RESUMEN
The anti-HIV-1 activities of butanol, hexane, chloroform and water extracts from four widely used folk medicinal plants (Sophora flavescens, Tulipa edulis, Herba ephedra, and Pachyma hoelen Rumph) were evaluated in this study. The hexane extract of Pachyma hoelen Rumph, PH-4, showed effective inhibition against HIV-1. The 50% effective concentration (EC(50)) of PH-4 was 37.3 microg/ml in the p24 antigen assay and 36.8% in the HIV-1 recombinant RT activity test (at 200 microg/ml). In addition, the PH-4 showed the protective effect on the infected MT-4 cells, with a 58.2% rate of protection. The 50% cytotoxic concentration (CC(50)) of PH-4 was 100.6 microg/ml. These results suggest that PH-4 from Pachyma hoelen Rumph might be the candidate for the chemotherapy agent against HIV-1 infection with further study.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales/química , Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/toxicidad , Línea Celular , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH , Humanos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/toxicidad , Replicación Viral/efectos de los fármacosRESUMEN
Various extracts of the aerial parts of Coleus forskohlii (Labiatae) were prepared and evaluated at their non cytotoxic concentration against HIV-1 NL4-3. Chloroform, ethyl acetate and n-butanol extracts showed 45.6, 66.5 and 37.7% inhibition of HIV, respectively in CEM-GFP cells infected with HIV-1(NL4-3) at 5 microg/mL. Four diterpenes, 1-deoxyforskolin, 1,9-dideoxyforskolin, forskolin and isoforskolin were isolated from the chloroform extract and tested against the virus. Six semi-synthetic derivatives of forskolin have been prepared to study SAR. 1-Deoxyforskolin and forskolin were found to be active against HIV(NL4-3). This is first report of anti HIV activity of this plant and its isolated constituents.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/farmacología , Coleus/química , Colforsina/análogos & derivados , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/química , Linfocitos T CD4-Positivos/virología , Línea Celular , Colforsina/química , Colforsina/aislamiento & purificación , Colforsina/farmacología , Ensayo de Inmunoadsorción Enzimática , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/tratamiento farmacológico , VIH-1/crecimiento & desarrollo , Humanos , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Componentes Aéreos de las Plantas/químicaRESUMEN
OBJECTIVE: To probe into the feasibility of screening anti-HIV compounds by using HIV-1 p24 detection kit made by Hebei Medical University. METHODS: The sensitivity, reproducibility and efficacy of the Hebei p24 kit were evaluated compared with the commercially available Vironostika HIV-1 Antigen Microelisa System (Biomerieux). RESULTS: Hebei p24 kit had high sensitivity and good reproducibility. In vitro screening demonstrated that there was no statistically significant difference (P greater than 0.05) between these two kits in assessing anti-HIV compounds. CONCLUSION: Hebei p24 kit could be used as an easily affordable alternative method for detection of HIV-1 in screening anti-HIV compounds.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteína p24 del Núcleo del VIH/análisis , VIH-1/efectos de los fármacos , Juego de Reactivos para Diagnóstico/normas , Fármacos Anti-VIH/aislamiento & purificación , Línea Celular , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Estudios de Factibilidad , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Humanos , Reproducibilidad de los ResultadosRESUMEN
We have isolated a substance with anti-retroviral activity from the freshly prepared crude extract of Chelidonium majus L. (greater celandine) by 9-aminoacridine precipitation method and ion exchange chromatography using Dowex-50W/H+ resin followed by the gel filtration on Sephadex-75 column. Elemental and phenol/sulfuric acid method analyses as well as the mass spectrometry of the purified substance indicated that it may represent a low-sulfated poly-glycosaminoglycan moiety with molecular weight of approximately 3800 Da. The substance prevented infection of human CD4+ T-cell lines AA2 and H9 with HIV-1 at concentration of 25 microg/mL as well as the cell-to-cell virus spread in H9 cells continuously infected with HIV-1, as determined by the measurement of reverse transcriptase activity and p24 content in cell cultures. Furthermore, we have shown in a murine AIDS model that the treatment with purified substance significantly prevented splenomegaly and the enlargement of cervical lymph nodes in C57Bl/6 mice chronically infected with the pool of murine leukemia retroviruses. The mechanism(s) of anti-retroviral activity of this substance have to be elucidated.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antirretrovirales/farmacología , Chelidonium/química , Glicosaminoglicanos/farmacología , VIH-1/efectos de los fármacos , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/aislamiento & purificación , Antirretrovirales/química , Antirretrovirales/aislamiento & purificación , Linfocitos T CD4-Positivos/virología , Línea Celular , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Modelos Animales de Enfermedad , Precipitación Fraccionada , Glicosaminoglicanos/química , Glicosaminoglicanos/aislamiento & purificación , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH/análisis , VIH-1/fisiología , Humanos , Virus de la Leucemia Murina/efectos de los fármacos , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Extractos Vegetales/química , Infecciones por Retroviridae/tratamiento farmacológico , Infecciones Tumorales por Virus/tratamiento farmacológicoRESUMEN
We evaluated the effect of vitamin E in controlling HIV-1 production upon activation of the patients' reservoir of resting CD4 lymphocytes in tissue culture experiments. The addition of vitamin E to patients' cultures resulted in significantly reduced levels of p24 virus production (P = 0.0015). These results suggest that vitamin E supplementation may interfere with the emergence of drug-resistant HIV-1 variants archived in the resting cell reservoir and delay or limit virus rebound upon treatment interruptions.
Asunto(s)
Antioxidantes/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Vitamina E/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Depresión Química , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/inmunología , Humanos , Latencia del Virus/efectos de los fármacosRESUMEN
We have previously described an in vitro model for the evaluation of the effects of different immunomodulatory agents and immunotoxins (ITs) on cells latently infected with human immunodeficiency virus (HIV). We demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25+ cells with an IT. Thus, by selectively killing the productively infected cells with an anti-CD25 IT we can generate a population of latently infected cells. CD25- cells generated in this manner were treated with nucleoside analog reverse transcriptase inhibitors and subsequently activated with phytohemagglutinin in the presence of the drugs. The antiviral activities of zidovudine (ZDV), lamivudine (3TC), and abacavir (ABC) were evaluated by using this model. 3TC and ABC demonstrated significant activity in decreasing HIV production from recently infected resting cells following their activation, whereas the effect of ZDV was more modest. These results suggest that the differences in antiviral activity of nucleoside analogs on resting cells should be considered when designing drug combinations for the treatment of HIV infection. The model presented here offers a convenient alternative for evaluating the mechanism of action of new antiretroviral agents (J. Saavedra, C. Johnson, J. Koester, M. St. Claire, E. Vitteta, O. Ramilo, 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. I-59, 1997).
Asunto(s)
Fármacos Anti-VIH/farmacología , Didesoxinucleósidos/farmacología , Infecciones por VIH/tratamiento farmacológico , Lamivudine/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/virología , Humanos , Inmunotoxinas/farmacología , Fitohemaglutininas/farmacología , Receptores de Interleucina-2/genéticaRESUMEN
Human immunodeficiency virus-1 (HIV-1) infection of the nervous system can result in neuroinflammatory events leading first to neuronal dysfunction then to cognitive and behavioral impairments in infected people. The multifaceted nature of the disease process, commonly called HIV-1-associated dementia (HAD), provides a number of adjunctive therapeutic opportunities. One proposed adjunctive therapy is sodium valproate (VPA), an anticonvulsant known to promote neurite outgrowth and increase beta-catenin through inhibiting glycogen synthase kinase 3beta activity and tau phosphorylation. We now show that VPA treatment of rat cortical neurons exposed to HIV-1 gp120 prevents resultant neurotoxic activities. This includes the induction of significant neurite outgrowth and microtubule-associated protein 2 (MAP-2) and neuron-specific nuclear protein (NeuN) antigens in affected neuronal cell bodies and processes. Similarly, VPA protects severe combined immunodeficient (SCID) mice against the neurodegeneration of HIV-1ADA infected monocyte-derived macrophages (MDMs). In SCID mice with HIV-1 MDM-induced encephalitis, VPA treatment significantly reduced neuronal phosphorylatedbeta-catenin and tau without affecting HIV-1 replication or glial activation. We conclude that VPA protects neurons against HIV-1 infected MDM neurotoxicity, possibly through its effects on the phosphorylation of tau and beta-catenin. The use of VPA as an adjuvant in treatment of human HAD is being pursued.
Asunto(s)
Complejo SIDA Demencia/tratamiento farmacológico , VIH-1 , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Ácido Valproico/uso terapéutico , Complejo SIDA Demencia/patología , Complejo SIDA Demencia/fisiopatología , Animales , Ganglios Basales/patología , Ganglios Basales/virología , Biomarcadores/análisis , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/toxicidad , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/aislamiento & purificación , Humanos , Macrófagos/patología , Macrófagos/virología , Masculino , Ratones , Ratones SCID , Monocitos/citología , Monocitos/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/patología , Neuronas/patología , Fosforilación/efectos de los fármacos , ARN Viral/análisis , ARN Viral/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas tau/metabolismoRESUMEN
Thai bitter gourd protein (MRK29) was isolated from Momordica charantia ripe fruit and seed. The purification was performed by ammonium sulfate fractionation and gel filtration chromatography. MRK29 possessed one isoelectric point of (pI) > or = 9, and the time of flight mass spectrum (TOFMS) indicated its molecular weight at 28.6 kD. The twenty amino acid sequence from the N-terminus was in the following order: 1Asp Val Asn Phe Arg Leu Ser Gly Ala 10Asp Pro Arg X Tyr Gly Met Phe Ile Glu 20Asp. MRK29 inhibited the HIV-1 reverse transcriptase with 50% IR at the concentration of 18 micrograms/ml. MRK29 was concentrated in the 30-60% salt precipitated fraction, at which the concentration of 0.175 microgram/ml exerted 82% reduction of viral core protein p24 expression in HIV-infected cells. MRK29 might have modulatory role on immune cells, because it increased 3-fold TNF activity.
Asunto(s)
Fármacos Anti-VIH/aislamiento & purificación , Cucurbitaceae/química , Frutas/química , N-Glicosil Hidrolasas , Proteínas de Plantas/aislamiento & purificación , Plantas Medicinales/química , Inhibidores de la Transcriptasa Inversa/aislamiento & purificación , Semillas/química , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/inmunología , Humanos , Linfocitos/inmunología , Macrófagos/inmunología , Espectrometría de Masas , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , ADN Polimerasa Dirigida por ARN/sangre , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 2 , Análisis de Secuencia de Proteína , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Inmunotoxinas/uso terapéutico , Animales , Fármacos Anti-VIH/efectos adversos , Anticuerpos Monoclonales , Ensayos Clínicos como Asunto , Contención de Riesgos Biológicos , Progresión de la Enfermedad , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Exotoxinas/efectos adversos , Exotoxinas/uso terapéutico , Proteína p24 del Núcleo del VIH/análisis , VIH-1/inmunología , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Inmunotoxinas/efectos adversos , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/patología , Linfoma de Células T/virología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Quimera por Radiación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Ricina , Bazo/virología , Trasplante Heterólogo , Células Tumorales Cultivadas/trasplante , Células Tumorales Cultivadas/virologíaRESUMEN
BACKGROUND AND PURPOSE: Autopsy series of patients with AIDS have found a 4% to 29% prevalence of cerebral infarction. Little is known of the prevalence of cerebral infarction when not associated with non-HIV central nervous system (CNS) infection, lymphoma, or cardioembolic sources. Clinical correlation has seldom been available. We describe the pathological and clinical features of patients from the Edinburgh HIV Cohort Study found to have had cerebral infarcts without evidence of non-HIV CNS infection, CNS lymphoma, or cardioembolic sources at autopsy. METHODS: From 183 autopsy cases, 26 without evidence of opportunistic cerebral infection or lymphoma were selected. These 26 cases went through a second selection process in which the presence of cerebral infarction, in the absence of the conditions mentioned, was verified. Histology and clinical records for the remaining patients were reviewed. RESULTS: Ten (5.5%) cases fulfilled the inclusion criteria and demonstrated similar hypoxic-ischemic lesions. Small-vessel thickening was seen in all cases, and perivascular space dilatation, rarefaction, and pigment deposition, with vessel wall mineralization and perivascular inflammatory cell infiltrates, were seen in some cases. Vasculitis was not found. One patient had had a transient ischemic attack, and no patient had had a stroke. CONCLUSIONS: Cerebral infarcts in HIV-infected patients are not common in the absence of cerebral non-HIV infection, lymphoma, or embolic sources. We found an HIV-associated vasculopathy with similar features in all risk groups. In AIDS patients presenting with stroke or transient ischemic attack, potentially treatable causes, such as cerebral coinfection or tumor, should be sought.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Infarto Encefálico/etiología , Encéfalo/patología , Síndrome de Inmunodeficiencia Adquirida/patología , Adulto , Autopsia , Encéfalo/irrigación sanguínea , Encéfalo/virología , Infarto Encefálico/epidemiología , Infarto Encefálico/patología , Estudios de Cohortes , Lóbulo Frontal/virología , Proteína p24 del Núcleo del VIH/análisis , Humanos , Inmunohistoquímica , Microcirculación/patología , Persona de Mediana Edad , Prevalencia , Escocia/epidemiología , Carga ViralRESUMEN
We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/virología , Evaluación Preclínica de Medicamentos/métodos , VIH-1/efectos de los fármacos , Membranas Artificiales , Resinas Acrílicas , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/trasplante , Supervivencia Celular , Efecto Citopatogénico Viral , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH/metabolismo , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Humanos , Ratones , Permeabilidad , Polímeros , Cloruro de Polivinilo , Prótesis e Implantes , Zidovudina/farmacologíaRESUMEN
OBJECTIVE: Several small short-term intervention studies have suggested that beta-carotene supplementation in HIV-infected patients can increase the number of various immune cells including CD4 cells. This prospective double-blinded study was designed to investigate whether beta-carotene supplementation would result in this immuno-enhancement in a larger number of patients over a longer time period. METHODS: HIV-positive patients were randomly assigned to receive either 60 mg beta-carotene orally three times daily or a matched placebo. In addition, all patients received a multivitamin supplement. Patients were evaluated at baseline, 1 month, and 3 months for T-cell quantitative subsets, natural killer cells, HIV p24 antigen, beta-carotene levels, complete blood counts and chemistry batteries. Body weights and Karnofsky scores were evaluated at each visit. RESULTS: Seventy-two patients signed informed consent forms and entered the study. Except for serum beta-carotene concentration, there were no statistically significant differences (P < 0.05) between the treatment (60 mg beta-carotene three times daily and multivitamins) and placebo (placebo and multivitamins) groups at baseline or after either 1 or 3 months of treatment. DISCUSSION: Earlier studies suggesting that beta-carotene supplementation increased levels of immune cells in HIV-infected patients were not replicated in this study. The addition of a multivitamin supplement to both arms of this study may have masked any difference between the two groups. However, on the basis of the results of this study, we would not recommend supplementation with high doses of beta-carotene for HIV-infected patients.
Asunto(s)
Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Subgrupos de Linfocitos T/patología , Linfocitos T/patología , beta Caroteno/administración & dosificación , Administración Oral , Método Doble Ciego , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Humanos , Recuento de Linfocitos , Estudios Prospectivos , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunologíaAsunto(s)
Seropositividad para VIH/sangre , Selenio/sangre , Adulto , Recuento de Linfocito CD4 , Femenino , Proteína p24 del Núcleo del VIH/análisis , Seropositividad para VIH/fisiopatología , VIH-1/inmunología , Humanos , Masculino , Pronóstico , Espectrometría por Rayos X , Microglobulina beta-2/análisisRESUMEN
Previous studies demonstrated that human cell lines can be cultivated in hollow fibers in the subcutaneous and intraperitoneal compartments of mice. We have extended the range of cell lines to include cells infected with the human immunodeficiency virus (HIV). Furthermore, these HIV-infected cells have been shown to replicate in the hollow fibers located in both physiologic compartments (intraperitoneal and subcutaneous) of SCID mice. Treatment of the host mice with antiviral agents can suppress virus replication in these hollow fiber cultures. The potential use of this system for early in vivo screening of anti-HIV compounds is discussed.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , VIH/efectos de los fármacos , Animales , Células Cultivadas , Proteína p24 del Núcleo del VIH/análisis , Humanos , Ratones , Ratones SCID , ADN Polimerasa Dirigida por ARN/metabolismo , Cultivo de Virus , Replicación Viral/efectos de los fármacos , Zalcitabina/farmacología , Zidovudina/farmacologíaRESUMEN
An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/análisis , Células Cultivadas , ADN Complementario/análisis , ADN Viral/análisis , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH , VIH-1/aislamiento & purificación , Humanos , Leucocitos/virología , Sensibilidad y EspecificidadRESUMEN
Heteropolyoxotungstates of the Keggin class containing different heteroatoms were tested for inhibition of two strains of human immunodeficiency virus 1 (HIV-1); they exhibited varying antiviral activity. Compounds containing boron were inactive, only one of those containing phosphorus showed selective anti-viral activity, whereas all silicon-containing compounds exhibited significant anti-viral activity in C8166 cells infected with the IIIB strain. Their effectiveness was some 10-fold higher in JM cells with selectivity indices of about 2000. The silicotungstates were effective inhibitors of HIV reverse transcriptase, showing greater inhibition with RNA/DNA template primers than with DNA/DNA template.primer. Kinetic analysis demonstrated that they inhibit the enzyme by different mechanisms, as, of the four compounds examined, two competed with template.primer and two competed with deoxynucleoside triphosphate. Inhibition of DNA polymerase activity by these compounds was compared using polymerases from different sources, including human; although not necessarily most specific for HIV-1 reverse transcriptase, they did not inhibit all DNA polymerases to a similar degree.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa , Compuestos de Tungsteno/farmacología , Boro/farmacología , Cartilla de ADN , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Diseño de Fármacos , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH , VIH-1/enzimología , VIH-1/fisiología , Humanos , Cinética , Inhibidores de la Síntesis del Ácido Nucleico , Fósforo/farmacología , ARN , Compuestos de Silicona/síntesis química , Compuestos de Silicona/química , Compuestos de Silicona/farmacología , Relación Estructura-Actividad , Moldes Genéticos , Compuestos de Tungsteno/síntesis química , Compuestos de Tungsteno/química , Replicación Viral/efectos de los fármacosRESUMEN
The human colonic epithelial cell line HT-29 can be productively infected with various HIV-1 and HIV-2 isolates that are highly cytopathic for T lymphocytes. In each case, a chronically infected HT-29 cell line can be established, and progeny viruses retain their original properties including high cytopathogenicity for T cells. Inasmuch as AIDS vaccines should include viral isolates capable of infecting mucosal epithelial cells, it may be useful to produce these isolates in such cells at a large scale. We describe here a microcarrier-based culture system allowing the production of infectious viruses from HT-29 cells grown in a chemically defined serum-free medium (Dulbecco's modified Eagle's medium/F12, HEPES 15 mM, pH 7.4, transferrin 5 micrograms/ml, selenium 10 ng/ml). The yield of HIV-1 from microcarrier cultures (275 ng of p24gag/ml) was greater than the yield from conventional culture flasks (122 ng of p24gag/ml). This virus, produced in serum-free medium, can be used either as a viral stock or as a source for HIV-1 proteins.
Asunto(s)
Medios de Cultivo , VIH-1/crecimiento & desarrollo , Mucosa Intestinal/virología , Microesferas , Cultivo de Virus/métodos , Adenocarcinoma , Neoplasias del Colon , Epitelio/virología , Técnica del Anticuerpo Fluorescente , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH , Humanos , Microscopía Electrónica , ADN Polimerasa Dirigida por ARN/análisis , Selenio/farmacología , Transferrina/farmacología , Células Tumorales CultivadasRESUMEN
GAP31 (gelonium anti-HIV protein of 31 kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity. The ability of GAP31 to interrupt both DNA and RNA functions may be related to its multiple antiviral actions. To define the roles of these activities in the anti-HIV action of GAP31, a series of peptides corresponding to the N-terminal segment of GAP31 were synthesized and assayed for the aforementioned activities of the parent molecule. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) designated as K10-K42 is the shortest peptide necessary and sufficient for HIV-1 inhibition, DNA and RNA binding, and ribosome inactivation. The peptides were 2-5 orders of magnitude less active than GAP31. Truncation of 19 aa from the C terminus of K10-K42 resulted in the loss of all of these activities. On the other hand, deletion of N-terminal residues to give E23-K42 did not alter ribosome-inactivation activity but eliminated the other activities. These findings permit identification of a 7-aa sequence, KGATYIT, at the N terminus of K10-K42 that is critical for DNA binding and RNA binding, whereas a 9-aa sequence, SHGIPSLRK, at the C terminus is important to ribosome inactivation. Both regions contribute to anti-HIV activity. Histidine at position 35 is critical for all of these activities. The disparity of sequence requirements for inhibition of HIV infection and replication and for ribosome-inactivation activity suggests that the anti-HIV activity of most ribosome-inactivating proteins may not be the result of N-glycosidase activity alone. Mapping the minimal domain of GAP31 offers insights into the rational design of molecular mimetics of anti-HIV drugs.
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Antivirales/farmacología , ADN Viral/efectos de los fármacos , VIH-1/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas de Plantas/farmacología , ARN Viral/efectos de los fármacos , Ribosomas/efectos de los fármacos , Secuencia de Aminoácidos , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN Viral/metabolismo , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/biosíntesis , Transcriptasa Inversa del VIH , VIH-1/fisiología , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/metabolismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: Laboratory data document the activation of the HIV-1 genome on exposure to UV radiation, including PUVA. The overall effects of UV radiation exposure on HIV-1 infection in human beings are unknown. OBJECTIVE: Our purpose was to observe CD4 cell counts and quantitative markers of HIV-1 load in late-stage HIV-1-infected human beings receiving PUVA for various cutaneous diseases. METHODS: Samples of peripheral blood were obtained on days 0, 14, 30, and 60 of PUVA administered in therapeutic doses. Number of CD4+ T lymphocytes was determined by flow cytometry, and HIV-1 load was measured by semiquantitative polymerase chain reaction for viral genome in peripheral blood mononuclear cells, semiquantitative RNA-polymerase chain reaction for HIV-1 RNA in serum, and determination of p24 in serum. RESULTS: No significant changes in the measurements were observed. CONCLUSION: This study did not detect a deleterious effect on CD4 cell count or HIV-1 load during 2 months of PUVA treatment for patients in late stages of infection, with low CD4 cell counts and high HIV-1 loads.
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Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , VIH-1/efectos de los fármacos , Terapia PUVA , Enfermedades de la Piel/tratamiento farmacológico , Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , ADN Viral/análisis , ADN Viral/sangre , Genoma Viral , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/sangre , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Proyectos Piloto , ARN Viral/análisis , ARN Viral/sangre , Enfermedades de la Piel/inmunología , Activación Viral/efectos de los fármacosRESUMEN
Suksdorfin (1), which is isolated from the fruit of Lomatium suksdorfii, was found to be able to inhibit HIV-1 replication in the T cell line, H9, with an average EC50 value of 2.6 +/- 2.1 microM. In addition, suksdorfin was also suppressive during acute HIV-1 infections of peripheral blood mononuclear cells, monocyte/macrophages and the promonocytic cell line, U937. Combinations of 1 and the anti-HIV nucleosides ddI and ddC demonstrated statistical synergy in inhibiting HIV-1 replication (ddC > ddI). However, the viral inhibition mediated by combining 1 with AZT was not statistically synergistic. Furthermore, the presence of suksdorfin did not antagonize the suppression mediated by the three nucleoside reverse transcriptase inhibitors. Comparison of the structure and activity of 1 with those of ten related compounds indicated that the dihydroseselin type of pyranocoumarin possessing a 4'-isovaleryl group is important to suksdorfin's enhanced anti-HIV activity.