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1.
Mucosal Immunol ; 11(4): 1230-1238, 2018 07.
Article in English | MEDLINE | ID: mdl-29855550

ABSTRACT

Epigallocatechin-3-gallate (EGCG), a natural and major ingredient of green tea, has been shown to have anti-inflammation and anti-HIV-1 properties. We demonstrated that the intrarectal administration of EGCG could protect rhesus macaques from repetitive, intrarectal challenges with low-dose SHIVSF162P3N. This protection has a per-exposure risk reduction of 91.5% (P = 0.0009; log-rank test) and a complete protection of 87.5% (P < 0.001; Fisher's exact test). All protected animals showed no evidence of systemic and mucosal SHIV infection as demonstrated by the absence of viral RNA, DNA and antibodies. In contrast, all controls became infected after repeated SHIV challenges (a median of 2.5 times, range of 1-8 times). Mechanistically, EGCG could block the binding of HIV-1 gp120 to CD4 receptor and suppress the macrophage infiltration/activation in the rectal mucosa of macaques. These data support further clinical evaluation and development of EGCG as a novel, safe and cost-effective microbicide for preventing sexual transmission of HIV-1.


Subject(s)
Antiviral Agents/therapeutic use , Catechin/analogs & derivatives , HIV Infections/drug therapy , HIV-1/physiology , Macrophages/drug effects , Simian Acquired Immunodeficiency Syndrome/drug therapy , Simian Immunodeficiency Virus/physiology , Animals , CD4 Antigens/metabolism , Catechin/therapeutic use , Cell Movement , Disease Transmission, Infectious , HIV Envelope Protein gp120/metabolism , Humans , Macaca , Macrophages/immunology , Macrophages/virology , Protein Binding/drug effects , Risk , Sexually Transmitted Diseases, Viral , Tea
2.
Cancer Gene Ther ; 19(2): 77-83, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21959967

ABSTRACT

Clinical efficacy of current therapies for hepatocellular carcinoma (HCC) treatment is limited. Indole-3-acetic acid (IAA) is non-toxic for mammalian cells. Oxidative decarboxylation of IAA by horseradish peroxidase (HRP) leads to toxic effects of IAA. The purpose of this study was to investigate the effects of a novel gene-targeted enzyme prodrug therapy with IAA on hepatoma growth in vitro and in vivo mouse hepatoma models. We generated a plasmid using adenovirus to express HRP isoenzyme C (HRPC) with the HCC marker, alpha-fetoprotein (AFP), as the promoter (pAdv-AFP-HRPC). Hepatocellular cells were infected with pAdv-AFP-HRPC and treated with IAA. Cell death was detected using MTT assay. Hepatoma xenografts were developed in mice by injection of mouse hepatoma cells. The size and weight of tumors and organs were evaluated. Cell death in tumors was assessed using hematoxylin and eosin-stained tissue sections. HRPC expression in tissues was detected using Reverse Transcriptase-Polymerase Chain Reaction. IAA stimulated death of hepatocellular cells infected with pAdv-AFP-HRPC, in a dose- and time-dependent manner, but not in control cells. Growth of hepatoma xenografts, including the size and weight, was inhibited in mice treated with pAdv-AFP-HRPC and IAA, compared with that in control group. pAdv-AFP-HRPC/IAA treatment induced cell death in hepatoma xenografts in mice. HRPC gene expressed only in hepatoma, but not in other normal organs of mice. pAdv-AFP-HRPC/IAA treatment did not cause any side effects on normal organs. These findings suggest that pAdv-AFP-HRPC/IAA enzyme/prodrug system may serve as a strategy for HCC therapy.


Subject(s)
Carcinoma, Hepatocellular/therapy , Genetic Therapy/methods , Horseradish Peroxidase/genetics , Indoleacetic Acids/pharmacology , Liver Neoplasms/therapy , alpha-Fetoproteins/genetics , Adenoviridae/genetics , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Growth Processes/drug effects , Cell Growth Processes/genetics , Cell Line, Tumor , Disease Models, Animal , Genetic Vectors , Hep G2 Cells , Horseradish Peroxidase/biosynthesis , Horseradish Peroxidase/metabolism , Humans , Indoleacetic Acids/pharmacokinetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Plasmids/genetics , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Xenograft Model Antitumor Assays , alpha-Fetoproteins/biosynthesis
3.
Transpl Infect Dis ; 13(1): 24-32, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21040279

ABSTRACT

Hepatitis C virus (HCV) infection is the leading indication for liver transplantation and a major cause of graft failure. This study investigated whether cyclosporin A (CsA), a widely used immunosuppressant for organ transplantation, inhibits full cycle HCV replication and restores type I interferon (IFN) signaling pathway in human hepatocytes. CsA treatment of hepatocytes before, during, and after HCV infection significantly inhibited full cycle viral replication, which is evidenced by decreased expression of HCV RNA, protein, and infectious viruses in human hepatocytes. The suppression of HCV replication by CsA was associated with elevated levels of endogenous IFN-α in infected hepatocytes. Although CsA had little effect on IFN-α signaling pathway in uninfected hepatocytes, CsA treatment of HCV-infected hepatocytes specifically upregulated the expression of IFN regulatory factor-1 and inhibited the expression of suppressor of cytokine signaling-1 and protein inhibitor of activated signal transducers and activators of transcription-x, the primary negative regulators of IFN signaling pathway. These findings provide additional evidence to support the development of CsA-based prevention/treatment of HCV infection for transplant recipients.


Subject(s)
Cyclosporine/pharmacology , Hepacivirus/drug effects , Hepatocytes/virology , Immunosuppressive Agents/pharmacology , Interferon-alpha/metabolism , Virus Replication/drug effects , Cell Line , Gene Expression Regulation , Hepacivirus/genetics , Hepacivirus/physiology , Hepatitis C/drug therapy , Hepatitis C/prevention & control , Hepatocytes/immunology , Hepatocytes/metabolism , Humans , Immunity, Innate , Interferon Regulatory Factor-1/genetics , Interferon Regulatory Factor-1/metabolism , Liver Transplantation/adverse effects , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation , Viral Proteins/metabolism
4.
Neuroscience ; 159(2): 629-37, 2009 Mar 17.
Article in English | MEDLINE | ID: mdl-19166911

ABSTRACT

We examined the gene expression and regulation of type III human interferon (IFN), IFN-lambda, in human neuronal cells. Human neuronal cells expressed endogenous IFN-lambda1 but not IFN-lambda2/3. Upon the activation of Toll-like receptor (TLR)-3 expressed in the neuronal cells by polyriboinosinic polyribocytidylic acid (PolyI:C), both IFN-lambda1 and IFN-lambda2/3 expression was significantly induced. The activation of TLR-3 also exhibited antiviral activity against pseudotyped human immunodeficiency virus (HIV)-1 infection of the neuronal cells. Human neuronal cells also expressed functional IFN-lambda receptor complex, interleukin-28 receptor alpha subunit (IL-28Ralpha) and IL-10Rbeta, as evidenced by the observations that exogenous IFN-lambda treatment inhibited pseudotyped HIV-1 infection of the neuronal cells and induced the expression of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC)3G/3F, the newly identified anti-HIV-1 cellular factors. These data provide direct and compelling evidence that there is intracellular expression and regulation of IFN-lambda in human neuronal cells, which may have an important role in the innate neuronal protection against viral infections in the CNS.


Subject(s)
Gene Expression Regulation/physiology , Interferons/metabolism , Neurons/metabolism , Toll-Like Receptor 3/metabolism , APOBEC Deaminases , Antineoplastic Agents/pharmacology , Brain/cytology , Cells, Cultured , Cytidine Deaminase , Cytosine Deaminase/metabolism , Dose-Response Relationship, Drug , Fetus , Gene Expression/drug effects , Gene Expression/genetics , Gene Expression Regulation/drug effects , HIV-1/immunology , Humans , Interferon Inducers/pharmacology , Interleukin-10 Receptor beta Subunit/genetics , Interleukin-10 Receptor beta Subunit/metabolism , Neurons/drug effects , Poly I-C/pharmacology , RNA, Messenger/metabolism , Receptors, Cytokine/genetics , Receptors, Cytokine/metabolism , Time Factors , Tretinoin/pharmacology
5.
J Viral Hepat ; 15(12): 855-64, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18637071

ABSTRACT

The role of natural killer (NK) cells in controlling hepatitis C virus (HCV) infection and replication has not been fully delineated. We examined NK cell-mediated noncytolytic effect on full cycle HCV infection of human hepatocytes. Human hepatocytes (Huh7.5.1 cells) co-cultured with NK cells or treated with supernatants (SN) from NK cells cultures had significantly lower levels of HCV RNA and protein than control cells. This NK cell-mediated anti-HCV activity could be largely abolished by antibody to interferon-gamma (IFN-gamma). The investigation of the mechanisms for NK cell-mediated anti-HCV activity showed that NK SN-treated hepatocytes expressed higher levels of IFN-alpha/beta than the control cells. NK SN also enhanced IFN regulatory factor-3 and 7 expression in the hepatocytes. In addition, NK SN enhanced the expression of signal transducer and activator of transcription 1 and 2, the nuclear factors that are essential for the activation of IFN-mediated antiviral pathways. These data provide direct evidence at cellular and molecular levels that NK cells have a key role in suppressing HCV infection of and replication in human hepatocytes.


Subject(s)
Down-Regulation , Hepacivirus/immunology , Hepatitis C/immunology , Hepatocytes/immunology , Killer Cells, Natural/immunology , Adult , Cells, Cultured , Coculture Techniques , Female , Gene Expression , Hepacivirus/physiology , Hepatitis C/virology , Hepatocytes/virology , Humans , Interferons/genetics , Interferons/immunology , Male , Signal Transduction , Virus Replication
6.
J Viral Hepat ; 15(4): 261-70, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18307590

ABSTRACT

Very little is known about the interactions between hepatitis C virus (HCV) and methamphetamine, which is a highly abused psychostimulant and a known risk factor for human immunodeficiency virus (HIV)/HCV infection. This study examined whether methamphetamine has the ability to inhibit innate immunity in the host cells, facilitating HCV replication in human hepatocytes. Methamphetamine inhibited intracellular interferon alpha expression in human hepatocytes, which was associated with the increase in HCV replication. In addition, methamphetamine also compromised the anti-HCV effect of recombinant interferon alpha. Further investigation of mechanism(s) responsible for the methamphetamine action revealed that methamphetamine was able to inhibit the expression of the signal transducer and activator of transcription 1, a key modulator in interferon-mediated immune and biological responses. Methamphetamine also down-regulated the expression of interferon regulatory factor-5, a crucial transcriptional factor that activates the interferon pathway. These in vitro findings that methamphetamine compromises interferon alpha-mediated innate immunity against HCV infection indicate that methamphetamine may have a cofactor role in the immunopathogenesis of HCV disease.


Subject(s)
Hepacivirus/growth & development , Hepatocytes/drug effects , Hepatocytes/virology , Immunosuppressive Agents/pharmacology , Methamphetamine/pharmacology , Virus Replication/drug effects , Blotting, Western , Cell Line , Down-Regulation , Humans , Interferon Regulatory Factors/antagonists & inhibitors , Interferon-alpha/antagonists & inhibitors , Interferon-alpha/immunology , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , STAT1 Transcription Factor/antagonists & inhibitors , STAT1 Transcription Factor/immunology
7.
Proc Natl Acad Sci U S A ; 103(20): 7771-6, 2006 May 16.
Article in English | MEDLINE | ID: mdl-16675550

ABSTRACT

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.


Subject(s)
Cell Differentiation/physiology , Macrophages/physiology , Monocytes/physiology , Protein Isoforms/metabolism , Receptors, Neurokinin-1/metabolism , Animals , Calcium/metabolism , Cell Line , Chemokine CCL5 , Chemokines, CC/metabolism , Humans , Macrophages/drug effects , Monocytes/drug effects , Protein Isoforms/genetics , Receptors, Neurokinin-1/genetics , Substance P/metabolism , Tetradecanoylphorbol Acetate/pharmacology
8.
J Neuroimmunol ; 121(1-2): 67-75, 2001 Dec 03.
Article in English | MEDLINE | ID: mdl-11730941

ABSTRACT

Substance P (SP) is a potent modulator of neuroimmunoregulation. SP receptors are present on human monocytes and T lymphocytes, and SP alters the function of these immune cells. We investigated the effects of SP on HIV-1 replication in latently infected human immune cells. SP significantly enhanced HIV-1 replication in the latently infected promonocytic cell line (U1) and T lymphocyte line (ACH-2) stimulated with tumor necrosis factor (TNF-alpha). When added to these cells in combination with TNF-alpha, SP also enhanced HIV-1 gag gene expression in U1 and ACH-2 cells. This stimulatory effect of SP was associated with the activation of HIV-LTR (long terminal repeat) driven chloramphenicol acetyltransferase (CAT) gene expression, and could be blocked by pretreatment of U1 and ACH-2 cells with an SP receptor antagonist RP-67,580, indicating specific SP receptor-mediated regulation. Furthermore, the addition of SP to the cultures of latently infected peripheral blood mononuclear cells isolated from HIV-1-infected patients enhanced HIV-1 gag gene expression. Thus, SP may play a potentially important role as a positive regulator of HIV-1 replication in latently infected monocytes and lymphocytes. These observations may have significant implications toward understanding the role of neuropeptide SP in the immunopathogenesis of HIV-1 infection and AIDS.


Subject(s)
HIV Infections/immunology , HIV-1 , Substance P/pharmacology , T-Lymphocytes/virology , Virus Replication/drug effects , Adult , Antineoplastic Agents/pharmacology , Carcinogens/pharmacology , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/immunology , Gene Products, gag/genetics , HIV Long Terminal Repeat/genetics , Humans , Monocytes/cytology , Monocytes/immunology , Monocytes/virology , Neuroimmunomodulation/immunology , RNA, Messenger/analysis , Receptors, Neurokinin-1/genetics , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Virus Replication/immunology
9.
Gene ; 279(1): 63-7, 2001 Nov 14.
Article in English | MEDLINE | ID: mdl-11722846

ABSTRACT

L-Lactate dehydrogenase (LDH) cDNAs encoding for LDH-A(4) (muscle) and LDH-B(4) (heart) isozymes from caiman (Caiman crocodilus apaporiensis) belonging to the order Crocodilia and Chinese soft-shelled turtle (Pelodiscus sinensis) belonging to the order Chelonia were sequenced. The phylogenetic relationships of the newly determined cDNA and their deduced protein sequences, as well as the previously published sequences of vertebrate LDH isozymes, were analyzed by various phylogenetic tree construction methods. These results indicated that Chelonia is indeed more closely related to Crocodilia. The divergent times between caiman and alligator, turtle and soft-shelled turtle, and Chelonia and Crocodilia were estimated to be approximately 36, 100 and 177 million years, respectively.


Subject(s)
Alligators and Crocodiles/genetics , Evolution, Molecular , L-Lactate Dehydrogenase/genetics , Phylogeny , Turtles/genetics , Animals , Birds/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Genetic Variation , Isoenzymes/genetics , Lactate Dehydrogenase 5 , Molecular Sequence Data , Sequence Analysis, DNA , Time Factors
10.
AIDS ; 15(15): 2043-5, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11600835

ABSTRACT

The neuropeptide, substance P, is a potent modulator of neuroimmunoregulation. Substance P and its receptor modulate HIV infection. HIV-seropositive men had significantly higher plasma substance P levels compared with uninfected controls, which were associated with decreased CD16 and CD56 natural killer (NK) cell populations. The changes in plasma substance P levels and decreases in NK subsets did not correlate with CD4 cell levels, but a diurnal pattern was suggested for substance P. The balance between substance P expression and functions of immune cells may be important in the immunopathogenesis of HIV infection.


Subject(s)
HIV Infections/blood , Substance P/blood , Cohort Studies , Flow Cytometry , HIV Infections/immunology , HIV Seronegativity , Homosexuality , Humans , Killer Cells, Natural , Male
11.
J Hematother Stem Cell Res ; 10(5): 609-20, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11672507

ABSTRACT

Dendritic cells (DC), the most potent antigen-presenting cells (APC), have been implicated as the initial targets of HIV infection in skin and mucosal surfaces. DC can be generated in vitro from blood-isolated CD14(+) monocytes or CD34(+) hematopoietic progenitor cells in the presence of various cytokines. In this study, we investigated whether monocytes obtained from placental cord blood are capable of differentiation into dendritic cells when cultured with a combination of cytokines - granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha). We then examined HIV infection, HIV receptor (CD4, CCR5) expression, and beta-chemokine [macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha, MIP-1beta)] production by placental cord monocyte-derived dendritic cells (MDDC) as compared to that of autologous cord monocyte-derived macrophages (MDM). Monocytes isolated from placental cord blood differentiate into DC after 7 days in culture with the mixture of cytokines, as demonstrated by development of characteristic DC morphology, loss of CD14 expression, and gain of CD83, a marker for mature DC. Mature cord MDDC had significantly lower susceptibility to M-tropic ADA (CCR5-dependent) envelope-pseudotyped HIV infection in comparison to autologous placental cord MDM, whereas there was no significant difference in virus replication in cord MDDC and MDM infected with murine leukemia virus envelope-pseudotyped HIV (HIV receptor-independent). This limited susceptibility of cord MDDC to M-tropic HIV infection may be due to lower expression of CD4 and CCR5 on the cell membrane and higher production of MIP-1alpha and MIP-1beta. These data provide important information toward our understanding of the biological properties of cord MDDC in relation to HIV infection.


Subject(s)
Dendritic Cells/virology , Fetal Blood/cytology , HIV Infections/virology , Monocytes/cytology , Antigens, CD , Antigens, CD34/immunology , CD4 Antigens/genetics , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Line , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Female , Fetal Blood/drug effects , Fetal Blood/immunology , Gene Expression Regulation/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HIV Infections/metabolism , HIV-1/genetics , Humans , Immunoglobulins/immunology , Interleukin-4/pharmacology , Lipopolysaccharide Receptors/immunology , Luciferases/genetics , Luciferases/metabolism , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Membrane Glycoproteins/immunology , Monocytes/drug effects , Monocytes/immunology , Placenta , Pregnancy , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CCR5/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Tumor Necrosis Factor-alpha/pharmacology , CD83 Antigen
12.
Antiviral Res ; 52(1): 63-75, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11530189

ABSTRACT

Peptide T, which is derived from the V2 region of HIV-1, inhibits replication of R5 and dual-tropic (R5/X4) HIV-1 strains in monocyte-derived macrophages (MDMs), microglia, and primary CD4(+)T cells. Little to no inhibition by peptide T was observed with lab adapted X4 viruses such as IIIB, MN, or NL4-3 propagated in CD4(+) T cells or in the MAGI entry assay. The more clinically relevant R5/X4 early passage patient isolates were inhibited via either the X4 or R5 chemokine receptors, although inhibition was greater with R5 compared to X4 receptors. Virus inhibition ranged from 60 to 99%, depending on the assay, receptor target, viral isolate and amount of added virus. Peak inhibitory effects were detected at concentrations from 10(-12) to 10(-9) M. Peptide T acted to block viral entry as it inhibited in the MAGI cell assay and blocked infection in the luciferase reporter assay using HIV virions pseudotyped with ADA envelope. These results using early passage virus grown in primary cells, together with two different entry reporter assays, show that peptide T selectively inhibits HIV replication using chemokine receptor CCR5 compared to CXC4, explaining past inconsistencies of in vitro antiviral effects.


Subject(s)
HIV-1/physiology , Peptide T/physiology , Receptors, CCR5/physiology , Virus Replication/drug effects , Antibodies, Monoclonal/immunology , Antiviral Agents/metabolism , Biological Assay , Cells, Cultured , Chemokines/antagonists & inhibitors , Chemokines/metabolism , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Fetus , Flow Cytometry , Genes, Reporter , HIV Core Protein p24/immunology , HIV-1/metabolism , HeLa Cells , Humans , Luciferases/analysis , Luciferases/genetics , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Microglia/cytology , Microglia/metabolism , Microglia/virology , Peptide T/immunology , Time Factors
13.
Proc Natl Acad Sci U S A ; 98(7): 3970-5, 2001 Mar 27.
Article in English | MEDLINE | ID: mdl-11274418

ABSTRACT

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP-neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.


Subject(s)
Anti-HIV Agents/pharmacology , Biphenyl Compounds/pharmacology , HIV-1/drug effects , Phagocytes/drug effects , Substance P/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drug Interactions , Genes, Reporter , HIV Long Terminal Repeat/drug effects , HIV-1/physiology , Humans , In Vitro Techniques , Neuroimmunomodulation , Neurokinin-1 Receptor Antagonists , Phagocytes/virology , Receptors, Neurokinin-1/metabolism , Substance P/pharmacology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/drug effects , Virus Replication/drug effects
14.
Neuroscience ; 101(4): 1137-44, 2000.
Article in English | MEDLINE | ID: mdl-11113362

ABSTRACT

Substance P, the most abundant neurokinin in the CNS, is a major modulator of the immune system. We have examined the gene expression of substance P and its receptor in human fetal brain microglia. Using reverse transcription-polymerase chain reaction and Southern blotting assay, the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma and delta) were detected in the microglia. The human fetal microglia produced significantly higher levels of endogenous substance P protein (640-850 pg/10(6) cells) than did human peripheral blood monocyte-derived macrophages (25-50 pg/10(6) cells), as determined by an enzyme immunoassay. Using immunohistochemical staining with an anti-substance P antibody, cell membrane substance P immunoreactivity was observed. In addition, we identified the presence of messenger RNA for neurokinin-1 receptor, a primary receptor for substance P in human fetal microglia.From these data, we propose that substance P and its receptor are biologically involved in regulating the functions of microglia, and potentially play an important role in host defense of the central nervous system.


Subject(s)
Brain/embryology , Microglia/metabolism , Receptors, Neurokinin-1/metabolism , Substance P/metabolism , Brain/cytology , Cells, Cultured , Fetus/metabolism , Gene Expression , Humans , Immunohistochemistry/methods , Macrophages/metabolism , Protein Precursors/genetics , RNA, Messenger/metabolism , Receptors, Neurokinin-1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Staining and Labeling , Tachykinins/genetics
15.
Cell Immunol ; 205(2): 120-7, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11104584

ABSTRACT

In vitro and in vivo studies have indicated that there is an important relationship between morphine and neuropeptide substance P (SP). We therefore investigated the interaction of morphine and cultured human immune cells on the expression of SP, a neuropeptide which we have recently demonstrated to be produced by human monocytes and lymphocytes. Morphine up-regulated SP production in human mononuclear phagocytes and lymphocytes at both the mRNA and the protein level. In addition, morphine induced SP receptor (NK-1R) expression in human lymphocytes. The specific morphine receptor antagonist (naltrexone) blocked morphine-induced SP expression in human mononuclear phagocytes, supporting the concept of authentic morphine receptor-mediated regulation. Since SP modulates neurogenic inflammation and immunologic events, these data suggest that morphine-induced SP expression in cells of the immune system may be of importance in the pathogenesis of immune-mediated diseases, including neuroimmunologic diseases and AIDS.


Subject(s)
Morphine/pharmacology , Receptors, Neurokinin-1/genetics , Substance P/genetics , Up-Regulation/drug effects , Adult , Gene Expression/drug effects , Humans , Leukocytes, Mononuclear/drug effects , Lymphocytes/drug effects , Monocytes/drug effects , Phagocytes/drug effects , RNA, Messenger , Substance P/biosynthesis , U937 Cells
16.
Clin Diagn Lab Immunol ; 7(3): 371-6, 2000 May.
Article in English | MEDLINE | ID: mdl-10799448

ABSTRACT

Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.


Subject(s)
Adenocarcinoma/immunology , Colonic Neoplasms/immunology , Monocytes/immunology , Receptors, Neurokinin-1/analysis , Receptors, Neurokinin-1/genetics , Adenocarcinoma/pathology , Cell Line , Colon/chemistry , Colon/immunology , Colon/pathology , Colonic Neoplasms/pathology , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic/immunology , Humans , Intestinal Mucosa/chemistry , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Lymphocytes/chemistry , Lymphocytes/cytology , Lymphocytes/immunology , Macrophages, Peritoneal/chemistry , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/immunology , Monocytes/chemistry , Monocytes/cytology , RNA, Messenger/analysis
17.
Gene Ther ; 7(4): 321-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10694813

ABSTRACT

Among potential genetic targets for intervention in the HIV-1 life cycle, the tat gene product is a key target. We investigated the ability of an antitat gene to inhibit HIV-1 activation and replication in chronically infected promonocyte (U1) and T cell (ACH-2) lines in vitro. U1 and ACH-2 cells were transduced with an antitat gene expressing RNA with dual (polymeric Tat activation response element and antisense-tat) function that interferes with HIV-1 replication. Tumor necrosis factor-alpha (TNF-alpha) plus phorbol 12- myristate 13-acetate (PMA)-induced HIV-1 expression, as determined by reverse transcribed PCR and reverse transcriptase (RT) assays, was significantly inhibited in U1 and ACH-2 cells transduced with the antitat gene, compared with the cells transduced with control vector and untransduced cells. This resistance to TNF-alpha plus PMA-induced HIV-1 expression was demonstrated in antitat gene-transduced U1 and ACH-2 cells maintained in G418-free media for 5 months, suggesting that functional antitat gene may persist for many months in transduced cells and their progeny. Most importantly, we demonstrate that the antitat gene, when introduced into peripheral blood mononuclear cells (PBMC) isolated from patients with HIV-1 infection, inhibited TNF-alpha plus PMA-induced viral replication as determined by RT-PCR and RT activity. In addition, the antitat gene enhanced the survival of CD4+ T lymphocytes from such patients. These data suggest the feasibility of utilizing antitat gene therapy to block activation and replication of HIV-1 in latently infected monocytes and T- lymphocytes in vivo. Gene Therapy (2000) 7, 321-328.


Subject(s)
Genes, tat/genetics , HIV-1/genetics , Virus Replication/genetics , Cell Line , Flow Cytometry , Gene Expression , Genetic Therapy/methods , HIV Infections/therapy , Humans , Leukocytes, Mononuclear/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Transduction, Genetic/genetics , Virus Activation
18.
J Immunol Methods ; 230(1-2): 149-57, 1999 Nov 19.
Article in English | MEDLINE | ID: mdl-10594362

ABSTRACT

We have recently demonstrated that human monocytes and lymphocytes express the substance P (SP) gene at both the mRNA and protein level [Ho, W.Z., Lai, J.P., Zhu, X.H., Uvaydova, M., Douglas S.D., 1997. Human monocytes and macrophages express substance P and neurokinin-1 receptor. Journal of Immunology, 159, p. 5654; Lai, J.P., Douglas, S. D., Ho, W.Z., 1998. Human lymphocytes express substance P and its receptor. Journal of Neuroimmunology, 86, p. 80; Lai, J.-P., Douglas, S.D., Rappaport, E., Wu, J., Ho, W.-Z., 1998. Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Using RT-PCR assay with several specific human SP primer pairs, we were able to differentiate four isoforms of preprotachykinin (PPT-A, the SP precursor) mRNA transcripts on ethidium bromide-stained agarose gels and clone the PCR amplified cDNA of the four isoforms (alpha, beta, gamma, and delta) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we have developed a mimic-based RT-PCR assay to analyze total PPT-A mRNA levels in human monocytes and lymphocytes. We designed a specific human SP primer pair (HSP4/HSP3) to amplify a single fragment of cDNA derived from all four isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules per reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be quantitatively analyzed using the mimic-based RT-PCR. The accuracy and reproducibility of this assay were confirmed by the plasmids containing alpha, beta, gamma and delta cDNA inserts and by in vitro synthesized mRNA from a plasmid containing beta isoform cDNA insert. Our data indicate that the SP mimic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens.


Subject(s)
RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Substance P/genetics , Adult , Alternative Splicing , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Evaluation Studies as Topic , Gene Expression , Humans , Lymphocytes/chemistry , Monocytes/chemistry , Neuroimmunomodulation , Plasmids/genetics , Protein Precursors/genetics , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Tachykinins/genetics
19.
Blood ; 93(4): 1137-44, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-9949155

ABSTRACT

The C-C chemokine receptors CCR5 and CCR3 are fusion coreceptors for human immunodeficiency virus (HIV) entry into macrophages. The regulation of their expression influences infectivity by HIV. We report here that interferon-gamma (IFN-gamma) a cytokine that has bidirectional effects on HIV infection of macrophages, significantly upregulated CCR5 and CCR3 cell surface expression in human mononuclear phagocytes isolated from placental cord blood and adult peripheral blood. Monocytes treated with IFN-gamma showed increased chemotaxis to the CCR5 ligands macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta, confirming the functional relevance of IFN-gamma-induced CCR5 expression. However, IFN-gamma suppressed HIV entry into macrophages. Interestingly, we demonstrated that IFN-gamma inhibited cell surface expression of CD4, the major receptor for HIV. This finding may explain the suppressive effect of IFN-gamma on HIV entry into macrophages, despite its enhancing effect on the expression of CCR5 and CCR3 by these cells. In addition, IFN-gamma-induced secretion of C-C chemokines (RANTES, MIP-1alpha, and MIP-1beta) by mononuclear phagocytes may also suppress HIV entry into macrophages. These data provide further evidence for cytokine-mediated regulation of CCR5 expression and are consistent with a novel paradigm in which cytokines regulate HIV infection and leukocyte migration by reciprocal and opposing effects on the expression of CD4 and chemokine receptors.


Subject(s)
Antineoplastic Agents/pharmacology , Interferon-gamma/pharmacology , Monocytes/metabolism , Receptors, CCR5/biosynthesis , Adult , Chemokine CCL3 , Chemokine CCL4 , Chemotaxis/drug effects , Female , Fetal Blood/cytology , Humans , Macrophage Inflammatory Proteins/pharmacology , Monocytes/cytology , Pregnancy , Receptors, CCR5/agonists , Up-Regulation/drug effects
20.
AIDS Res Hum Retroviruses ; 14(17): 1573-80, 1998 Nov 20.
Article in English | MEDLINE | ID: mdl-9840290

ABSTRACT

We investigated a strategy for gene therapy, intracellular expression of anti-HIV-1 Rev single-chain variable fragments (SFvs), in promonocytic (U1) and T (ACH-2) cell lines latently infected with HIV-1. The cellular and molecular mechanisms leading to activation of latent integrated HIV-1 provirus in U1 and ACH-2 cells have been well delineated. These cells produce HIV-1 in response to stimulation with certain cytokines. U1 and ACH-2 cells were transduced with a murine retroviral shuttle vector that expresses anti-Rev SFv (pLXSN-D8SFv-Rev) or with a control murine leukemia virus (MLV) vector (pLXSN). Tumor necrosis factor alpha (TFNalpha)-, interleukin 6 (IL-6)-, and phorbol myristate acid (PMA)-induced HIV-1 expression, as determined by reverse transcriptase (RT) assay, was significantly inhibited in cells transduced with pLXSN-D8SFv-Rev, compared with cells transduced with pLXSN. In addition, pLXSN-D8SFv-Rev-transduced cells, when incubated with monokine-enriched supernatants of human peripheral blood monocyte cultures, produced significantly less HIV-1 than did cells transduced with pLXSN. This resistance to cytokine-induced HIV-1 expression was demonstrated in SFv-transduced U1 and ACH-2 cells maintained in G418-free medium for 2 months. These data suggest that feasibility of utilizing various anti-HIV-1 SFvs to block activation of HIV-1 infection in vivo.


Subject(s)
Anti-HIV Agents/immunology , Gene Products, rev/immunology , Genetic Vectors , HIV-1/immunology , Leukemia Virus, Murine , Virus Replication , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Cell Culture Techniques , Cell Line , Gene Expression , Gene Products, rev/genetics , Gene Transfer Techniques , HIV-1/physiology , Humans , Immunoglobulin Variable Region , Interleukin-6/pharmacology , Lymphocytes/virology , Mitogens/pharmacology , Monocytes/virology , Phytohemagglutinins/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Proteins , Single-Chain Antibodies , Time Factors , Tumor Necrosis Factor-alpha/pharmacology , rev Gene Products, Human Immunodeficiency Virus
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