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Biomedical subjects

J A Turpin

Publications and source records attributed to J A Turpin.

At least 19 recordsLinked to original sources
PubMed1995

Inhibitors of HIV nucleocapsid protein zinc fingers as candidates for the treatment of AIDS.

Strategies for the treatment of human immunodeficiency virus-type 1 (HIV-1) infection must contend with the obstacle of drug resistance. HIV-1 nucleocapsid protein zinc fingers are prime antiviral targets because they are mutationally intolerant and are required both for acute infection and virion assembly. Nontoxic disulfide-substituted benzamides were identified that attack the zinc fingers, inactivate cell-free virions, inhibit acute and chronic infections, and exhibit broad antiretroviral activity. The compounds were highly synergistic with other antiviral agents, and resistant mutants have not been detected. Zinc finger-reactive compounds may offer an anti-HIV strategy that restricts drug-resistance development.

Amino Acid Sequence↗
PubMed1992

Enhanced HIV-1 replication in retinoid-treated monocytes. Retinoid effects mediated through mechanisms related to cell differentiation and to a direct transcriptional action on viral gene expression.

Vitamin A and other retinoids have profound effects on macrophage differentiation and function. Such effects could alter interactions between HIV and tissue macrophages, a principal target cell and reservoir for virus during HIV disease. Indeed, retinoids are used to treat various symptoms associated with HIV infection. We show that levels of virus replication in monocytes cultured 7 days before and continuously after HIV infection in 1 to 10 microM retinoic acid were 10- to 20-fold greater than those of control cells. No direct toxicity (detachment from substrate or cell death) was evident in infected or control monocytes treated with less than or equal to 10 microM retinoic acid. Maximum effects of retinoic acid (50% maximum effect was at 0.8 +/- 0.1 microM) required 5 to 7 days treatment before infection and persisted without additional treatment through more than 4 wk. RT activity in cultures of retinoic acid-treated monocytes reached maximum levels much earlier than those of control cultures, but the minimum tissue culture infectious doses for retinoic acid-treated and untreated monocytes were comparable. Retinoic acid treatment did not affect susceptibility of monocytes to HIV infection. Further, the frequency of infected cells in retinoic acid-treated and control cultures were also comparable: about 20% of cells in each culture expressed HIV proteins or RNA 2 wk after infection. In contrast, levels of HIV-specific RNA and DNA were 3- to 5-fold higher in the retinoic acid-treated over control monocytes 1 wk after infection. That retinoic acid increased levels of HIV gene expression in monocyte cultures without affecting the number of infected cells per culture suggested a transcriptional mechanism for the effect. This was confirmed in the U937 myeloid cell line transfected with HIV LTR linked to a chloramphenicol acetyl transferase reporter gene. Chloramphenicol acetyl transferase activity in lysates of retinoic acid-treated cells were 20-fold higher than that of control cells. These data show that retinoic acid significantly increased HIV replication in monocytes through mechanisms related to cell differentiation and to a direct transcriptional effect on viral gene expression.

Cell Differentiation↗
PubMed1992

Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells.

NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.

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

Characterization of a class 3 tyrosine kinase.

In an effort to identify unique tyrosine kinases found in human leukemia cell lines, we utilized polymerase chain reaction (PCR) technology and degenerate oligonucleotide primers to produce a cDNA library of kinase catalytic domains found in the human monocytic cell line AML-193. This search yielded a member of the class 3 tyrosine kinases closely related to the murine kinase FD-22. Previous work has identified this kinase as JAK1. This class of tyrosine kinases is characterized by being ubiquitously expressed, lacking both a ligand-binding domain and a SH2 domain, while containing a second domain similar to a degenerate kinase domain. Our studies focused on the further characterization of this class 3 tyrosine kinase using Northern blot analysis to demonstrate an increase in steady-state mRNA by interferon-gamma in human monocytes. A human-hamster somatic cell hybrid panel and linkage mapping was used to assign JAK1 (aml-116) to human chromosome 1.

Alleles↗
PubMed1991

Enhanced HIV replication in macrophage colony-stimulating factor-treated monocytes.

Monocytes cultured 7 to 10 days in recombinant human macrophage CSF (MCSF) were greater than 400-fold more susceptible to HIV infection than an equal number of cells cultured in medium alone. Levels of reverse transcriptase activity and p24 Ag in culture fluids of monocytes treated with MCSF 1 wk before and continuously after HIV infection were significantly greater than those of control cells cultured without MCSF. HIV-induced cytopathic effects in the MCSF-treated cultures also increased in both frequency and extent. At any given viral inoculum, the frequency of HIV-infected cells, the level of HIV mRNA/infected cell, and the level of proviral DNA/infected culture in MCSF-treated monocyte cultures were dramatically greater than those in control cultures. These differences were directly related to MCSF concentration to a maximum between 750 and 1000 U/ml MCSF, and were evident at all time points examined through 5 wk. None of the preceding effects was observed when MCSF was added at the time of or 1 wk after HIV infection. These data suggest that the predominant effect of MCSF for control of HIV infection is on the monocyte itself, not the virus. If these in vitro observations extend to the HIV-infected patient, then the variable levels of MCSF in tissue or blood may determine both the susceptibility of macrophages to virus infection and the extent of virus replication in infected cells.

Cells, Cultured↗
PubMed1991

Regulation of cytokine and viral gene expression in monocytes infected with the human immunodeficiency virus.

Monocytes treated with interferon-alpha (IFN-alpha) at virus challenge show no evidence of human immunodeficiency virus (HIV) infection: no p24 antigen or reverse transcriptase (RT) activity, no viral mRNA and no proviral DNA. Levels of p24 antigen and RT activity in monocytes infected with HIV 1-3 weeks before IFN-alpha treatment gradually decrease to baseline. HIV-induced cytopathic changes are markedly reduced, as are levels of HIV mRNA: the frequency of productively infected cells is less than or equal to 1%. But, levels of proviral DNA in the IFN-alpha-treated and control HIV-infected cells are indistinguishable, and remain so through 3 weeks. Large quantities of proviral DNA in IFN-alpha-treated cells with little active transcription suggest true microbiological latency. The major potential source for IFN-alpha in HIV-infected patients is the macrophage. With any of 15 virus isolates, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, IFN-omega or IFN-beta are not detected nor the mRNA expressed in HIV-infected or uninfected monocytes. Both uninfected and HIV-infected monocytes produce high levels of these cytokines after treatment with synthetic double-stranded RNA (poly-I:C). Uninfected monocytes also produce high levels of IFN-alpha after treatment with Poly-I:C, Newcastle disease virus or herpes simplex virus. In marked contrast, HIV-infected monocytes express no IFN-alpha activity or mRNA before or after treatment with any of these agents. The markedly diminished capacity of HIV-infected monocyte to produce IFN-alpha reflects a specific transcriptional block and may be an adaptive mechanism of virus to alter basic microbicidal functions of this cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines↗
PubMed1991

Tumor necrosis factor and c-fos expression in human peripheral-blood monocytes: expression is dependent on stage of in vitro differentiation.

The differentiation of monocytes and macrophages both in vitro and in vivo can be characterized by the modulation of specific functional and molecular phenotypes. We have determined in human peripheral-blood monocytes (HPBM) the role of in vitro differentiation on the expression of nonspecific tumoricidal activity, induction of soluble tumor necrosis factor (TNF) activity and TNF-specific mRNA transcription. HPBM were activatable by bacterial lipopolysaccharide (LPS; 1 microgram/ml) for nonspecific cytotoxicity to A375M (human malignant melanoma cell line) only during the first 24 h of in vitro differentiation. Activated supernatants of HPBM were found to be partially neutralizable (75 +/- 7%) by rabbit polyclonal anti-TNF antibody and, in freshly isolated HPBM, the release of soluble TNF activity determined by the L929 assay was found to occur only after activation with LPS. Maximal TNF release occurred at 8 h of LPS stimulation, and required both protein and RNA synthesis as evidenced by the ability of both actinomycin D and cycloheximide to inhibit its release. Neither control untreated HPBM nor recombinant interferon-gamma (rIFN-gamma; 1 U/ml)-treated HPBM alone released soluble TNF activity. Further in vitro culture determined that HPBM were activatable for TNF release out to 72 h of culture after which HPBM became resistant to LPS-mediated TNF release. The expression of TNF and c-fos mRNA was also determined during in vitro differentiation. Both TNF and c-fos mRNA were expressed in freshly isolated HPBM, and returned to baseline by 24 h of in vitro culture. Treatment of HPBM with LPS induced TNF transcription as late as 5 days of in vitro culture with maximal induction occurring during the first 48 h. rIFN-gamma significantly induced TNF transcription at 24 h in the absence of soluble TNF activity, but did not increase transcription at later times. The expression of nonspecific cytolytic activity, the release of soluble bioactive TNF and the induction of TNF and c-fos mRNA are regulated in HPBM by differentiation-determined processes.

Cell Differentiation↗
PubMed1990

A selective defect of interferon alpha production in human immunodeficiency virus-infected monocytes.

Interferon alpha (IFN-alpha) induces significant antiretroviral activities that affect the ability of human immunodeficiency virus (HIV) to infect and replicate in its principal target cells, CD4+ T cells and macrophages. A major endogenous source of IFN-alpha during any infection is the macrophage. Thus, macrophages have the potential to produce both IFN-alpha and HIV. In this study, we examined the production of IFN-alpha and other cytokines by macrophage colony-stimulating factor (M-CSF)-treated cultured monocytes during HIV infection. Tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), IL-6, IFN-omega, or IFN-beta were not detected nor was the mRNA expressed in either uninfected or HIV-infected monocytes. However, both uninfected and HIV-infected monocytes produced high levels of each of these cytokines after treatment with synthetic double-stranded RNA [poly(I).poly(C)]. Uninfected monocytes also produced high levels of IFN-alpha after treatment with poly(I).poly(C), Newcastle disease virus, or herpes simplex virus. In marked contrast to the preceding observations, HIV-infected monocytes produced little or no IFN-alpha before or after treatment with any of these agents. The absence of detectable IFN-alpha activity and mRNA in poly(I).poly(C)-treated HIV-infected monocytes was coincident with high levels of 2',5' oligoadenylate synthetase and complete ablation of HIV gene expression. The antiviral activity induced by poly(I).poly(C) may be a direct effect of this synthetic double-stranded RNA or secondary to the low levels of IFN-beta and IFN-omega produced by infected cells. The markedly diminished capacity of HIV-infected monocytes to produce IFN-alpha may reflect a specific adaptive mechanism of virus to alter basic microbicidal functions of this cell. The inevitable result of this HIV-induced cytokine dysregulation is virus replication and persistence in mononuclear phagocytes.

Base Sequence↗
PubMed1990

Restriction of HIV replication in infected T cells and monocytes by interferon-alpha.

Human recombinant interferon-alpha (IFN alpha) restricted viral replication in human immunodeficiency virus- (HIV) infected T cells and monocytes. With T cells, reverse transcriptase (RT) activity in culture fluids was reduced threefold from that of control infected cells by IFN treatment, but HIV p24 antigen levels were unchanged. In contrast, levels of p24 antigen and RT activity in lysates of IFN-treated infected cells were threefold greater than those of controls. These differences suggest that the mechanism for IFN-induced antiviral effects in HIV-infected T cells resides in the terminal events (assembly and release) of the virus replication cycle. Monocytes treated with IFN at the time of virus challenge showed no p24 antigen or RT activity, no HIV-specific mRNA, and no proviral DNA in cells for up to 3 weeks after infection. IFN treatment of chronically infected monocytes also decreased virus replication, as assessed by p24 antigen, mRNA and RT detection assays. However, levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.

DNA, Viral↗
PubMed1990

Macrophages as susceptible targets for HIV infection, persistent viral reservoirs in tissue, and key immunoregulatory cells that control levels of virus replication and extent of disease.

Although macrophages are major targets for human immunodeficiency virus (HIV) infection in vivo, study of HIV-macrophage interactions in vitro was hindered because many laboratory strains of HIV would not replicate in macrophages, and because survival of macrophages in culture was poor. Addition of purified macrophage colony-stimulating factor (M-CSF) to cultured macrophages markedly improves their survival, but does not induce proliferation. HIV isolates that replicate in macrophages will also replicate in lymphocytes; however, isolates adapted to lymphoid cells (such as HIV-HTLVIIIB) will not replicate in macrophages. The envelope gene appears to be a major determinant of the cell tropism of viral isolates. T-cell grown virus stocks synthesize abundant gp120, while virus grown in macrophages contains relatively much less gp120. Electron microscopy of virions from macrophages shows them to be depleted of gp120 surface "spikes." Recombination studies show that the portion of the genome coding for the envelope glycoprotein appears to determine cell tropism. Lastly, rsCD4 neutralized macrophage-tropic isolates less efficiently than T-cell tropic isolates. HIV replication in macrophages is partially under the control of cellular factors, although these have been less well characterized than they have in lymphocytes.

Disease Susceptibility↗
PubMed1990

Macrophage-HIV interaction: viral isolation and target cell tropism.

Viral isolates were recovered by cocultivation on macrophage colony-stimulatingfactor (MCSF)-treated monocyte target cells from peripheral blood mononuclear cells (PBMCs) in 25 out of 27 patients seropositive or at risk for HIV infection. Frequency of virus recovery was independent of the patient's age, sex, numbers of CD4+ T cells, clinical stage or zidovudine (azidothymidine) therapy. Sixteen out of 19 HIV isolates were serially passaged in MCSF- treated monocytes. Five out of five virus isolates were also passaged in phytohemagglutinin/interleukin-2 (PHA/IL-2)-treated lymphoblasts. In lymphoblasts, no qualitative or quantitative differences were observed between these isolates and human T-cell leukemia virus IIIB (HTLV-IIIB) for (1) release of p24 antigen reverse transcriptase, and infectious virus, (2) induction of typical cytopathic effects (cell syncytia in 3-10% of cells) and cell lysis, (3) frequency of infected cells (5-20% of PBMC) as detected by in situ hybridization for HIV RNA, (4) down-modulation of T cell plasma membrane CD4, and (5) site of progeny virion assembly and budding (plasma membrane only with no intracytoplasmic accumulation of virus). Progeny virus recovered from infected lymphoblasts was fully infectious for other lymphoblasts, but failed to infect MCSF-treated monocytes. Detailed analysis of target cell tropism among HIV isolates showed that HIV isolated in monocytes infected both monocytes and lymphoblasts; progeny virus isolated in lymphoblasts infected only T cells. HIV interacts differently with monocytes and T cells. Understanding this interaction may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.

Adult↗
PubMed1986

Depletion of T lymphocytes from human bone marrow by the use of counterflow elutriation centrifugation.

Normal donor bone marrow buffy coat (BMBC) cells were fractionated into three subsets by counterflow elutriation centrifugation. Fraction 1 (Fr-1) was lymphocyte-enriched, fraction 2 (Fr-2) was a mixture of lymphocytes and myelomonocytic cells, and fraction 3 (Fr-3) was enriched for myelomonocytic cells. The extent of T cell depletion was determined by limiting dilution analysis of the growth of T cells. The cells in each group were stimulated with phytohemagglutinin and cultured in the presence of interleukin-2. These conditions allowed clonogenic growth of one of three T cells purified from peripheral blood. After a 3-week culture period, replicate wells were scored for growth and precursor frequency was determined. The frequency of T cells in BMBC was 1/22 (n = 4), 1/16 in FR-1 (n = 4), and 1/246 in Fr-2 (n = 2), and in Fr-3 it ranged from 1/3,000 to 1/10,000 (n = 4). For comparison, depletion of T cells from bone marrow by cytofluorographic sorting with OKT-11 and twice E-rosetting yielded 98.1% and 97.4% removal of T cells, respectively. Thus, the clonogenic assay assured that more than 99.3% of the original T cells were depleted by elutriation; however, hematopoietic progenitor assay (CFU-GM, CFU-GEMM, and BFU-E) showed no enrichment in any particular fraction, implying that the progenitors measured in this assay are of diverse sizes. Elutriation represents a useful method for depletion of T cells from human bone marrow. Limiting dilution assay of T cell growth is a sensitive measure to monitor T cell depletion.

Bone Marrow Cells↗
PubMed1986

The alteration of hamster serum elastase inhibitory capacities by chloramine T, in vitro and in vivo.

The major elastase inhibitor of human serum, alpha-1 proteinase inhibitor (A1PI), is susceptible to oxidative inactivation by a variety of agents, including chloramine T. We have examined the effects of chloramine T on the catalytic activity of porcine pancreatic (PPE) and human leukocyte elastase (HLE) and on the elastase inhibitory capacity of hamster, rat, and human serum as well as pure human A1PI. Both PPE and HLE, but not trypsin, were inhibited in a concentration-dependent manner by concentrations of chloramine T greater than 0.1 mM. The abilities of rat and human serum and pure human A1PI to inhibit both PPE and HLE were inhibited in a concentration-dependent manner by chloramine T. In contrast only the ability of hamster serum to inhibit HLE was altered by exposure to chloramine T: inhibition of PPE was not effected. Gel exclusion chromatography disclosed the existence of two major peaks of elastase inhibitory activity in hamster plasma: one, with an approximate molecular weight of 55 K, eluting in the region of A1PI that was sensitive to chloramine T inactivation and one with a molecular weight of approximately 180 K which was chloramine T insensitive. The parenteral administration of chloramine T to hamsters resulted in a modest and transient diminution of the serum HLE inhibitory activity and an equally modest and transient elevation of PPE inhibitory activity.

Animals↗
PubMed1985

Lack of renal tubular and hemodynamic effects of non-selective and delta-opioid receptor antagonism.

The renal pharmacological actions of the non-selective opioid receptor antagonist naloxone and the selective delta (delta)-opioid receptor antagonist ICI 154,129 were examined in conscious dogs. Neither naloxone nor ICI 154,129 altered glomerular filtration rate, renal blood flow, blood pressure, heart rate, or renal excretion of water, Na+, K+, or Cl-. In addition, urine and plasma osmolality and electrolyte concentrations and hematocrit were unchanged, suggesting that neither agent produced physiologically significant alteration in plasma vasopressin levels. These data suggest that (a) naloxone and ICI 154,129 exert no renal pharmacological effects in dogs and (b) under resting physiological conditions, delta-opioid receptors, as well as other opioid receptor subtypes, probably are not involved in the tonic regulation of renal hemodynamics or tubular function.

Animals↗