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Biomedical subjects

S F Josephs

Publications and source records attributed to S F Josephs.

At least 37 records · Page 2Linked to original sources
PubMed1990

In vitro characterization of a biologically active molecular clone of HIV-2NIH-Z containing a nef deletion and expressing a full-length transmembrane protein.

We have previously described the cloning and sequencing of a novel stain of human immunodeficiency virus type 2 (HIV-2) called HIV-2NIH-Z. A plasmid clone, pHIV2Z, containing the full-length provirus has now been constructed, and virus particles have been obtained upon transfection into COS-1 and H-9 cells. These particles can infect a number of T-cell lines and exert a cytopathic effect on fresh human and macaque peripheral blood lymphocytes. The cloned virus is biologically and morphologically indistinguishable from its parental uncloned strain as shown by restriction enzyme analysis, electron microscopy, and kinetics of infection. However, as shown by radioimmunoprecipitation assays, the cloned virus-infected cells express a full-length gp41 protein as predicted by the nucleotide sequence, whereas the wild-type parental strain expresses a truncated gp33 protein. Both the parental strain and the cloned virus possess a deletion encompassing the end of the nef gene within the U3 region which apparently does not affect their in vitro cytopathic and replicative capacities.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells.

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.

Base Sequence↗
PubMed1990

Search for early lesions following human immunodeficiency virus type 1 infection. A study of six individuals who died a violent death after seroconversion.

Histologic studies supplemented by in situ hybridization for human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus, cytomegalovirus, and human herpesvirus type 6 were performed on tissues obtained from the autopsy of six patients who died either by homicide or suicide shortly after learning of their seroconversion. Except for mild nonspecific lymphoid tissue reactions, no lesions were noted that would indicate HIV-1 infection. DNA from all viruses was detected in some lymphoid cells. The amount of DNA for Epstein-Barr virus, cytomegalovirus, and human herpesvirus type 6 corresponded to that observed for clinically occult latent infection. Lymphoid cells carrying HIV-1 DNA were even less frequent. Cells positive for HIV-1 were noted in the lamina propria of the large intestine in three male homosexuals and in one female prostitute. The cells were arranged similar to antigen-presenting cells. The present findings are consistent with current theories regarding the pathogenesis of HIV-1-associated disease.

Adult↗
PubMed1989

Human herpes virus-6 increases HIV-1 expression in co-infected T cells via nuclear factors binding to the HIV-1 enhancer.

Human Herpes virus-6 (HHV-6) can co-infect with HIV-1 human CD4+ T-cells, leading to accelerated cell death, and factors in HHV-6-infected cells stimulate HIV-1 LTR directed gene expression. In this study, we have examined the mechanism of HIV-1 activation by HHV-6 and localized the cis-acting sequences of HIV-1 LTR responsive to trans-activation. Increased HIV-1 LTR directed gene expression is obtained in HIV-1 infected cells co-infected with HHV-6, or in HHV-6 infected cells co-transfected with the HIV-1 tat gene. Parallel increases of HIV-1-specific transcripts are seen by in situ hybridization in HHV-6/HIV-1 doubly infected cells as compared to single HIV-1 infection. Similarly, infection by HHV-6 increases the steady-state level of HIV-1 LTR mRNA that parallels CAT enzymatic activity, suggesting a transcriptional and/or post-transcriptional activation. Sequences necessary for HIV-1 LTR activation by HHV-6 are distinct from those required for that tat response and map to a region of the HIV-1 LTR from -103 to -48. The HIV-1 enhancer sequence (-105 to -80) is sufficient to confer HHV-6 inducibility to a heterologous promoter, and nuclear protein(s) activated or induced by HHV-6 infection specifically bind to the NF kappa B motifs of the HIV-1 enhancer region.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Augmentation of human immunodeficiency virus type 1 gene expression by tumor necrosis factor alpha.

It is now well established that cytokines are involved in the regulation of gene expression from HIV-1 LTR. The present study provides evidence that TNF-alpha stimulates HIV-1 gene expression and that the enhancer sequence within the HIV-1 LTR is involved in the stimulation. These results support the idea that immunologic stimulation and infection may trigger the development of clinical AIDS in individuals latently infected with HIV-1.

Animals↗
PubMed1989

Analysis of human KS biopsies and cloned cell lines for cytomegalovirus, HIV-1, and other selected DNA virus sequences.

Investigation into a possible role of several human viruses, including human cytomegalovirus (HCMV), human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), human herpesvirus 6 (HHV6), and Epstein-Barr virus (EBV) in the pathogenesis of Kaposi's sarcoma (KS) has resulted in the lack of an association of these viruses in KS biopsy and cloned KS cell line specimens. Since nearly all patients with KS, including those with HIV-associated KS, have a high incidence of HCMV infection, HCMV has been proposed to be etiologically associated with KS. Moreover, our previous studies showed the retention of HCMV morphological transforming region II (mtrII) in both transformed and tumor-derived cell lines. As a result, we focused on the nucleic acid hybridization analysis of both KS biopsies from AIDS patients as well as cloned KS endothelial cell lines for HCMV mtrII sequences. We also analyzed KS biopsy and KS cloned cell line specimens for HIV-1, HBV, HHV6, and EBV sequences, since these viruses have also been implicated in the etiology of KS. In one set of experiments, Southern blot analysis revealed the presence of HCMV mtrII sequences in two of six KS biopsies; in other experiments, HBV sequences were found in one of seven KS biopsies. No hybridization in any biopsy tissue was detected for HIV-1 DNA sequences. The analysis of six independently derived cloned KS cell lines was next studied. All these lines were negative for hybridization to the HCMV mtrII transforming fragment as well as to subgenomic fragments of HHV6 and EBV.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗
PubMed1989

HIV-1 expression is posttranscriptionally repressed in Drosophila cells.

The long terminal repeats (LTRs) of the human immunodeficiency virus (HIV) the Rous sarcoma virus (RSV) and the copia Drosophila retrotransposon were compared in their capacity to direct expression of the bacterial cat (chloramphenicol acetyltransferase) gene in human, murine, and Drosophila cell lines. The results indicate that HIV and RSV LTR expression is post transcriptionally repressed in the Drosophila cells while copia LTR expression is post-transcriptionally repressed in the human and murine cells.

Animals↗
PubMed1989

Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).

In 1986, we reported the discovery and isolation of a novel human herpesvirus (HBLV) from AIDS and other lymphoproliferative disorders. Because HBLV is distinct from other members of the herpesvirus family and can infect B- and T-lymphocytes and other human cells (megakaryocytes and glioblastoma cells), we suggested human herpesvirus-6 (HHV-6) as the taxonomic designation for this virus. In cultures from patients' peripheral blood, the evidence of HBLV can be recognized from the appearance of short-lived giant cells (2-10%), which are large, refractile, and are often mono- and binucleated. As these cells degenerate, extracellular virus particles are found in the culture medium. HBLV can infect fresh mononuclear cells, established B- and T-lymphoblastoid cell lines, megakaryocytes and glioblastoma cell lines. HBLV infection can be detected by: a. morphological changes; b. indirect immunofluorescence assay, in situ hybridization, southern blot analysis, polymerase chain reaction amplification; and c. electron microscopy. Because of its wide cell tropism, HBLV DNA sequences have been detected in B-cell lymphomas and short term cultured cells from Sjogren's patients. Expression of HBLV RNA was also detected in sarcoidosis. The etiological role of HBLV in human tumors is unclear. While in vitro data may not necessarily apply to in vivo conditions, the infection of various cell lines from tumors and fresh mononuclear cells suggests HBLV involvement in a variety of diseases.

Cell Line↗
PubMed1988

Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).

Details of the productive infection of established human cell lines of diverse origin by HBLV (also designated Human Herpesvirus 6) are described in this report. The infection and replication of HBLV in several T and B lymphoid and other cell lines was observed by electron microscopic examination, by the detection of viral antigen expression by indirect immunofluorescence assay (IFA) and by the presence of HBLV DNA by Southern blot hybridization. Several of these cell lines produced large amounts of virus. For this reason and because of the absence of other human herpesviruses, these lines have provided a valuable resource for the preparation of reagents and the development of assays for the detection and characterization of HBLV. The isolation and characterization of new HBLV isolates from patients with chronic fatigue syndrome were also facilitated by using some of the cell lines reported here. The host range of HBLV in established cell lines, therefore, does not appear to be limited to the B lymphocytes, as initially suggested by in vivo studies. The infection of T and B lymphocytes, megakaryocytes and neuronal cells in vitro suggests a need for the evaluation of diverse hematological and neurological disorders to shed light on a possible HBLV involvement.

Blotting, Southern↗
PubMed1988

The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites.

The Fos protein complex and several Fos-related antigens bind directly or indirectly to a common sequence element that is similar to the consensus binding site for HeLa cell activator protein 1 (AP-1). This element is present in a negative regulatory sequence in the differentiation-sensitive adipocyte gene, aP2; in a transcriptional enhancer for the Gibbon ape leukemia virus; and in a region of the human immunodeficiency virus (HIV) long terminal repeat partially characterized as a negative regulatory element. The protein level and binding activity of Fos and Fos-related antigens increase rapidly after calcium ionophore treatment of a CD4+ human lymphoblast cell line, H9. These data suggest that several proteins may associate with the AP-1 binding site. Moreover, temporally regulated control of the level of each protein could represent a mechanism for modulation of these putative mediators of gene expression.

Base Sequence↗
PubMed1988

Human B-lymphotropic virus (human herpesvirus-6).

Human B-lymphotropic virus (HBLV), also known as human herpesvirus-6 (HHV-6) was first isolated in 1986 from AIDS patients and patients with other lymphoproliferative disorders. HBLV is distinct from known human herpesviruses, biologically, immunologically and by molecular analysis. HBLV can infect and replicate in fresh and established lines of hemopoietic cells and cells of neural origin, suggesting wide tropism. The prevalence of HBLV antibody in the normal population was 26% though clear differences between different populations were observed. The prevalence of HBLV antibody an elevated antibody titer was higher in sera from certain malignancies, Sjögren's syndrome and sarcoidosis. Antibody to HBLV was also elevated in AIDS patients and patients with chronic fatigue syndrome. HBLV-DNA was detected in some B-cell lymphomas. The broad in vitro tropism, combined with immunological and molecular evidence of HBLV infection in individuals raise the question of the pathogenicity of this virus in some diseases. Because in vitro co-infection of CD4 cells by HBLV and HIV leads to enhanced degeneration, this raises the possibility that infection in AIDS patients by both viruses can aggravate the HIV-induced immunodeficiency. Specific reagents and immunological and molecular assays are currently being investigated, which will aid in virus detection in cells from patients, and in elucidating the possible pathogenesis of HBLV.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Antibody prevalence to HBLV (human herpesvirus-6, HHV-6) and suggestive pathogenicity in the general population and in patients with immune deficiency syndromes.

Detailed serologic screening showed an antibody prevalence to HBLV (HHV-6) in the general population of 26% if very strict criteria for antibody positivity were applied. Lower and borderline antibody titers yet may be found in up to 63% of the population. Only 17% of these persons have clinical symptoms; in the majority infection remains silent. HHV-6 infection apparently occurs already quite early in life, and initial symptoms can occur, such as short-term high fever, sore throat, local lymphadenopathy and skin rash. Lesions disappear without specific treatment. The frequency of positive antibody tests at higher titers rises in patients with immune deficiency and with atypical lymphoproliferative diseases to 60 and 75%. The rise in antibody titers is associated in patients with immune deficiency by characteristic shifts of blood lymphocyte populations, essentially by increase in immature T-lymphocytes. Highest titers are found in patients with lymphoproliferative syndromes, yet the percentage of atypical lymphoid cells harboring the viral genome is low (about 2% of seropositive patients). Thus it appears, that HBLV, similar to other herpesviruses such as Epstein-Barr virus, usually causes a silent seroconversion, yet may be associated with variable clinical pathology when persisting in an active state. Its pathogenic effect might be rather a cofactor contributing to immune disturbance than overt oncogenicity.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Molecular studies of HHV-6.

Methods for the purification of enveloped HHV-6 virions and the viral DNA are presented. The viral genome is estimated to be 170,000 base pairs in size and does not appear to contain inversions due to absence of submolar bands by restriction enzyme analyses. The genomes of two independent HHV-6 isolates, HHV-6GS and HHV-6Z29, showed restriction enzyme site pleomorphism. Large scale purification of enveloped HHV-6 was achieved by continuous flow centrifugation utilizing sucrose gradients, DNAse 1 treatment and banding on 10-30% Dextran T10 gradients. The viral proteins were visualized on high resolution two dimensional polyacrylamide gels and the proteins recognized by serum antibody from patient GS were detected by HR2D Western blot analysis and radioimmunoprecipitation assay. The major antigenic proteins were 200, 120, 80, 72, 30 and 19 kDa.

Blotting, Western↗
PubMed1988

Polymerase chain reaction amplification and in situ hybridization for the detection of human B-lymphotropic virus.

Polymerase chain reaction amplification (PCR) is a recently described technique that allows for the amplification of a given sequence of DNA. It can be used to reliably amplify sequences of up to 3 kb within hours. The amplified sequence can then be recognized by hybridization with a specific probe after transfer onto nitrocellulose or nylon paper. We used PCR to recognize human B-lymphotropic virus (HBLV or HHV-6) specific sequences in various tumors as well as in the blood of patients with AIDS. Sixty-three specimens of DNA extracted from peripheral blood of patients with AIDS as well as DNA extracted from various lymphoproliferative disorders were analysed; 52 out of 63 (83%) patients with AIDS were found to have amplification of the HHV-6 specific sequence; 2 out of the 63 (3%) had equivocal amplification and 9 (14%) were found to be negative. Twenty out of 23 tumors were found to have amplified HBLV-specific sequences. Only one of these tumors was positive by Southern hybridization on restriction enzyme digested genomic DNA. In situ hybridization of clinical specimens using radiolabelled RNA probes or hapten-labelled DNA probes was used to detect the presence of HBLV in tumors. Three tumors of B cell origin were found to be positive for HBLV.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Diagnosis and differential diagnosis of progressive lymphoproliferation and malignant lymphoma in persistent active herpesvirus infection.

Persistent active Epstein-Barr virus (EBV) and human herpesvirus-6 (HHV-6) infections may be accompanied by extensive lymphoproliferative reactions mimicking malignant lymphoma. Such atypical polyclonal lymphoproliferations (APL) can frequently be separated from malignant lymphoma only by detailed immunopathologic and virologic studies. Recommended procedures include immunotyping of lymphoid cells, extensive viral serology, in situ hybridization and Southern blotting for viral DNA, gene rearrangement and DNA ploidy studies. Clear separation of APL from malignant lymphoma is essential for therapeutic planning.

Diagnosis, Differential↗
PubMed1988

Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor.

Single nucleotide alterations were introduced into an infectious clone of human immunodeficiency virus type 1 to create a series of missense mutants in the tat coding region. Although mutations in a proline-rich region and a basic lysine-arginine-rich region resulted in wild-type phenotypes, five of six mutations in a cysteine-rich domain completely abolished tat activity and virus replication. One cysteine mutant retained tat activity but was negative for virus expression. Surprisingly, this mutant could not be complemented by tat, and virus expression was restored only by cotransfection with a plasmid expressing the rev gene. Another mutant with an alteration toward the C-terminal region showed significantly reduced tat activity and required complementation by a combination of tat and rev for virus replication. Further analysis revealed that a previously unrecognized splice acceptor site within this region, apparently used to generate the rev mRNA, had been altered. We provide evidence suggesting that tat and rev proteins are encoded by distinct mRNA species.

Amino Acid Sequence↗