Bacterial Infection and Non-Hodgkin B-Cell Lymphoma: Interactions between Pathogen, Host and the Tumor Environment.

Non-Hodgkin B-cell lymphomas (NHL) are a heterogeneous group of lymphoid neoplasms with complex etiopathology, rich symptomatology, and a variety of clinical courses, therefore requiring different therapeutic approaches. The hypothesis that an infectious agent may initiate chronic inflammation and facilitate B lymphocyte transformation and lymphogenesis has been raised in recent years. Viruses, like EBV, HTLV-1, HIV, HCV and parasites, like Plasmodium falciparum, have been linked to the development of lymphomas. The association of chronic Helicobacter pylori (H. pylori) infection with mucosa-associated lymphoid tissue (MALT) lymphoma, Borrelia burgdorferi with cutaneous MALT lymphoma and Chlamydophila psittaci with ocular adnexal MALT lymphoma is well documented. Recent studies have indicated that other infectious agents may also be relevant in B-cell lymphogenesis such as Coxiella burnettii, Campylobacter jejuni, Achromobacter xylosoxidans, and Escherichia coli. The aim of the present review is to provide a summary of the current literature on infectious bacterial agents associated with B-cell NHL and to discuss its role in lymphogenesis, taking into account the interaction between infectious agents, host factors, and the tumor environment.

Endoscopic ultrasound-guided diagnosis of Helicobacter pylori-associated gastric Burkitt's lymphoma in an adolescent patient: a rare case.

Primary gastric Burkitt's lymphoma (BL) is rare in the pediatric population. Furthermore, the association of Burkitt's lymphoma with Helicobacter pylori is not well defined. We report a case of primary gastric Burkitt's lymphoma associated with Helicobacter pylori diagnosed in a pediatric patient. This diagnosis was made with the aid of endoscopic ultrasound (EUS)-guided fine-needle biopsy (FNB). This is one of the first pediatric cases of EUS-guided FNB for the diagnosis of H. pylori-associated gastric BL.

Relation between Epstein-Barr viral and cell membrane immunofluorescence of Burkitt tumor cells. I. Dependence of cell membrane immunofluorescence on presence of EB virus.

A comparison was made of the immunofluorescence tests for detection of cell membrane and Epstein-Barr virus antigens in cells from Burkitt tumor biopsies or continuous cultures derived therefrom. On the whole, cell membrane fluorescence in established lines appeared to depend not only upon the presence of EBV but to a considerable degree also upon the extent of the persistent viral infection. There was no constant relationship, however, between the results of the two tests and exceptions to the rule were noted. These observations indicate that different antigens are involved in the two tests. Biopsy cells in general and young cultures may reveal strong MIF activity but few, if any, EBV-positive cells. The reverse, the presence of relatively large numbers of EBV antigen-containing cells in the absence of significant MIF reactions, was also noted on occasion in a few established cultures. The possible interpretations of these findings have been discussed.

T cell responses and virus evolution: loss of HLA A11-restricted CTL epitopes in Epstein-Barr virus isolates from highly A11-positive populations by selective mutation of anchor residues.

Epstein-Barr virus (EBV) is a B lymphotropic herpesvirus of humans that elicits strong HLA class I-restricted cytotoxic T lymphocyte (CTL) responses. An influence of such responses on virus evolution was first suggested by our finding that EBV isolates from the highly HLA A11-positive Papua New Guinea (PNG) population carried a lys-thr mutation at residue 424 of the nuclear antigen EBV-encoded nuclear antigen (EBNA4) that destroyed the immunodominant target epitope for A11-restricted CTL recognition. Here we turn to a much larger population, Southern Chinese, where the A11 allele is again present in over 50% of the individuals. Each of 23 EBV isolates analyzed from this population were also mutated in the EBNA4 416-424 epitope, the mutations selectively involving one of the two anchor residues in positions 2 (417 val-leu) or 9 (424 lys-asp, -arg or -thr) that are critical for A11-peptide interaction. The majority of the Chinese isolates and all 10 PNG isolates also carried mutations affecting positions 1 and 2 of the next most immunodominant A11-restricted epitope, EBNA4 residues 399-408. These changes clearly affected antigenicity since A11-positive lymphoblastoid cell lines (LCLs) carrying these mutant EBV strains were not recognized by A11-restricted CTLs raised against the prototype B95.8 virus. Furthermore, Chinese donors naturally infected with these mutant viruses did not mount detectable A11-restricted CTL responses on in vitro stimulation with autologous LCL cells carrying either the B95.8 or their endogenous EBV strain. In two different highly A11-positive populations, therefore, immune pressure appears to have selected for resident EBV strains lacking immunodominant A11-restricted CTL epitopes.

Inhibition of Epstein-Barr virus (EBV) release from the P3HR-1 Burkitt's lymphoma cell line by a monoclonal antibody against a 200,000 dalton EBV membrane antigen.

In raising murine hybridoma antibodies against Epstein-Barr virus (EBV)-induced membrane antigens (MA), we found one antibody that blocked the release of infectious EBV from cultured P3HR-1 cells. This monoclonal antibody (mAb) recognized a 200 kD, phosphonoacetic acid-sensitive (late) MA, and did not directly neutralize virus without complement. When this mAb was added to 33 degrees C-cultured, spontaneously EBV-producing P3HR-1 cells, the intracellular expression of viral capsid antigen and infectious virus was not inhibited, but the appearance of infectious virus in the culture medium was significantly reduced. The duration of this suppression was dependent upon the concentration of the mAb, an effect being observed to a 1:4 X 10(5) titer of the ascites mAb preparation. A more acute effect of suppression of EBV release was observed in a second model of 12-o-tetradecanoyl phorbol-13-acetate and n-butyrate induction of EBV in 37 degrees C-cultured P3HR-1 cells. Again, intracellular infectious virus production was not inhibited, but the level of infectious virus in the culture medium was significantly reduced as early as 1 and 2 d of culture with antibody. This effect was reversed within 31 h after replacement of mAb-containing medium with fresh medium. This description of antibody-mediated inhibition of EBV release might lead to the characterization of another form of immune defense for the control of EBV infections.

Infectious mononucleosis: complement-fixing antibodies to herpes-like virus associated with Burkitt lymphoma.

Complement-fixing antibodies to a herpes-like virus derived from a Burkitt tumor-cell line developed in each of 21 patients with infectious mononu-cleosis. These antibodies were absent in all serums before the patients became ill, appeared during the early phases of illness, and persisted for long periods of time. These antibodies are distinct from heterophile antibodies. None of the patients developed immune responses to herpes simplex, cytomegalo-, or reoviruses in the course of their illness. The data suggest that the development of complement-fixing antibodies to this herpes-like virus in these patients may be linked to infectious mononucleosis.

Persistence of the entire Epstein-Barr virus genome integrated into human lymphocyte DNA.

The entire Epstein-Barr virus genome is integrated into Burkitt tumor cell DNA at the terminal direct repeat sequence of the virus. There is no homology between the GC-rich (G, guanine; C, cytosine) terminal repeat and the AT-rich (A, adenine; T, thymine) cell sequences with which it has recombined. More than 15 kilobases of cell DNA have been deleted and 236 base pairs are duplicated at one virus-cell junction site.

Hybridization of Burkitt lymphoblastoid cells.

Burkitt lymphoblastoid cell lines have been fused to mouse and human cell lines with the use of inactivated Sendai virus. The heterokaryons have developed into somatic cell hybrids of both parental cell types. Chromosome analyses confirm that cells now growing in selective medium are hybrids. Initial observations of preparations of the hybrid cells reveal that 5'-iododeoxyuridine can induce continued synthesis of Epstein-Barr virus antigens by these hybrid cells.

Individual tumors of multifocal EB virus-induced malignant lymphomas in tamarins arise from different B-cell clones.

Cotton-top tamarins were inoculated with sufficient Epstein-Barr virus to induce multiple tumors in each animal within 14 to 21 days. The tumors consisted of large-cell lymphomas that contained multiple copies of the Epstein-Barr virus genome and generated Epstein-Barr virus-carrying cell lines showing no detectable consistent chromosomal abnormality. Hybridization of tumor DNA with immunoglobulin gene probes revealed that each lymphoma was oligo- or monoclonal in origin and that individual tumors from the same animal arose from different B-cell clones. Thus the virus induced multiple transformation events in tamarins in vivo to cause malignant tumors resembling the Epstein-Barr virus-associated lymphomas of patients with organ transplants.

A second nuclear protein is encoded by Epstein-Barr virus in latent infection.

A region of the Epstein-Barr virus (EBV) genome that is important in inducing cell proliferation includes a single long open reading frame. Part of this open reading frame has been fused to the lacZ gene and expressed in Escherichia coli. Antisera to the fusion protein identify a protein in the nuclei of latently infected growth-transformed lymphocytes and in Burkitt tumor cells grown in vitro. This nuclear protein is encoded by a different virus-gene than that which encodes the previously described EBV nuclear antigen, EBNA.

Evidence for exposure to HTLV-III in Uganda before 1973.

Fifty of 75 serum samples collected in the West Nile district of Uganda between August 1972 and July 1973 contained antibodies reactive with human T-cell leukemia (lymphotropic) virus type 3 (HTLV-III; mean titer, 601), while 12 of 75 samples were positive in a similar test for HTLV type 1 (HTLV-1) antibodies (mean titer, 236). The samples were screened by enzyme-linked immunosorbent assay and positive results were confirmed by a newly developed unlabeled antibody-peroxidase procedure with enhanced sensitivity for detection of antibody binding to immunoblots of HTLV-III antigen, demonstrating antibodies to proteins with molecular weights of 24,000, 41,000, and 76,000 in nearly all positive samples. Analysis of titration data indicated enhanced titers of antibody against HTLV-III and HTLV-I when coinfection occurred. The high prevalence and relatively low titers [compared to serum from patients with acquired immune deficiency syndrome (AIDS)] of antibodies recognizing HTLV-III proteins in sera from this population at a time that may predate or coincide with the appearance or spread of the AIDS agent (HTLV-III) suggest that the virus detected may have been a predecessor of HTLV-III or is HTLV-III itself but existing in a population acclimated to its presence. It further suggests an African origin of HTLV-III.

Epstein-Barr virus: detection of genome in somatic cell hybrids of Burkitt lymphoblastoid cells.

Somatic cell hybrids of Burkitt lymphoblastoid cells, from which Epstein-Barr virus can be recovered, were examined for the presence of virus DNA by DNA-RNA hybridization. Four clones of hybrid cells, each negative for virus antigens by immunofluorescence, contained virus DNA in varying genomic equivalents. The number of virus genome equivalents increased in the hybrid cells after induction of virus with iododeoxyuridine.

An Epstein-Barr virus transforming protein associates with vimentin in lymphocytes.

The Epstein-Barr virus (EBV) latent infection membrane protein (LMP) is likely to be an important mediator of EBV-induced cell proliferation, since it is one of the few proteins encoded by the virus in latent infection and since production of this protein in Rat-1 cells results in their conversion to a fully transformed phenotype. LMP was previously noted to localize to patches at the cell periphery. In this paper we examine the basis of LMP patching in EBV-infected, transformed lymphocytes. Our data indicate that LMP is associated with the cytoskeletal protein vimentin. Although LMP is fully soluble in isotonic Triton X-100 buffer, only 50% of it is extracted from cells in this solution. The rest remains bound to the cytoskeleton. LMP undergoes phosphorylation, and phosphorylated LMP is preferentially associated with the cytoskeleton. As judged by both immunofluorescence and immunoelectron microscopy, the vimentin network in EBV-transformed lymphocytes or EBV-infected Burkitt tumor lymphocytes is abnormal. Vimentin and LMP often colocalize in a single patch near the plasma membrane. In response to Colcemid treatment of EBV-infected cells, vimentin reorganizes into perinuclear rings, as it does in uninfected cells. LMP is associated with these perinuclear rings. Vimentin (or a vimentin-associated protein) may be a transducer of an LMP transmembrane effect in lymphoproliferation.

Inhibitory effect of adult bovine serum on release of infectious Epstein-Barr virus from a virus-carrier cell line.

When certain bovine sera (BS) were used to culture P3HR-1 cells, the amount of infectious Epstein-Barr virus (EBV) released into the culture fluid was significantly less than that obtained when calf serum (CS) or fetal calf serum (FCS) was used. A direct dose-response relationship was noted between the concentration of BS and the degree of inhibition of virus production. Cell growth was not inhibited nor was virus infectivity neutralized by BS. Similarly, EBV infectivity was not reduced by spent medium containing BS. The inhibition of virus production by BS could be reversed by replacement of the serum supplement with CS. The formation of viral capsid antigen and of infectious virus within the cells was not inhibited by BS, whereas the amount of virus released was significantly decreased in comparison with culture media containing FCS or CS. These findings suggested the presence of a factor(s) in the BS which inhibited the release of EBV from the cell.

State of Epstein-Barr virus DNA in an American Burkitt's lymphoma line.

A human lymphoma cell line, positive for the Epstein-Barr virus (EBV)-associated nuclear antigen, was recently established from a North American Burkitt's lymphoma. This cell line, SU-AmB-2, contained EBV DNA both in the form of circular, nonintegrated DNA molecules of viral genome length, present in multiple copies per cell, and as integrated sequences. Having DNA present in both of these forms, it resembled cell lines established from African Burkitt's lymphomas. In studies on EBV strain differences, the episomal viral DNA in Burkitt's lymphoma cells may now be compared with viral DNA in nonmalignant cells with the use of cell lines from Burkitt's lymphoma patients of similar geographic origin.

Epstein-Barr virus in an American patient with Burkitt's lymphoma: detection of viral genome in tumor tissue and establishment of a tumor-derived cell line (NAB).

Epstein-Barr virus (EBV) DNA (17.7 genome equivalents/cell) was found in tumor tissue from an American patient with Burkitt's lymphoma who had never traveled outside the United States. A lymphoid cell line (NAB) containing the EBV genome was established from tumor tissue from this patient; characteristics of this cell line were described. Previous Burkitt's tumors found in Americans and examined by molecular hybridization were negative for EBV DNA. Our results suggested that EBV is associated with at least some American Burkitt's tumors.

Establishment of lymphomatous cell lines from bone marrow samples from patients with Burkitt's lymphoma.

A total of 233 bone marrow aspirates were obtained from 43 patients with Burkitt's lymphoma (BL). Lymphoma cells were absent and lymphoblastoid cell lines could not be established from 197 samples, which were characterized by limited initial cell proliferation and development of an adherent population, followed by cell death after 2-4 weeks. In 14 aspirates, after a similar pattern of growth, cell proliferation began again after about 6 weeks, with a rapid appearance and growth of cell clumps from the feeder layer--a type of growth typical of spontaneous lymphoblastoid cell lines. In 22 aspirates, growth of malignant cells was observed in culture and cytocentrifuged, stained smears, including marrow samples from 9 patients in whom the presence of BL cells had not been ascertained or even suspected by cytology. Karyotypic anomalies characteristic of BL were found in these cells: t(8;14) in the majority, two t(8;22), two t(2;8), and one t(2;8;9).

Activation of Epstein-Barr virus in hybrid cells.

Human-primate hybrid cell lines were established by fusion of African green monkey kidney cells (VERO) with lymphoblastoid cells from patients with infectious mononucleosis (IM)(IMK101) and from Burkitt's lymphoma culture (HR1K). Both Epstein-Barr virus (EBV)-specific antigens and EBV particle-containing cells increased in the hybrid lines (IMK1-1/VERO,HR1K/VERO). Treatment of the hybrids with 5-bromodeoxyuridine induced more antigen-positive and more virus-containing cells. EBV could be activated from IM lymphoblastoid cells by fusion of the lymphoblastoid cells with the VERO cells. The increase of viral antigens and virus particles may have been due to the cellular interaction between VERO cells and the lymphoblastoid cells or to a possible derepressor supplied by the VERO component of the hybrid. Virus derived from the HR1K cell line was replicated in the human-primate hybrid, but further investigation may be necessary to determine if it was identical to the EBV derived from the human cell line.

B-cell maturation stages of Burkitt's lymphoma cell lines according to Epstein-Barr virus status and type of chromosome translocation.

This study addressed the possible relationship between B-cell maturation stage of Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV) status, ethnic group, or type of chromosome translocation. Fifty-seven cell lines obtained at the International Agency for Research on Cancer from 51 patients were studied. Cytogenetic analyses of 54 cells lines were available. Cell size, surface immunoglobulins (sIgs), cytoplasmic immunoglobulins (cIgs), mouse red blood cell receptors, and reactivity with various monoclonal antibodies were assessed. Immunoglobulin (Ig) class secretions were measured in the supernatant of 2- and 5-day cultures from 33 cell lines, with the use of a sensitive enzyme-linked immunosorbent assay technique. From this study, BL appears to cover a broad range of the B-cell differentiation sequence, since the following Ig phenotypes were observed: null cells (sIg-, cIg-), large pre-B-cells (intracytoplasmic mu-chains), small B-cells (sIg+, cIg-), and various types of secreting B-cells (sIg+, cIg+). Among the latter, various patterns of cIg could be defined (perinuclear, paranuclear, and vesicular). B-cell maturation stages were correlated with the amount of secreted Ig. In sIg+ cell lines, different classes of Ig were found: 35 IgM, 10 IgM plus IgD, 4 IgG, and 1 IgA. None of the different monoclonal antibodies used was specific to a precise stage of maturation. The stages of maturation were correlated with neither the type of chromosome translocations of BL nor the presence of EBV genome, but the most immature cell lines were all EBV positive and most of them originated from African patients. In contrast with acute lymphoblastic leukemia, the common acute lymphocytic leukemia antigen (CD10) was expressed on nearly all BL cell lines of intermediate maturation stages but only on half of the pre-B ones. In addition, none of the cell lines tested was found to react with CD5 antibodies, which recognize most of the chronic lymphocytic leukemia of the same stage of maturation as that in the B-lymphocyte lineage.