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Biomedical subjects

P M Pitha

Publications and source records attributed to P M Pitha.

At least 19 recordsLinked to original sources
PubMed1992

In-vitro resistance to zidovudine and alpha-interferon in HIV-1 isolates from patients: correlations with treatment duration and response.

OBJECTIVE: To measure in-vitro antiviral drug susceptibilities of human immunodeficiency virus type 1 (HIV-1) isolates recovered from patients treated with alpha-interferon or zidovudine and patients not treated with these drugs and to examine the relation of these susceptibility measurements to duration of therapy, disease stage, and response to alpha-interferon therapy. DESIGN: Cross-sectional study. SETTING: Outpatient HIV clinic. PATIENTS: Twenty-six ambulatory HIV-1-infected patients: Fifteen of these patients were receiving alpha-interferon therapy, and 11 had never received such therapy. Nine patients were participating in a clinical trial of combination therapy with zidovudine and alpha-interferon. MEASUREMENTS: The 50% inhibitory concentration (IC50) of zidovudine and alpha-interferon was determined for HIV-1 isolates recovered from each patient. Plasma concentrations of HIV-1 p24 antigen in the nine patients in the clinical trial were measured monthly after alpha-interferon was added to zidovudine monotherapy. RESULTS: Zidovudine IC50 (range, 0.01 to 4.87 microM) increased steadily with duration of zidovudine therapy (r = 0.57, P = 0.003). In contrast, alpha-interferon IC50 (range, 0.8 to 415 units/mL) was not related to duration of alpha-interferon treatment; in fact, high IC50s were found in isolates from patients who had never received exogenous alpha-interferon therapy. Resistance to alpha-interferon was greater in isolates from the 15 patients with the acquired immunodeficiency syndrome (AIDS) (median, 104 units/mL) than in those from the 10 patients without AIDS (median, 50 units/mL). Interferson activity was detected in plasma samples from 23 of 24 patients and was also at higher levels in patients with AIDS than in HIV-infected patients without AIDS. Reductions in plasma concentrations of HIV-1 p24 antigen in nine patients after beginning alpha-interferon therapy were greater in those with more susceptible isolates (r = -0.72, P = 0.03). CONCLUSIONS: Interferon resistance, possibly due to endogenous interferon, is not related to duration of interferon therapy but may limit the effectiveness of interferon therapy. Determinations of interferon susceptibility may identify patients most likely to benefit from this agent.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Distinct activation of murine interferon-alpha promoter region by IRF-1/ISFG-2 and virus infection.

Virus infection in mouse L929 cells activates expression of interferon-alpha 4 (IFN-alpha 4), but not IFN-alpha 6. The integrity of a symmetrical sequence, GTAAAGAAAGT (alpha F1 site); (-103 to -93), present in the 35 nucleotide (nt) long inducible element (IE) (-109 to -75) of the alpha 4 promoter region is essential for the virus-induced expression. In the present study, we have shown that the interferon regulatory factor 1 (IRF-1) can induce expression of both IFN-alpha 4 and -alpha 6 in a transient expression assay. Virus infection cooperates with IRF-1 and further enhances transcription from the alpha 4 promoter, but inhibits the IRF-1-mediated expression from the alpha 6 promoter. The virus-mediated induction is determined by both IRF-1 and alpha F1 sites, while activation by IRF-1 in a cotransfection assay is not greatly influenced by the alpha F1 sequence. The activation of IFN-alpha gene promoters by IRF-1 was limited to the transient expression assay. The integrated alpha 4 promoter or the endogenous IFN-alpha genes could not be induced by transfection with IRF-1 expressing plasmid and IRF-1 did not up-regulate expression of the endogenous IRF-1 gene. However, expression of IRF-1 alone was sufficient to up-regulate the expression of two IFN stimulated genes, 2',5' oligoadenylate synthetase (OAS) and interferon stimulated (ISG)-15 gene. These results suggest that induction of IFN-alpha gene expression by virus infection requires cooperation between IRF-1 and another factor(s) that binds to the alpha F1 sequence.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Mouse genotype affects inducible expression of cytokine genes.

The levels of circulating IFN in mice infected with Newcastle disease virus (NDV) are regulated by the If-1 locus. In this study we show that in NDV-infected C57BL/6 mice, which carry the If-1h allele and produce high levels of IFN, high levels of both IFN-alpha and -beta mRNA can be detected in the spleen. In contrast, only very low levels of IFN mRNA could be detected in spleens of infected BALB/c mice containing the If-1l allele and producing low levels of IFN or in B6.C-H28c mice that are congenic for the If-1l allele. The relative levels of all individual IFN-alpha 1, alpha 4, and alpha 6 mRNA in spleens of infected BALB/c were lower than in spleens of infected C57BL/6 mice, indicating that the If-1 locus affects the expression of all IFN-alpha subtypes and is not associated with the deletion or inactivation of a specific IFN gene. The relative levels of IFN regulatory factor-1 mRNA in infected mice carrying the If-1l and If-1h loci were comparable, suggesting that the If-1 regulation is not associated with the altered expression of the IFN regulatory factor-1 gene. Quantitative difference in the expression of IFN-alpha and -beta genes was also observed in in vitro-infected peritoneal macrophages isolated from either C57BL/6 or BALB/c mice. A surprise finding was that the If-1 locus also affected the NDV-induced expression of two other cytokine genes, TNF-alpha and IL-6. Priming of the macrophage cultures with murine IFN enhanced the expression of all cytokine genes, and the relative levels of IFN, TNF-alpha, and IL-6 mRNA induced by NDV in macrophages derived from C57BL/6 and BALB/c mice were comparable. We propose that the If-1 locus affects the early stages of a signal transduction pathway which are common to the virus-mediated induction of IFN, TNF-alpha, and IL-6 genes.

Animals↗
PubMed1992

Poly(A) removal is the kinase-regulated step in tumor necrosis factor mRNA decay.

Tumor necrosis factor (TNF) is a pleiotropic biomodulator and an important inducer of certain pathophysiologic immune reactions such as granuloma formation, cachexia, and septic shock. The production of TNF by astrocytes, which may figure prominently in the development of immune responses within the central nervous system, is subject to post-transcriptional regulation. We have previously shown that in virus-stimulated astrocytes, inhibition of protein kinase C results in a specific, 10-fold decrease in TNF mRNA half-life. Here we show that the decay of TNF messages induced in the macrophage-like cell line RAW 264.7 by either virus or lipopolysaccharide was subject to similar regulation, and that this pathway influenced the amount of TNF protein released by stimulated cells. Using a modified RNase protection assay, we demonstrate that inhibition of protein kinase C significantly enhanced the rate of poly(A) removal from TNF mRNA, thus facilitating an early event in the process of mRNA degradation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Activation of human immunodeficiency virus type 1 provirus in T-cells and macrophages is associated with induction of inducer-specific NF-kappa B binding proteins.

We have analyzed the limiting factors involved in the induction of human immunodeficiency virus type 1 (HIV-1) provirus expression by tumor necrosis factor-alpha (TNF-alpha), phorbol-12-myristate-13-acetate (PMA), and bryostatin-1 in T-cells (ACH-2) and monocytes (U1). We have demonstrated that, while there is a correlation among the increase of 9.2-kilodalton (kDa) HIV-1 RNA, the increase of viral proteins (p24) in the cells, and the release of HIV-1 virions into the medium, there is no direct correlation between the levels of induced NF-kappa B binding proteins and the expression of HIV-1 provirus. The presence of nuclear NF-kappa B-specific proteins appears to be essential only for the initiation of viral replication, since the HIV-1 transcripts could be detected in TNF-alpha or bryostatin-1-stimulated cells also at later times postinduction, times when no NF-kappa B proteins could be detected in the nucleus. The uv crosslinking of DNA and proteins has shown that TNF-alpha, PMA, and bryostatin-1 induce different sets of NF-kappa B binding proteins with distinct kinetics of binding.

Blotting, Northern↗
PubMed1992

Transcriptional activation of the tat-defective human immunodeficiency virus type-1 provirus: effect of interferon.

The effect of human interferon-alpha 2 (HuIFN-alpha 2) on the activation of HIV-1 provirus was studied in cell lines containing either an integrated tat-defective HIV-1 provirus (HIV-1 (-tat)) (HNHIVdt4 cells) or the HIV-1 (-tat) provirus and a plasmid in which the expression of HuIFN-alpha 2 was under the control of HIV LTR (HNHIV alpha 1 cells). In both cell lines, the expression of HIV-1 RNA was below the limit of detection, but transcription of the HIV-1 (-tat) provirus could be induced either by transfection with Tat-expressing plasmid or by treatment with TPA and cycloheximide (CHX). By contrast, stimulation with TPA alone induced HIV-1 transcription only in HNHIVdt4 cells, but not in HNHIV alpha 1 cells that produced low levels of IFN-alpha constitutively. Similarly in a transient expression assay, TPA upregulated transcription of the transfected HIV-1 CAT plasmid only in HNHIVdt4 cells, but not in HNHIV alpha 1 cells. UV-crosslinking analysis of NF-kappa B-specific proteins induced in TPA-treated cells showed the presence of 45 and 55 kDa NF-kappa B-binding protein in TPA-induced HNHIVdt4 cells while, in HNHIV alpha 1 cells, we detected only 55-, 110-, and 200-kDa proteins, but no 45-kDa protein. The transcriptional effects of IFN could not, however, be seen in the presence of Tat protein, suggesting that the virus developed a mechanism to overcome the IFN-mediated restrictions.

Gene Expression↗
PubMed1992

Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle.

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Herpes simplex virus type 1-mediated induction of human immunodeficiency virus type 1 provirus correlates with binding of nuclear proteins to the NF-kappa B enhancer and leader sequence.

Herpes simplex virus type 1 (HSV-1) infection induces expression of the human immunodeficiency virus type 1 (HIV-1) provirus in the chronically infected T-cell line ACH-2. The HSV-1-mediated induction correlates with the appearance of two NF-kappa B-specific proteins of 55 and 85 kDa in the nucleus and with the binding of 50-kDa nuclear protein to the LBP-1 binding site of the untranslated leader sequence of the HIV-1 long terminal repeat. The HSV-1-induced LBP-1 binding protein, designated HLP-1, is present exclusively in HSV-1-infected, but not in phorbol-12-myristate-13-acetate- or tumor necrosis factor alpha-treated ACH-2 cells. Both the NF-kappa B and LBP-1 target sequences, when inserted either alone or together 5' of a heterologous minimal promoter (thymidine kinase), confer inducibility by HSV-1 infection in a transient transfection assay. Thus, it appears that the HSV-1-mediated activation of HIV-1 provirus is brought about by the binding of both NF-kappa B and HLP-1 specific proteins to two distinct regions of HIV-1 long terminal repeat.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Herpes simplex virus infection selectively stimulates accumulation of beta interferon reporter gene mRNA by a posttranscriptional mechanism.

To study the mechanism of a novel herpes simplex virus (HSV) activity that stimulates expression of reporter genes containing beta interferon (IFN-beta)-coding sequences, we have established permanent DNA-transfected cell lines that each contain two distinct hybrid genes encoding mRNA species with different half-lives. These reporter genes comprised either the human IFN-beta- or bacterial chloramphenicol acetyltransferase (CAT)-coding and 3' untranslated regions placed under the transcriptional control of the powerful major immediate-early promoter-enhancer region (IE94) from simian cytomegalovirus. Most of the dual-transfected cell lines yielded significant levels of steady-state IE94-CAT mRNA and abundant constitutive synthesis of CAT enzyme activity, whereas no accumulation of IE94-IFN mRNA could be detected. However, infection with HSV type 1 resulted in a 300-fold increase in IE94-IFN-specific mRNA transcripts, compared with no more than 3- to 5-fold stimulation of IE94-CAT-specific mRNA. In contrast, cycloheximide treatment increased stable mRNA levels and transcription initiation rates from both the IE94-IFN and IE94-CAT hybrid genes. Run-on transcription assays in isolated nuclei suggested that induction of IE94-IFN gene expression by HSV type 1 occurred predominantly at the posttranscriptional level. Enhancement of the unstable IFN mRNA species after HSV infection was also observed in cell lines containing a simian virus 40 enhancer-driven IFN gene (SV2-IFN). Similarly, in transient-transfection assays, both SV2-IFN and IE94-IFN gave only low basal mRNA synthesis, but superinfection with HSV again led to high-level accumulation of IFN mRNA. Finally, substitution of the SV2-IFN gene 3' region with poly(A) and splicing signals from the SV2-CAT gene cassette led to stabilization of the IFN mRNA even in the absence of HSV. Therefore, we conclude that HSV infection leads to selective accumulation of IFN-beta mRNA by a posttranscriptional mechanism that is reporter gene specific and promoter independent.

Chloramphenicol O-Acetyltransferase↗
PubMed1991

Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus.

The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.

Cells, Cultured↗
PubMed1991

Identification of a novel virus-responsive sequence in the promoter of murine interferon-alpha genes.

We have previously shown that the infection of mouse L-cells with Newcastle disease virus activates transcription of the alpha 4 but not the alpha 6 interferon gene and that the induction is mediated by a 35-base pair inducible element (IE) found in the alpha 4 promoter (-109 to -75). In the present study, we show that the inactivity of the alpha 6 promoter can be mapped to 2 out of 6 nucleotides in which the alpha 6 differs from alpha 4 IE. The symmetrical sequence, GTAAAGAAAGT (-103 to -93), present in the alpha 4 IE is essential for its inducibility and binding of nuclear protein(s) to the alpha 4 IE.

Animals↗
PubMed1991

Aberrant expression of cytokine genes in peritoneal macrophages from mice infected with LP-BM5 MuLV, a murine model of AIDS.

Mice infected with LP-BM5 murine leukemia virus (MuLV) develop a syndrome denoted as murine AIDS. Macrophages harvested from the peritoneal cavities of these mice at 4 or 9 wk postinoculation with LP-BM5 MuLV were analyzed by Northern hybridization for the presence of the defective LP-BM5 virus and their ability to synthesize various cytokines upon induction with Newcastle disease virus (NDV) or (LPS). Neither IFN-alpha or IFN-beta was found to be constitutively expressed in LP-BM5-infected macrophages and in NDV induction studies, and the levels of biologically active IFN-alpha and its mRNA were found to be lower in LP-BM5 MuLV-infected macrophages than in the macrophages from uninfected controls. Similarly, after NDV or LPS induction, the levels of TNF mRNA and TNF protein were significantly lower in LP-BM5-infected macrophages than in macrophages from uninfected mice. The LP-BM5 MuLV-infected macrophages constitutively expressed low levels of IL-1 beta, and when induced with LPS, the relative levels of IL-1 beta were significantly higher in infected than in uninfected macrophages. Although no constitutive expression of IL-6 was detected, the levels of IL-6 mRNA induced with NDV were higher in LP-BM5 MuLV-infected macrophages than in controls. Thus, we found alterations in the expression of selected cytokines in macrophages from mice inoculated with LP-BM5 MuLV rather than a general deregulation of all cytokine expression. These results show that macrophages infected with the defective LP-BM5 virus respond differently to NDV- or LPS-stimulation and suggest that aberrant expression of certain cytokine genes may play a role in the immunopathologic condition in mice with murine AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Relative antiviral activity of in vitro-synthesized murine interferon-alpha 4 and -alpha 1.

Murine interferon-alpha 4 (MuIFN-alpha 4) is notable among the MuIFN-alpha subtypes because it lacks 5 amino acids corresponding to positions 103-107 of the other subtypes, yet is the most highly expressed subtype. Site-directed in vitro mutagenesis has been used to modify the genes coding for MuIFN-alpha 4 and MuIFN-alpha 1. The modifications have allowed (i) the in vitro expression of the mature form of each MuIFN-alpha subtype and (ii) the insertion of five amino acids, corresponding to amino acid positions 103-107 of MuIFN-alpha 1, into the MuIFN-alpha 4 sequence. In contrast to previously published data MuIFN-alpha 4 and MuIFN-alpha 1 show only a twofold difference in antiviral activity, with MuIFN-alpha 4 being the more active subtype. In keeping with this observation, it was also found that insertion of the five "missing" amino acids into MuIFN-alpha 4 resulted in an analogue MuIFN-alpha with antiviral activity equivalent to MuIFN-alpha 1. It may be inferred from this work that the deletion of amino acids 103-107 (QVGVQ) is solely responsible for the difference in antiviral activity between MuIFN-alpha 4 and MuIFN-alpha 1.

Animals↗
PubMed1991

Expression of defective virus and cytokine genes in murine AIDS.

A syndrome characterized by severe immunodeficiency and lymphoproliferation develops in susceptible strains of mice infected with a mixture of murine leukemia viruses (MuLVs) designated LP-BM5 MuLV. The etiologic agent in this mixture has been shown to be a replication-defective virus (BM5d) with a 4.8-kb genome that required replication-competent helper viruses, primarily ecotropic (BM5e), for cell-to-cell spread in the host. In the present study, we studied the expression of BM5d and BM5e in tissues of infected mice at various times after inoculation in relation to the expression of cytokine genes that may contribute to the pathogenesis of this disorder. Northern (RNA) analysis of total RNA showed that BM5d was expressed at significant levels in lymphoid tissues within 1 week of infection and that the levels of expression increased with time after inoculation. By 16 weeks postinfection, BM5d was expressed in all tissues examined. Expression of BM5e was relatively more restricted to lymphoid tissues and was detected at lower levels than expression of BM5d at early times after infection, but this virus was expressed in all tissues by 16 weeks. Infection with the virus mixture was associated with constitutive expression of tumor necrosis factor in all tissues examined and of interleukin-1 (IL-1) in lymphoid tissues within 1 week of infection, and at later times with widespread expression of these cytokines and gamma interferon. Also, the levels of interferon regulatory factor 1 mRNA were significantly increased in all infected tissues during the infection. In contrast, expression of IL-3, IL-4, IL-5, and IL-6 was not detectable by Northern analysis of the respective mRNAs in any infected tissue at early or late times postinfection.

Animals↗
PubMed1990

Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes.

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency.

Infection of cells by HIV can result in a period of quiescence or latency which may be obviated by treatment with inducing agents such as 5-azacytidine. Evidence from these experiments demonstrate the existence of two CpG sites in the HIV LTR which can silence transcription of both reporter genes (CAT) and infectious proviral DNA when enzymatically methylated. This transcriptional block was consistently overcome by the presence of the trans-activator tat without significant demethylation of the HIV LTR. These results suggest that DNA hypermethylation of the HIV LTR may change the binding characteristics between LTR sequences and cellular proteins, thereby suppressing HIV LTR transcription and modulating viral expression.

Animals↗
PubMed1990

Retrovirus vector-targeted inducible expression of human beta-interferon gene to B-cells.

We have introduced the human beta-interferon gene with its promoter region into murine B-cell and fibroblast cell lines via a Moloney murine leukemia virus (M-MuLV) vector and have studied the inducible expression of the beta-interferon gene as a function of the various retroviral vector designs. By deleting the enhancer within the 3' viral long terminal repeat (LTR), inserting the human beta-interferon gene, and varying placement of the immunoglobulin heavy chain enhancer, we were able to construct vectors which yielded proviruses with various cell type-specific regulation. One of the vectors (pT154) led to a greater than 21-fold increase in beta-interferon protein synthesis after viral infection in the two B-cell lines analyzed, while no inducibility was seen in the fibroblast cells. The data show that inducible beta-interferon expression within a MuLV vector was highly dependent on the absence of the viral enhancer region in the long terminal repeat and the orientation of the beta-interferon gene within the proviral transcriptional unit; the insertion of the immunoglobulin enhancer elevated both constitutive and (or) inducible expression of beta-interferon in B-cells but inhibited constitutive expression of this gene in fibroblasts.

Animals↗