Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Linfocitos T , Ratones , Animales , Linfocitos T/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Transducción de Señal , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11RESUMEN
Fluorescent proteins can be used to visualize cells and their constituents by various imaging techniques. Adoptive transfer of T cells from C57Bl/6 (B6) mice that expressed DsRed.T3 under the actin promoter lead to frequent rejection of transferred cells. In short term in vivo cytotoxicity assays these mice showed detectable, but weak lysis of DsRed spleen cells but their responses could be boosted by re-challenge with DsRed spleen cells. To determine whether DsRed protein may contain an H-2(b) MHC I-restricted T cell epitope, B6 mice immune to DsRed spleen cells were examined for in vivo lysis of target cells coated with various DsRed-derived peptides selected by the SYFPEITHI epitope prediction program. This analysis identified one D(b)-restricted peptide sequence within DsRed (SSLQDGCFI) that acted as an epitope for B6 target lysis. Knowledge of this epitope could allow DsRed to be used as a model antigen in B6 mice and cautions against using this fluorochrome, as well as several others containing the immunogenic sequence, in adoptive transfer studies where rejection is not desirable.
Asunto(s)
Epítopos de Linfocito T/inmunología , Proteínas Luminiscentes/inmunología , Péptidos/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/química , Colorantes Fluorescentes/química , Expresión Génica , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas Luminiscentes/química , Ratones , Ratones Endogámicos C57BL , Imitación Molecular , Datos de Secuencia Molecular , Péptidos/química , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
Single nucleotide polymorphisms in the gene encoding the protein tyrosine phosphatase TCPTP (encoded by PTPN2) have been linked with the development of autoimmunity. Here we have used Cre/LoxP recombination to generate Ptpn2(ex2-/ex2-) mice with a global deficiency in TCPTP on a C57BL/6 background and compared the phenotype of these mice to Ptpn2(-/-) mice (BALB/c-129SJ) generated previously by homologous recombination and backcrossed onto the BALB/c background. Ptpn2(ex2-/ex2-) mice exhibited growth retardation and a median survival of 32 days, as compared to 21 days for Ptpn2(-/-) (BALB/c) mice, but the overt signs of morbidity (hunched posture, piloerection, decreased mobility and diarrhoea) evident in Ptpn2(-/-) (BALB/c) mice were not detected in Ptpn2(ex2-/ex2-) mice. At 14 days of age, bone development was delayed in Ptpn2(-/-) (BALB/c) mice. This was associated with increased trabecular bone mass and decreased bone remodeling, a phenotype that was not evident in Ptpn2(ex2-/ex2-) mice. Ptpn2(ex2-/ex2-) mice had defects in erythropoiesis and B cell development as evident in Ptpn2(-/-) (BALB/c) mice, but not splenomegaly and did not exhibit an accumulation of myeloid cells in the spleen as seen in Ptpn2(-/-) (BALB/c) mice. Moreover, thymic atrophy, another feature of Ptpn2(-/-) (BALB/c) mice, was delayed in Ptpn2(ex2-/ex2-) mice and preceded by an increase in thymocyte positive selection and a concomitant increase in lymph node T cells. Backcrossing Ptpn2(-/-) (BALB/c) mice onto the C57BL/6 background largely recapitulated the phenotype of Ptpn2(ex2-/ex2-) mice. Taken together these results reaffirm TCPTP's important role in lymphocyte development and indicate that the effects on morbidity, mortality, bone development and the myeloid compartment are strain-dependent.
Asunto(s)
Desarrollo Óseo , Remodelación Ósea , Eritropoyesis , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Cruzamientos Genéticos , Hiperplasia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Especificidad de la EspecieRESUMEN
Persisting infections are often associated with chronic T cell activation. For certain pathogens, this can lead to T cell exhaustion and survival of what is otherwise a cleared infection. In contrast, for herpesviruses, T cells never eliminate infection once it is established. Instead, effective immunity appears to maintain these pathogens in a state of latency. We used infection with HSV to examine whether effector-type T cells undergoing chronic stimulation retained functional and proliferative capacity during latency and subsequent reactivation. We found that latency-associated T cells exhibited a polyfunctional phenotype and could secrete a range of effector cytokines. These T cells were also capable of mounting a recall proliferative response on HSV reactivation and could do so repeatedly. Thus, for this latent infection, T cells subjected to chronic Ag stimulation and periodic reactivation retain the ability to respond to local virus challenge.
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Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/toxicidad , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Proteínas del Envoltorio Viral/toxicidad , Activación Viral/inmunología , Latencia del Virus/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/trasplante , Linfocitos T CD8-positivos/virología , Enfermedad Crónica , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Ganglios Sensoriales/enzimología , Ganglios Sensoriales/inmunología , Ganglios Sensoriales/patología , Granzimas/biosíntesis , Herpes Simple/patología , Herpesvirus Humano 1/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Envoltorio Viral/administración & dosificaciónRESUMEN
Many autoimmune diseases exhibit familial aggregation, indicating that they have genetic determinants. Single nucleotide polymorphisms in PTPN2, which encodes T cell protein tyrosine phosphatase (TCPTP), have been linked with the development of several autoimmune diseases, including type 1 diabetes and Crohn's disease. In this study, we have identified TCPTP as a key negative regulator of TCR signaling, which might explain the association of PTPN2 SNPs with autoimmune disease. We found that TCPTP dephosphorylates and inactivates Src family kinases to regulate T cell responses. Using T cell-specific TCPTP-deficient mice, we established that TCPTP attenuates T cell activation and proliferation in vitro and blunts antigen-induced responses in vivo. TCPTP deficiency lowered the in vivo threshold for TCR-dependent CD8(+) T cell proliferation. Consistent with this, T cell-specific TCPTP-deficient mice developed widespread inflammation and autoimmunity that was transferable to wild-type recipient mice by CD8(+) T cells alone. This autoimmunity was associated with increased serum levels of proinflammatory cytokines and anti-nuclear antibodies, T cell infiltrates in non-lymphoid tissues, and liver disease. These data indicate that TCPTP is a critical negative regulator of TCR signaling that sets the threshold for TCR-induced naive T cell responses to prevent autoimmune and inflammatory disorders arising.
Asunto(s)
Enfermedades Autoinmunes/etiología , Tolerancia Inmunológica/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 2/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/enzimología , Animales , Anticuerpos Antinucleares/biosíntesis , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Inflamación/sangre , Inflamación/genética , Inflamación/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Procesamiento Proteico-Postraduccional , Proteína Tirosina Fosfatasa no Receptora Tipo 2/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Quimera por Radiación , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Timocitos/patología , Proteína Tirosina Quinasa ZAP-70/fisiología , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Rapid urease test (CLO-test) is an inexpensive and quick method for diagnosis of Helicobacter pylori infection with controversial results in children. We evaluated the performance of CLO-test in relation to endoscopic and histological findings in children with H. pylori infection. MATERIALS AND METHODS: We studied the medical records of children with H. pylori infection who were diagnosed between 1989 and 2009. Noninfected children were used as controls. H. pylori infection was defined by positive culture or by two other positive tests (histology and CLO-test, or urea breath test when a single test was positive). All children had histology together with CLO-test. Tissue culture was performed whenever possible. RESULTS: Five hundred thirty infected children (10.4 +/- 3.0 years) and 1060 controls (7.3 +/- 4.4 years) were studied. Sensitivity of CLO-test was 83.4% (95% CI, 79.9-86.3%), of culture 84.6% (95% CI, 78.7-89.1%), of histology 93.2% (95% CI, 90.7-95.1%), and specificity 99% (95% CI, 98.2-99.4%), 100%, and 100% respectively. CLO-test positivity was correlated with higher bacterial density (p < .001), activity (p < .001) and severity of gastritis (p < .01), older age (p < .01), and the presence of antral nodularity (p < .001). When CLO-test was positive, the concordance with histology and culture was high (95.5 and 89.2% respectively), whereas low concordance was observed when CLO-test was negative (17.05 and 45.83% respectively). CONCLUSIONS: CLO-test had lower sensitivity and comparable specificity with histology. Both tests should be performed concurrently to accurately diagnose H. pylori infection in children.
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Endoscopía Gastrointestinal , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/enzimología , Ureasa/análisis , Adolescente , Pruebas Respiratorias , Niño , Preescolar , Femenino , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/metabolismo , Histocitoquímica , Humanos , Masculino , Sensibilidad y EspecificidadRESUMEN
The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-kappaB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal-regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Fosfatasas/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Familia-src Quinasas/metabolismo , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-6/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Factor 2 Asociado a Receptor de TNF/metabolismo , Regulación hacia ArribaRESUMEN
The four mammalian golgins, p230/golgin-245, golgin-97, GCC88 and GCC185 are targeted to trans-Golgi network (TGN) membranes by their C-terminal GRIP domain in a G-protein-dependent process. The Arf-like GTPase, Arl1, has been shown to mediate TGN recruitment of p230/golgin245 and golgin-97 by interaction with their GRIP domains; however, it is not known whether all the TGN golgins bind to Arl1 and whether they are all recruited to the same or different TGN domains. Here we demonstrate differences in membrane binding properties and TGN domain recruitment of the mammalian GRIP domain proteins. Overexpression of full-length GCC185 resulted in the appearance of small punctate structures dispersed in the cytoplasm of transfected cells that were identified as membrane tubular structures by immunoelectron microscopy. The cytoplasmic GCC185-labelled structures were enriched for membrane binding determinants of GCC185 GRIP, whereas the three other mammalian GRIP family members did not colocalize with the GCC185-labelled structures. These GCC185-labelled structures included the TGN resident protein alpha2,6 sialyltransferase and excluded the recycling TGN protein, TGN46. The Golgi stack was unaffected by overexpression of GCC185. Overexpression of both full-length GCC185 and GCC88 showed distinct and nonoverlapping structures. We also show that the GRIP domains of GCC185 and GCC88 differ in membrane binding properties from each other and, in contrast to p230/golgin-245 and golgin-97, do not interact with Arl1 in vivo. Collectively these results show that GCC88, GCC185 and p230/golgin245 are recruited to functionally distinct domains of the TGN and are likely to be important for the maintenance of TGN subdomain structure, a critical feature for mediating protein sorting and membrane transport.
Asunto(s)
Proteínas de Transporte de Membrana/fisiología , Red trans-Golgi/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Autoantígenos/fisiología , Membrana Celular/metabolismo , Citoplasma/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Immunoblotting , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Microscopía Electrónica , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Sialiltransferasas/metabolismo , Transfección , Tirosina/química , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, integrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations.