Biomedical subjects

B Fleckenstein

Publications and source records attributed to B Fleckenstein.

At least 37 records · Page 2Linked to original sources

PubMed1999

Herpesvirus ateles gene product Tio interacts with nonreceptor protein tyrosine kinases.

Herpesvirus ateles is a gamma-2-herpesvirus which naturally infects spider monkeys (Ateles spp.) and causes malignant lymphoproliferative disorders in various other New World primates. The genomic sequence of herpesvirus ateles strain 73 revealed a close relationship to herpesvirus saimiri, with a high degree of variability within the left terminus of the coding region. A spliced mRNA transcribed from this region was detected in New World monkey T-cell lines transformed by herpesvirus ateles in vitro or derived from T cells of infected Saguinus oedipus. The encoded viral protein, termed Tio, shows restricted homology to the oncoprotein StpC and to the tyrosine kinase-interacting protein Tip, two gene products responsible for the T-cell-transforming and oncogenic phenotype of herpesvirus saimiri group C strains. Tio was detectable in lysates of the transformed T lymphocytes. Dimer formation was observed after expression of recombinant Tio. After cotransfection, Tio was phosphorylated in vivo by the protein tyrosine kinases Lck and Src and less efficiently by Fyn. Stable complexes of these Src family kinases with the viral protein were detected in lysates of the transfected cells. Binding analyses indicated a direct interaction of Tio with the SH3 domains of Lyn, Hck, Lck, Src, Fyn, and Yes. In addition, tyrosine-phosphorylated Tio bound to the SH2 domains of Lck, Src, or Fyn. Thus, herpesvirus ateles-encoded Tio may contribute to viral T-cell transformation by influencing the function of Src family kinases.

Amino Acid Sequence↗

PubMed1998

Suppression of HHV-8 viremia by foscarnet in an HIV-infected patient with Kaposi's sarcoma and HHV-8 associated hemophagocytic syndrome.

Human herpes virus 8 (HHV-8) seems to be involved in the pathogenesis of Kaposi s sarcoma. In vitro, antiviral drugs with activity against herpes viruses also can suppress HHV-8, however, little is known about the antiviral activity against HHV-8 in vivo. In this report we describe the effects of foscarnet on HHV-8 viremia in an HIV-infected patient with disseminated Kaposi s sarcoma and a presumably HHV-8 associated hemophagocytic syndrome. HHV-8 DNA could be detected in this patient by PCR in peripheral blood mononuclear cells (PBMC), in bronchoalveolar fluid and tumor biopsies. After initiation of foscarnet because of a severe hemophagocytic syndrome HHV-8 PCR turned negative in PBMC, but stayed positive in pleural effusions and in a tumor biopsy. After termination of foscarnet therapy HHV-8 DNA in PBMC persistently reappeared. Under treatment with foscarnet the hemophagocytic syndrome dramatically improved, suggesting that HHV-8 had a pathogenetic role in this syndrome.

AIDS-Related Opportunistic Infections↗

PubMed1998

New ligands for HLA DRB1*0301 by random selection of favourable amino acids ranked by competition studies with undecapeptide amide sublibraries.

An efficient screening procedure for the identification of high affinity HLA class II ligands and their binding pattern has been established to characterize peptide specificities for the HLA allele DRB1*0301. The method is based on the screening of 209 synthetic undecapeptide amide sublibraries O/X10-NH2 representing collections of 19(10) individual peptides in a competition ELISA using HLA DRB1*0301 protein and the biotinylated natural ligand ApoB 2877-2894. Screening results represent the effect on competition induced by an individual amino acid residue in its sequence position of undecapeptide amides. Amino acids clustered as active in their position were randomly selected for the same position of a restricted set of 96 individual undecapeptide amides. This novel approach for the design of ligands was introduced to compensate for the inaccuracy induced by the translational invariance of amino acids in peptide libraries characterized by one defined amino acid. Translational invariance is facilitated by shifted docking of O/X10-NH2 libraries in the binding cleft and protrusion from the ends of the cleft. A second more directed deduced set of 24 peptides was obtained by combination of the most favourable residues in each position. All individual peptides were investigated in the competition assay. The most active HLA DRB1*0301 ligands were obtained by random selection of favourable amino acids and six of them showed improved affinity in comparison to the model ligand alpha AChR 310-325.

Alleles↗

PubMed1998

Increased HLA-DQ2-affinity of a synthetic gliadin peptide by acid-induced deamidation of glutamine residues.

Presentation of antigenic gliadin peptides by the HLA-DQ2 molecule is considered as a key event in celiac disease pathogenesis. Chemical deamidation of the side chains of glutamine residues might have a strong influence on gliadin peptide binding to the DQ2 molecule. Glutamine deamidation of A-gliadin peptide (45-56) under acidic conditions corresponding to the gastric environment was studied using RP-HPLC, Edman degradation, capillary electrophoresis and electrospray mass spectrometry. Deamidation resulted in peptides with increased DQ2-affinities as assessed in a cell-free binding assay.

Amides↗

PubMed1998

New eukaryotic semaphorins with close homology to semaphorins of DNA viruses.

Semaphorins were initially described as a family of repulsive guidance molecules in embryonal development. Their basic structure consists of an N-terminal signal sequence, the defining semaphorin domain ofapproximately 500 amino acids, an Ig-like domain,and a variable carboxy-terminus. We recently described a viral semaphorin homologue encoded by the alcelaphine herpesvirus type 1. Less conserved, truncated homologues were also identified in poxviruses. Here we describe new human and murine semaphorin homologues. The respective genes were cloned and sequenced, and they were termed H-Sema-L and M-Sema-L (HGMW-approved symbols SEMAL and Semal, respectively). A multiply spliced mRNA of 3.2 kb is expressed in human placenta, spleen, thymus, and gonadal tissue. H-Sema-L maps to chromosome 15q22.3-q23 and M-Sema-L to the homologous locus 9A3.3-B in the mouse genome. The expression patterns and the presence of related genes in large DNA viruses suggest that this new semaphorin has a relevant function in the immune system.

Adjuvants, Immunologic↗

PubMed1998

Human herpesvirus type 8 interleukin-6 homologue is functionally active on human myeloma cells.

Seroepidemiology and polymerase chain reaction studies have strongly suggested that human herpesvirus type 8 (HHV-8) is associated with Kaposi's sarcoma, Castleman's disease, and body cavity-based lymphoma. The genome of HHV-8 harbors a viral analogue of the interleukin-6 (IL-6) gene. The amino acid sequence of the viral IL-6 (vIL-6) protein is 24.7% identical to human IL-6 (hIL-6). IL-6 as a B-cell growth and differentiation factor is known to play an essential role in the pathophysiology of B-cell tumors. Thus, it seems possible that virus-encoded IL-6 contributes to malignant growth of HHV-8-positive B-cell lymphatic tumors. We have tested a preparation of HHV-8-derived IL-6 for the ability to promote the proliferation of the human myeloma cell line INA-6, which is strictly dependent on exogenous IL-6 for growth and survival. Viral IL-6 significantly induced DNA synthesis of INA-6 cells, but required much more protein on a weight basis when compared with hIL-6 for maximal proliferation. The proliferative effect of vIL-6 was almost completely inhibited by a combination of anti-IL-6 receptor (IL-6R) and anti-gp130 antibodies or IL-6R superantagonist Sant7 and anti-gp130 antibodies. This report demonstrates that vIL-6 has proliferative activity on human cells and that the IL-6R and gp130 are involved in vIL-6 signaling in the myeloma cell line INA-6.

Antibodies, Monoclonal↗

PubMed1998

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

OBJECTIVE: The purpose of the study was to identify varicella zoster virus (VZV) DNA and viral antigen in human eyes at various intervals after clinical onset of herpes zoster ophthalmicus (HZO). DESIGN: A retrospective case series. PARTICIPANTS: There were 9 eyes and 4 corneal buttons surgically obtained from 13 patients with HZO at the University Eye Hospital of Erlangen-Nürnberg between 1984 and 1994. Specimens were examined at different timepoints after clinical onset of HZO (range, 1 day-19 years; median, 36 months). METHODS: Histopathologic evaluation was performed on formalin-fixed and paraffin-embedded tissue by routine histology, immunohistochemistry (5-B-7 murine monoclonal antibody to VZV; peroxidase-antiperoxidase method), and DNA-in situ hybridization (35S deoxyadenosine triphosphate-labeled HindIII fragments [A and C] of VZV). RESULTS: Typical histopathologic changes associated with HZO were identified: vascularization of the corneal stroma (11 of 13), granulomatous reaction to Descemet's membrane (8 of 13), fusiform-shaped ciliary scarring (5 of 9), optic neuritis (4 of 9), and perineuritis (8 of 9) and perivasculitis (8 of 9) of the long posterior ciliary nerves and arteries. VZV antigen was detected in two patients with acute infection 1 and 7 days after onset of HZO, respectively. VZV-DNA was identified in seven patients up to 10 years after onset of HZO in corneal epithelial cells (2 of 13), corneal stroma (5 of 13), inflammatory infiltrate of the anterior chamber (1 of 9), episclera (2 of 9), posterior ciliary nerves (1 of 9) and arteries (5 of 9), optic nerve (5 of 9), and adjacent leptomeninges (2 of 9). CONCLUSION: Persistence of viral genomes, most likely accompanied by gene expression or slow viral replication, appears to be responsible for the often smoldering panophthalmitis and the chronic recurrent keratouveitis in patients with HZO. Localization of viral DNA in vascular structures suggests a role for vasculitis in the pathogenesis of some ocular findings associated with HZO.

Adult↗

PubMed1998

Anti-apoptotic strategies of lymphotropic viruses.

Induction of apoptosis of virus-infected cells is an important host cell defence mechanism. However, some viruses have incorporated genes that encode anti-apoptotic proteins or modulate the expression of cellular regulators of apoptosis. Here, Edgar Meinl and colleagues discuss recent evidence that viral interference with host cell apoptosis leads to enhanced viral replication, and to evasion of cytotoxic T-cell effects.

Apoptosis↗

PubMed1998

Deregulation of cell growth by the K1 gene of Kaposi's sarcoma-associated herpesvirus.

At a position equivalent to the gene encoding the saimiri transforming protein (STP) of herpesvirus saimiri (HVS), Kaposi's sarcoma-associated herpesvirus (KSHV) contains a distinct open reading frame called K1. Although KSHV and HVS are related members of the rhadinovirus subgroup of gamma herpesviruses, K1 and STP exhibit no similarity in amino acid sequence or in structural organization. Since STP is required for the oncogenic potential of HVS, we investigated the functional consequence of K1 expression. Expression of the K1 gene in rodent fibroblasts produced morphologic changes and focus formation indicative of transformation. A recombinant herpesvirus in which the STP oncogene of HVS was replaced with K1, immortalized primary T lymphocytes to IL-2 independent growth and induced lymphoma in common marmosets. These results demonstrate the transforming potential of the K1 gene of KSHV.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Human herpesvirus 8--the first human Rhadinovirus.

Kaposi's sarcoma (KS)-associated herpesvirus, also known as human herpesvirus 8 (HHV-8), is the first known human member of the genus Rhadinovirus. It is regularly found by polymerase chain reaction in all forms of KS, in certain types of Castleman's disease, and in body cavity-based B-cell lymphoma. Other members of this virus group occur in nonhuman primates, ungulates, rabbits, and mice and cause in part fulminant lymphomas and other neoplastic disorders of the hematopoietic system. Rhadinoviruses share a typical genome structure; most characteristically, they contain numerous sequences that appear to be sequestered from cellular DNA. We cloned and sequenced almost the complete genome of HHV-8 from a single KS biopsy specimen. Although this procedure revealed collinear organization and extensive homologies with the open reading frames of herpesvirus saimiri, genes with homology to the known oncoproteins (Stp, Tip) were not identified in the HHV-8 genome. However, HHV-8 reading frame K1, the positional analogue of Stp/Tip, was found to be significantly variable between different strains. We found, in addition, the reading frames for homologues of cellular interleukin 6, macrophage inflammatory proteins alpha and beta (MIP1 alpha and MIP1 beta, respectively), an interferon-responsive factor, and two inhibitors of apoptosis. Several of these cell-homologous genes of HHV-8 have already been shown to code for functional proteins.

Animals↗

PubMed1998

Herpesvirus saimiri transforms human T-cell clones to stable growth without inducing resistance to apoptosis.

Herpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-X(L), Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-alpha. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways.

Animals↗

PubMed1998

T-cell lymphoma caused by herpesvirus saimiri C488 independently of ie14/vsag, a viral gene with superantigen homology.

The immediate-early gene ie14/vsag of herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with either ie14/vsag deletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viral ie14/vsag gene.

Animals↗

PubMed1998

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor.

Interferons (IFNs) are a family of multifunctional cytokines with antiviral activities. The K9 open reading frame of Kaposi's sarcoma-associated herpesvirus (KSHV) exhibits significant homology with cellular IFN regulatory factors (IRFs). We have investigated the functional consequence of K9 expression in IFN-mediated signal transduction. Expression of K9 dramatically repressed transcriptional activation induced by IFN-alpha, -beta, and -gamma. Further, it induced transformation of NIH 3T3 cells, resulting in morphologic changes, focus formation, and growth in reduced-serum conditions. The expression of antisense K9 in KSHV-infected BCBL-1 cells consistently increased IFN-mediated transcriptional activation but drastically decreased the expression of certain KSHV genes. Thus, the K9 gene of KSHV encodes the first virus-encoded IRF (v-IRF) which functions as a repressor for cellular IFN-mediated signal transduction. In addition, v-IRF likely plays an important role in regulating KSHV gene expression. These results suggest that KSHV employs an unique mechanism to antagonize IFN-mediated antiviral activity by harboring a functional v-IRF.

3T3 Cells↗

PubMed1998

The interleukin-17 gene of herpesvirus saimiri.

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.

Animals↗

PubMed1998

Antiapoptotic activity of the herpesvirus saimiri-encoded Bcl-2 homolog: stabilization of mitochondria and inhibition of caspase-3-like activity.

Viruses have evolved different strategies to interfere with host cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in the human leukemia cell line Jurkat and in the murine T-cell hybridoma DO to assess its antiapoptotic spectrum and to gain further insight into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as a result of a disturbance of intracellular homeostasis by, for example, DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with CD95-mediated apoptosis but blocked dexamethasone-induced cell death. Mitochondrial damage is a central coordinating event in apoptosis induced by different stimuli. To assess the integrity of mitochondria, we used rhodamine 123, which is released upon disturbance of the mitochondrial membrane potential, and determined the release of cytochrome c into the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the cleavage of poly(ADP-ribose) polymerase, a natural substrate of caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a great variety of apoptotic stimuli, stabilizes mitochondria, and acts upstream of the generation of caspase-3-like activity.

Apoptosis↗

PubMed1998

The immunogenic glycoprotein gp35-37 of human herpesvirus 8 is encoded by open reading frame K8.1.

Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi's sarcoma (KS) and body cavity-based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in BCBL-derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters (R. Renne, W. Zhang, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. E. Ganem, Nat. Med. 2:342-346, 1996). A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently and intensively recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular mass is reduced to 30 kDa by digestion with peptide-N-glycosidase F. By searching the known HHV-8 genomic sequence for open reading frames (ORF) with the potential to encode such a glycoprotein, an additional, HHV-8-specific reading frame was identified adjacently to ORF K8. This ORF, termed K8.1, was found to be transcribed primarily into a spliced mRNA encoding a glycoprotein of 228 amino acids. Recombinant K8.1 was regularly recognized by KS patient sera in Western blots, and immunoaffinity-purified antibodies to recombinant K8.1 reacted with gp35-37. This shows that the immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1, which will be a useful tool for studies of HHV-8 epidemiology and pathogenesis.

Amino Acid Sequence↗

PubMed1997

Herpes viral cyclin/Cdk6 complexes evade inhibition by CDK inhibitor proteins.

The passage of mammalian cells through the restriction point into the S phase of the cell cycle is regulated by the activities of Cdk4 and Cdk6 complexed with the D-type cyclins and by cyclin E/Cdk2. The activities of these holoenzymes are constrained by CDK inhibitory proteins. The importance of the restriction point is illustrated by its deregulation in many tumour cells and upon infection with DNA tumour viruses. Here we describe the properties of cyclins encoded by two herpesviruses, herpesvirus saimiri (HVS) which can transform blood lymphocytes and induce malignancies of lymphoid origin in New World primates, and human herpesvirus 8 (HHV8) implicated as a causative agent of Kaposi's sarcoma and body cavity lymphomas. Both viral cyclins form active kinase complexes with Cdk6 that are resistant to inhibition by the CDK inhibitors p16(Ink4a), p21Cip1 and p27Kip1. Furthermore, ectopic expression of a viral cyclin prevents G1 arrest imposed by each inhibitor and stimulates cell-cycle progression in quiescent fibroblasts. These results suggest a new mechanism for deregulation of the cell cycle and indicate that the viral cyclins may contribute to the oncogenic nature of these viruses.

3T3 Cells↗

PubMed1997

Interactions between autologous CD4+ and CD8+ T lymphocytes and human squamous cell carcinoma of the head and neck.

The autotumor (AuTu)-specific cytolytic T lymphocyte (CTL) line established from the peripheral blood of a patient with oral squamous cell carcinoma (Cancer Res. 53, 1461, 1993) contained >95% of CD8+ and <5% of CD4+ T cells. This CTL line was infected with Herpesvirus saimiri to increase its life span in culture. Two transformed T cell sublines were obtained: the monoclonal CD4+ line (TCR Vbeta2+ V alpha15+) and the oligoclonal CD8+ line (TCR Vbeta6+, Vbeta7+ and Vbeta9+) both of which were maintained in culture for >6 months without AuTu restimulation and which did not produce any virus. The virus-transformed and untransformed T cell lines were compared for phenotypic and functional characteristics, including the ability to kill AuTu, induce expression of the major histocompatibility complex (MHC) antigens on AuTu, and respond to AuTu by cytokine production and/or proliferation. The H. saimiri-transformed CD4+ T cells expressed higher levels of surface adhesion molecules and CD45RO than untransformed cells and lysed AuTu by inducing DNA fragmentation as well as necrosis. This lysis was inhibited by antibodies to CD4 but not to class I or II MHC molecules. The CD4+ T cells produced IL2, TNF-alpha, and GM-CSF and proliferated in response to AuTu. They induced and sustained proliferation of CD8+ T cells in cocultures with AuTu. Supernatants obtained from cocultures of the CD4+ T cells with AuTu also induced proliferation of the CD8+ T cell line. In contrast, the H. saimiri-transformed CD8+ T cells did not kill AuTu or release cytokines in response to AuTu. However, upon pretreatment of AuTu with IFN-gamma to increase expression of MHC antigens, these T cells regained the ability to recognize and kill AuTu targets. Coincubation of AuTu with the CD4+ or CD8+ T cells significantly augmented expression of class I and II MHC antigens on AuTu. These data indicate that H. saimiri-transformed tumor-reactive T cell lines provide a useful model of interactions between immune effector cells and AuTu, and that CD4+ T cells play a critical role in the regulation of immune responses to squamous cell carcinoma of the head and neck.

CD4-Positive T-Lymphocytes↗