RESUMEN
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.
Asunto(s)
Ciclosporina/farmacología , Hepacivirus/efectos de los fármacos , Hepatocitos/virología , Inmunosupresores/farmacología , Interferón-alfa/metabolismo , Replicación Viral/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Hepatitis C/prevención & control , Hepatocitos/inmunología , Hepatocitos/metabolismo , Humanos , Inmunidad Innata , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Trasplante de Hígado/efectos adversos , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Interferones/metabolismo , Neuronas/metabolismo , Receptor Toll-Like 3/metabolismo , Desaminasas APOBEC , Antineoplásicos/farmacología , Encéfalo/citología , Células Cultivadas , Citidina Desaminasa , Citosina Desaminasa/metabolismo , Relación Dosis-Respuesta a Droga , Feto , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , VIH-1/inmunología , Humanos , Inductores de Interferón/farmacología , Subunidad beta del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/metabolismo , Neuronas/efectos de los fármacos , Poli I-C/farmacología , ARN Mensajero/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
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.
Asunto(s)
Hepacivirus/crecimiento & desarrollo , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Inmunosupresores/farmacología , Metanfetamina/farmacología , Replicación Viral/efectos de los fármacos , Western Blotting , Línea Celular , Regulación hacia Abajo , Humanos , Factores Reguladores del Interferón/antagonistas & inhibidores , Interferón-alfa/antagonistas & inhibidores , Interferón-alfa/inmunología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/inmunologíaRESUMEN
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.
Asunto(s)
Regulación hacia Abajo , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatocitos/inmunología , Células Asesinas Naturales/inmunología , Adulto , Células Cultivadas , Técnicas de Cocultivo , Femenino , Expresión Génica , Hepacivirus/fisiología , Hepatitis C/virología , Hepatocitos/virología , Humanos , Interferones/genética , Interferones/inmunología , Masculino , Transducción de Señal , Replicación ViralRESUMEN
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.
Asunto(s)
Antivirales/uso terapéutico , Catequina/análogos & derivados , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Macrófagos/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Antígenos CD4/metabolismo , Catequina/uso terapéutico , Movimiento Celular , Transmisión de Enfermedad Infecciosa , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Macaca , Macrófagos/inmunología , Macrófagos/virología , Unión Proteica/efectos de los fármacos , Riesgo , Enfermedades Virales de Transmisión Sexual , TéRESUMEN
We investigated the effects of glutathione (GSH), the major naturally occurring thiol, and a pharmacologic thiol precursor of GSH, N-acetyl cysteine (NAC), on the expression of human immunodeficiency type 1 (HIV-1) in primary cord blood and adult donor monocyte-derived macrophages (MDM). HIV-1 infection of cord blood and adult MDM was accomplished after incubating 10-15-d-old cultures for 4 h with a monocyte-tropic strain of HIV-1 (Bal). After 1 wk in culture cell supernatants were tested for reverse transcriptase (RT) activity. MDM were exposed to 5, 10 and 20 mM concentrations of both GSH and NAC before infection, during infection, and after infection was established. GSH and NAC suppressed the replication of HIV-1 in both primary cord blood and adult donor MDM in a concentration dependent fashion. These suppressive effects were more pronounced in cord-derived cells than in adult-derived cells. In cells treated with GSH or NAC before infection, there was no significant rise in RT activity as compared with controls. Similarly, when cells were treated with GSH and NAC and simultaneously infected, there was also no significant rise in RT activity after 1 wk in culture. In cells treated after infection was established, RT values were suppressed 80-90% that of untreated controls. This effect persisted for 1-2 wk after exposure to GSH and NAC. Untreated controls demonstrated syncytium formation and lost characteristics of spreading and elongation 2 wk after HIV-1 infection, whereas most of the treated cells remained free of syncytium and retained cytoplasmic spreading, adherence, and elongation. These data are consistent with other studies of thiol suppression of HIV-1 replication and demonstrate a similar observation for primary cultured cord MDM. These results may offer new approaches toward cellular protection after infection with HIV-1.
Asunto(s)
Acetilcisteína/farmacología , Sangre Fetal/microbiología , Glutatión/farmacología , VIH-1/efectos de los fármacos , Macrófagos/microbiología , Replicación Viral/efectos de los fármacos , Adulto , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Transcriptasa Inversa del VIH , VIH-1/patogenicidad , Humanos , Recién Nacido , ADN Polimerasa Dirigida por ARN/análisisRESUMEN
In an effort to facilitate the efficiency of human immunodeficiency virus type 1 (HIV-1) and/or human cytomegalovirus (HCMV) infection in primary monocyte/macrophages in vitro, the effect of low-speed centrifugation was studied. The infectivity of three strains (Bal, Ada-M, and IIIB) of HIV-1 tested was significantly enhanced by centrifugal inoculation at a force of 1500g for 60 min. Reverse transcriptase activity and HIV-1 p24 antigen in primary monocyte/macrophages infected by a centrifugal inoculation technique were detectable 3-7 days earlier and were more than 10-fold greater in magnitude (at an early stage of the infection) than those of control cells infected by the conventional inoculation technique. Examination of the cells by indirect immunofluorescence revealed higher expression of HIV-1 p24 protein in the monocyte/macrophages infected by the centrifugal inoculation technique. These differences were directly related to centrifugal inoculation and were evident up to 3 weeks after infection. Enhancement was not observed when centrifugation was carried out before or after HIV-1 infection. Centrifugal inoculation of HCMV also enhanced its immediate-early and early gene expression up to 30- to 50-fold, although neither late nuclear antigens and glycoproteins of HCMV nor infectious virus was detected in HCMV-infected monocyte/macrophage cultures. These results show that centrifugal inoculation is a useful technique for improving the efficiency of HCMV and HIV-1 infection in vitro.
Asunto(s)
Citomegalovirus/genética , VIH-1/fisiología , Macrófagos , Monocitos , Replicación Viral , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH , VIH-1/enzimología , VIH-1/genética , Humanos , ADN Polimerasa Dirigida por ARN/análisisRESUMEN
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.
Asunto(s)
Infecciones por VIH/sangre , Sustancia P/sangre , Estudios de Cohortes , Citometría de Flujo , Infecciones por VIH/inmunología , Seronegatividad para VIH , Homosexualidad , Humanos , Células Asesinas Naturales , MasculinoRESUMEN
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.
Asunto(s)
Caimanes y Cocodrilos/genética , Evolución Molecular , L-Lactato Deshidrogenasa/genética , Filogenia , Tortugas/genética , Animales , Aves/genética , ADN Complementario/química , ADN Complementario/genética , Variación Genética , Isoenzimas/genética , Lactato Deshidrogenasa 5 , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
In order to investigate the hypothesis that human cytomegalovirus (HCMV) influences HIV-1 infection of brain cells, we studied primary astrocytes derived from human fetal brains and a neuronal cell line (SK-N-MC). Infection of these cells with two strains of HCMV resulted in expression of immediate early, early, and late antigens and production of infectious virus. HCMV infection of primary astrocytes also led to cytopathic effects and cell death. SK-N-MC cells were infected with HIV-1 strains with or without HCMV. HIV LTR-directed CAT activities and the expression of HIV p24 antigen from the SK-N-MC culture coinfected with both HIV-1 and HCMV were higher than those from the cells infected with HIV-1 alone. The primary astrocytes were cotransfected with HIV-1 proviral DNAs and HIV LTR-CAT with or without HCMV infection. HCMV-infected astrocytes produced greater amounts of CAT activity and higher p24 than the cells transfected with HIV-1 proviral DNAs alone. When both primary astrocytes and SK-N-MC cells were transfected with (a) HIV LTR-CAT alone, (b) HIV LTR-CAT plus HCMV-IE gene, or (c) HIV LTR-CAT plus HCMV infection 2 days before the transfection, both HCMV infection and its IE gene trans-activated the HIV LTR promoter. HCMV-IE gene 2 may play a critical role in trans-activation of HIV-1 LTR.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/genética , Encéfalo/microbiología , Infecciones por Citomegalovirus/genética , Duplicado del Terminal Largo de VIH , VIH-1/genética , Activación Transcripcional , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/microbiología , Astrocitos/inmunología , Astrocitos/microbiología , Encéfalo/inmunología , Encéfalo/patología , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Infecciones por Citomegalovirus/complicaciones , Infecciones por Citomegalovirus/microbiología , Genes Virales , Antígenos VIH/análisis , Humanos , Replicación ViralRESUMEN
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.
Asunto(s)
ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sustancia P/genética , Adulto , Empalme Alternativo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Estudios de Evaluación como Asunto , Expresión Génica , Humanos , Linfocitos/química , Monocitos/química , Neuroinmunomodulación , Plásmidos/genética , Precursores de Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad , Taquicininas/genéticaRESUMEN
We have investigated the effects of SP on the constitutive and/or lipopolysaccharide (LPS)-induced expression of interleukin-10 (IL-10) and tumor necrosis factor (TNF-alpha) in both freshly isolated cord blood monocytes (FICBM) and cord blood monocyte-derived macrophages (CBMDM). The cells were treated with SP at various concentrations (10(-14) to 10(-6) M) in the presence or absence of LPS and culture supernatants were analyzed for IL-10 and TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). FICBM and CBMDM treated with SP alone increased TNF-alpha secretion. The stimulatory effects of SP on TNF-alpha secretion are inhibited by a anti-SP polyclonal antibody and SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). Although the treatment with SP alone did not enhance IL-10 secretion by both freshly isolated and cultured cord monocytes, treatment with SP in combination with LPS leads to a synergistic interaction in upregulation of IL-10 secretion. Fragments of SP (SP1-4 and SP5-11) in the presence or absence of LPS show little effects on IL-10 secretion by FICBM. SP reverses the inhibitory effect of IFN-gamma on LPS-induced IL-10 secretion by FICBM. In addition, the two SP antagonists and the anti-SP polyclonal antibody blocked the SP effect on IL-10 secretion by FICBM, indicating that these effects are specific and SP receptor mediated. Thus, SP is likely to play an important role in certain inflammatory conditions in the immune and nervous systems.
Asunto(s)
Sangre Fetal/citología , Interleucina-10/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Sustancia P/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , HumanosRESUMEN
We present data demonstrating the gene expression of substance P (SP) and its receptor in human peripheral blood-isolated lymphocytes. Using reverse transcribed polymerase chain reaction (RT-PCR) assay, preprotachykinin-A (substance-P) mRNA is detected in human peripheral blood-isolated lymphocytes. Among the alpha, beta, and gamma transcripts of the SP gene, only the beta and gamma transcripts are detectable in these cells. These RT-PCR amplified transcripts are recognized by Southern blot assay using a specific SP probe. Direct DNA sequence analysis of the RT-PCR products from lymphocytes also confirmed the structure of these transcripts which are identical to those found in human neuronal cells. At the protein level, human lymphocytes produced endogenous SP as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released preformed SP from lymphocytes. In addition, using RT/nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (the receptor for SP) in human peripheral blood-isolated lymphocytes, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human lymphocytes express SP and its receptor support the notion that SP is biologically involved in regulating the functions of these cells in an autocrine fashion.
Asunto(s)
Linfocitos/química , Linfocitos/metabolismo , Receptores de Neuroquinina-1/genética , Sustancia P/genética , Adulto , Comunicación Autocrina/inmunología , Southern Blotting , Capsaicina/farmacología , Expresión Génica/inmunología , Humanos , Linfocitos/efectos de los fármacos , Comunicación Paracrina/inmunología , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/genética , ARN Mensajero/análisis , Taquicininas/genéticaRESUMEN
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.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1 , Sustancia P/farmacología , Linfocitos T/virología , Replicación Viral/efectos de los fármacos , Adulto , Antineoplásicos/farmacología , Carcinógenos/farmacología , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/inmunología , Productos del Gen gag/genética , Duplicado del Terminal Largo de VIH/genética , Humanos , Monocitos/citología , Monocitos/inmunología , Monocitos/virología , Neuroinmunomodulación/inmunología , ARN Mensajero/análisis , Receptores de Neuroquinina-1/genética , Linfocitos T/citología , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/inmunologíaRESUMEN
We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (alpha, beta, gamma and delta) in human peripheral blood-isolated monocytes and PBL. The delta-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of delta-PPT transcript completely match those of beta-PPT transcript. The alpha-PPT and beta-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C-->T change at amino acid position 87 (CCT-->CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.
Asunto(s)
Linfocitos/inmunología , Neuroinmunomodulación/inmunología , Fagocitosis/inmunología , Precursores de Proteínas/genética , Taquicininas/genética , Adulto , Secuencia de Aminoácidos , Southern Blotting , Química Encefálica/inmunología , Humanos , Datos de Secuencia Molecular , Monocitos/inmunología , Precursores de Proteínas/química , Precursores de Proteínas/inmunología , Estructura Secundaria de Proteína , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Sustancia P/genética , Taquicininas/química , Taquicininas/inmunología , Transcripción Genética/inmunologíaRESUMEN
We have investigated the effects of the substance P C-terminal octapeptide analogues [Pro4, Glu (OBzl)11] SP4-11, [Hyp4, Glu(OBzl)11] SP4-11, [cHyp4, Glu(OBzl)11] SP4-11 and [kPro4, Glu(OBzl)11] SP4-11 on the constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF-alpha) in both freshly isolated human blood monocytes (FIBM) and monocyte-derived macrophages (MDM). The cells were treated with substance P and the substance P analogues at various concentrations (10-14 to 10-6 M) in the presence or absence of LPS and culture supernatants were analyzed for TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). Monocytes and macrophages treated with the substance P analogues alone increased TNF-alpha secretion at a magnitude similar to the effect of entire undecapeptide substance P. The stimulatory effects of the substance P analogues on TNF-alpha secretion are inhibited by substance P antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the substance P receptor), indicating that these effects are specific and substance P receptor-mediated. Treatment of monocytes and macrophages with the substance P analogues in combination with LPS, however, showed no synergistic interaction in upregulation of TNF-alpha. These data indicate that the biological effect of substance P on TNF-alpha production by human monocytes and macrophages depends mainly on the sequence of the C-terminal region of the molecule.
Asunto(s)
Macrófagos/metabolismo , Monocitos/metabolismo , Fragmentos de Péptidos/farmacología , Sustancia P/análogos & derivados , Factor de Necrosis Tumoral alfa/metabolismo , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Encéfalo/embriología , Microglía/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Encéfalo/citología , Células Cultivadas , Feto/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica/métodos , Macrófagos/metabolismo , Precursores de Proteínas/genética , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Coloración y Etiquetado , Taquicininas/genéticaRESUMEN
Glutathione (GSH), its derivatives and N-acetylcysteine (NAC) inhibit the induction of HIV-1 expression in a chronically HIV-1-infected promonocytic cell line (U1/HIV) and peripheral blood mononuclear cells (PBMC). We have examined the effects of GSH and NAC on HIV-1 replication in human primary monocyte/macrophages cultured in vitro. Ficoll-gradient purified human monocytes were cultivated in vitro for 7-10 days and then infected with HIV-1 (Bal and Ada-M). Infection was blocked or substantially reduced by GSH or NAC (5-20 mM). Significant reduction (greater than or equal to 90%) in the amount of virus released, as determined by measuring supernatant reverse transcriptase activity and secreted p24 protein, was obtained when the cells were treated for 4 h with greater than or equal to 10 mM of GSH or NAC. The inhibitory effects of GSH and NAC were concentration dependent. This anti-HIV-1 effect persisted in these cultures for at least 35 days without evidence of significant increase in HIV-1 expression. Thus, a single pulse exposure of HIV-1-infected monocyte/macrophages with GSH or NAC led to a sustained, concentration-dependent decrease in HIV-1 p24 antigen levels, as well as, reverse transcriptase activity without producing detectable cellular toxicity in monocyte/macrophages.
Asunto(s)
Acetilcisteína/farmacología , Glutatión/farmacología , VIH-1/efectos de los fármacos , Macrófagos/microbiología , Monocitos/microbiología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , VIH-1/fisiología , Humanos , Cinética , Replicación Viral/efectos de los fármacosRESUMEN
Clonal lines of human rhabdomyosarcoma (RD) cells, constitutively expressing human immunodeficiency virus type 1 (HIV-1) tat gene (RD tat cell lines) showed enhanced expression of human cytomegalovirus (HCMV) immediate-early (IE) and late (L) proteins upon HCMV infection, as compared with control RD cells. One of the RD tat cell lines produced infectious HCMV. The RD-tat cell lines, following transfection with recombinant plasmids containing the full length of the HCMV-IE enhancer/promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, exhibited an increased CAT expression by the tat product. A chronically HIV-1-infected human T-lymphoid cell line, SupT1, superinfected with HCMV, expressed HCMV-IE proteins while the parental SupT1 cells infected with HCMV were negative. Parental SupT1 cells coinfected with HIV-1 and HCMV also expressed HCMV-IE proteins, indicating that HIV-1-encoded proteins exert a positive regulatory effect on HCMV expression.
Asunto(s)
Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Genes tat/fisiología , VIH-1/genética , Antígenos Virales/biosíntesis , Antígenos Virales/inmunología , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Clonación Molecular , Citomegalovirus/inmunología , Citomegalovirus/fisiología , ADN Viral , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transfección , Células Tumorales Cultivadas , Replicación Viral/genéticaRESUMEN
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.