The Epstein-Barr Virus (EBV) in T Cell and NK Cell Lymphomas: Time for a Reassessment.

While Epstein-Barr virus (EBV) was initially discovered and characterized as an oncogenic virus in B cell neoplasms, it also plays a complex and multifaceted role in T/NK cell lymphomas. In B cell lymphomas, EBV-encoded proteins have been shown to directly promote immortalization and proliferation through stimulation of the NF-κB pathway and increased expression of anti-apoptotic genes. In the context of mature T/NK lymphomas (MTNKL), with the possible exception on extranodal NK/T cell lymphoma (ENKTL), the virus likely plays a more diverse and nuanced role. EBV has been shown to shape the tumor microenvironment by promoting Th2-skewed T cell responses and by increasing the expression of the immune checkpoint ligand PD-L1. The type of cell infected, the amount of plasma EBV DNA, and the degree of viral lytic replication have all been proposed to have prognostic value in T/NK cell lymphomas. Latency patterns of EBV infection have been defined using EBV-infected B cell models and have not been definitively established in T/NK cell lymphomas. Identifying the expression profile of EBV lytic proteins could allow for individualized therapy with the use of antiviral medications. More work needs to be done to determine whether EBV-associated MTNKL have distinct biological and clinical features, which can be leveraged for risk stratification, disease monitoring, and therapeutic purposes.

Microgeographic variations in Burkitt's lymphoma incidence correlate with differences in malnutrition, malaria and Epstein-Barr virus.

BACKGROUND: Endemic Burkitt's lymphoma (eBL) has been associated with Epstein-Barr virus (EBV) and holoendemic Plasmodium falciparum malaria. But recent evidence suggests that other risk factors are involved. METHODS: We hypothesised that selenoprotein glutathione peroxidase (GPx), a surrogate of nutritional status, is an important biomarker for eBL risk. We measured plasma GPx, anthropometric markers of malnutrition, EBV viral loads and malaria parasitaemia in children aged 1-9 years (n=258) from two locations in Nyanza Province, Kenya, with higher-than-expected and lower-than-expected incidence of eBL. The study participants were malaria asymptomatic children from the community. RESULTS: Children from eBL high-incidence areas had significantly lower GPx levels, high EBV viral load and more evidence of chronic malnutrition than children from eBL low-incidence areas (all P<0.001). Additionally, GPx levels were significantly lower in children with the highest EBV viral load and for those with P. falciparum infections (P=0.035 and P=0.004, respectively). CONCLUSIONS: These results suggest that selenium deficiency may be a risk factor for eBL.

Gammaherpesvirus-induced lung pathology is altered in the absence of macrophages.

The purpose of this study was to examine the lung pathogenesis of murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemokine receptor CCR2, an important receptor for macrophage recruitment to sites of inflammation. BALB/c and CCR2(-/-) mice were inoculated intranasally (i.n.) with MHV-68 and samples were collected during acute infection (6 dpi) and following viral clearance (12 dpi). Immunohistochemistry was used to determine which cells types responded to MHV-68 infection in the lungs. Lung pathology in infected BALB/c mice was characterized by a mixed inflammatory cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi. Immunohistochemistry showed intense positive staining for macrophages. CCR2(-/-) mice showed greater inflammation in the lungs at 12 dpi than did BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the alveoli. Immunohistochemistry demonstrated much less macrophage infiltration in the CCR2(-/-) mice than in the BALB/c mice. These studies show that CCR2 is involved in macrophage recruitment in response to MHV-68 infection and illustrates how impairments in macrophage function affect the normal inflammatory response to this viral infection.

Clinical characteristics of Burkitt's lymphoma from three regions in Kenya.

OBJECTIVES: To describe the clinical characteristics of Burkitt's lymphoma (BL) from three regions in Kenya at different altitudes with a view towards understanding the contribution of local environmental factors. DESIGN: Prospective cross-sectional study. SETTING: Kenyatta National Hospital and seven provincial hospitals in Kenya. METHOD: Histologically proven cases of Burkitt's lymphoma in patients less than 16 years of age were clinically examined and investigated. MAIN OUTCOME MEASURES: For every case the following parameters were documented: chief complaint(s); physical examination, specifically pallor, jaundice, oedema, lymphadenopathy, presence of masses, splenomegaly and hepatomegaly. Reports of evaluation of chest radiograph, abdominal ultrasound/scan, bone marrow aspiration, cerebral spinal fluid cytology, liver and kidney function tests, urinalysis, stool occult blood and full blood count results. Stage of disease was assigned A, B, C or D. Cases of BL from three provinces of Kenya with diverse geographical features were analysed: Central, Coast, and Western. RESULTS: This study documented 471 BL cases distributed as follows: Central 61 (males 39 and 22 females), M:F ratio 1.8:1; Coast 169 (111 males and 58 females), M:F ratio 1.9:1; and Western 241 (140 males and 101 females), M:F ratio 1.4:1. The major presenting complaints were: abdominal swelling--Central 36%, Coast 4% and Western 26%; swelling on the face--Central 31%, Coast 81% and Western 64%; and proptosis--Central 3%, Coast 1% and Western 9%. The mean duration of these complaints in weeks were Central 6.9, Coast 6.08, and Western 5.05. The initial physical finding was a tumour mass in 39%, 72% and 54% of cases for Central, Coast and Western respectively. Tumour stage at diagnosis was: stage A--Central 21%, Coast 43% and Western 34%; stage B--Central 10%, Coast 5% and Western 10%; stage C--Central 41%, Coast 34% and Western 30%; and stage D--Central 28%, Coast 17% and Western 26%. For the age and sex matched cases the results show that commonly involved sites were: abdomen--Central 35%, Coast 9% and Western 14%; jaw (mandible)--Central 24%, Coast 22% and Western 31%; maxilla--Central 6%, Coast 24% and Western 11%; and lymph nodes--Central 10%, Coast 4% and Western 8%. The disease stage was A--Central 33%, Coast 44% and Western 36%; stage B--Central 11%, Coast 10% and Western 27%; stage C--Central 39%, Coast 34% and Western 27%; and stage D--Central 21%, Coast 13% and Western 37%. CONCLUSION: This study shows that clinical features of childhood BL vary with geographical region. The variations are documented in proportion of jaw, maxilla, abdominal and lymph nodal sites involvement. The differences observed are potentially due to the local environmental factors within these provinces. BL cases from Western province had features, intermediate between endemic and sporadic. Coastal province BL cases were similar to endemic BL, while BL cases from Central province resembled more or less sporadic BL subtypes. Strategies to explain and investigate the local environmental factors associated with the observed differences may certainly contribute towards improved understanding and clinical management of BL.

Strategies to overcome myelotoxic therapy for the treatment of Burkitt's and AIDS-related non-Hodgkin's lymphoma.

BACKGROUND: Strategies to circumvent or lessen the myelotoxicity associated with combination chemotherapy may improve the overall outcome of the management of patients particularly in resource poor settings. OBJECTIVES: To develop effective non-myelotoxic therapies for Burkitt's Lymphoma (BL) and AIDS-related non-Hodgkin's lymphoma. DATA SOURCES: Publications, original and review articles, conference abstracts searched mainly on Pubmed indexed for medline. DATA EXTRACTION: A systematic review of the clinical problem of combination chemotherapy. Identification of clinical strategies that circumvent or lessen the myelotoxicity of combination cytotoxic chemotherapy. Length of survival, lack of clinically significant (> grade 3) myelosuppression and weight loss were used as markers of myelotoxicity. DATA SYNTHESIS: Review of published experience with some of these strategies including dose-modification of multi-agent chemotherapy; rationale for targeted therapies, and the preclinical development of a mouse model exploring the role of metronomic scheduling substantiate pragmatism and feasibility of these approaches. CONCLUSION: Myelotoxic death rates using multi-agent induction chemotherapy approach 25% for endemic Burkitt's lymphoma and range between 20% to 60% for AIDS-related malignancy. This is mostly explained by the paucity of supportive care compounded by wasting and inanition attributable to advanced cancer and HIV infection making patients more susceptible to myelosuppressive side effects of cytotoxic chemotherapy. Investigations and alternative approaches that lessen or circumvent myelotoxicity of traditional cytotoxic chemotherapy for the management of Burkitt's lymphoma and AIDS-related non-Hodgkin's lymphoma in the resource-constrained setting are warranted. Pertinent pre-clinical and clinical data are emerging to support the need for abrograting the myelosuppressive effects of traditional cytotoxic chemotherapy. This can be achieved by developing targeted anti-viral and other strategies, such as the use of bryostatin 1 and vincristine, and by developing a preclinical mouse model to frame the clinical rationale for a pilot trial of metronomic therapy for the treatment of Burkitt's and AIDS-related lymphoma. Implementation of these investigational approaches must be encouraged as viable anti-cancer therapeutic strategies particularly in the resource-constrained settings.

Burkitt's lymphoma in Kenya: geographical, age, gender and ethnic distribution.

OBJECTIVE: To show the geographical (Provincial), age, gender and ethnic distribution of Burkitt's lymphoma in patients in Kenya. DESIGN: A retrospective review of patients' records for the years 1988-1992 and a prospective evaluation of patients with BL between 1993 and 1997. These were descriptive and hospitals based studies. SETTING: Kenyatta National Hospital; Kenya's main referral and teaching hospital and seven provincial hospitals. MAIN OUTCOME MEASURES: For each tissue proven Burkitt's lymphoma case the following were required; province of birth and residence, tribe, age, sex, chief complains, physical examination findings, investigation results and tissues result confirming the diagnosis of BL. STATISTICAL METHOD: Mainly proportions were used to compare variables, however Pearson's liner correlation was used to assess the time trends. RESULTS: This study registered 1005 patients; 961 (95.6%) children and 44 (4.4%) adults. 0-14 years the age standardized incidence rate (ASR) of 0.83. Variations documented in the provinces' BL ASR range; 1.8 Coast to 0.23 Rift Valley and increasing yearly trend for both children and adults. The major tribes in Kenya consisted; Luo 29.5%. Luhya (24.1%) and Coastal (16.5%). No patient of Asian or European or Arab extraction was recorded in the study. The age distribution showed no case below two years, a rapid rise from three year 3 (5.6%), and peak at 6 (19.5%) for children and at 17 years (13.6%) years for the adult. Age group 5-9 years had the highest ASR. The male to female (M:F) ratios were; 1.5:1 and 1:1 in children and adults respectively, provincial ratios range; 2.6:1 in Nairobi to 1.2:1 in Nyanza, the tribes range; 3.5:1 in Somali to 1:1 in other tribes between 2 and 14 years old when also males were more than females. Peak time of presentation of symptoms was 4 weeks. Tumour sites were in children; jaw 51.6%, abdomen (25%), combined jaw and abdomen 13.8% and others 9.6% and adults; jaw (4.5%), abdomen (43.2%), combined jaw and abdomen (25%) and other sites (27.3%) 67.6% males and 42.4% female adults had HIV infection and disseminated BL disease. CONCLUSION: The study demonstrates that Burkitt's lymphoma is a childhood disease. The disease distribution is consistent with intermediate risk Burkitt's lymphoma level. Furthermore the distribution varied by province, tribe, age and gender. The variations could be due to environmental factors.

Challenges in the epidemiological and clinical aspects of Burkitt's lymphoma in Kenya: linking evidence and experience.

BACKGROUND: In a series of 1005 cases of Burkitt's lymphoma studied for epidemiological and clinical characteristics, some features remain less obvious contrary to what is commonly held about this disease. OBJECTIVES: To use the case series to document the challenges in the epidemiological and clinical characteristics of Burkitt's lymphoma (BL) in Kenya. DESIGN: Cross sectional study involving clinical review of case series. SETTING: Kenyatta National Hospital and the seven provincial hospitals in Kenya during the period between 1986 and 1996. DATA SOURCES: Systematic review of the epidemiological and clinical features of the 1005 cases enrolled in the case study and review of reference lists of retrieved articles to identify original research dealing with the epidemiological and clinical features of Burkitt's lymphoma. DATA EXTRACTION: The investigators and research assistants screened both the case series and published information and data to yield relevant information. CONCLUSION: The majority of Burkitt's lymphoma cases between the age group three and nine years of age coincide with the established epidemiological and clinical characteristics. The adult BL cases and some childhood cases however do not conform entirely to the established characteristics. Therefore, making the diagnosis of Burkitt's lymphoma require that; geographical, demographical, clinical features as well as any underlying infections for instance, Human Immunodeficiency Virus be taken into consideration.

Activation of the Epstein-Barr virus lytic cycle by the latex of the plant Euphorbia tirucalli.

Exposure to the plant Euphorbia tirucalli has been proposed to be a cofactor in the genesis of endemic Burkitt's lymphoma (eBL). The purpose of this study was to examine the effects of unpurified E. tirucalli latex on Epstein-Barr virus (EBV) gene expression. A Burkitt lymphoma cell line was treated with varying dilutions of the latex and the effects on EBV gene expression were measured. We observed that the latex was capable of reactivating the EBV lytic cycle in a dose-dependent manner and at dilutions as low as 10(-6). Simultaneous treatment of cells with E. tirucalli latex and the protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride blocked lytic cycle activation. These data suggest that environmental exposure to the latex of E. tirucalli could directly activate the EBV lytic cycle and provide further evidence of a role for E. tirucalli in the aetiology of eBL.

Kinetics of murine gammaherpesvirus 68 gene expression following infection of murine cells in culture and in mice.

A model system to study the pathogenesis of gammaherpesvirus infections is the infection of mice with murine gammaherpesvirus 68 (MHV-68). To define the kinetics of infection, we developed an RNase protection assay to quantitate gene expression from lytic (K3, Rta, M8, DNA polymerase [DNA pol], and gB) and candidate latency (M2, M3, M9, M11, ORF73, and ORF74) genes. All candidate latency genes were expressed during lytic infection of 3T3 cells. Four kinetic classes of transcripts were observed following infection of 3T3 cells: immediate-early (K3, Rta, M8, and ORF73), early (DNA pol), early-late (M3, M11, and ORF74), and late (M2, M9, and gB). To assess the kinetics of viral gene expression in vivo, lungs, spleens, and mediastinal lymph nodes (MLN) were harvested from MHV-68-infected mice. All transcripts were expressed between 3 and 6 days postinfection (dpi) in the lungs. In the spleen, K3, M3, M8, and M9 transcripts were expressed between 10 and 16 dpi when latency is established. The K3, M3, M8, M9, and M11 transcripts were detected in the MLN from 2 through 16 dpi. This is the first demonstration of MHV-68 gene expression in the MLN. Importantly, our data showed that MHV-68 has different kinetics of gene expression at different sites of infection. Furthermore, we demonstrated that K3, a gene recently shown to encode a protein that downregulates major histocompatibility complex class I on the surface of cells, is expressed during latency, which argues for a role of K3 in immune evasion during latent infection.

Malaria enhances expression of CC chemokine receptor 5 on placental macrophages.

Malaria and human immunodeficiency virus (HIV) coinfections are common in pregnant women in sub-Saharan Africa. The current study shows that placentas of malaria-infected women contain 3 times as much CC chemokine receptor 5 (CCR5) RNA as placentas of women without malaria. By immunohistochemistry, CCR5(+) maternal macrophages were seen in placentas from malaria-infected women but not in placentas from malaria-uninfected women. In addition, CCR5 also was found on fetal Hofbauer cells in placentas from both groups. Thus, malaria infections increase the potential reservoir for HIV in the placenta by increasing the number of HIV target cells.

Malaria and pregnancy: placental cytokine expression and its relationship to intrauterine growth retardation.

Malaria infections during pregnancy can lead to the delivery of low-birth-weight infants. In this study, cytokine mRNA was measured in placentas from 23 malaria-infected and 21 uninfected primigravid women who had delivered in Mangochi, Malawi, a region with a high rate of transmission of falciparum malaria. Significantly increased expression of interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha and decreased expression of IL-6 and transforming growth factor-beta1 were found in malaria-infected compared with uninfected placentas. TNF-alpha and IL-8 were produced by maternally derived hemozoin-laden placental macrophages. Increased TNF-alpha expression was associated with increased placental hemozoin concentrations. Increased TNF-alpha or IL-8 expression in the placenta was associated with intrauterine growth retardation but not with preterm delivery. The results suggest that malaria infections induce a potentially harmful proinflammatory response in the placenta.

TNF-alpha inhibits HIV-1 replication in peripheral blood monocytes and alveolar macrophages by inducing the production of RANTES and decreasing C-C chemokine receptor 5 (CCR5) expression.

The pathogenesis of HIV-1 infection is influenced by the immunoregulatory responses of the host. Macrophages present in the lymphoid tissue are susceptible to infection with HIV-1, but are relatively resistant to its cytopathic effects and serve as a reservoir for the virus during the course of disease. Previous investigators have demonstrated that increased serum levels of TNF-alpha contribute to the clinical symptoms of AIDS and that TNF-alpha stimulates the production of HIV-1 in chronically infected lymphocytic and monocytic cell lines by increasing HIV-1 gene expression. Although previous studies have suggested that TNF-alpha may increase HIV-1 infection of primary human mononuclear cells, some recent studies have indicated that TNF-alpha suppresses HIV-1 infection of macrophages. We now demonstrate that TNF-alpha suppresses HIV-1 replication in freshly infected peripheral blood monocytes (PBM) and alveolar macrophages (AM) in a dose-dependent manner. As TNF-alpha has been shown to increase the production of C-C chemokine receptor (CCR5)-binding chemokines under certain circumstances, we hypothesized that TNF-alpha inhibits HIV-1 replication by increasing the expression of these HIV-suppressive factors. We now show that TNF-alpha treatment of PBM and AM increases the production of the C-C chemokine, RANTES. Immunodepletion of RANTES alone or in combination with macrophage inflammatory protein-1alpha and -1beta block the ability of TNF-alpha to suppress viral replication in PBM and AM. In addition, we found that TNF-alpha treatment reduces CCR5 expression on PBM and AM. These findings suggest that TNF-alpha plays a significant role in inhibiting monocytotropic strains of HIV-1 by two distinct, but complementary, mechanisms.

Viral clearance without destruction of infected cells during acute HBV infection.

Viral clearance during hepatitis B virus (HBV) infection has been thought to reflect the destruction of infected hepatocytes by CD8(+) T lymphocytes. However, in this study, HBV DNA was shown to largely disappear from the liver and the blood of acutely infected chimpanzees long before the peak of T cell infiltration and most of the liver disease. These results demonstrate that noncytopathic antiviral mechanisms contribute to viral clearance during acute viral hepatitis by purging HBV replicative intermediates from the cytoplasm and covalently closed circular viral DNA from the nucleus of infected cells.

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders.

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.

Lipoarabinomannan of Mycobacterium tuberculosis promotes protein tyrosine dephosphorylation and inhibition of mitogen-activated protein kinase in human mononuclear phagocytes. Role of the Src homology 2 containing tyrosine phosphatase 1.

Lipoarabinomannan (LAM) is a putative virulence factor of Mycobacterium tuberculosis that inhibits monocyte functions, and this may involve antagonism of cell signaling pathways. The effects of LAM on protein tyrosine phosphorylation in cells of the human monocytic cell line THP-1 were examined. LAM promoted tyrosine dephosphorylation of multiple cell proteins and attenuated phorbol 12-myristate 13-acetate-induced activation of mitogen-activated protein kinase. To examine whether these effects of LAM could be related to activation of a phosphatase, fractions from LAM-treated cells were analyzed for dephosphorylation of para-nitrophenol phosphate. The data show that LAM induced increased phosphatase activity associated with the membrane fraction. The Src homology 2 containing tyrosine phosphatase 1 (SHP-1) is important for signal termination and was examined as a potential target of LAM. Exposure of cells to LAM brought about (i) an increase in tyrosine phosphorylation of SHP-1, and (ii) translocation of the phosphatase to the membrane. Phosphatase assay of SHP-1 immunoprecipitated from LAM-treated cells, using phosphorylated mitogen-activated protein kinase as substrate, indicated that LAM promoted increased activity of SHP-1 in vivo. LAM also activated SHP-1 directly in vitro. Exposure of cells to LAM also attenuated the expression of tumor necrosis factor-alpha, interleukin-12, and major histocompatibility class II molecules. These results suggest that one mechanism by which LAM deactivates monocytes involves activation of SHP-1.

In vivo growth of Epstein-Barr virus transformed B cells with mutations in latent membrane protein 2 (LMP2).

Epstein-Barr virus (EBV) causes infectious mononucleosis in adolescents and is associated with malignant B lymphocyte proliferation in AIDS patients, patients undergoing immune suppression for organ transplantation, and SCID mice. In vitro, EBV transformed, latently infected lymphoblastoid B cell lines (LCLs) contain EBV episomes and express nine virus encoded proteins. Six are nuclear proteins (EBNAs) and three are the integral membrane proteins, LMP1, LMP2A, and LMP2B. To determine if LMP2 was essential for in vivo growth, SCID mice were injected with LCLs containing wild-type EBV (LMP2+) or with LCLs transformed with EBV containing mutations in either LMP2A or LMP2B (LMP2-). SCID mice injected with the LMP2+ or LMP2- LCLs were monitored for tumor development, length of time to tumor development, and phenotypic characterization of the resulting tumors. No difference was observed in any of the above parameters between LMP2+ and LMP2- LCLs demonstrating that LMP2 is not essential for the in vivo growth of EBV transformed B lymphocytes in SCID mice.

Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines.

Epstein-Barr virus (EBV) transformed human B cells proliferate indefinitely in vitro, and it has been proposed that cytokine-mediated autocrine loops contribute to the maintenance of the lymphoblastoid phenotype. We used a novel multiprobe RNase protection assay to quantify cytokine mRNA species expressed by EBV-transformed lymphoblastoid cell lines (LCL), derived either by the transformation of B cells with B95-8 or wild-type EBV or by the in vitro outgrowth of EBV-associated B cell lymphomas to identify cytokines that are commonly expressed in all LCL and thus more likely to be essential for immortalization of B cells. All 16 LCL expressed high levels of tumor necrosis factor (TNF)alpha, TNFbeta, and transforming growth factor (TGF)beta1 mRNA, while interleukin (IL)-10 transcripts were detected in most LCL but at a lower level. Expression of IL-1alpha, IL-1beta, IL-6, IL-12p35, IL-12p40, IL-13 and IFNgamma mRNA was variable among the LCL tested. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, and IL-5 mRNA were undetectable in all LCL. Furthermore, we found that IL-10, TNFalpha, and TNFbeta mRNA were induced in EBV-negative B cell lines after infection with EBV. These data define common versus idiosyncratic patterns of cytokine expression by LCL and, in the former case, such cytokines as TNFalpha, TNFbeta, and IL-10 emerge as strong candidates that are essential for the autocrine regulation of EBV-immortalized B cells.

Differential Epstein-Barr virus gene expression in B-cell subsets recovered from lymphomas in SCID mice after transplantation of human peripheral blood lymphocytes.

We have analyzed the human B-cell tumors that arise spontaneously in SCID mice who have been given transplants of peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors to determine if patterns of EBV gene expression are correlated with phenotypic changes in the tumor B cells. Tumor cells were separated into two B-cell subsets by cell sorting on the basis of differential coexpression of membrane CD23 and CD38. One subset showed intermediate levels of CD23 and CD38 expression (CD23intCD38int), while a second subset had low-level CD23 but high-level CD38 expression (CD23loCD38hi). The CD23intCD38int cells had a high proliferative index and secreted little immunoglobulin in vitro; the CD23loCD38hi cells had a low proliferative index and high-level immunoglobulin secretion. We next analyzed the sorted cells for viral transcripts associated with latency (EBNA-1, EBNA-2, and LMP-1) or lytic cycle replication (ZEBRA and gp350 envelope protein). Only latent cycle transcripts were found in CD23intCD38int cells, whereas lytic cycle transcripts and transforming virus were present in the CD23loCD38hi cells. Finally, we generated short-term cell lines from the sorted CD23intCD38int cells and transferred these cells to SCID recipients. The resulting secondary tumors were predominantly CD23loCD38hi, suggesting that the CD23intCD38int lymphoblastoid cells are precursors to the well-differentiated, plasmacytoid CD23loCD38hi cells. These observations are discussed in the context of a three-step model for EBV-associated lymphomagenesis in humans.

Proteolytic removal of the C-terminal transmembrane region of cytochrome f during extraction from turnip and charlock leaves generates a water-soluble monomeric form of the protein.

Water-soluble, monomeric cytochrome f purified from leaves of turnip (Brassica rapa) and charlock (Sinapis arvensis) is approximately 3 kDa smaller than the protein in chloroplast thylakoid membranes determined by SDS/PAGE. Sequencing the N-terminal and C-terminal regions of the monomeric protein, by automated Edman degradation and carboxypeptidase P digestion, suggested the loss of 33 amino acid residues at the C-terminus by comparison to sequences of cytochrome f from other higher plants. This was confirmed by the isolation and nucleotide sequencing of the turnip petA gene and by determination of the molecular mass of the monomeric turnip protein by electrospray mass spectrometry. The turnip petA gene encodes a protein of 320 amino acid residues consisting of a presequence of 35 amino acid residues and a mature protein of 285 amino acid residues. The molecular mass of the monomeric turnip protein was 28,160.2 +/- 5.4 Da, indicating cleavage after Gln252 of the mature protein. Electrospray mass spectrometry of the monomeric charlock protein indicated the presence of two main forms with molecular masses of 28,135.1 +/- 5.5 Da and 27,750.7 +/- 4.3 Da corresponding to cleavage after Gln252 and Leu249, respectively. Cleavage in this region of the cytochrome f polypeptide during extraction with butanone removes the single transmembrane span of the protein and liberates the water-soluble globular domain of cytochrome f.