RESUMEN
A significant splenomegaly and lymphadenopathy develops during the progressive growth of Lewis Lung (3LL) tumors in mice. Enlarged spleen and lymph nodes occur because of a pronounced increase in granulocytes in these organs. This granulocytosis in spleen and lymph node was not simply due to recruitment of granulocytes from peripheral blood to spleen and lymph nodes, but also a result of development and/or differentiation of granulocytes from the bone marrow. There was a marked increase in development of myeloid lineage cells, whereas lymphoid populations including T cells and B cells, were dramatically decreased in bone marrow and peripheral blood of 3LL tumor-bearing mice. These data demonstrate that host hematopoiesis shifts from lymphoid to granulocytic development in the 3LL tumor-bearing mice. Interestingly, a somatic mutation of N-Ras gene was found in 3LL tumor cells at codon 61, suggesting that mutated N-Ras may contribute to induction of granulocytosis in 3LL tumor-bearing mice.
Asunto(s)
Carcinoma Pulmonar de Lewis/patología , Granulocitos/patología , Hematopoyesis , Linfocitos/patología , Animales , Carcinoma Pulmonar de Lewis/complicaciones , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Diferenciación Celular/genética , Linaje de la Célula , Quimiocinas/biosíntesis , Quimiocinas/genética , Codón/genética , Regulación Neoplásica de la Expresión Génica , Genes ras , Inmunofenotipificación , Interferón gamma/deficiencia , Interferón gamma/genética , Enfermedades Linfáticas/etiología , Enfermedades Linfáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neutrófilos/patología , Organismos Libres de Patógenos Específicos , Esplenomegalia/etiología , Esplenomegalia/patologíaRESUMEN
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.
Asunto(s)
VIH-1/fisiología , Péptido T/fisiología , Receptores CCR5/fisiología , Replicación Viral/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Antivirales/metabolismo , Bioensayo , Células Cultivadas , Quimiocinas/antagonistas & inhibidores , Quimiocinas/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Feto , Citometría de Flujo , Genes Reporteros , Proteína p24 del Núcleo del VIH/inmunología , VIH-1/metabolismo , Células HeLa , Humanos , Luciferasas/análisis , Luciferasas/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Microglía/citología , Microglía/metabolismo , Microglía/virología , Péptido T/inmunología , Factores de TiempoRESUMEN
DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4(+) cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of the p16(INK4A) gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as the p16(INK4A) gene, silencing their expression.
Asunto(s)
Metilasas de Modificación del ADN/metabolismo , Genes p16 , VIH-1/fisiología , Linfocitos/virología , Línea Celular , Metilación de ADN , Metilasas de Modificación del ADN/genética , Regulación Enzimológica de la Expresión Génica , VIH-1/enzimología , VIH-1/genética , Humanos , Integrasas/deficiencia , Integrasas/genética , Linfocitos/metabolismo , Mutación , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Factores de TiempoRESUMEN
Transforming growth factor beta (TGF-beta) is a pleiotropic regulator of all stages of hematopoieis. Depending on the differentiation stage of the target cell, the local environment, and the concentration of TGF-beta, TGF-beta can be proproliferative or antiproliferative, proapoptotic or antiapoptotic, and/or prodifferentiative or antidifferentiative. TGF-beta is the major regulator of stem cell quiescence and can act directly or indirectly through effects on the marrow microenvironment. In addition, paracrine and autocrine actions of TGF-beta have overlapping but distinct regulatory effects on hematopoietic stem/progenitor cells. Neutralization of autocrine TGF-beta has therapeutic potential.
Asunto(s)
Células Madre Hematopoyéticas/citología , Factor de Crecimiento Transformador beta/fisiología , Animales , Comunicación Autocrina , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Sinergismo Farmacológico , Sangre Fetal/citología , Vectores Genéticos/genética , Hematopoyesis/efectos de los fármacos , Hematopoyesis/fisiología , Factores de Crecimiento de Célula Hematopoyética/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Retroviridae/genética , Transfección , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Trasplante HomólogoRESUMEN
In order to assess the prevalence rate of HTLV-1-associated T-cell lymphomas and human retrovirus infection in general, approximately 21,000 individuals representing various patient populations, retroviral risk groups, and blood donors were examined for HTLV-I, HTLV-II, HIV-1, or HIV-2 infection using serologic and PCR assays. The prevalence rates among volunteer blood donors were 0.02% and 0% for HTLV and HIV, respectively. Significantly increased HTLV prevalence rates were observed among paid blood donors, African American health care clinic patients, Amerindians, recipients of HTLV-positive cellular blood products, intravenous drug users, sexual contacts and family members of HTLV-positive people, and patients with primary thrombocytosis and other-than-low-grade non-Hodgkin's lymphoma (NHL). Among some of these groups there were significant differences in the prevalence of HTLV-I versus HTLV-II. The eight HTLV-positive NHL patients all had mature, high-grade, CD4+ T-cell lymphomas with clonally integrated HTLV-I, for a prevalence of 4% among other-than-low-grade NHL patients. Seven of the eight died from their disease within 2 years despite treatment. Interestingly, two groups at risk for HTLV infection, namely needle stick victims and recipients of HTLV-infected and/or pooled plasma products, showed no evidence for infection. Significantly increased HIV-1 prevalence was observed among paid blood donors, African Americans, homosexuals, female prostitutes, hemophiliacs, and other-than-low-grade NHL patients. Only one patient was infected with HIV-2. Of the nine HIV-positive, other-than-low-grade NHL patients, seven HIV-1 positives had B-cell lymphomas, one HIV-1 positive had an HTLV-I-positive CD4+ T-cell lymphoma, and one infected with HIV-2 had a CD4+ T-cell lymphoma that was HTLV negative. The data indicate that HTLV-I lymphoma, while uncommon, is not necessarily rare among other-than-low-grade NHL cases in the United States and, given its poor prognosis, should probably be studied separately in clinical trials.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/epidemiología , Infecciones por Retroviridae/epidemiología , Negro o Afroamericano , Agammaglobulinemia/epidemiología , Donantes de Sangre , Comorbilidad , ADN de Neoplasias/análisis , ADN Viral/análisis , Salud de la Familia , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , Infecciones por HTLV-I/epidemiología , Infecciones por HTLV-II/epidemiología , Hemofilia A/epidemiología , Indígenas Norteamericanos , Leucemia/epidemiología , Leucemia-Linfoma de Células T del Adulto/etnología , Linfoma/clasificación , Linfoma/epidemiología , Linfoma/etnología , Linfoma/virología , Linfoma Relacionado con SIDA/epidemiología , Linfoma Relacionado con SIDA/etnología , Linfoma Relacionado con SIDA/virología , Lesiones por Pinchazo de Aguja/complicaciones , Prevalencia , Infecciones por Retroviridae/etnología , Infecciones por Retroviridae/virología , Enfermedades Reumáticas/epidemiología , Factores de Riesgo , Estudios Seroepidemiológicos , Conducta Sexual , Abuso de Sustancias por Vía Intravenosa , Trombocitosis/epidemiología , Reacción a la Transfusión , Estados Unidos/epidemiologíaRESUMEN
The G protein-coupled 7 transmembrane (STM) chemoattractant receptors can be inactivated by heterologous desensitization. Earlier work showed that formly peptide receptor-like 1 (FPRL1), an STM receptor with low affinity for the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalamine (fMLF), is activated by peptide domains derived from the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 and its activation results in desensitization and down-regulation of the chemokine receptors CCR5 and CXCR4 from monocyte surfaces. This study investigated the possibility of interfering with the function of CCR5 or CXCR4 as HIV-1 coreceptors by activating FPRL1. Cell lines were established expressing FPRL1 in combination with CD4/CXCR4 or CD4/CCR5 and the effect of a synthetic peptide, WKYMVm, a potent activator of formyl peptide receptors with preference for FPRL1 was determined. Both CXCR4 and CCR5 were desensitized by activation of the cells with WKYMVm via a staurosporine-sensitive pathway. This desensitization of CXCR4 and CCR5 also attenuated their capacity as the fusion cofactors for HIV-1 envelope glycoprotein and resulted in a significant inhibition of p24 production by cell lines infected with HIV-1 that use CCR5 or CXCR4 as coreceptors. Furthermore, WKYMVm inhibited the infection of human peripheral monocyte-derived macrophages and CD4(+) T lymphocytes by R5 or X4 strains of HIV-1, respectively. These results indicate that heterologous desensitization of CCR5 and CXCR4 by an FPRL1 agonist attenuates their major biologic functions and suggest an approach to the development of additional anti-HIV-1 agents. (Blood. 2001;97:2941-2947)
Asunto(s)
Proteínas Quimioatrayentes de Monocitos/farmacología , Oligopéptidos/farmacología , Receptores CCR5/efectos de los fármacos , Receptores CXCR4/efectos de los fármacos , Receptores Inmunológicos/efectos de los fármacos , Receptores Inmunológicos/fisiología , Receptores de Lipoxina , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/fisiología , Antivirales/farmacología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Macrófagos/virología , Osteosarcoma , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Receptores de Formil Péptido , Receptores del VIH/genética , Receptores Inmunológicos/genética , Receptores de Péptidos/genética , Transfección , Células Tumorales CultivadasRESUMEN
Transforming growth factor (TGF)-beta1 plays an important role during hematopoiesis. Previously, we had shown that the growth of a v-Src-transformed myeloid cell line was markedly more inhibited by TGF-beta treatment when compared with the wild-type myeloid cell line. To investigate the increased growth sensitivity of the v-Src-transformed myeloid cell line, 32D-src, to TGF-beta, we examined expression of the TGF-beta type II receptor (TGF-beta RII) gene in myeloid cell lines. Northem blot analysis showed that expression of approximately 8- and 6-kb species of TGF-beta RII transcripts was markedly increased in the 32D-src cell line. The expression of the TGF-beta RII promoter linked to a reporter gene was increased 23-fold by v-Src. DNA transfection and electrophoretic mobility shift assay revealed that v-Src induces TGF-beta RII promoter activity through an AP1/ATF2-like sequence (-219 to -172), ETS binding sites (+1 to +36), and the inverted CCAAT box (-81 to -77). Novel DNA-protein complexes with ETS binding sites are significantly increased in v-src-transformed cell lines compared with the control cell line. These results suggest that v-Src induces activity of the TGF-beta RII promoter through multiple elements by inducing expression of nuclear proteins interacting with these elements.
Asunto(s)
Regulación de la Expresión Génica , Células Mieloides/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Northern Blotting , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Genes Reporteros , Luciferasas/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Serina-Treonina Quinasas , ARN Mensajero/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Elementos de Respuesta , Activación Transcripcional , TransfecciónRESUMEN
IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.
Asunto(s)
Rayos gamma , Mucosa Intestinal/inmunología , Mucosa Intestinal/efectos de la radiación , Intestino Delgado/inmunología , Intestino Delgado/efectos de la radiación , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/efectos de la radiación , Animales , Apoptosis/genética , Apoptosis/inmunología , Apoptosis/efectos de la radiación , Trasplante de Médula Ósea/inmunología , Trasplante de Médula Ósea/patología , Relación Dosis-Respuesta Inmunológica , Relación Dosis-Respuesta en la Radiación , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Intestino Delgado/metabolismo , Intestino Delgado/patología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/efectos de la radiación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/efectos de la radiación , Receptores de Interleucina-7/biosíntesis , Receptores de Interleucina-7/deficiencia , Trasplante Homólogo , Pérdida de Peso/genética , Pérdida de Peso/inmunología , Pérdida de Peso/efectos de la radiación , Irradiación Corporal Total , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína bcl-XRESUMEN
Expression profiling of cellular genes was performed using a 10,000 cDNA human gene array in order to identify expression changes following chronic infection of human hematopoietic cells with Kaposi's Sarcoma-associated Virus (KSHV) also known as Human Herpesvirus 8 (HHV8) and Human T cell leukemia virus-1 (HTLV-1). We performed cell-free in vitro infection of primary bone marrow derived CD34+ cells using semi-purified HHV8 and a mature IL-2 dependent T cell line, KIT 225, using highly concentrated viral stocks prepared from an infectious molecular clone of HTLV-1. Thirty days post infection, mRNA was isolated from infected cultures and uninfected controls and submitted for microarray analysis. More than 400 genes were differentially expressed more than two-fold following HHV8 infection of primary bone marrow derived CD34+ cells. Of these 400, interferon regulatory factor 4 (IRF4), cyclin B2, TBP-associated factor, eukaryotic elongation factor and pim 2 were up-regulated more than 3.5 fold. In contrast, less than 100 genes were differentially expressed more than two-fold following chronic infection of a mature T cell line with HTLV-1. Of these, only cdc7 was up-regulated more than 3.5 fold. These data may provide insight into cellular signatures of infection useful for diagnosis of infection as well as potential targets for therapeutic intervention.
Asunto(s)
Regulación de la Expresión Génica , Infecciones por HTLV-I/genética , Células Madre Hematopoyéticas/virología , Infecciones por Herpesviridae/genética , Línea Celular , Herpesvirus Humano 8 , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Aberrant expression of homeobox genes has been described in primary leukemia blasts. We recently cloned a new cDNA, BP1, which is a member of the homeobox gene family. BP1 expression was investigated in bone marrow samples from acute myeloid leukemia (AML), acute T cell lymphocytic leukemia (ALL) and pre-B cell ALL. Expression levels of two apparent isoforms of BP1, DLX7 and DLX4, were measured in the same samples. They are weakly if at all detectable in normal bone marrow, PHA-stimulated T cells or B cells. BP1 RNA was highly expressed in 63% of AML cases, including 81% of the pediatric and 47% of the adult cases, and in 32% of T-ALL cases, but was not found in any of the pre-B ALL cases. Coexpression of BP1, DLX7 and DLX4 occurred in a significant number of leukemias. Our data, including co-expression of BP1 with c-myb and GATA-1, markers of early progenitors, suggest that BP1 expression occurs in primitive cells in AML. Analysis of CD34+ and CD34- normal bone marrow cells revealed BP1 is expressed in CD34- cells and virtually extinguished in CD34+ cells. Ectopic expression of BP1 in the leukemia cell line K562 increased clonogenicity, consistent with a role for BP1 in leukemogenesis. The presence of BP1 RNA in leukemic blasts may therefore be a molecular marker for primitive cells and/or may indicate that BP1 is an important upstream factor in an oncogenic pathway.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio/genética , Leucemia/genética , Proteínas de Neoplasias/genética , Proteínas Oncogénicas , Isoformas de Proteínas/genética , Factores de Transcripción , Enfermedad Aguda , Factores de Edad , Empalme Alternativo , Biomarcadores de Tumor/genética , Examen de la Médula Ósea , Diferenciación Celular/genética , Transformación Celular Neoplásica/genética , ADN Complementario/genética , ADN de Neoplasias/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/aislamiento & purificación , Humanos , Células K562/citología , Leucemia/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/aislamiento & purificación , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/aislamiento & purificación , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas Recombinantes de Fusión/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayo de Tumor de Célula MadreRESUMEN
Baicalin (BA) is a flavonoid compound purified from medicinal plant Scutellaria baicalensis Georgi and has been shown to possess anti-inflammatory and anti-HIV-1 activities. In an effort to elucidate the mechanism of the anti-inflammatory effect of BA, we recently found that this flavonoid compound was able to form complexes with selected chemokines and attenuated their capacity to bind and activate receptors on the cell surface. These observations prompted us to investigate whether BA could inhibit HIV-1 infection by interfering with viral entry, a process known to involve interaction between HIV-1 envelope proteins and the cellular CD4 and chemokine receptors. We found that BA at the noncytotoxic concentrations, inhibited both T cell tropic (X4) and monocyte tropic (R5) HIV-1 Env protein mediated fusion with cells expressing CD4/CXCR4 or CD4/CCR5. Furthermore, presence of BA at the initial stage of HIV-1 viral adsorption blocked the replication of HIV-1 early strong stop DNA in cells. Since BA did not inhibit binding of HIV-1 gp120 to CD4, we propose that BA may interfere with the interaction of HIV-1 Env with chemokine coreceptors and block HIV-1 entry of target cells. Therefore, BA can be used as a basis for developing novel anti-HIV-1 agents.
Asunto(s)
Antivirales/farmacología , Flavonoides/farmacología , VIH-1/efectos de los fármacos , Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Humanos , Células Tumorales CultivadasRESUMEN
The use of the neuroendocrine hormones growth hormone (GH) and prolactin (PRL) in preclinical models, demonstrating promotion of hematopoietic recovery and immune function, offers promise for several clinical situations. These hormones do not appear to produce the same extent of immune/hematopoietic effects when compared to conventional hematopoietic and immune stimulating cytokines (i.e. G-CSF or interleukin-2). However, their pleiotropic effects and limited toxicity after systemic administration makes them attractive to test in myeloablative situations. More work needs to be performed to understand the mechanism(s) of GH and PRL action, particularly with regard to hematopoietic progenitor cell expansion and differentiation both in normal and pathologic situations.
Asunto(s)
Hormona del Crecimiento/farmacología , Hormona del Crecimiento/fisiología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/fisiología , Prolactina/farmacología , Prolactina/fisiología , Animales , Humanos , Transducción de SeñalRESUMEN
Dendritic cells (DC) that have been genetically modified to express cytokine genes may be novel tools for inducing antitumor immune responses. In the present study, the pMX retroviral vector was modified to express the mouse IL-2 (mIL-2pMX) and mouse IL-12 (mIL-12pMX) genes. Supernatants from 293 cells transfected with pMX retroviral vectors were harvested and used to transduce mouse lin- bone marrow (BM) progenitor cells. After 48 h co-culture with pseudotype retrovirus, BM cells were cultured for 12 days in the presence of mGM-CSF, mSCF and mTNF-alpha to obtain a DC-enriched fraction. Flow cytometric analysis showed that GFP protein expression in these cultures was 20-40% and that 40-50% of the cultured BM cells were positive for the DC marker, DEC205. About 60% of cells sorted for DEC205 also expressed GFP. The supernatants of DC-mIL-2 and DC-mIL-12 cultured for 48 h contained 5.2 +/- 0.15 and 33.9 +/- 2.6 ng cytokine protein per milliliter, respectively. Intratumoral injection of DC-mIL-2 or DC-mIL-12 on days 8 and 15 after the intradermal injection of 1 x 105 B16F10 cells, resulted in a significant reduction in tumor size by day 21, as compared with mice treated with unmodified DC or DC-GFP. Longer term analysis as assessed at day 42 revealed that B16 tumor-bearing mice treated with cytokine gene-modified DC survived significantly longer than mice from other groups. Spleen cells obtained from DC-treated mice were specifically sensitized for the generation of CTL by subsequent restimulation with gene-modified DC. These results suggested that DC genetically modified to express IL-2 or IL-12 can induce potent antitumor responses against well-established, poorly immunogenic B16F10 tumors. Gene Therapy (2000) 7, 2113-2121.
Asunto(s)
Células Dendríticas/inmunología , Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Interleucina-12/genética , Interleucina-2/genética , Melanoma Experimental/terapia , Animales , Expresión Génica , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/inmunología , Inyecciones Intralesiones , Interleucina-12/inmunología , Interleucina-2/inmunología , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Retroviridae/genética , Transducción GenéticaAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD4-Positivos/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , VIH-1/fisiología , Receptores de Quimiocina/antagonistas & inhibidores , Sitios de Unión , Quimiocinas/metabolismo , Humanos , Monocitos/fisiología , Receptores de Quimiocina/fisiologíaRESUMEN
Induction and maintenance of Ag-specific tolerance are pivotal for immune homeostasis, prevention of autoimmune disorders, and the goal of transplantation. Recent studies suggest that certain cytokines, notably IL-10 and TGF-beta, may play a role in down-regulating immune functions. To further examine the role of cytokines in Ag-specific hyporesponsiveness, murine CD4+ T cells were exposed ex vivo to alloantigen-bearing stimulators in the presence of exogenous IL-10 and/or TGF-beta. Primary but not secondary alloantigen proliferative responses were inhibited by IL-10 alone. However, the combined addition of IL-10 + TGF-beta markedly induced alloantigen hyporesponsiveness in both primary and secondary MLR cultures. Alloantigen-specific hyporesponsiveness was observed also under conditions in which nominal Ag responses were intact. In adoptive transfer experiments, IL-10 + TGF-beta-treated CD4+ T cells, but not T cells treated with either cytokine alone, were markedly impaired in inducing graft-vs-host disease alloresponses to MHC class II disparate recipients. These data provide the first formal evidence that IL-10 and TGF-beta have at least an additive effect in inducing alloantigen-specific tolerance, and that in vitro cytokines can be exploited to suppress CD4+ T cell-mediated Ag-specific responses in vivo.
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Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica/inmunología , Interleucina-10/fisiología , Isoantígenos/inmunología , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Aminoácidos , Animales , Antígenos/inmunología , Células Cultivadas , Combinación de Medicamentos , Epítopos de Linfocito T/inmunología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Inmunosupresores/farmacología , Interleucina-10/farmacología , Prueba de Cultivo Mixto de Linfocitos/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
There are a number of problems associated with the development of standards suitable for use in the most commonly used assays to detect cytokines in biological fluids. These problems include: (i) the failure of some MoAbs used in immunoassays to detect all different <
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Citocinas/análisis , Sustancias de Crecimiento/análisis , Inmunoensayo/métodos , Animales , Bioensayo , Líquidos Corporales/química , Citocinas/normas , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Sustancias de Crecimiento/normas , Humanos , Inmunoensayo/normas , Inmunoensayo/estadística & datos numéricos , Interferón gamma/análisis , Interferón gamma/normas , Interleucina-1/análisis , Interleucina-1/normas , Interleucina-4/análisis , Interleucina-4/normas , Ratones , Estándares de ReferenciaRESUMEN
While the majority of purified pluripotential hematopoietic stem cells (PHSC) express c-Kit, the receptor for steel factor, we have phenotypically and functionally separated a distinct class of PHSC that does not express c-Kit. In contrast to c-Kit-positive (c-Kit(pos)) PHSC, the c-Kit-negative (c-Kit(neg)) PHSC do not proliferate in response to multiple hematopoietic growth factors in vitro and do not radioprotect or form macroscopic spleen colonies (CFU-s) when transplanted into lethally irradiated recipients. However, the c-Kit(neg) PHSC show delayed or slow reconstitution kinetics when cotransplanted with radioprotective bone marrow cells. c-Kit(neg) PHSCs cells can give rise to c-Kit(pos) cells with CFU-s activity, radioprotective activity, and PHSC activity. Thus, constitutive hematopoiesis is maintained by c-Kit(pos) PHSCS cells that are recruited from a more primitive quiescent c-Kit(neg) PHSC population, which represents a critical developmental stage in definitive hematopoiesis.
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Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas c-kit/fisiología , Animales , Células de la Médula Ósea/fisiología , Línea Celular , Separación Celular , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la PolimerasaRESUMEN
We compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2-8) or late (weeks 21-137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located approximately 40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrent in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MA nef) nef on the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnef contained a large proportion of cells carrying provirus with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were "corrected" in vivo.
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Genes nef , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Secuencia de Aminoácidos , Animales , Clonación Molecular , Productos del Gen nef/genética , Macaca fascicularis , Macaca nemestrina , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Provirus/genética , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación Viral/genéticaRESUMEN
Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.
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Antígenos CD4/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Monocitos/inmunología , Monocitos/virología , Receptores CXCR4/inmunología , Células Cultivadas , Quimiocinas/inmunología , Quimiocinas/farmacología , Regulación hacia Abajo , Proteína gp120 de Envoltorio del VIH/farmacología , Humanos , Proteínas Recombinantes/farmacología , Transducción de Señal/inmunologíaRESUMEN
We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.