformed cells in the co-cultures were of mixed origin with foetal cells usually predominating. Neutralizing antisera to EB virus markedly reduced or abolished the incidence of transformation in IM leukocyte cultures. This effect was not due to cytotoxicity and followed the pattern seen with cultures where transformation was known to depend on the intercellular transfer of infectious EB virus. The findings suggest that EB virus is harboured in peripheral lymphocytes of IM patients as a non-productive unexpressed infection which is activated to produce virus in vitro, the particles released then infecting neighbouring cells to give transformed lines. The differences between this mechanism and the one whereby lines arise in culture from the malignant cells of Burkitt's lymphoma are considered, and their significance is discussed.

A 12-year-old patient with acute lymphoblastic leukemia received a bone-marrow transplant (BMT) from a matched sibling donor. Nine weeks prior to transplant the donor experienced Epstein-Barr virus (EBV)-induced infectious mononucleosis. The bone-marrow recipient was EBV-negative at the time of transplant; however, 4 weeks post transplant the recipient developed clinical symptoms of graft-verus-host disease (GVHD) coincident with serological evidence of acute EBV infection. In addition, a lymphoblastoid cell line positive for Epstein-Barr nuclear antigen was established from a bone-marrow sample obtained at the onset of symptoms compatible with GVHD. Sera obtained from the recipient over the ensuing 2 months showed the appearance of antibodies to specific EBV antigens consistent with a primary immune response to EBV infection. This association of acute EBV infection with symptoms of GVHD in a BMT recipient suggests a need for further investigation of the epidemiology of EBV infections in human bone-marrow transplantation and the relationship between EBV infection and GVHD.

Peripheral blood lymphocytes from 20 patients with acute infectious mononucleeosis (IM) were studied for cell aggregation and for cap formation by concanavalin A (Con A). The lymphocytes from these patients showed 5.2+/-1.5% cells with a Con-A-induced cap and a high degree of cell aggregation without Con A, compared to 27.7+/-3.2% caps and a low degree of cell aggregation with normal lymphocytes. The lymphocytes from IM patients were fractionated to enrich for T and B cells. There was a low frequency of cap formation in both T and B cells, but the high degree of celll aggregation without Con A only occurred with B cells. Studies with four patients in clinical remission from acute IM have shown that the frequency of Con-A-induced cap formation only returned to normal more than 3 months after the beginning of clinical remission and that even at 6 months the cells still showed a high degree of cell aggregation. The results indicate that a high degree of B-cell aggregation and a low percentage of B and T cells with a Con-A-induced cap were associated with acute IM and that the changes associated with a high degree of B-cell aggregation were by themselves not sufficient to cause the disease.

Under appropriate culture conditions, EB virus infection of lymphocytes from seropositive donors leads to regression of transformation, and this was shown previously to be due to activation in a secondary immune response to T lymphocytes inhibitory for the autologous lymphoblastoid cell line. Regression can be quantified by determining the number of cells required for its expression. To investigate the development of memory T cells with EB-virus specificity in the primary infection, a comparison was made of the capacity for regression of lymphocytes from 16 cases of infectious mononucleosis (IM) and 13 normal donors. With 9 normal seropositive donors a mean lymphocyte concentration of 4.6 X 10(5)/ml was required to achieve 50% regression. In contrast, with 8 cases of IM tested within 1 week of onset, a much higher mean lymphocyte concentration (3.7 X 10(6)/ml) was necessary. Six of these IM cases, and another not tested in the first week, were tested on several occasions between 5 and 23 weeks after onset, and showed a slight reduction in the mean cell concentration required for regression (1.5 X 10(6)/ml). Six additional were tested 23--83 weeks after onset by which time the cell concentration required for 50% regression (mean = 4.5 X 10(5)/ml) had reached the level shown by normal seropositive donors. Regression did not occur with lymphocytes from seronegative donors, even at the highest cell concentration. Recombination cultures of T-cell-depleted and T-cell-enriched lymphocyte populations from 3 IM cases in ratios of 1:7 to 7:1 showed that the failure of regression in acute IM was not due simply to lack of sufficient numbers of T cells. The results indicate that EB-virus-specific memory T-cell activity as detected by the regression test is absent in the acute phase of IM, becomes evident at low levels 5--23 weeks after onset, and reaches a maximum after about 6 months.