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Biomedical subjects

B Fleckenstein

Publications and source records attributed to B Fleckenstein.

At least 91 records · Page 5Linked to original sources
PubMed1994

Karyotypic characterization of human T-cell lines immortalized by Herpesvirus saimiri.

Herpesvirus saimiri subgroup-C strains transform non-neoplastic human T lymphocytes in vitro to antigen-independent continuous growth. The karyotypes of 8 of these T-cell lines, derived from peripheral blood or bone marrow, are analyzed here. In general, these lines showed a normal diploid karyotype, although one of them had acquired a clonal chromosomal rearrangement after 21 months in culture. Chromosomally aberrant cell clones were found more frequently in cell lines derived from blood lymphocytes of leukemia patients than in cultures from healthy donors. However, the observed aberrations, some of which were defined in detail using chromosome in situ suppression hybridization (CISS) with chromosome-specific recombinant DNA libraries, did not show any relation between the various cell lines nor to the respective types of leukemia.

Cell Line↗
PubMed1994

Engagement of the CD4 receptor inhibits the interleukin-2-dependent proliferation of human T cells transformed by Herpesvirus saimiri.

Infection with Herpesvirus saimiri, a tumor virus of non-human primates, transformed human CD4+ T cell clones to permanent interleukin (IL)-2-dependent growth without need for restimulation with antigen and accessory cells. The IL-2-dependent proliferation of these cells was dramatically inhibited by soluble anti-CD4 whole antibodies, F(ab')2 and Fab fragments, and also by gp 120 of human immunodeficiency virus. The inhibition was not due to cell death and could be overcome by high concentrations of exogenous IL-2. Cell surface expression of CD4, and to a lesser degree the density of the IL-2 receptor alpha chain, were reduced upon anti-CD4 treatment. After long lasting (> 12 h) incubation with anti-CD4, abundance and activity of CD4-bound p56lck were diminished while the free fraction of p56lck remained unchanged. Since IL-2 binding to its receptor activated only the CD4-bound fraction of p56lck, the IL-2-induced p56lck activity was diminished after long-term CD4 ligation. Taken together, our results suggest a cross talk between CD4- and IL-2 receptor-mediated signaling via p56lck.

Antibodies, Monoclonal↗
PubMed1994

Cytokine dependence of human to mouse graft-versus-host disease.

Human peripheral blood leucocytes (PBL) induce chronic graft versus-host disease (GvHD) in non-conditioned severe combined immunodeficient mice. Chronic GvHD was observed in such animals after transplantation of 6 x 10(7) human PBL per g body weight. However, acute xenogeneic GvHD results from grafting at least 2 x 10(7) human PBL per g body weight to heavily conditioned murine hosts. The large numbers of human PBL were thought to be required to produce above threshold amounts of certain cytokines. We show that treatment of the recipient mice with human interleukin 2 reduces the number of cells to inflict acute GvHD by a factor of ten. Human T cells and not B cells or macrophages, were previously shown to generate acute xenogeneic GvHD, when selected cell types from peripheral blood were grafted. Most of the infiltrating cells had the CD4+ phenotype. We demonstrate that CD4+ T cells are the main mediator, as the disease is abrogated by treating the mice with cytotoxic CD4 antibodies, but not with CD8 antibodies. A survival pattern, similar to that seen in GvHD, was induced by transplantation of a Herpesvirus saimiri transformed human CD4+ clonal T cell line in conjunction with daily interleukin 2 injections. Herpesvirus saimiri transformed human T cells allow easily reproducible graft properties in chimeric mouse models for human diseases.

Acute Disease↗
PubMed1994

Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri.

Herpesvirus saimiri (HVS) is a lymphotropic herpesvirus that induces T-cell transformation in vitro and causes lymphomas and leukemias in New World primates other than its natural host, the squirrel monkey. Nucleotide sequence analysis of the HVS genome revealed two open reading frames with significant homology to genes for human complement regulatory molecules. One of these genes encodes a predicted protein (designated HVSCD59) with 48% amino acid sequence identity to the human terminal complement regulatory protein CD59 (HuCD59). The CD59 homolog from squirrel monkey (SMCD59) was cloned, and the corresponding amino acid sequence showed 69% identity with HVSCD59. BALB/3T3 cells stably expressing HVSCD59, SMCD59, or HuCD59 were equally protected from complement-mediated lysis by human serum. However, only HVSCD59-expressing cells were effectively protected from complement-mediated lysis when challenged with rat serum, suggesting that HVSCD59 was less species restrictive. The complement regulatory activity of HVSCD59 and SMCD59 occurred after C3b deposition, indicating terminal complement inhibition. Treatment of BALB/3T3 stable transfectants with phosphatidylinositol-specific phospholipase C prior to complement attack decreased the complement regulatory function of HVSCD59, suggesting cell surface attachment via a glycosyl-phosphatidylinositol anchor. Cells expressing HVSCD59 effectively inhibited complement-mediated lysis by squirrel monkey serum in comparison with SMCD59-expressing cells. Finally HVSCD59-specific transcripts were detected in owl monkey cells permissive for lytic HVS replication but not in T cells transformed by HVS, which failed to produce virions. These data are the first to demonstrate a functional, virally encoded terminal complement inhibitor and suggest that HVSCD59 represents a humoral immune evasion mechanism supporting the lytic life cycle of HVS.

3T3 Cells↗
PubMed1994

Structure and transcription of an immediate-early region in the human herpesvirus 6 genome.

The unique segment of the human herpesvirus 6 (HHV-6) genome is essentially collinear to the unique long DNA segment of another betaherpesvirus, the human cytomegalovirus (HCMV). However, the HHV-6 genomic section that is analogous in position to the major immediate-early (IE) locus of HCMV does not exhibit recognizable sequence homologies. The respective HHV-6 region of 5.5 kbp is flanked on one side by 25 to 28 incomplete tandem repeats of 105 to 110 bp that contain, with one exception, a single KpnI restriction site (KpnI repeats). About 250 reiterations of the sequence motif CACATA are located on the other end. We identified two open reading frames of 375 and 2,595 nucleotides, respectively, on one strand. Strand-specific Northern blot analyses with RNA harvested from HHV-6 (strain U1102)-infected HSB-2 cells or cord blood lymphocytes revealed two transcripts of about 3.5 and 4.7 kb in the corresponding orientation. Sequence analyses of the respective cDNA clones and primer extension experiments were used to map the mRNAs. The two transcripts are coterminal and multiply spliced and code for the same putative 104.6-kDa protein, but they are initiated from different promoters. Characterization of smaller cDNA clones and Northern blotting with other strand-specific probes showed that singly spliced mRNAs of 1.0 and 1.5 kb are transcribed from the opposite strand; they could code for a 17.2-kDa polypeptide. Blocking experiments with cycloheximide led to the conclusion that only the 3.5-kb mRNA is synthesized in the absence of protein biosynthesis upon infection with cell-free virus. This identifies a single IE gene of HHV-6 at the genomic position corresponding to the major IE region of HCMV, although the coding content and transcriptional regulation are quite different for these two herpesvirus IE regions.

Base Sequence↗
PubMed1993

Transformation of human T-cell clones by Herpesvirus saimiri: intact antigen recognition by autonomously growing myelin basic protein-specific T cells.

Herpesvirus saimiri has recently been shown to immortalize human T cells. It was unknown, however, whether Herpesvirus saimiri transformation affects T-cell receptor (TCR) expression and signal transduction. In the present study, we have transformed CD4+ human T-cell clones specific for human myelin basic protein. The transformed T cells were grown in interleukin 2 and divided in the absence of antigen and antigen-presenting cells. They retained the membrane phenotype of activated T cells and secreted the cytokines interferon gamma and lymphotoxin, but interleukin 4 was not detected. Further, the transformed T cells continued to express the original TCR as demonstrated by TCR variable-region-V beta-specific monoclonal antibodies and TCR sequencing. Antigen-specific recognition and signal transduction by the TCR were demonstrated by myelin-basic-protein-induced HLA-DR-restricted secretion of interferon gamma and lymphotoxin and by myelin-basic-protein-specific proliferation. Antigen specificity and reactivity have been maintained for > 1 year after transformation. Transformation with Herpesvirus saimiri now allows the production of virtually unlimited numbers of (auto)antigen-specific T cells expressing functional TCR and a stable membrane phenotype. This technology will facilitate studies of the pathogenesis of putative autoimmune diseases, such as multiple sclerosis, and may be of help in TCR-targeted immunotherapy.

Antigens, CD↗
PubMed1993

Immortalization with herpesvirus saimiri modulates the cytokine secretion profile of established Th1 and Th2 human T cell clones.

T blasts of six established human CD4+ T cell clones with defined Ag specificity and cytokine secretion profile (3 Th1 and 3 Th2) were immortalized with Herpesvirus saimiri (HVS) and compared with their uninfected counterparts for their ability to proliferate, produce cytokines, and express cytolytic activity. HVS-transformed Th1 and Th2 clones neither substantially changed their original surface markers nor lose their ability to proliferate in response to their specific Ag but did acquire the ability to proliferate in response to contact signals delivered by SRBC or autologous APC alone. In addition, transformation by HVS substantially enhanced the lectin-dependent cytolytic activity of Th1 clones and enabled noncytolytic Th2 clones to exert cytolytic activity. HVS-transformed Th1 clones but not their uninfected counterparts spontaneously transcribed and secreted Th1-type cytokines (IL-2, IFN-gamma, and TNF-beta) and such a production was further enhanced by stimulation with either SRBC or PMA plus anti-CD3 mAb. HVS transformed but not uninfected Th2 clones constitutively expressed both IL-4 and IL-2 mRNA and secreted IFN-gamma. Stimulation with PMA plus anti-CD3 mAb induced uninfected Th2 clones to secrete high amounts of IL-4 and IL-5 but not Th1-type cytokines, whereas the same HVS-transformed Th2 showed minimal IL-4 and IL-5 secretion with concomitant high production of IL-2, IFN-gamma, and TNF-beta. Transformation by HVS also resulted in up-regulation of TNF-alpha and IL-3 production by both Th1 and Th2 clones. The ongoing proliferation of HVS-transformed clones was partially inhibited by either anti-IL-2 or anti-IL-3 antibodies and virtually abolished by the combined addition of the two anticytokine antibodies, suggesting that both IL-2 and IL-3 can function as autocrine growth factors for HVS-transformed Th1 and Th2 clones.

Cell Transformation, Viral↗
PubMed1993

Immortalization of human T cell clones by Herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.

Investigation of human activated T cells has been complicated by the need for periodic restimulation with Ag/mitogen and accessory cells and by the limited life span of most human T cell clones. To overcome these problems, we have transformed established human T cell clones to permanent growth with Herpesvirus saimiri, a lymphoma-inducing virus of nonhuman primates. Three human CD4+ T cell clones were investigated in detail. They have been growing in the presence of exogenous IL-2 but without restimulation with mitogen or feeder cells for more than 11 mo with doubling times between 2 and 4 days. In contrast, their nontransformed parent clones needed to be restimulated with PHA and feeder cells every 14 to 21 days. To compare responses of H. saimiri-transformed clones with those of their parent clones, we stimulated the cells with IL-2 or with anti-CD3 and/or anti-CD4 mAb with and without cross-linking on the cell surface. Transformed and nontransformed T cell clones were strikingly similar in parameters of early signal transduction, namely, tyrosine phosphorylation and mobilization of calcium. Ligation of their TcR/CD3 complexes by mAb or by Ag in the presence of autologous accessory cells increased the proliferation and the secretion of IFN-gamma. Taken together, we have shown that human T cell clones immortalized with H. saimiri express functional CD3, CD4, and IL-2R. They constitute a simple, stable, reproducible and accessory cell-free model system for the investigation of signal transduction events in activated human T cells.

Antigens, Bacterial↗
PubMed1993

Mouse monoclonal antibodies recognizing the activation domain of HIV-1 rev transactivator.

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. So far, at least four necessary functional domains have been assigned to the HIV-1 rev protein: (1) one for RNA binding; (2) a second for nuclear/nucleolar localization that may be indistinguishable from the RNA binding motif; (3) two domains for multimerization; and (4) a putative activation domain (AD) that is suppressed in trans by dominant-negative mutant rev protein. We report three IgG1 kappa mouse monoclonal antibodies (mabs) that were independently raised against rev protein expressed in Escherichia coli. Epitopes are mapped by immunoprecipitation and Western blot screening with 40 different rev mutant peptides. Surprisingly, monoclonal antibodies from all three hybridomas recognized the activation domain of HIV-1 rev.

Amino Acid Sequence↗
PubMed1993

Herpesvirus saimiri transformed human T cell lines: a permissive system for human immunodeficiency viruses.

Human peripheral blood T lymphocytes are readily transformed to continuous growth by Herpesvirus saimiri subgroup C strains. The immortalized cells express the phenotype of mature activated T cells and bear either CD4 or CD8 surface markers. Here we report that Herpesvirus saimiri transformed CD4+ cell lines are highly susceptible to infection with human immunodeficiency viruses types 1 and 2. The prototype viruses HIV-1IIIB and HIV-2ROD replicated rapidly and caused cell death within 14 days. These cell lines did also support growth of a poorly replicating HIV-2 strain (HIV-2NEP) and of primary clinical isolates. Thus H. saimiri transformed T cells represent a new system for HIV propagation and isolation, especially for HIV-2 strains with restricted cell tropism. These cells should be considered as an alternative approach in cases where conventional attempts fail.

Cell Line, Transformed↗
PubMed1993

Herpes virus saimiri-transformed human T lymphocytes: normal functional phenotype and preserved T cell receptor signalling.

Herpes virus saimiri (HVS), a primate herpes virus, transforms human CD4+ and CD8+ T lymphocytes to continuous growth in vitro. We have previously shown that HVS-transformed human T cells (HVS-T cells) respond to stimulation via CD2 with autocrine growth. In the present study we have investigated the functional characteristics of HVS-T cells. We describe that these cells can perform all the functions of normal T cells, i.e. cytokine secretion, cytotoxicity, and exocytosis of granule esterases. All these activities can be triggered via CD2 by binding to its natural ligand or via the TCR, e.g. by anti-TCR antibodies, by recognition of a bacterial superantigen and by MHC-restricted recognition of specific antigen. The pattern of tyrosine-phosphorylated proteins after TCR triggering was identical in HVS-T cells and normal T cells. We conclude that HVS-T cells can respond to TCR-mediated signals with the functions of normal T lymphocytes. Furthermore, HVS-T cells are the only transformed human T cells that can be specifically triggered by cytotoxicity and esterase release. The finding that the TCR functions normally in these cells will make HVS a convenient means to immortalize antigen-specific human T lymphocytes.

Antigens↗
PubMed1993

Detection of varicella zoster virus DNA and viral antigen in human cornea after herpes zoster ophthalmicus.

This article describes the histopathology, immunohistochemistry, and varicella zoster virus DNA in situ hybridization of 14 corneal buttons obtained from 14 patients (average age 69.0 years) after perforating keratoplasty (four patients) or surgical enucleation (10 patients) at different times after the clinical onset of herpes zoster ophthalmicus (average 58.7 months). The main histopathologic features were intense stromal vascular scarring (12 patients) and granulomatous reaction to Descemet's membrane (nine patients). Using the peroxidase-antiperoxidase method, varicella zoster virus (VZV) antigen could be detected by immunohistochemistry in two patients within epithelial cells of the cornea and in the limbal episclera during the active phase of herpes zoster ophthalmicus. For in situ hybridization we used the 35S-labeled HindIII A and C fragment of VZV and identified viral DNA in five corneal buttons obtained 1 day to 8 years after the clinical onset of infection. Viral DNA was mainly found in mononuclear cells with eosinophilic intracytoplasmic inclusions within vascular stromal scars, in keratocytes, and in epithelial cells of the cornea. Our results show that VZV DNA is detectable in human cornea even 8 years after the clinical onset of herpes zoster ophthalmicus and may indicate VZV persistence in a latent form in corneal tissue or reactivation of the virus from an endogenous or exogenous source causing a severe and often recurrent keratitis in the progress of herpes zoster ophthalmicus.

Adult↗
PubMed1993

The glycoprotein B homologue of human herpesvirus 6.

The gene for the homologue of herpesvirus glycoprotein B (gB) has been identified in the genome of human herpesvirus 6 (HHV-6), strain U1102, and the nucleotide sequence was determined. The open reading frame encodes a protein of 830 amino acids (93.2K) with the characteristics of a transmembrane glycoprotein and close similarity to the gp58/116 complex of human cytomegalovirus (HCMV). Monoclonal antibodies 2D10 and 2B9 have been shown previously to react with an HHV-6 glycoprotein of apparent M(r) 112K, and its proteolytic cleavage products of M(r) 64K and 58K. We show that both monoclonal antibodies detect prokaryotically expressed carboxy-terminal fragments of the HHV-6 gB homologue. This indicates that the HHV-6 gB homologue is probably processed by proteolytic cleavage similar to its equivalents in HCMV and various other herpesviruses.

Amino Acid Sequence↗