Elevated vitamin B12 levels in autoimmune lymphoproliferative syndrome attributable to elevated haptocorrin in lymphocytes.

OBJECTIVE: Identify the etiology of elevated B(12) in autoimmune lymphoproliferative syndrome (ALPS). DESIGN: Peripheral blood of ALPS patients with elevated B(12) and controls were evaluated. RESULTS: Total and holo-haptocorrin (HC) levels were 26- and 23-fold higher in ALPS patients, respectively. No abnormal B(12)-binding proteins were found. Western blot revealed HC in lymphocyte lysates only from ALPS patients. CONCLUSION: Elevated concentrations of B(12) found in ALPS patients were due to increased lymphocyte expression of HC.

Xenotropic murine leukemia virus-related virus is not associated with chronic fatigue syndrome in patients from different areas of the us in the 1990s.

BACKGROUND: In 2009, xenotropic murine leukemia virus-related virus (XMRV) was reported in 67% of patients with chronic fatigue syndrome (CFS) compared to 4% of controls. Since then numerous reports failed to detect XMRV in other cohorts of CFS patients, and some studies suggested that XMRV sequences in human samples might be due to contamination of these samples with mouse DNA. RESULTS: We determined the prevalence of XMRV in patients with CFS from similar areas in the United States as the original 2009 study, along with patients with chronic inflammatory disorders and healthy persons. Using quantitative PCR, we initially detected very low level signals for XMRV DNA in 15% of patients with CFS; however, the frequency of PCR positivity was no different between patients with CFS and controls. Repeated attempts to isolate PCR products from these reactions were unsuccessful. These findings were supported by our observations that PHA and IL-2 stimulation of peripheral blood mononuclear cells from patients with apparently low levels of XMRV, which induced virus replication in the 2009 report, resulted in the disappearance of the signal for XMRV DNA in the cells. Immunoprecipitation of XMRV-infected cell lysates using serum from patients from whom we initially detected low levels of XMRV DNA followed by immunoblotting with antibodies to XMRV gp70 protein failed to detect antibody in the patients, although one control had a weak level of reactivity. Diverse murine leukemia virus (MLV) sequences were obtained by nested PCR with a similar frequency in CFS patients and controls. Finally, we did not detect XMRV sequences in patients with several chronic inflammatory disorders including rheumatoid arthritis, Bechet's disease, and systemic lupus erythematosus. CONCLUSIONS: We found no definitive evidence for XMRV DNA sequences or antibody in our cohort of CFS patients, which like the original 2009 study, included patients from diverse regions of the United States. In addition, XMRV was not detected in a cohort of patients with chronic inflammatory disorders.

Characterization and treatment of chronic active Epstein-Barr virus disease: a 28-year experience in the United States.

Chronic active EBV disease (CAEBV) is a lymphoproliferative disorder characterized by markedly elevated levels of antibody to EBV or EBV DNA in the blood and EBV RNA or protein in lymphocytes in tissues. We present our experience with CAEBV during the last 28 years, including the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients. Most patients presented with lymphadenopathy and splenomegaly; fever, hepatitis, and pancytopenia were common. Most patients died of infection or progressive lymphoproliferation. Unlike cases reported from Japan, our patients often showed a progressive loss of B cells and hypogammaglobulinemia. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many of our patients had reduced NK-cell numbers. Although immunosuppressive agents, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active lymphoproliferative disease that was unresponsive to chemotherapy. These studies are registered at http://www.clinicaltrials.gov as NCT00032513 for CAEBV, NCT00062868 and NCT00058812 for EBV-specific T-cell studies, and NCT00578539 for the hematopoietic stem cell transplantation protocol.

Exploratory analysis of the relationships between aerobic capacity and self-reported fatigue in patients with rheumatoid arthritis, polymyositis, and chronic fatigue syndrome.

OBJECTIVE: To determine if self-reported levels of physical activity and fatigue are related to peak oxygen uptake (VO(2peak)) and whether these relationships differ among the patient groups (rheumatoid arthritis [RA], polymyositis [PM], and chronic fatigue syndrome [CFS]). DESIGN: Correlational investigation. SETTING: Two ambulatory research clinics at the National Institutes of Health, Clinical Center, Bethesda, MD. PARTICIPANTS: There were 9 patients with PM, 10 with RA, and 10 with CFS. All patients met case criteria for their respective diagnoses. METHODS/MAIN OUTCOME MEASUREMENTS: VO(2peak) during bicycle ergometry and self-reported fatigability, fatigue, and physical activity. VO(2peak) was used as the criterion measurement of physiological fatigue with which the self-reported variables were compared. RESULTS: The Pearson r revealed that self-reported physical activity correlated with VO(2peak) (r = 61, P = .01). However, fatigability and fatigue did not correlate with VO(2peak). Linear regression analysis was performed to assess the effects of diagnosis group, self-reported activity level or fatigue, and their interaction. A trend in the data showed a distinctive relationship between fatigue/fatigability within the 3 groups. In addition, when controlling for group status, self-reported activity predicted aerobic capacity as measured by VO(2peak). CONCLUSIONS: This study confirms that patients with chronic, but stable RA, PM, or CFS are fatigued and have significantly decreased aerobic capacity. Self-reports of physical activity predicted VO(2peak), and may be used as an indicator of activity-based aerobic capacity. Self-reports of fatigue, however, did not correlate with VO(2peak) and hence are assessing something other than an index of aerobic capacity, and provide additional information about patients' perceptions, which will require further investigation.

Use of rituximab for refractory cytopenias associated with autoimmune lymphoproliferative syndrome (ALPS).

BACKGROUND: ALPS is a disorder of apoptosis resulting in accumulation of autoreactive lymphocytes, leading to marked lymphadenopathy, hepatosplenomegaly, and multilineage cytopenias due to splenic sequestration and/or autoimmune destruction often presenting in childhood. We summarize our experience of rituximab use during the last 8 years in 12 patients, 9 children, and 3 adults, out of 259 individuals with ALPS, belonging to 166 families currently enrolled in studies at the National Institutes of Health. METHODS: Refractory immune thrombocytopenia (platelet count <20,000) in nine patients and autoimmune hemolytic anemia (AIHA) in three patients led to treatment with rituximab. Among them, seven patients had undergone prior surgical splenectomy; three had significant splenomegaly; and two had no palpable spleen. RESULTS: In seven out of nine patients with ALPS and thrombocytopenia, rituximab therapy led to median response duration of 21 months (range 14-36 months). In contrast, none of the three children treated with rituximab for AIHA responded. Noted toxicities included profound and prolonged hypogammaglobulinemia in three patients requiring replacement IVIG, total absence of antibody response to polysaccharide vaccines lasting up to 4 years after rituximab infusions in one patient and prolonged neutropenia in one patient. CONCLUSION: Toxicities including hypogammaglobulinemia and neutropenia constitute an additional infection risk burden, especially in asplenic individuals, and may warrant avoidance of rituximab until other immunosuppressive medication options are exhausted. Long-term follow-up of ALPS patients with cytopenias after any treatment is necessary to determine relative risks and benefits.

Critical role for BIM in T cell receptor restimulation-induced death.

BACKGROUND: Upon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals. RESULTS: Upon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption. CONCLUSION: Our data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis. REVIEWERS: This article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr.

Pyrimethamine treatment does not ameliorate lymphoproliferation or autoimmune disease in MRL/lpr-/- mice or in patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte apoptosis leading to childhood onset of marked lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, and increased risk of lymphoma. Most cases are associated with heterozygous mutations in the gene encoding Fas protein. Prolonged use of immunosuppressive drugs that do ameliorate its autoimmune complications fail to consistently lessen lymphoproliferation in ALPS. A case series had described children with ALPS, whose spleens (SPL) and lymph nodes decreased in size when treated weekly with pyrimethamine and sulfadoxine; parallel in vitro studies showed only pyrimethamine to promote apoptosis. On the basis of that experience, we undertook additional in vitro lymphocyte apoptosis assays, and measured SPL weights, lymphocyte numbers, and immunophenotypes in Fas-deficient MRL/lpr-/- mice to gain further insights into the utility of combined pyrimethamine/sulfadoxine or pyrimethamine alone. Moreover, seven children with ALPS enrolled in a study of escalating dose of pyrimethamine alone given twice weekly for 12 weeks to determine if their lymphadenopathy and/or splenomegaly would diminish, as assessed by standardized computerized tomography. Neither pyrimethamine alone or with sulfadoxine in the MRL/lpr-/- mice, nor pyrimethamine alone in ALPS patients proved efficacious. We conclude that these drugs do not warrant further use empirically or as part of clinical trials in ALPS Type Ia as a lympholytic agent.

Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus. METHODS: Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death. RESULTS: We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis. CONCLUSION: Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.

Eosinophilia is associated with a higher mortality rate among patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder associated with heritable defects in lymphocyte apoptosis that result in chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity. To examine the prevalence, mechanisms, and potential implications of eosinophilia in ALPS, we reviewed data retrospectively from 187 consecutive ALPS patients and their family members studied at the National Institutes of Health. ALPS patients with eosinophilia were compared with ALPS patients without eosinophilia with respect to their clinical and immunologic phenotype. Potential mechanisms for the eosinophilia, including abnormal Fas-mediated eosinophil apoptosis, increased production of eosinophilopoietic cytokines, and presence of anti-eosinophilic autoantibodies were also explored in a small number of patients from whom samples were available. Analysis of data from 68 ALPS patients and 119 of their relatives identified a distinct subgroup of patients with prominent and persisting eosinophilia that proved to be associated with increased numbers of peripheral blood leukocytes (PBL) of multiple lineages and a trend towards increased serum IgE levels. Eosinophilic ALPS patients also had a significantly higher risk of death due to infectious complications. Although the specific etiology of the eosinophilia in these patients remains uncertain, it does not appear to be associated with an altered serum cytokine profile, increased survival responsiveness of eosinophils to IL-5, defective Fas-mediated eosinophil apoptosis, or anti-eosinophil antibodies. Eosinophilia defines a distinct subgroup of ALPS patients with increased serum IgE levels, increased numbers of PBL of multiple lineages, and higher mortality from infectious complications.

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with alphabeta-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.

Fluorodeoxyglucose positron emission tomography (FDG-PET) for monitoring lymphadenopathy in the autoimmune lymphoproliferative syndrome (ALPS).

Autoimmune lymphoproliferative syndrome (ALPS) is associated with mutations that impair the activity of lymphocyte apoptosis proteins, leading to chronic lymphadenopathy, hepatosplenomegaly, autoimmunity, and an increased risk of lymphoma. We investigated the utility of fluorodeoxyglucose positron emission tomography (FDG-PET) in discriminating benign from malignant lymphadenopathy in ALPS. We report that FDG avidity of benign lymph nodes in ALPS can be high and, hence, by itself does not imply presence of lymphoma; but FDG-PET can help guide the decision for selecting which of many enlarged nodes in ALPS patients to biopsy when lymphoma is suspected.

HLA B44 is associated with decreased severity of autoimmune lymphoproliferative syndrome in patients with CD95 defects (ALPS type Ia).

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte apoptosis characterized by non-malignant lymphadenopathy and splenomegaly, expansion of T cells without either CD4 or CD8 surface markers, and increased incidence of autoimmune diseases and lymphoma. Most patients with ALPS have dominant, heterozygous mutations in tumor necrosis factor receptor superfamily member 6 (TNFRSF6), which encodes CD95, also known as Fas, a mediator of apoptosis. Penetrance and range of disease manifestations in ALPS are highly variable, even among family members who share the same dominant TNFRSF6 mutation. To evaluate HLA as a candidate modifier locus, we typed HLA A, B (including subtypes), and DQB alleles in 356 individuals from 63 unrelated families with defined TNFRSF6 mutations associated with ALPS. We also developed a quantitative severity score and performed statistical analysis. Among the healthier, mutation-bearing individuals, transmission of HLA B44 was significantly overrepresented (nominal P<0.0074) as compared to transmission in patients with severe clinical features of ALPS. The B44 allele may exert a protective role in ALPS.

Histologic features of sinus histiocytosis with massive lymphadenopathy in patients with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder associated with defects in Fas-mediated apoptosis, characterized most often by childhood onset of lymphadenopathy, splenomegaly, hypergammaglobulinemia, and autoimmune phenomena. Children with sinus histiocytosis with massive lymphadenopathy (SHML) have a somewhat similar clinical phenotype in which prominent adenopathy also is associated with hypergammaglobulinemia, and autoimmune phenomena are reported in 10-15% of cases. We observed histopathological features of SHML in the lymph nodes of some of our ALPS patients, further suggesting an association between these two disorders. We, thus, reviewed the lymph nodes from 44 patients ALPS type Ia, all of whom were confirmed to have germline mutations in the TNFRSF6 gene encoding Fas (CD95/Apo-1). Eighteen of 44 (41%) patients had a histiocytic proliferation resembling SHML. The affected patients included 15 males and 3 females ranging in age from 11 months to 30 years at the time of the LN biopsy. The lymph nodes contained S-100+ histiocytes with characteristic nuclear features of SHML, and showed evidence of emperipolesis in both hematoxylin and eosin (H and E) and immunostained sections. The extent of the histiocytic proliferation was variable, being confluent in 2 cases, multifocal in 13, and only evident as isolated SHML-type histiocytes in 3. In lymph nodes without confluent SHML changes, increased numbers of CD3+CD4-CD8+ (double negative) alphabeta T-cells, also negative for CD45RO, a feature of ALPS, could be identified in the paracortex. Furthermore, because SHML shares many clinical features with ALPS, we sought evidence of ALPS in sporadic SHML. We attempted to sequence TNFRSF6 DNA from archived tissue of 14 cases of Rosai-Dorfman disease. Full sequencing of the gene was successful in 4 of the cases; no mutations were identified. Nevertheless, our observations suggest that histologic features of SHML are part of the pathologic spectrum of ALPS type Ia. It remains to be determined if some cases of apparently sporadic SHML may be associated with heritable defects in Fas-mediated apoptosis.

Use of mycophenolate mofetil for chronic, refractory immune cytopenias in children with autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of apoptosis associated most often with heritable FAS mutations leading to lymphadenopathy, hypersplenism and chronic refractory autoimmune cytopenias. Mycophenolate mofetil (MMF) was used to treat cytopenias in 13 ALPS patients aged 9 months to 17 years from a cohort of 118 children (aged < 18 years) and 82 adults. Twelve responded for a median follow-up of 49 weeks (range 38-240 weeks), defined by maintenance of adequate blood counts and reduction in dosage or cessation of other immunosuppressive agents. This preliminary experience suggests that MMF may spare steroid usage in patients with ALPS-associated cytopenias.

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds.

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.

Autoimmune lymphoproliferative syndrome: a cause of chronic splenomegaly, lymphadenopathy, and cytopenias in children-report on diagnosis and management of five patients.

Autoimmune lymphoproliferative syndrome (ALPS) usually manifests in early childhood with splenomegaly, lymphadenopathy, and cytopenias. In most patients, it results from mutations in genes that regulate lymphocyte apoptosis via the Fas pathway. Here, we report five children with ALPS. All five children had splenomegaly, cytopenias, and hypertriglyceridemia at presentation; four had lymphadenopathy. Mutations in the Fas receptor gene were demonstrated in three children. Clinical picture is variable: in only one child manifestations are severe enough to require immunosuppressive therapy. Diagnosis of ALPS can be challenging and increased awareness of the disease can result in more directed diagnostic approaches as well as earlier initiation of treatment.

Chronic active Epstein-Barr virus infection associated with mutations in perforin that impair its maturation.

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient's stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.

Autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome arises early in childhood in people who inherit mutations in genes that mediate lymphocyte apoptosis, or programmed cell death. In the immune system, antigen-induced lymphocyte apoptosis maintains immune homeostasis by limiting lymphocyte accumulation and minimizing reactions against self-antigens. In autoimmune lymphoproliferative syndrome, defective lymphocyte apoptosis manifests as chronic, nonmalignant adenopathy and splenomegaly; the expansion of an unusual population of CD4-CD8- T cells; and the development of autoimmune disease. Most cases of autoimmune lymphoproliferative syndrome involve heterozygous mutations in the lymphocyte surface protein Fas (CD95, Apo1) that impair a major apoptotic pathway. Prospective evaluations of patients and their families have revealed an ever-expanding spectrum of autoimmune lymphoproliferative syndrome and its major complications.

Discrete alterations in the BZLF1 promoter in tumor and non-tumor-associated Epstein-Barr virus.

BACKGROUND: Although the Epstein-Barr virus (EBV) is associated with malignant and nonmalignant diseases, its lytic replication is predominantly associated with nonmalignant diseases such as acute infectious mononucleosis (IM) or chronic active EBV infection. Lytic replication is also associated with type B EBV more than with type A EBV. Sustained lytic replication, however, is not compatible with tumor growth. We investigated whether control of an EBV lytic regulatory gene, BZLF1, differed in these diseases. METHODS: Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequence analyses were used to characterize the promoter sequence of BZLF1 (Zp) in 52 tumors (34 non-Hodgkin's lymphomas, 13 post-transplant lymphoproliferative disease samples, and five nasopharyngeal carcinomas), and in peripheral blood lymphocytes from seven patients with chronic active EBV, six with IM, and 40 healthy, EBV-seropositive individuals. All sequences were compared with the prototype EBV strain B95.8 sequence. All statistical tests were two-sided. RESULTS: Three polymorphic Zp sequences were detected. Among the malignant samples, sequence Zp-P, associated with 84% of type A EBV, was identical to that of EBV strain B95.8, whereas a second sequence (Zp-V3), associated exclusively with type B EBV (P<.001), contained three base substitutions. Among the nonmalignant samples, a distinct polymorphism, Zp-V4, containing the substitutions detected in Zp-V3 and an additional base change, was identified in all samples from chronic active EBV, IM, and healthy individuals, but in none of the malignant samples (P<.001). Zp-V4 was independent of the EBV type. CONCLUSIONS: Polymorphisms in the regulatory sequences of BZLF1 are differentially distributed among malignant and nonmalignant cells and may identify EBV subtypes with various lytic activities, including those not associated with malignancies.