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2.
J Clin Invest ; 111(6): 851-7, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12639991

RESUMEN

Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2-deficient 129 mice onto the NOD background along with 16 genetic markers (including I-A(g7) MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2(-/-) mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2(-/-) mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti-glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2(-/-) mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control beta cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2(-/-) NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.


Asunto(s)
Diabetes Mellitus Tipo 1/etiología , Proinsulina/fisiología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/inmunología , Anticuerpos Insulínicos/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Datos de Secuencia Molecular , Proinsulina/deficiencia
3.
Oncotarget ; 8(20): 32608-32617, 2017 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-28427233

RESUMEN

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.


Asunto(s)
Análisis Mutacional de ADN/normas , Janus Quinasa 2/genética , Laboratorios/normas , Trastornos Mieloproliferativos/genética , Análisis Mutacional de ADN/métodos , Humanos , Italia , Janus Quinasa 2/metabolismo , Laboratorios/estadística & datos numéricos , Mutación , Trastornos Mieloproliferativos/enzimología , Variaciones Dependientes del Observador
4.
J Immunol ; 168(7): 3251-8, 2002 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-11907079

RESUMEN

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-gamma. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vbeta repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vbeta8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-gamma, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-gamma than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.


Asunto(s)
Inmunofenotipificación , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Subgrupos de Linfocitos T/inmunología , Vísceras/citología , Vísceras/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Galactosilceramidas/farmacología , Inflamación/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Hígado/citología , Hígado/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos , Mesenterio/citología , Mesenterio/inmunología , Mesenterio/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Páncreas/citología , Páncreas/inmunología , Páncreas/patología , Receptores de Antígenos de Linfocitos T/biosíntesis , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Vísceras/patología
5.
Immunity ; 17(6): 725-36, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12479819

RESUMEN

To determine the precise regulatory effect of NKT cells on CD4(+) T cells involved in autoimmune diabetes, we developed an in vivo model in which transferred naive transgenic T cells are stimulated by their antigen in the presence or absence of NKT cells or in the presence of another conventional transgenic alphabeta T cell. The presence of NKT cells did not block the initial activation and expansion of the CD4(+) T cells but did inhibit their IL-2 and IFN-gamma production and later proliferation, resulting in an anergic phenotype. These CD4(+) T cells did not induce significant insulitis and were unable to destroy the beta cells. Thus, NKT cells prevent alphabeta CD4 T cell differentiation into effector cells.


Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Células Asesinas Naturales/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inmunidad Celular , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología
6.
Proc Natl Acad Sci U S A ; 100(5): 2663-8, 2003 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-12598649

RESUMEN

Steady-state numbers of peripheral lymphocyte are tightly controlled. For conventional T cells, signals delivered through the interaction of the T cell receptor (TCR) with antigen-loaded MHC molecules are required for the peripheral survival of naive T cells and for their homeostatic expansion in lymphopenic hosts. Cytokines, including IL-7, are also essential for survival of peripheral naive T cells. CD1d-restricted, V alpha 14(+) natural killer (NK)-T cells are a specialized autoreactive T subset with immunoregulatory activity. The relative roles of TCR engagement and cytokine signaling in the peripheral homeostasis of V alpha 14(+) NK-T cells were investigated. After adoptive transfer, the survival and expansion of peripheral V alpha 14(+) NK-T cells was independent of CD1d expression in the host. In contrast, IL-15 (but not IL-7) was required for maintenance of peripheral CD1d-reactive V alpha 14(+) T cells. Comparison of V alpha 14(+) T cell transfers into NK-proficient vs. deficient hosts suggests that NK-T cells and NK cells compete for peripheral resources. Our results indicate that IL-15 maintains the homeostasis of peripheral V alpha 14(+) NK-T cells. In contrast, TCR "tickling" of NK-T cells, if it occurs under steady-state conditions, does not by itself provide a sufficient signal for their peripheral survival.


Asunto(s)
Interleucina-15/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Traslado Adoptivo , Animales , Antígenos CD1/metabolismo , Antígenos CD1d , Bromodesoxiuridina/farmacología , División Celular , Supervivencia Celular , Citometría de Flujo , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Hígado/inmunología , Hígado/metabolismo , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Bazo/citología , Timo/citología , Factores de Tiempo
7.
J Immunol ; 168(12): 6007-11, 2002 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-12055208

RESUMEN

Although deficiencies in the NKT cell population have been observed in multiple sclerosis and mouse strains susceptible to experimental autoimmune encephalomyelitis (EAE), little is known about the function of these cells in CNS autoimmunity. In this work we report that TCR Valpha14-Jalpha281 transgenic nonobese diabetic mice, which are enriched in CD1d-restricted NKT cells, are protected from EAE. The protection is associated with a striking inhibition of Ag-specific IFN-gamma production in the spleen, implying modulation of the encephalitogenic Th1 response. This modulation is independent of IL-4 because IL-4-deficient Valpha14-Jalpha281 mice are still protected against EAE and independent of NKT cell-driven Th1 to Th2 deviation, because no increased autoantigen-specific Th2 response was observed in immunized Valpha14-Jalpha281 transgenic mice. Our findings indicate that enrichment and/or stimulation of CD1d-dependent NKT cells may be used as a novel strategy to treat CNS autoimmunity.


Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/inmunología , Encefalomielitis Autoinmune Experimental/genética , Epítopos de Linfocito T/inmunología , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Glicoproteínas/administración & dosificación , Glicoproteínas/inmunología , Inyecciones Subcutáneas , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-4/fisiología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Datos de Secuencia Molecular , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/genética
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