RESUMEN
The CBA mouse model was used to investigate the immunopathology induced in the lung by the pathogenic equine herpesvirus 1 (EHV-1) strain RacL11 in comparison to infection with the attenuated vaccine candidate strain KyA. Intranasal infection with KyA resulted in almost no inflammatory infiltration in the lung. In contrast, infection with the pathogenic RacL11 strain induced a severe alveolar and interstitial inflammation, consisting primarily of lymphocytes, macrophages, and neutrophils. Infection with either EHV-1 strain resulted in the accumulation of similar numbers and ratios of CD4 and CD8 T lymphocytes in the lung and bronchoalveolar lavage (BAL) fluid. Further analysis of these T-cell populations revealed identical EHV-1-specific cytotoxic T-lymphocyte responses. RNase protection analysis of RNA isolated from the BAL fluid of RacL11-infected mice on day 3 postinfection revealed much higher levels of RNA specific for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and MIP-2 than were observed for KyA-infected mice. Furthermore, significantly higher levels of transcripts specific for tumor necrosis factor alpha were induced on day 3 postinfection with RacL11 compared with KyA. These findings suggest that the early production of proinflammatory beta chemokines plays a major role in the severe, most often lethal, respiratory inflammatory response induced by the pathogenic EHV-1 strain RacL11.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Herpesvirus Équido 1/patogenicidad , Pulmón/inmunología , Pulmón/patología , Proteínas Inflamatorias de Macrófagos/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Équido 1/inmunología , Vacunas contra Herpesvirus/inmunología , Inflamación/fisiopatología , Ratones , Ratones Endogámicos CBA , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Cutaneous infection in the footpads of C57BL/6 mice with HSV-1 results in an accumulation of activated (CD44high CD25+) CD8+ T cells within the draining popliteal lymph node (PLN). These studies were undertaken to evaluate the frequency and phenotype of the CD8+ T cell population within the PLN, recognizing the single immunodominant HSV-1 epitope derived from the viral envelope glycoprotein, glycoprotein B (gB), using an intracellular IFN-gamma-staining assay. It revealed that approximately 6% of the CD8+ T cells were specific for the gB epitope. Phenotypic analysis of the IFN-gamma-producing gB-specific CD8+ T cells generated in the PLN during the course of the acute infection expressed the CD44high CD25+ phenotype on days 3-5 postinfection. Surprisingly, IFN-gamma-producing CD8+ T cells expressed the CD44high CD25- phenotype on days 5-8 postinfection, in contrast to expectations for a CD8+ effector T cell. IFN-gamma-producing CD25- CD8+ T cells were detected in the PLN on day 21 postinfection, long after infectious virus had been cleared. Throughout the response, the spleen was found to be the major reservoir of gB-specific CD8+ T cells, even during the peak of the response. In contrast to the gB-specific CD8+ T cell population within the PLN, the entire gB-specific CD8+ T cell population within the spleen was CD25-. Collectively, these results suggest the generation of subpopulations of virus-specific CD8+ T cells, distinguished by the expression of CD25, during the acute phase of the primary response to a localized viral infection.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Epítopos de Linfocito T/inmunología , Herpesvirus Humano 1/inmunología , Interferón gamma/biosíntesis , Líquido Intracelular/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/virología , Animales , Antígenos de Superficie/biosíntesis , Linfocitos T CD8-positivos/química , Linfocitos T CD8-positivos/inmunología , Miembro Posterior , Memoria Inmunológica , Inmunofenotipificación , Interferón gamma/análisis , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Líquido Intracelular/virología , Cinética , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Coloración y Etiquetado , Subgrupos de Linfocitos T/química , Subgrupos de Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate-buffered saline (PBS) developed clinical evidence of colitis at 6-8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Th1 and macrophage-derived cytokines including interferon gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-12, and IL-18 lymphotoxin-beta. In addition, message levels and vascular surface expression of ICAM-1, VCAM-1, and MAdCAM-1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Citocinas/biosíntesis , Enfermedades Inflamatorias del Intestino/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Moléculas de Adhesión Celular/análisis , Citocinas/análisis , Modelos Animales de Enfermedad , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/inmunología , Ratones , Ratones SCIDRESUMEN
Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line "JOK-l." These cells express conventional epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherins, beta-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epithelial-like barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line "MDCK" also expresses several epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherin, beta-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.
Asunto(s)
Línea Celular Transformada/citología , Células Epiteliales/citología , Estómago/citología , Transactivadores , Antígenos Transformadores de Poliomavirus/metabolismo , Western Blotting , Cadherinas/metabolismo , Línea Celular Transformada/metabolismo , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos , Humanos , Queratinas/metabolismo , Riñón/citología , Proteínas de la Membrana/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Virus 40 de los Simios/genética , Transfección , Vimentina/metabolismo , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2 , beta CateninaRESUMEN
The antibody responses of CBA/J mice infected intranasally (i.n.) with either the attenuated KyA strain or the pathogenic RacL11 strain of equine herpesvirus 1 (EHV-1) or immunized with recombinant glycoprotein D (rgD) were investigated using the ELISPOT assay to measure EHV-1-specific antibody-secreting cells (ASC) in the regional lymphoid tissue of the respiratory tract. IgG, IgA, and IgM ASC specific for EHV-1 were detected in the mediastinal lymph nodes (MLN) and lungs 2 weeks after i.n. infection with EHV-1 strain KyA or RacL11, or immunization with heat-killed KyA or rgD. EHV-1-specific ASC were present in the MLN and lungs at 4 and 8 weeks, but declined in frequency by fivefold in the lung at 8 weeks. However, i.n. immunized (2 x 10(6) pfu KyA or 50 microgram rgD/mouse) mice infected at 8 weeks with pathogenic EHV-1 RacL11 resisted challenge and showed eight- and tenfold increases in MLN ASC and lung ASC, respectively, by 3 days after challenge. In contrast to the intranasal route of immunization, intraperitoneal immunization yielded ASC frequencies in the MLN and lungs that were only slightly above those of nonimmunized control mice. These data indicate that immunization with infectious or heat-killed EHV-1 KyA, or rgD, induces significant levels of virus-specific ASC both in the MLN and lungs, a specific memory B-cell response, and long-term protective immunity. The finding that the numbers of ASC induced by the pathogenic strain versus the attenuated strain of EHV-1, which were virtually identical, indicated that the ability to generate a B-cell response is independent of and does not contribute to EHV-1 virulence.
Asunto(s)
Anticuerpos Antivirales/análisis , Herpesvirus Équido 1/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Células Productoras de Anticuerpos/citología , Células Productoras de Anticuerpos/inmunología , Células Productoras de Anticuerpos/virología , Linfocitos B/inmunología , Linfocitos B/virología , Femenino , Herpesvirus Équido 1/patogenicidad , Memoria Inmunológica , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/virología , Ganglios Linfáticos/citología , Ganglios Linfáticos/virología , Recuento de Linfocitos , Mesenterio/citología , Mesenterio/virología , Ratones , Ratones Endogámicos CBA , Factores de TiempoRESUMEN
We have shown that C57BL/6-derived CD8(+) CTL specific for an immunodominant herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) determinant express a highly conserved Vbeta10/junctional sequence combination. This extreme T cell receptor beta-chain bias can be used to track the activation of gB-specific CTL in lymph nodes draining the site of HSV-1 infection. In this report we have examined the accumulation of gB-specific CTL in the primary and secondary or recall CTL responses to HSV-1 infection. We found that gB-specific cytolytic activity present within popliteal lymph nodes draining HSV-infected foot-pads peaked at day 5 post-infection during the primary response. As found previously, this correlates with the accumulation of Vbeta10(+)CD8(+) CTL in the activated T cell subset. Lymph node-derived cytotoxicity peaked between days 3 and 4 on secondary challenge with virus and, somewhat surprisingly, was considerably below that seen in the primary response. This reduced gB-specific cytolytic activity mirrored a near absence of Vbeta10(+)CD8(+) T cell enrichment found within the draining lymph nodes during this recall response, consistent with the overall diminution of gB-specific CTL accumulation in this site. Finally, there was a second wave of biased accumulation of Vbeta10(+)CD8(+) activated T cells within the popliteal lymph nodes well after the resolution of infection in both the primary and secondary responses. These results are discussed in terms of preferential activation of virus-specific memory T cells directly in infected tissues during a secondary CTL response at the expense of draining lymphoid organs.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Citotoxicidad Inmunológica , Herpes Simple/virología , Epítopos Inmunodominantes , Memoria Inmunológica , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Piel/inmunología , Células Vero , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Optimal immunological control of cutaneous herpes simplex virus type 1 (HSV-1) infections initiated in the hind footpad of C57BL/6 (B6, H-2b) mice is dependent upon the presence of functional HSV-1-specific T lymphocytes. The class I MHC-restricted, CD8+ T cell subpopulation is involved in the clearance of infectious HSV-1 from the skin and limiting HSV-1 replication and spread within the peripheral nervous system. However, the frequency of HSV-1-specific CTL precursors (CTLp), as a measure of potential anti-viral CD8+ T cell function, is relatively low compared with other acute viral infections. To gain insight into the basis for this low functional frequency, changes in the CD8+ T cell subpopulation phenotype associated with activation and differentiation were investigated. Analysis of the phenotypic changes showed that HSV-1-specific CTLp were found predominantly within a subpopulation of CD8+ T cells expressing high levels of CD44 (CD44high) and high levels of the IL-2 receptor alpha-chain (CD25high). A second activated subpopulation of CD8+ T cells expressing the CD44high CD25low phenotype did not contain detectable HSV-1-specific CTLp, even after the addition of HSV-1-infected stimulator cells as a source of an exogenous Ag. These data suggested that HSV-1-specific CD8+ T cells must increase expression of CD25 before attaining the potential to become CTL effector cells. These findings also indicated that the up-regulation of CD44 alone is not sufficient to identify precisely HSV-1-specific CD8+ T cells.
Asunto(s)
Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Ganglios Linfáticos/inmunología , Células Madre/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Enfermedad Aguda , Animales , Línea Celular , Chlorocebus aethiops , Citotoxicidad Inmunológica , Herpes Simple/patología , Receptores de Hialuranos/biosíntesis , Inmunofenotipificación , Ganglios Linfáticos/patología , Ganglios Linfáticos/virología , Recuento de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-2/análisis , Células Madre/patología , Células Madre/virología , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/virología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virología , Regulación hacia Arriba/inmunologíaRESUMEN
Class I MHC-restricted, HSV-1-specific CD8(+) cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8(+) T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8(+) population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8(+) T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8(+) CD44(hi) CD25(hi) fraction of cells in culture, but, surprisingly, was not detectable in CD8(+) CD44(hi) CD25(lo) T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8(+) CD25(hi) T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Herpesvirus Humano 1/inmunología , Animales , Biomarcadores , Línea Celular Transformada , Chlorocebus aethiops , Citotoxicidad Inmunológica , Citometría de Flujo , Humanos , Receptores de Hialuranos/biosíntesis , Inmunofenotipificación , Activación de Linfocitos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Interleucina-2/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células VeroRESUMEN
The effects of in utero alcohol exposure on neonatal lymphopoiesis were examined in a murine model of fetal alcohol syndrome. At birth, both immature and mature B cells were decreased in the spleens of neonatal animals and these subpopulations of B cells did not recover to normal levels until 3-4 weeks of life. Pre-B cells and total B cells were decreased as well in the bone marrow of ethanol-exposed animals. By 3-4 weeks of life, the number of B cells in the bone marrow recovered to normal levels, but the pre-B cells remained below normal levels through 5 weeks of age. Furthermore, a recently described early B cell progenitor was reduced in frequency in ethanol-exposed neonates. Together, these data suggest that in utero exposure to ethanol can result in abnormalities in B cell development that may initiate at an early stage of B cell development.
Asunto(s)
Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Trastornos del Espectro Alcohólico Fetal/inmunología , Hematopoyesis , Bazo/inmunología , Animales , Animales Recién Nacidos , Linfocitos B/citología , Biomarcadores , Células de la Médula Ósea/citología , Linaje de la Célula , Femenino , Células Madre Hematopoyéticas , Ratones , Embarazo , Bazo/citologíaRESUMEN
Although neutrophils have been implicated in the hepatic injury elicited by gut ischemia/reperfusion (I/R), the contribution of other leukocyte populations to this injury process remains unclear. The objective of this study was to determine whether lymphocytes contribute to gut I/R-induced microvascular dysfunction and inflammatory responses in the liver. Intravital videomicroscopy was used to monitor leukocyte recruitment, the number of nonperfused sinusoids and pyridine nucleotide (NADH) autofluorescence in livers of wild-type, SCID, and interferon-gamma (IFN-gamma) knockout mice exposed to 15 min of gut ischemia and 1 h of reperfusion. In wild-type mice, gut I/R elicited significant increases in the number of stationary leukocytes, nonperfused sinusoids, NADH autofluorescence (indicating hypoxia), and elevated plasma alanine aminotransferase (ALT) and TNF-alpha levels. All of these responses were profoundly attenuated in SCID mice, while only some of the responses (in the midzonal region) were blunted in IFN-gamma knockout mice. Reconstitution (24 h before ischemia) of the circulating lymphocyte pool with T-cell enriched splenocytes, but not T cell deficient (from nude mice), CD4+ T-cell depleted splenocytes or splenocytes derived from IFN-gamma knockout mice, allowed the SCID mice to respond to gut I/R in a manner similar to wild-type mice. Some of the responses were restored following reconstitution with CD8+ T-cell depleted splenocytes. These findings implicate CD4+ T-lymphocytes and IFN-gamma in the hepatic microvascular dysfunction and inflammatory cell accumulation elicited by gut I/R.
Asunto(s)
Sistema Digestivo/irrigación sanguínea , Hipoxia/etiología , Leucostasis/etiología , Hígado/irrigación sanguínea , Daño por Reperfusión/etiología , Linfocitos T/fisiología , Animales , Interferón gamma/sangre , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The ability of recombinant preparations of equine herpesvirus type 1 (EHV-1) glycoprotein D (gD) to elicit specific antibody and T lymphocyte responses in the BALB/c mouse model of respiratory infection was investigated. Recombinant gD (rgD) expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli elicited both high titer neutralizing antibody (nAb) and CD4 T cell proliferative responses following subcutaneous or intranasal immunization, but elicited only a weak antibody response after intraperitoneal immunization. Protection against respiratory tract infection with pathogenic EHV-1 RacL11 was observed in mice immunized subcutaneously with GST-gD. Furthermore, the degree of protection correlated to the titer of nAb and the T cell response observed. Finally, GST-gD was more effective in protecting against respiratory RacL11 infection if delivered intranasally. These results confirm that gD plays an important role in eliciting the protective immune response against EHV-1 infection, and indicate that subunit vaccines containing preparations of gD may be very effective if delivered directly to the upper respiratory tract.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Herpesvirus Équido 1/inmunología , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Clonación Molecular , Femenino , Herpesvirus Équido 1/aislamiento & purificación , Caballos , Ratones , Ratones Endogámicos BALB C , Infecciones del Sistema Respiratorio/inmunología , Vacunación , Proteínas Virales de Fusión/genéticaRESUMEN
A low-molecular-weight recombinant Brucella abortus protein reactive with antibodies from a variety of naturally and experimentally infected hosts and T lymphocytes from experimentally infected mice was identified and given the designation BA14K. The gene encoding BA14K was cloned and characterized, and the predicted amino acid sequence of this immunoreactive protein showed no significant homology with previously described proteins. Sequences homologous to the cloned fragment encoding BA14K were identified by Southern blot analysis of genomic DNAs from representatives of all of the currently recognized Brucella species. Studies employing BA14K should contribute to our efforts to better understand the antigenic specificity of protective immunity to brucellosis.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucelosis/inmunología , Proteínas Recombinantes/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Secuencia de Bases , Brucella abortus/inmunología , ADN Bacteriano , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia MolecularRESUMEN
The cytolytic T-lymphocyte (CTL) response to respiratory infection with equine herpesvirus 1 (EHV-1) in CBA (H-2(k)) mice was investigated. Intranasal (i.n.) inoculation of mice with the attenuated EHV-1 strain KyA resulted in the generation of a primary virus-specific CTL response in the draining mediastinal lymph nodes 5 days following infection. EHV-1-specific CTL could be restimulated from the spleen up to 26 weeks after the resolution of infection, indicating that a long-lived memory CTL population was generated. Depletion of CD8+ T cells by treatment with antibody and complement prior to assay eliminated CTL activity from both primary and memory populations, indicating that cytolytic activity in this model was mediated by class I major histocompatibility complex-restricted, CD8+ T cells. A single i.n. inoculation with KyA induced protective immunity against infection with the pathogenic EHV-1 strain, RacL11. The adoptive transfer of splenocytes from KyA-immune donors into sublethally irradiated recipients resulted in a greater than 250-fold reduction in RacL11 in the lung. The elimination of both CD4+ and CD8+ T cells from the transferred cells abrogated clearance of RacL11, while the selective depletion of either subpopulation alone had little effect. These results suggested that both lymphocyte subpopulations contribute to viral clearance, with either subpopulation alone being sufficient.
Asunto(s)
Herpesvirus Équido 1/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas Virales/inmunología , Traslado Adoptivo , Animales , Femenino , Memoria Inmunológica , Ratones , Ratones Endogámicos CBARESUMEN
Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/efectos de los fármacos , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/efectos de los fármacos , Interleucina-7/farmacología , Linfocitos T/inmunología , Animales , Células de la Médula Ósea , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inmunofenotipificación , Interleucina-3/farmacología , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , ARN Mensajero/genética , Factor de Células Madre/farmacología , Transcripción GenéticaRESUMEN
The equine herpesvirus type-1 (EHV-1) strain Kentucky A (KyA) has a long history of repeated passage either in vivo in the Syrian hamster or in vitro in mouse L-M fibroblast tissue culture. This repeated passage in cells other than those of the natural host has caused genomic alterations of the KyA chromosome resulting in deletion of several genes or portions of open reading frames (ORFs). This report presents in vivo data from a mouse model of EHV-1 infection demonstrating the attenuated nature of EHV-1 strain KyA and that intranasal infection with KyA protects animals from subsequent challenge with a pathogenic strain, RacL, by reducing RacL viral titers in the lungs of the challenged animals. Mice infected with KyA exhibit no clinical manifestations of EHV-1 disease and do not experience the wasting that occurs with RacL infection. KyA-infected mice clear virus from the lung by day 5 post-infection (p.i.), whereas RacL infected mice have substantial virus titers (5 x 10(5) pfu/lung) at this time point. Intranasal infection with KyA followed by a challenge with RacL 4 weeks post-KyA infection resulted in a significant (P = 0.0079) reduction in the lung titers of the RacL virus. RacL was identified as the virus present in the lungs of the challenged mice by a PCR assay employing primers to amplify the EUS4 gene which differs in size by 1.2 kilobase pairs (kbp) in the two strains. Importantly, the protection afforded by KyA is long lasting in that challenge with RacL 15 months after KyA infection, results in reduced virus titers and viral clearance by day 5 post-challenge. These results support the further consideration of EHV-1 KyA as a live virus vaccine.
Asunto(s)
Infecciones por Herpesviridae/prevención & control , Herpesvirus Équido 1/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Línea Celular , ADN Viral/análisis , Modelos Animales de Enfermedad , Femenino , Herpesvirus Équido 1/genética , Herpesvirus Équido 1/patogenicidad , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Conejos , Factores de TiempoRESUMEN
The immune response to HSV infection in C57BL/6 mice includes a CTL population that recognizes the virion envelope glycoprotein gB. However, previous studies showed that CTL specific for other viral determinants were also responding to HSV infection. These studies demonstrate that an additional determinant is the HSV immediate-early protein ICP27. During the primary response, both gB- and ICP27-specific CTL were detected in the draining lymph node. In response to reinfection, ICP27-specific CTL were present early in the lymph node, while the appearance of gB-specific CTL activity was delayed. Analysis of the primary amino acid sequence of ICP27 predicted two potential Kb-binding epitopes, one of which sensitized uninfected cells for lysis by HSV-specific CTL. In addition, ICP27 epitope-specific CTL activity was detected in the splenic memory CTL pool. These results show that CTL which recognize different antigens may also exhibit differences in how they respond to HSV reinfection in vivo.
Asunto(s)
Herpes Simple/inmunología , Proteínas Inmediatas-Precoces , Memoria Inmunológica/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Epítopos/inmunología , Inmunización , Inmunización Secundaria , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Bazo/citología , Células Vero , Proteínas Virales/inmunologíaRESUMEN
Previous studies (C. C. Flowers and D. J. O'Callaghan, 1992, Virology 190, 307-315) employed peptide-specific antibodies to identify the product of the glycoprotein D (gD) gene of equine herpesvirus 1 strain Kentucky A (KyA). gD polypeptides of 55 and 58 kDa were detected in EHV-1-infected L-M cells, and the 58-kDa protein was observed in the membrane fraction of EHV-1 virions. In this report, the kinetics of synthesis and processing of gD polypeptides are described. One-hour pulse-labeling of EHV-1-infected L-M cells revealed that gD proteins are first detected at 6 hr after infection and that maximal synthesis of gD occurs between 5 and 8 hr postinfection. gD polypeptides accumulate progressively with time of infection as shown by immunoprecipitation analysis of gD proteins. Pulse-chase analysis of gD revealed that the 55-kDa protein is a precursor to the 58-kDa species and that processing of all pulse-labeled precursor protein requires approximately 2.5 hr. Analysis of the carbohydrate content of gD proteins, as judged by their sensitivity to digestion with endoglycosidases, revealed that the 55-kDa gD precursor contains high-mannose N-linked oligosaccharides, while the 58-kDa gD mature polypeptide possesses complex type oligosaccharides. Expression of the mature form of gD on the cell surface, as determined by fluorescent flow cytometric analysis, is delayed compared to the accumulation of the mature form of gD within the cell. The gD ORF encodes a potential protein of 442 amino acids but analysis of the translated sequence of gD indicated that the gD polypeptide is 392 amino acids, a size predicted by previous mapping of the transcription start site of the gD mRNA. Coupled in vitro transcription/translation of a pGEM-3Z construct containing the 392-amino-acid gD ORF, in the absence or presence of canine pancreatic microsomes, demonstrated that the 43-kDa gD polypeptide undergoes processing in vitro. These studies demonstrate that the EHV-1 strain KyA gD is processed in a fashion similar to that of the gD proteins of other alphaherpesviruses.
Asunto(s)
Herpesvirus Équido 1/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Citometría de Flujo , Regulación Viral de la Expresión Génica , Células L , Ratones , Plásmidos , Procesamiento Proteico-Postraduccional , Transfección , Proteínas del Envoltorio Viral/genéticaRESUMEN
The effect of intrauterine exposure to ethanol on lymphocyte development in the neonatal period was studied in C57BI/6J mice. Mice were bred, and then the female mice were assigned to 1 of 3 diet groups, 25% ethanol-derived calories (EDC), pair-fed control, or ad libitum laboratory chow. At birth, all offspring were cross-fostered to surrogate mothers who had been fed laboratory chow. At weekly intervals, the neonatal mice were weighed, and 4 mice from each group were used to assess the development of splenic lymphocytes. The total number of splenocytes was similar in all three groups at each sampling. The number of T-cells, B-cells, and natural killer (NK) cells was measured by flow cytometry. T-cells and NK cells did not vary significantly among the three diet groups. However, the total number of B-cells was decreased for the first 3 weeks of life in the ethanol-exposed animals. The function of the T-cells and B-cells was determined by assessing the response to lipopolysaccharide, pokeweed mitogen, phytohemagglutinin, and concanavalin A. The response to all four mitogens was significantly reduced in the ethanol-exposed animals and did not recover to control levels until 4-5 weeks of life. Ethanol exposure had no significant effect on the kinetics of acquisition of NK lytic function, as assessed by determining the killing of chromium-51 labeled YAC-1 tumor target cells. These data show that prenatal exposure to ethanol causes a transient immunodeficiency in some, but not all compartments of the immune system.
Asunto(s)
Linfocitos B/inmunología , Trastornos del Espectro Alcohólico Fetal/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos/inmunología , Línea Celular , Citotoxicidad Inmunológica/inmunología , Femenino , Tolerancia Inmunológica/fisiología , Leucemia Experimental , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Ratones , EmbarazoRESUMEN
Analyses of the synthesis and processing of recombinant full-length glycoprotein D of equine herpesvirus type 1 (EHV-1; gD392) or recombinant truncated gD (gD352) expressed in baculovirus-infected Sf9 cells revealed the following: (1) gD polypeptides encoded by both recombinant baculoviruses react with gD-specific antibodies including peptide-specific antiserum that neutralizes EHV-1 in a plaque reduction assay, (2) both the full-length recombinant gD392 and the truncated gD352 are expressed predominantly as gD species that contain high mannose-type oligosaccharides (55 kDa and 52 kDa, respectively), (3) both the full-length recombinant gD392 and the truncated gD352 are also expressed in lesser amounts as gD species that contain complex-type oligosaccharides (58 kDa and 55 kDa, respectively) as well as the unglycosylated forms of gD (43 kDa and 37 kDa, respectively), (4) flow cytometric analyses of cells expressing gD392 revealed that gD first appears on the cell surface at 24 h post infection; by 60 h, 95% of the cells express high levels of cell surface gD, (5) cells expressing gD352, in contrast to cells expressing gD392, secrete gD into the extracellular medium. This initial demonstration that immunoreactive EHV-1 glycoprotein D can be produced as a secreted polypeptide in the baculovirus system should provide reagents to assess the potential use of gD as a subunit vaccine in an animal model.
Asunto(s)
Vectores Genéticos/genética , Herpesvirus Équido 1/genética , Proteínas de la Membrana/biosíntesis , Nucleopoliedrovirus/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas del Envoltorio Viral/biosíntesis , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Glicosilación , Herpesvirus Équido 1/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Oligosacáridos/análisis , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The immune response to herpes simplex virus type 1 (HSV-1) infection in C57BL/6 mice includes a population of major histocompatibility complex class I-restricted cytolytic T lymphocytes (CTL) that recognize the structural glycoprotein gB. To gain insight into the importance of this CTL subpopulation in vivo, gB-specific CTL present in the regional lymph nodes after a primary infection and after a reinfection of convalescent animals were analyzed. In a primary infection, gB-specific CTL precursors (CTLp) that recognized either a cell line constitutively expressing gB or cells pulsed with the optimal Kb-restricted gB epitope 498SSIEFARL505 were present at an estimated frequency of 1/12,000 compared with a frequency of 1/3,000 for CTLp which recognized cells infected with HSV-1 itself. In convalescent mice responding to reinfection, HSV-specific CTLp were present at an estimated frequency of 1/4,000 to 1/14,000. However, gB-specific CTLp could not be detected at this site. These findings suggest that CTL specific for an immunodominant epitope contribute substantially to the primary response but may not be a component of the HSV-specific CTL population that responds rapidly to reinfection in vivo.