RESUMEN

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 x 10(6) activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 x 10(6) cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 x 10(6) L-selectin(low) T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 x 10(6) T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/terapia , Inmunoterapia Adoptiva , Neoplasias Pulmonares/terapia , Glicoproteínas de Membrana/inmunología , Receptores del Factor de Necrosis Tumoral , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Movimiento Celular/inmunología , Femenino , Fibrosarcoma/inmunología , Fibrosarcoma/patología , Fibrosarcoma/terapia , Inmunoterapia Adoptiva/métodos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Interleucina-2/administración & dosificación , Interleucina-2/efectos adversos , Selectina L/biosíntesis , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ganglios Linfáticos/trasplante , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/trasplante , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ligando OX40 , Receptores OX40 , Linfocitos T/inmunología , Linfocitos T/patología , Linfocitos T/trasplante , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Factores de Necrosis Tumoral , Regulación hacia Arriba/inmunología

RESUMEN

We report on the characterization of monoclonal antibodies against Plasmodium falciparum schizonts, which recognize parasite proteins of 130 kDa and 20 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatment, suggesting that the protein is a peripheral membrane protein, while the 20-kDa protein remained associated with the membranes following alkali treatment, suggesting it may be an integral membrane protein. Both proteins were localized to large cytoplasmic vesicles within the cytoplasm of trophozoite and schizont-infected erythrocytes by immunofluorescence assay and confocal microscopy. Both proteins colocalized with Bodipy-ceramide in trophozoite and immature schizont-infected erythrocytes, but not in segmenters. The 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maurer's clefts underneath knobs in a knobby and cytoadherent (K +/ C+) P. falciparum strain. No IEM reactivity was obtained in a knobless and non-cytoadherent (K-/C-)

Asunto(s)

Antígenos de Protozoos/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Anticuerpos Monoclonales , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Técnicas In Vitro , Microscopía Confocal , Microscopía Inmunoelectrónica , Peso Molecular , Plasmodium falciparum/ultraestructura

RESUMEN

We reported previously that non-neoplastic astrocytes (derived from brain tissues of patients with epilepsy) expressed interleukin 4 receptor alpha (IL-4Ralpha) and responded to interleukin 4 (IL-4) in culture. To determine whether reactivity of cultured astrocytes was relevant to primary tissue, we investigated IL-4Ralpha expression in specimens of non-neoplastic cerebral cortex removed for surgical treatment of intractable epilepsy compared to specimens of glial tumours, which have been reported to contain IL-4Ralpha. Freshly frozen tissues from eight cases (four epilepsy, four malignant astrocytoma) were evaluated for IL-4Ralpha expression by reverse-transcriptase polymerase chain reaction (RT-PCR), Southern blotting, and double-labelled immunohistochemistry with antibodies to IL-4Ralpha and glial fibrillary acidic protein (GFAP). IL-4Ralpha mRNA was detectable in both non-neoplastic and neoplastic tissues, whereas interleukin 2 receptor gamma chain (IL-2Rgammac) mRNA was not found. By immunohistochemistry, IL-4Ralpha protein co-localized to cells displaying GFAP and astrocytic morphology in epilepsy tissues. As anticipated, IL-4Ralpha was detectable in astrocytoma, but, surprisingly, was also observed in GFAP-positive, non-neoplastic "reactive" astrocytes adjacent to tumour. Results are consistent with the concept that non-neoplastic epilepsy astrocytes express IL-4Ralpha in situ, thus confirming in vitro studies and implying IL-4 sensitivity in vivo.

Asunto(s)

Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Epilepsia/metabolismo , Interleucina-4/farmacología , Receptores de Interleucina-4/metabolismo , Actinas/metabolismo , Southern Blotting , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Células Cultivadas , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Inmunohistoquímica , ARN Mensajero/metabolismo , Receptores de Interleucina-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.

Asunto(s)

Astrocitoma/metabolismo , Proteínas de Ciclo Celular , Ciclinas/genética , Interleucina-4/antagonistas & inhibidores , Interleucina-4/fisiología , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteínas Supresoras de Tumor , Astrocitoma/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Mutación/genética , Mutación Missense , Oligonucleótidos Antisentido/genética , ARN Mensajero/análisis , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética

RESUMEN

BACKGROUND: Endothelial cells that line microvascular blood vessels have an important role in inflammation through their ability to bind and recruit circulating leucocytes. Endothelial cells from the intestines of patients with chronically inflamed Crohn's disease and ulcerative colitis--the two forms of inflammatory bowel disease--display an increased leucocyte-binding capacity in vitro. We investigated whether this enhanced leucocyte binding is a primary or an acquired defect. METHODS: We cultured human intestinal microvascular endothelial cells (HIMEC) from the uninvolved intestine and chronically inflamed bowel of three patients with inflammatory bowel disease (two Crohn's disease, one ulcerative colitis). We assessed HIMEC binding to polymorphonuclear leucocytes and U937 cells by means of an adhesion assay. FINDINGS: After activation with interleukin-1beta or lipopolysaccharide, HIMEC from the chronically inflamed tissue in all three patients with inflammatory bowel disease bound twice as many polymorphonuclear leucocytes and U937 cells as endothelial cells from uninvolved tissue. INTERPRETATION: Enhanced leucocyte binding by HIMEC from chronically inflamed tissue in patients with inflammatory bowel disease is an acquired defect since it is not found in the uninvolved intestinal segments from the same individuals. Because interaction between endothelial cells and leucocytes is a key regulatory step in the inflammatory process, this enhanced binding may contribute to the pathophysiology of chronic intestinal inflammation.

Asunto(s)

Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Endotelio Vascular/inmunología , Leucocitos/inmunología , Receptores de Adhesión de Leucocito/inmunología , Adulto , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Colitis Ulcerosa/patología , Enfermedad de Crohn/patología , Endotelio Vascular/patología , Femenino , Humanos , Técnicas In Vitro , Mucosa Intestinal/irrigación sanguínea , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Prueba de Inhibición de Adhesión Leucocitaria , Leucocitos/patología , Masculino , Microcirculación/inmunología , Microcirculación/patología , Neutrófilos/inmunología , Neutrófilos/patología

RESUMEN

Demyelination of the central nervous system is a hallmark of multiple sclerosis and its widely used animal model, experimental autoimmune encephalomyelitis (EAE). Recent studies using magnetic resonance imaging and spectroscopy on multiple sclerosis patients have revealed abnormalities of central nervous system normal-appearing white matter suggesting that micro-demyelination and/or extensive membrane turnover accompanies and perhaps precedes the appearance of manifest inflammatory lesions. In the present study, we induced EAE in SWXJ mice and analyzed digitized images of immunocytochemically stained spinal cord for detection of myelin proteolipid protein (PLP). We found that digitized image analysis is a highly sensitive, objective methodology for measuring the extent of myelin loss during EAE. Our data show that two-thirds of the measured reduction of myelin PLP occurring in EAE spinal cord could be attributed to a loss of myelin in normal-appearing white matter. The marked decrease in detection of PLP was accompanied by a corresponding decrease in PLP mRNA in the central nervous system. Our results indicate that during acute EAE, diffuse myelin abnormalities extend far beyond visibly detectable inflammatory foci and are characterized by a global decrease in the expression of myelin genes and their encoded proteins.

Asunto(s)

Encefalomielitis Autoinmune Experimental/patología , Procesamiento de Imagen Asistido por Computador , Proteína Proteolipídica de la Mielina/análisis , Médula Espinal/patología , Animales , Northern Blotting , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/inmunología , Fragmentos de Péptidos/inmunología , ARN Mensajero/análisis , ARN Mensajero/genética , Médula Espinal/química

RESUMEN

The 3' ends of the genome and antigenome RNA of vesicular stomatitis virus (VSV) serve as the promoter sites for the RNA-dependent RNA polymerase in the initiation of transcription and replication, respectively. The leader RNA, the first transcript synthesized during the RNA synthetic step, contains sequences to initiate encapsidation with the nucleocapsid protein, which is a prerequisite for replication. It also plays a role in the inhibition of cellular RNA synthesis. To search for a specific cellular factor(s) which may interact with the leader RNA sequences and regulate these processes, we used a gel mobility shift assay to identify such a protein(s). By using nuclear extract, it was found that in addition to the previously reported La protein, a 120-kDa nuclear protein specifically interacts with the leader RNA. Biochemical and immunological studies identified the 120-kDa protein as heterogeneous nuclear ribonucleoprotein particle U (hnRNP U), which is involved in pre-mRNA processing. We also demonstrate that hnRNP U is associated with the leader RNA in the nuclei of VSV-infected cells and also packaged within the purified virions. By double immunofluorescence labeling and confocal microscopy, hnRNP U appears to colocalize with the virus in the cytoplasm of infected cells. These results strongly suggest that hnRNP U plays an important role in the life cycle of VSV.

Asunto(s)

Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Ribonucleoproteínas/metabolismo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo U , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Microscopía Confocal , Datos de Secuencia Molecular , Unión Proteica , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas Virales/metabolismo , Replicación Viral

RESUMEN

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective (NG-monomethyl-L-arginine) and specific (N-iminoethyl-L-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substrate L-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.

Asunto(s)

Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Regulación Enzimológica de la Expresión Génica , Mucosa Intestinal/irrigación sanguínea , Leucocitos/fisiología , Microcirculación , Óxido Nítrico Sintasa/metabolismo , Arginina/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Lisina/análogos & derivados , Lisina/farmacología , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Reacción en Cadena de la Polimerasa , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , omega-N-Metilarginina/farmacología

RESUMEN

Stimulation of beta-adrenergic receptors activates type I and II cyclic AMP-dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase-anchoring proteins (AKAPs) regulates cyclic AMP-dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus alpha-actinin-specific antibodies or AKAP100 plus ryanodine receptor-specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Miocardio/metabolismo , Proteínas/metabolismo , Proteínas de Anclaje a la Quinasa A , Adulto , Animales , Especificidad de Anticuerpos , Secuencia de Bases , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN/genética , Humanos , Inmunohistoquímica , Miocardio/ultraestructura , Fosforilación , Proteínas/genética , Proteínas/inmunología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Retículo Sarcoplasmático/metabolismo , Fracciones Subcelulares/metabolismo

RESUMEN

Several neurotransmitter receptors have been identified on axons, and emerging evidence suggests that central axonal conduction may be modulated by neurotransmitters. We have recently demonstrated the presence of extra-synaptic adenosine Al receptors along rat hippocampal axons. We now present immunocytochemical evidence for Al receptors on rat corpus callosum axons and show that these receptors actively modulate axon physiology. Using rat brain coronal slices, we stimulated the corpus callosum and recorded the evoked extracellular compound action potential. The lipid-soluble, Al-specific adenosine receptor agonist cyclopentyladenosine, dose-dependently decreased the compound action potential amplitude, an effect reversed by the specific Al antagonist 8-cyclopentyl-1, 3-dipropylxanthine. These data provide the first direct evidence that axonal Al adenosine receptors modulate axon physiology in the adult mammalian brain. Influencing axonal transmission is a potentially powerful mechanism of altering information processing in the nervous system.

Asunto(s)

Axones/fisiología , Cuerpo Calloso/metabolismo , Receptores Purinérgicos P1/metabolismo , Potenciales de Acción/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Axones/efectos de los fármacos , Cafeína/farmacología , Cuerpo Calloso/citología , Cuerpo Calloso/efectos de los fármacos , Electrofisiología , Inmunohistoquímica , Masculino , Inhibidores de Fosfodiesterasa/farmacología , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología

RESUMEN

Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Virus de la Parainfluenza 3 Humana/fisiología , Replicación Viral , Animales , Línea Celular , Chlorocebus aethiops , Citocalasina D/farmacología , Citoesqueleto/metabolismo , Humanos , Líquido Intracelular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Virus de la Parainfluenza 3 Humana/genética , Virus de la Parainfluenza 3 Humana/crecimiento & desarrollo , Virus de la Parainfluenza 3 Humana/metabolismo , ARN Viral/metabolismo , Ribonucleoproteínas/metabolismo , Virión

RESUMEN

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.

Asunto(s)

Astrocitos/patología , Proteínas de Ciclo Celular , Interleucina-4/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de Tumor , Antígenos CD/metabolismo , Astrocitos/metabolismo , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Humanos , Interleucina-4/genética , Proteínas Asociadas a Microtúbulos/genética , Oligonucleótidos Antisentido/farmacología , Receptores de Interleucina/metabolismo , Receptores de Interleucina-4 , Células Tumorales Cultivadas

RESUMEN

The migratory properties of memory T cells provide a model vector system for site-specific delivery of therapeutic transgene factors to autoimmune inflammatory lesions. Lymph node cells from (SWRxSJL)F1 mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with a DNA construct that placed the anti-inflammatory cytokine interleukin-10 (IL-10) cDNA under control of an antigen-inducible IL-2 promoter region. Isolated T cell clones demonstrated antigen-inducible expression of transgene IL-10 and expressed cell surface markers consistent with the phenotype of normal memory T cells. Upon adoptive transfer, transfected T cell clones were able to inhibit onset of experimental autoimmune encephalomyelitis (EAE) and to treat EAE animals therapeutically after onset of neurologic signs. Semiquantitative immunocytochemistry showed a significant correlation between decreased demyelination and treatment with the transfected T cells. Taken together, these data indicate the autoreactive T cells can be genetically designed to produce therapeutic factors in an antigen-inducible manner resulting in a decreased severity of clinical and histological autoimmune demyelinating disease.

Asunto(s)

Encefalomielitis Autoinmune Experimental/terapia , Terapia Genética , Inmunoterapia Adoptiva , Interleucina-10/genética , Linfocitos T/inmunología , Animales , ADN/genética , Encefalomielitis Autoinmune Experimental/inmunología , Técnicas de Transferencia de Gen , Memoria Inmunológica/genética , Ratones , Ratones Transgénicos

RESUMEN

We have previously used antibodies to the NG2 proteoglycan and the alpha receptor for platelet-derived growth factor (PDGF alpha receptor) to identify oligodendroglial progenitor cells in vivo and in vitro. It has recently become evident that the GD3 antigen, which has been widely used as a marker for oligodendrocyte progenitor cells, is also expressed by microglial cells. In this study we have examined the relationship between the NG2+/PDGF alpha receptor+ glial progenitor cells and microglial cells in normal developing and mature rat brain and in inflammatory lesions in mice with experimental autoimmune encephalomyelitis (EAE). Double-labeling of sections from normal rat brain using anti-NG2 antibodies and lectin from Griffonia simplicifolia (GSA I-B4) or monoclonal antibody 4H1 indicated that there is no overlap between NG2+ glial progenitor cells and microglia in the parenchyma of the central nervous system. In EAE lesions, both NG2+ cells and microglia, identified by antibodies to F4/80 and CD45, displayed reactive changes characterized by increased cell number and staining intensity and shortening and thickening of cell processes. Both cell types were found surrounding perivascular infiltrates of lymphocytes. Double-labeling EAE sections for NG2 and F4/80 or CD45 failed to reveal cells that co-expressed both antigens, suggesting that reactive NG2+ cells are distinct from activated microglia. However, a close spatial relationship between NG2+ cells and microglia was observed in the normal brain and to a greater extent in EAE, where processes of an activated microglial cell were sometimes seen to encircle an NG2+ cell. These observations are indicative of a functional interaction between microglia and the NG2+ glial cells.

Asunto(s)

Encéfalo/citología , Microglía/citología , Neuroglía/citología , Animales , Encéfalo/ultraestructura , Inmunohistoquímica , Ratones , Microscopía Confocal , Ratas

RESUMEN

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.

Asunto(s)

Autoantígenos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Virus de la Parainfluenza 3 Humana/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Humanos , Conformación de Ácido Nucleico , Unión Proteica , ARN Viral/química , ARN Viral/genética , Células Tumorales Cultivadas

RESUMEN

The nucleoside adenosine exerts potent biological effects via specific receptors, including the inhibitory A1 adenosine receptor (A1AR). In the hippocampus A1ARs play an important role in regulating neuronal activity. However, the cellular sites of hippocampal A1ARs are undefined. Using in situ hybridization, receptor autoradiography, and single- and double-label immunocytochemistry techniques, we have characterized the cellular sites of A1AR expression in the rat hippocampus. In situ hybridization and receptor autoradiography studies revealed strikingly different patterns of labeling. In situ hybridization studies revealed heaviest labeling of cell bodies in the granular layer of the dentate gyrus and the pyramidal layers of Ammon's horn. In contrast, using [3H]DPCPX, we observed heavy specific labeling over the neuropil in the dentate hilus stratum moleculare, stratum lacunosum-moleculare, stratum radiatum, and stratum oriens, and little labeling over cell bodies. Using single-label immunocytochemistry, A1AR immunoreactivity was found to be heaviest over fibers in regions corresponding with heavy [3H]DPCPX labeling. Double-label florescent confocal microscopy was then used to determine the identity of labeled fibers. A1AR immunoreactivity was found to co-localize with SMI-31 that labels axons, but not with MAP2a,b that labels cell bodies and dendrites, or with synaptophysin that labels synapses. These data identify axons as the predominant site of A1AR expression in hippocampus. Activation of A1ARs may be a powerful mechanism by which adenosine alters axonal transmission to inhibit neurotransmitter release.

Asunto(s)

Axones/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Receptores Purinérgicos P1/metabolismo , Animales , Autorradiografía , Axones/ultraestructura , ADN/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Fibras Nerviosas/metabolismo , Fibras Nerviosas/ultraestructura , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P1/biosíntesis , Xantinas