Faruk Sinangil

Non-Hodgkin's Lymphoma After Treatment of Hodgkin's Disease: Association with Epstein-Barr Virus

Non-Hodgkins lymphoma occurs infrequently as a late complication of obscure cause after treatment of Hodgkins disease. We investigated the possible role of Epstein-Barr virus in the pathogenesis of such secondary malignancies of B-cell lineage. Two patients, aged 25 and 43 years, developed high-grade non-Hodgkins lymphomas 12 and 8 years after radiation therapy for Hodgkins disease. Serologic profiles in these patients showed evidence of acute and past Epstein-Barr virus infections, respectively. Molecular hybridization analysis showed the presence of multiple cellular equivalents of virus genome in tumor specimens from each patient. Our findings suggest that Epstein-Barr virus may play an integral role in the pathogenesis of non-Hodgkins lymphoma of B-cell lineage that develops after treatment of Hodgkins disease.

ANTIBODIES TO HTLV-III/LAV AMONG ABORIGINAL AMAZONIAN INDIANS IN VENEZUELA

Serum samples from 224 aboriginal Amazonian Indians were tested for antibodies to HTLV-III/LAV by an indirect immunofluorescence (IF) assay. 9 individuals (4%), 5 of them female, were seropositive by IF and by confirmatory western blotting and radioimmunoprecipitation tests. 3 of the positive sera were collected in 1968. HTLV-III/LAV seropositivity rates varied among the ethnic groups and ranged from 13.3% among the Pemon Indians to 3.3% among the Yanoama tribe. The titres of HTLV-III/LAV antibodies ranged from 1/40 to 1/320. All individuals tested were apparently healthy at the time of the study. None of 211 randomly chosen, healthy blood donors from Venezuelan cities had antibodies to HTLV-III/LAV. The prevalence of specific antibodies among Amazonian Indians suggests the HTLV-III/LAV or a closely related cross-reactive virus may be endemic in this area. The findings also indicate that this virus is indigenous in non-negroid Latin American and negroid tropical populations.

Antibodies to HTLV-III/LAV in Venezuelan patients with acute malarial infections.

Malarial infections have been associated with an immunosuppressive state similar to that seen in acquired immunodeficiency syndrome (AIDS). Its manifestations include severe lymphopenia depletion of the T4-lymphocyte subset activation of B cells and diminished in vitro lymphocyte responses. Because of these similarities the authors tested Venezuelan patients with acute malaria for antibodies to human T-lymphotropic virus type III (HTLV-III). The sample included 12 patients with acute Plasmodium vivax infection and 12 with P. falciparum infection. None of the patients were receiving antimalarial drugs and none belonged to any of the recognized risk groups for AIDS. 3 of the patients with P. falciparum infection (25%) and 5 with P. vivax (41%) were found to be positive for HTLV-III antibodies by indirect immunofluorescence. Western blot and radioimmunoprecipitation tests. The pattern of the Western blot and radioimmunoprecipitation bands obtained with samples from malaria patients was similar to that noted in AIDS patients. Since acute malaria does not evolve into an AIDS-like syndrome it is concluded that exposure to HTLV-III and the occurrence of severe immunoregulatory disturbances are not always sufficient for the induction of AIDS.

Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

ABSTRACT The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals.

Transfection of Lymphoblastoid Cells Using DNA-Loaded Reconstituted Sendai Virus Envelopes: Expression of Transfected DNA and Selection of Transfected Cells

The American Burkitts lymphoma cell line Loukes was cotransfected with cloned BamHI K fragment of EBV DNA and a vector pSV2neo. Reconstituted Sendai virus envelopes (RSVE) loaded with DNA were used for efficient gene transfer. Two cell lines have been obtained following culture in the presence of geneticin sulfate (G-418). Messenger RNA from both transfected DNAs was expressed during the whole period of observation, 42 days after transfection. This method provides a relatively simple and efficient means for selection of lymphoblastoid cells expressing a transfected gene.

Pattern of Epstein-Barr virus (EBV) -specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.

Infection of human epithelial cells by Epstein-Barr Virus (EBV): II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells☆

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.

Transformation of Human Lymphocytes by Coinfection with EBV DNA and Transformation-Defective Virions

EBV DNA from B95-8 strain was introduced into human cord blood lymphocytes (CBL) using reconstituted Sendai virus envelopes (RSVE) as gene transfer vehicles. The DNA- treated CBL expressed EB virus nuclear antigen (EBNA) and synthesized cellular DNA at an increased rate but were not immortalized. However, when EBV DNA transfer was followed by exposure to UV-in activated virions, full transformation was achieved. The resulting lympho-blastoid cell lines, termed NEB, were of B-cell origin, contained 25–50 EBV genome equivalents/cell, but were not capable of expressing EA or VCA, or releasing infectious virus. Permanent cell lines were also established when CBL were coinfected with purified EBV DNA and EBV of the HR-1 strain. These cells, termed HBD, were of T-cell origin, did not express EBNA, but contained EBV genomes as determined by nucleic acid hybridization. The HBD cells also expressed the early and virus capsid antigens of EBV as determined by immunofluorescence and radio immunoprecipitation. The NEB and HBD cell lines will be useful for analyzing the mechanism of cell transformation by EBV.