RESUMO
The function and structure of the TCR proteins of intraepithelial lymphocytes (IEL) were examined using a panel of mAbs specific for TCR-gamma/delta. Three subsets of TCR-gamma/delta+ IEL could be detected with five mAbs, termed GL1-GL5. The mAbs were able to trigger lysis via crosslinking of the IEL TCR and all of the subsets were constitutively cytolytic. Immunoprecipitation of IEL TCR proteins revealed that the GL2 mAb reacted only with gamma, delta heterodimers containing high Mr delta chains, while the other mAbs precipitated all of the observed gamma and delta proteins. Two-color fluorescence analysis showed that the GL2+ subset was contained within the larger GL1+ subset. The GL3 and GL4 mAbs appear to be specific for all TCR-gamma/delta while GL2 was V delta 4 specific. Analysis of IEL for TCR-alpha/beta expression demonstrated that approximately 20% of B6 IEL were TCR-alpha/beta+. Interestingly, this population of IEL contained Thy-1- and CT1+ cells, indicating that the unique phenotype of IEL was not restricted to TCR-gamma/delta+ cells. Moreover, the TCR-alpha/beta+ IEL were also constitutively cytolytic, suggesting that the intestinal milieu was controlling the functional programming of IEL regardless of TCR type. The mAbs reported here as well as the ability to exploit the distinct phenotype of IEL should prove useful in determining the function of IEL and the TCR-gamma/delta.
Assuntos
Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Superfície , Ligação Competitiva , Células Epiteliais , Epitélio/imunologia , Citometria de Fluxo , Mucosa Intestinal/citologia , Camundongos , Peso Molecular , Receptores de Antígenos de Linfócitos T gama-delta , Antígenos Thy-1RESUMO
We have investigated which T cell subclass defined by cytolysis with monoclonal anti-Lyt-1.2 and anti-Lyt-2.2 antibodies is required to adoptively transfer the ability to reject skin grafts. B6.Thy-1.1 spleen cells immune to graft antigens were fractionated with antibody plus C' and transferred to adult thymectomized, irradiated, bone marrow-reconstituted (ATXBM) B6.Thy-1.2 hosts that were simultaneously grafted with BALB.B skin. We found that when the ATXBM hosts were used 6 wk after irradiation and marrow reconstitution, both Lyt-1-depleted and Lyt-2-depleted immune spleen cells could transfer the ability to promptly reject skin grafts. However, such ATXBM recipients of Lyt-2-depleted cells that had rejected skin grafts were found to contain graft-specific CTL that were largely of host (B6.Thy-1.2) origin. When ATXBM hosts were used for the experiment 1 wk after irradiation and marrow reconstitution, no host-derived graft-specific CTL could be detected. However, graft rejection occurred in recipients of anti-Lyt-1- or anti-Lyt-2 plus C'-treated immune cells and specific CTL were generated from spleen cells of both groups. Thus, in the absence of a host-derived response, adoptively transferred immune Lyt-2+ cells, either resistant to, or that escaped from, antibody plus C' treatment, are able to expand in response to the antigenic stimulus provided by the graft. A more complete elimination of specific T cell subclasses is therefore needed to assess the relative contribution of a particular subset to the graft rejection process.
Assuntos
Antígenos Ly/imunologia , Rejeição de Enxerto , Transplante de Pele , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Ly/genética , Transplante de Medula Óssea , Citotoxicidade Imunológica , Epitopos , Imunização Passiva , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quimera por Radiação , Linfócitos T Citotóxicos/fisiologiaRESUMO
The CT antigenic determinants have previously been shown to be present on the T200 glycoproteins and other proteins of murine cytotoxic T cell clones but not of T helper clones or nonactivated lymphocytes (1, 2). Two determinants recognized by mAbs CT1 and CT2 are also expressed on thymocytes in a developmentally regulated fashion during fetal thymus ontogeny and are found in a subset of Lyt-2+ intraepithelial lymphocytes in the intestinal mucosa (3-5). Previous studies of the biosynthesis of CT+ proteins suggested that these determinants were composed of carbohydrate (8). We now demonstrate that the anti-CT mAbs react with a carbohydrate determinant at the nonreducing terminus of O-linked oligosaccharides that has the configuration GalNAc beta 1,4[SA alpha 2,3]-galactose. The CT antibodies detected this determinant not only on CTL clones but also in the human blood group antigens Cad and Sda+. Variant CTL lines, non-Cad erythrocytes, and Sda- glycoproteins that lacked the GalNAc residue did not bind the CT mAb. Sialic acid was essential for CT antigen expression since neuraminidase or mild periodate treatment abrogated CT antibody binding. In addition, other carbohydrate structures with terminal GalNAc residues such as the A or Tn blood group antigens were not recognized. The CT antibodies thus define GalNAc and sialic acid containing carbohydrate antigens that are expressed on discrete subsets of T lymphocytes and may also be useful reagents for the detection of Cad and Sda+ blood group antigens.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Carboidratos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Especificidade de Anticorpos , Reações Cruzadas , Epitopos/imunologia , Humanos , Camundongos , Ácido N-Acetilneuramínico , Ácidos Siálicos/imunologiaRESUMO
The role of CD40 ligand (CD40L) in CD8 T cell activation was assessed by tracking antigen-specific T cells in vivo using both adoptive transfer of T cell receptor transgenic T cells and major histocompatibility complex (MHC) class I tetramers. Soluble antigen immunization induced entry of CD8 cells into the intestinal mucosa and cytotoxic T lymphocyte (CTL) differentiation, whereas CD8 cells in secondary lymphoid tissue proliferated but were not cytolytic. Immunization concurrent with CD40L blockade or in the absence of CD40 demonstrated that accumulation of CD8 T cells in the mucosa was CD40L dependent. Furthermore, activation was mediated through CD40L expressed by the CD8 cells, since inhibition by anti-CD40L monoclonal antibodies occurred after adoptive transfer to CD40L-deficient mice. However, mucosal CD8 T cells in normal and CD40(-/-) mice were equivalent killers, indicating that CD40L was not required for CTL differentiation. Appearance of virus-specific mucosal, but not splenic, CD8 cells also relied heavily on CD40-CD40L interactions. The mucosal CTL response of transferred CD8 T cells was MHC class II and interleukin 12 independent. The results established a novel pathway of direct CD40L-mediated CD8 T cell activation.
Assuntos
Antígenos CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Ligante de CD40 , Divisão Celular , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Mucosa Intestinal/imunologia , Linfonodos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Knockout , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Cytotoxic T cell (CTL)-specific activation antigens, termed CT determinants, have been detected by monoclonal antibodies (mAb) that inhibit CTL function. At the cell surface, the CT antigens are associated with the T200 glycoproteins and two other proteins of Mr 140,000 and 85,000 and are present on a secreted protein, gp155. Periodate treatment followed by binding analysis and immunoprecipitation experiments using tunicamycin-treated cells indicated that carbohydrate is necessary for CT antigen expression. Furthermore, gp155 is secreted in the presence of tunicamycin while retaining the CT antigens, and the CT determinants are added late in T200 biosynthesis, suggesting that the CT glycans are O-linked. Finally, interleukin 2 was shown to dramatically influence the expression of the CT mAb-reactive oligosaccharides present at the CTL cell surface.
Assuntos
Antígenos de Superfície/biossíntese , Carboidratos/análise , Epitopos/análise , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/análise , Glicoproteínas/biossíntese , Interleucina-2/fisiologia , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Peso Molecular , Linfócitos T Auxiliares-Indutores/imunologia , Tunicamicina/farmacologiaRESUMO
The leukocyte common antigen, T200, is expressed on all white blood cells but not on other differentiated cells. Within the hematopoietic lineage, specific cell types display characteristic structural forms of the molecule on their surface. We show that murine cytotoxic T lymphocyte clones and helper T cell clones contain different size mRNA for this molecule, and that the early precursors of T200 glycoprotein made in the helper and cytotoxic T cells differ in Mr. Thus, in addition to differences in posttranslational modifications, it is highly likely that a difference in protein structure contributes to the distinct forms of T200 glycoprotein found on these functional T cell subsets.
Assuntos
Antígenos de Histocompatibilidade/genética , Linfócitos T/fisiologia , Animais , Linhagem Celular , Glicoproteínas/genética , Glicoproteínas/imunologia , Antígenos Comuns de Leucócito , Ativação Linfocitária , Camundongos , RNA Mensageiro/genética , Linfócitos T/classificação , Linfócitos T Citotóxicos/fisiologia , Linfócitos T Auxiliares-Indutores/fisiologiaRESUMO
gamma/delta cells were at least four times more frequent in lactating mouse mammary glands than among T cells of the most proximal lymph nodes. Two-color staining of freshly isolated T cells and a study of clonally expressed gamma/delta receptors on hybridomas further revealed that the mammary gamma/delta population is heterogeneous, including at least three different subsets, among them cells expressing V gamma 5, V gamma 4 together with V delta 4, or none of these V regions.
Assuntos
Lactação , Linfócitos/imunologia , Glândulas Mamárias Animais/imunologia , Receptores de Antígenos de Linfócitos T/análise , Linfócitos T/imunologia , Animais , Sequência de Bases , Feminino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
It has generally been assumed that most if not all CTL specific for vesicular stomatitis virus (VSV)-infected cells recognize the viral glycoprotein (G), an integral membrane protein abundantly expressed on infected cell surfaces. Using recombinant vaccinia viruses containing copies of cloned VSV genes to examine CTL recognition of VSV, we have confirmed that G is recognized by VSV-specific CTL. More interestingly, however, we have also found that nucleocapsid protein (N), an internal virion protein, can be detected on infected cell surfaces using mAb, and serves as a major target antigen for VSV-specific CTL. In contrast to the highly serotype-specific recognition of G, N is recognized by a major population of CTL able to lyse cells infected with either the Indiana or New Jersey VSV serotypes. Using target cells expressing a cloned MHC class I gene, we could directly show that CTL recognition of N occurs in the context of the MHC Ld molecule.
Assuntos
Capsídeo/imunologia , Vírus da Influenza A/imunologia , Glicoproteínas de Membrana , Linfócitos T Citotóxicos/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vesiculovirus , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Capsídeo/genética , Linhagem Celular , Sarcoma de Mastócitos , Camundongos , Camundongos Endogâmicos DBA , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vaccinia virus/genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Core Viral/genética , Proteínas Virais/genéticaRESUMO
The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.
Assuntos
Enterócitos/imunologia , Interleucina-7/imunologia , Intestino Delgado/imunologia , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/imunologia , Animais , Proteínas de Transporte/genética , Células Dendríticas/imunologia , Células Epidérmicas , Epiderme/imunologia , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Centro Germinativo/citologia , Interleucina-7/genética , Intestino Delgado/citologia , Camundongos , Camundongos Transgênicos , Proteína P2 de Mielina/genética , Nódulos Linfáticos Agregados/citologia , Proteínas Recombinantes de Fusão/imunologia , Distribuição Tecidual , TransgenesRESUMO
The requirement of beta7 integrins for lymphocyte migration was examined during an ongoing immune response in vivo. Transgenic mice (OT-I) expressing an ovalbumin-specific major histocompatibility complex class I-restricted T cell receptor for antigen were rendered deficient in expression of all beta7 integrins or only the alphaEbeta7 integrin. To quantitate the relative use of beta7 integrins in migration in vivo, equal numbers of OT-I and OT-I-beta7(-/-) or OT-I-alphaE-/- lymph node (LN) cells were adoptively transferred to normal mice. Although OT-I-beta7(-/-) LN cells migrated to mesenteric LN and peripheral LN as well as wild-type cells, beta7 integrins were required for naive CD8 T cell and B cell migration to Peyer's patch. After infection with a recombinant virus (vesicular stomatitis virus) encoding ovalbumin, beta7 integrins became critical for migration of activated CD8 T cells to the mesenteric LN and Peyer's patch. Naive CD8 T cells did not enter the lamina propria or the intestinal epithelium, and the majority of migration of activated CD8 T cells to the small and large intestinal mucosa, including the epithelium, was beta7 integrin-mediated. The alphaEbeta7 integrin appeared to play no role in migration during a primary CD8 T cell immune response in vivo. Furthermore, despite dramatic upregulation of alphaEbeta7 by CD8 T cells after entry into the epithelium, long-term retention of intestinal intraepithelial lymphocytes was also alphaEbeta7 independent.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cadeias beta de Integrinas , Integrinas/imunologia , Linfonodos/imunologia , Animais , Linfócitos T CD8-Positivos/virologia , Movimento Celular , Camundongos , Camundongos Transgênicos , Ovalbumina , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Regulação para Cima/imunologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
The present study examined mechanisms of tolerance for T cell receptor gamma/delta (TCR-gamma/delta) cells. Using a transgenic (Tg) model, we demonstrate that although alloantigen (Ag)-specific TCR-gamma/delta cells are deleted in the thymus and spleen of Ag-bearing mice, intraepithelial lymphocytes (IELs) expressing normal levels of the Tg TCR were present. However, Tg+ IELs from Ag-bearing mice were unresponsive to activation. Furthermore, self-reactive Tg+ IELs decreased in number over time. Thus, in epithelial tissue, Tg TCR-gamma/delta cells are eliminated subsequent to and most likely as a result of the induction of clonal anergy.
Assuntos
Tolerância Imunológica , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Cultivadas , Cruzamentos Genéticos , Feminino , Antígenos H-2/imunologia , Isoantígenos/imunologia , Ativação Linfocitária , Linfocinas/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/imunologia , Timo/imunologiaRESUMO
Although the functional aspects of the alpha beta T cell antigen receptor (TCR) found on most peripheral T cells are well described, the function of the gamma delta TCR remains unclear. Murine intraepithelial lymphocytes (IEL) of the small intestine are CD8+, express the gamma delta TCR, and are constitutively lytic. Fresh IEL from germ-free mice had no lytic activity. Moreover, whereas IEL from normal mice are 30 to 50 percent Thy-1+, IEL from germ-free did not express Thy-1. Acclimation of germ-free mice to nonsterile conditions resulted in the generation of Thy-1+ IEL and induction of lytic activity. Thus CD8+ TCR-gamma delta IEL were regulated by externally derived stimuli via a specific functional interaction between IEL and gut-associated antigens.
Assuntos
Antígenos de Superfície/análise , Citotoxicidade Imunológica , Intestino Delgado/citologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Superfície/imunologia , Antígenos CD8 , Células Epiteliais , Vida Livre de Germes , Técnicas de Imunoadsorção , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/análise , Transdução de Sinais , Antígenos Thy-1RESUMO
Many intracellular pathogens infect a broad range of host tissues, but the importance of T cells for immunity in these sites is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Here, we show that in response to viral or bacterial infection, antigen-specific CD8 T cells migrated to nonlymphoid tissues and were present as long-lived memory cells. Strikingly, CD8 memory T cells isolated from nonlymphoid tissues exhibited effector levels of lytic activity directly ex vivo, in contrast to their splenic counterparts. These results point to the existence of a population of extralymphoid effector memory T cells poised for immediate response to infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Listeriose/imunologia , Infecções por Rhabdoviridae/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Movimento Celular , Células Cultivadas , Citometria de Fluxo , Antígenos H-2/imunologia , Intestino Delgado/imunologia , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Fígado/imunologia , Pulmão/imunologia , Ativação Linfocitária , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fenótipo , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Dysregulation of growth factors and their receptors is central to human hepatocellular carcinoma (HCC). We previously demonstrated that the Frizzled-7 membrane receptor mediating the Wnt signalling can activate the beta-catenin pathway and promotes malignancy in human hepatitis B virus-related HCCs. Expression patterns of all the 10 Frizzled receptors, and their extracellular soluble autoparacrine regulators (19 Wnt activators and 4 sFRP inhibitors) were assessed by real-time RT-PCR in 62 human HCC of different etiologies and their matched peritumorous areas. Immunostaining was performed to localise Frizzled on cell types in liver tissues. Regulation of three known Frizzled-dependent pathways (beta-catenin, protein kinase C, and C-Jun NH(2)-terminal kinase) was measured in tissues by western blot. We found that eight Frizzled-potentially activating events were pleiotropically dysregulated in 95% HCC and 68% peritumours as compared to normal livers (upregulations of Frizzled-3/6/7 and Wnt3/4/5a, or downregulation of sFRP1/5), accumulating gradually with severity of fibrosis in peritumours and loss of differentiation status in tumours. The hepatocytes supported the Wnt/Frizzled signalling since specifically overexpressing Frizzled receptors in liver tissues. Dysregulation of the eight Frizzled-potentially activating events was associated with differential activation of the three known Frizzled-dependent pathways. This study provides an extensive analysis of the Wnt/Frizzled receptor elements and reveals that the dysregulation may be one of the most common and earliest events described thus far during hepatocarcinogenesis.
Assuntos
Carcinoma Hepatocelular/genética , Receptores Frizzled/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Receptores Frizzled/biossíntese , Regulação da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologiaRESUMO
Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A 'murinized' Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the 'gut-homing' integrin α4ß7 and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27- Ly6C- and CD69+ CD103- while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69-. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.
Assuntos
Memória Imunológica , Mucosa Intestinal/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Células Th1/imunologia , Administração Oral , Animais , Antígenos CD/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/imunologia , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Integrina alfa4/metabolismo , Cadeias beta de Integrinas/metabolismo , Mucosa Intestinal/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Retorno de Linfócitos/metabolismoRESUMO
Sal-like protein 4 (SALL4), an embryonic stem cell transcriptional regulator, is re-expressed by an unknown mechanism in poor prognosis hepatocellular carcinoma (HCC), often associated with chronic hepatitis B virus (HBV) infection. Herein, we investigated the mechanism of SALL4 re-expression in HBV-related HCCs. We performed bisulfite sequencing PCR of genomic DNA isolated from HBV-related HCCs and HBV replicating cells, and examined DNA methylation of a CpG island located downstream from SALL4 transcriptional start site (TSS). HBV-related HCCs expressing increased SALL4 exhibited demethylation of specific CpG sites downstream of SALL4 TSS. Similarly, SALL4 re-expression and demethylation of these CpGs was observed in HBV replicating cells. SALL4 is also re-expressed in poor prognosis HCCs of other etiologies. Indeed, increased SALL4 expression in hepatitis C virus-related HCCs correlated with demethylation of these CpG sites. To understand how CpG demethylation downstream of SALL4 TSS regulates SALL4 transcription, we quantified by chromatin immunoprecipitation (ChIP) assays RNA polymerase II occupancy of SALL4 gene, as a function of HBV replication. In absence of HBV replication, RNA polymerase II associated with SALL4 exon1. By contrast, in HBV replicating cells RNA polymerase II occupancy of all SALL4 exons increased, suggesting CpG demethylation downstream from SALL4 TSS influences SALL4 transcriptional elongation. Intriguingly, demethylated CpGs downstream from SALL4 TSS are within binding sites of octamer-binding transcription factor 4 (OCT4) and signal transducer and activator of transcription3 (STAT3). ChIP assays confirmed occupancy of these sites by OCT4 and STAT3 in HBV replicating cells, and sequential ChIP assays demonstrated co-occupancy with chromatin remodeling BRG1/Brahma-associated factors. BRG1 knockdown reduced SALL4 expression, whereas BRG1 overexpression increased SALL4 transcription in HBV replicating cells. We conclude demethylation of CpGs located within OCT4 and STAT3 cis-acting elements, downstream of SALL4 TSS, enables OCT4 and STAT3 binding, recruitment of BRG1, and enhanced RNA polymerase II elongation and SALL4 transcription.
Assuntos
Carcinoma Hepatocelular/patologia , Metilação de DNA , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Neoplasias Hepáticas/patologia , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Ilhas de CpG/genética , DNA Helicases/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Most studies of injury and repair to mucosal tissue have used nonspecific mediators to induce injury. Damage to the mucosal epithelium resulting from chemical or radiation treatment associated with cancer therapy may fall into this category of injury. When such treatments are applied, it is generally not possible to predict or control the extent of possible injury. This fact makes analysis of inductive and reparative processes difficult. In addition, the role of the immune system in the etiology and subsequent healing of mucosal tissue following cancer therapy with or without bone marrow transplantation remains unclear. To study tissue- and antigen-specific immune damage of intestinal mucosal tissue, we generated transgenic mice that express a nominal antigen exclusively in intestinal epithelial cells. The transfer of antigen-specific CD8 T cells with concomitant virus infection resulted in the destruction of intestinal epithelial cells and disease. The destructive phase in some cases was followed by complete recovery and tolerance induction. This model will provide a system that can be regulated for analysis of the mediators of mucosa-specific tissue damage and repair.
Assuntos
Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Camundongos Transgênicos , Animais , Peso Corporal , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , DNA Complementar/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Citometria de Fluxo , Doenças Inflamatórias Intestinais/patologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucosa/metabolismo , RNA/metabolismo , Transgenes/genéticaRESUMO
We have investigated the effect of 5'-guanylylimidodiphosphate (Gpp(NH)p) and two disulfide bond reducing agents, reduced glutathione (GSH) and dithiothreitol (DTT), on the modulation of [125I-Tyr10]hGRF(1-44)NH2 binding to GRF receptor binding sites, in pituitaries of young and aging rats. In pituitaries from 2-month-old rats, Gpp(NH)p (0.1-1.0 mM), GSH and DTT (1-50 mM) exhibited a partial but concentration-dependent inhibitory effect on GRF specific binding. These effects were associated with a conversion of the high affinity GRF binding sites to lower affinity sites and to a reduction of the apparent number of total binding sites (high and low). No potentiation of these effects was observed when Gpp(NH)p (1 mM) and DTT (1 mM) were combined. In pituitaries from 14-month-old rats, Gpp(NH)p (1 mM) was capable of modulating GRF binding parameters in a similar fashion to that in pituitaries from 2-month-old rats. In pituitaries from 18-month-old rats, the high affinity GRF binding sites were already blunted and neither Gpp(NH)p nor Gpp(NH)p plus DTT significantly altered GRF binding parameters. In addition, in 20-month-old rats, the affinity of hGRF(1-29)NH2 and that of the full antagonist N alpha-Ac-[D-Arg2,Ala15]rGRF(1-29)NH2 were respectively decreased 9.3- and 9.9-fold. Our results suggest that in aging, alterations of GRF receptor binding sites could involve disulfide bond reduction or other structural modifications leading to conformational changes, similar to those induced by GSH or DTT. Such structural changes may prevent an efficient coupling of the GRF receptor with its ligands and G-protein, leading to a loss of somatotroph responsiveness.
Assuntos
Envelhecimento/fisiologia , Guanilil Imidodifosfato/farmacologia , Receptores de Hormônios Reguladores de Hormônio Hipofisário/fisiologia , Fatores Etários , Animais , Sítios de Ligação , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Glutationa/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Hormônios Reguladores de Hormônio Hipofisário/efeitos dos fármacosRESUMO
The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.
Assuntos
Endopeptidases/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hipotálamo/metabolismo , Fragmentos de Peptídeos/metabolismo , Hipófise/metabolismo , Receptores de Neuropeptídeos , Receptores de Hormônios Reguladores de Hormônio Hipofisário , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Sistema Hipotálamo-Hipofisário/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/metabolismoRESUMO
A report of a program which performs the initial computational reduction of the raw data from a cytotoxicity assay and outputs the reduced data as an image of the arrangement of the assay(s) upon the microwell plate. The program accepts the raw data either as manual or diskette file input.