RESUMO
Lymphocytes in different states of activation use different intracellular signalling pathways and may therefore differ in their susceptibility to immunosuppressive agents. In this study we examined the proliferation and production of interleukin-2 (IL-2) by unprimed/naive CD4+CD45RA+ T cells and previously activated/memory CD4+CD45RO+ T cells from human peripheral blood when stimulated in vitro in the presence of cyclosporin A (CsA). Further, the dependency of the IL-2 response on calcium (Ca2+) ions was analysed by the addition of the chelating agent EGTA. The CD4+CD45RO+ memory T cells were shown to be less susceptible to CsA and less dependent on the level of Ca+ ions than the naive CD4+CD45RA+ T cells. The subcellular mechanisms involved in this difference and the potential clinical implications are discussed.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Ciclosporina/farmacologia , Interleucina-2/biossíntese , Antígenos Comuns de Leucócito , Anticorpos Monoclonais , Linfócitos T CD4-Positivos/metabolismo , Ácido Egtázico/farmacologia , Humanos , Técnicas In Vitro , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de SinaisRESUMO
A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR-ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST-SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST-SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST-SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST-SSRs each. This unexpectedly high incidence of EST-SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.
Assuntos
Cromossomos Artificiais Bacterianos , Etiquetas de Sequências Expressas , Marcadores Genéticos , Genoma de Planta , Hordeum/genética , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
Stimulation of resting human T cells by crosslinked CD28 monoclonal antibodies (mAb) induces some early signaling events but does not lead to IL-2 secretion and proliferation. The induction of these functions usually requires the delivery of additional signals such as that provided by costimulation of the T cell receptor (TCR). We analyzed the capacity of a panel of different CD28 mAb to induce cellular functions in purified human T cells. Two patterns of reactivity were observed. "Costimulatory" CD28 mAb like 9.3 required coengagement of the CD3/TCR complex for the induction of IL-2 gene transcription and proliferation. On the other hand, a "stimulatory" pathway could be defined by the use of the CD28 mAb BW 828, which triggered IL-2 synthesis, IL-2R expression, and proliferation without further requirement for additional stimuli. BW 828-induced proliferation was sensitive to inhibition by cyclosporin A and was mainly found in the CD4+CD45R0+ ("memory") T cell subset. These data suggest that T cell stimulation with mAb BW 828 defines a CD28-associated signaling pathway which leads to the induction of effector functions without the need for CD3/TCR coengagement. This pathway might play a role in antigen-independent activation and expansion of T cells.
Assuntos
Antígenos CD28/imunologia , Complexo CD3/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ciclosporina/farmacologia , Humanos , Técnicas In Vitro , Interleucina-2/biossíntese , Receptores de Interleucina-2/biossíntese , Linfócitos T/efeitos dos fármacosRESUMO
The reaction of (eta 5-C5Me5)TaMe4 with tert-butylphosphonic acid leads to the formation of a mixture of compounds: [[(eta 5-C5Me5)TaMe][t-BuP(O)(OH)][t-BuP(O)(OH)2]]2(t-BuPO3)2 (1) and [[(eta 5-C5Me5)Ta][t-BuP(O)(OH)2]]2(t-BuPO3)2(mu-O)2 (2). Compound 2 was also obtained by recrystallization of 1 from a THF/hexane mixture. Reaction of (eta 5-C5Me5)MCl4 (M = Mo, W) with PhP(O)(OH)2 yields the binuclear phosphonates [[(eta 5-C5Me5)M][PhP(O)(OH)2]]2(PhPO3)2(mu-O)2 (M = Mo (3); M = W (4)). Compounds 2.THF and 3(.)2.5THF were characterized by single-crystal X-ray studies. The tantalum and molybdenum phosphonates 2.THF and 3(.)2.5THF have different structures as compared to those of the previously reported titanophosphonate cages.
RESUMO
CD45RA+ cells have been described to be less responsive to CD3/T cell receptor (TcR)-mediated activation than CD45R0+ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR-triggered tyrosine phosphorylation in the two subsets and studied the role of co-stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA+ and CD45R0+ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co-stimulatory signals, strong expression of the 55-kDa chain of the interleukin-2 (IL-2) receptor (CD25), significant IL-2 production and vigorous proliferation were observed in CD45R0+ cells, whereas CD45RA+ cells responded weakly. However, when CD3/TcR-mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA+ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA+ and CD45R0+ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co-stimulatory signals determine whether CD3/TcR-initiated activation results in strong or weak responses.
Assuntos
Antígenos Comuns de Leucócito/fisiologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Células Apresentadoras de Antígenos/fisiologia , Western Blotting , Células Cultivadas , Humanos , Interleucina-2/fisiologia , Fosforilação , Proteína Quinase C/fisiologia , Tirosina/metabolismoRESUMO
Stimulation of resting human T cells with the CD28-specific mAb BW 828 induces proliferation and cytokine synthesis without further requirement for TCR coengagement. This observation prompted us to postulate that signal 2 (costimulatory signal) alone without signal 1 (TCR signal) can activate T cells. To test whether this putative function of CD28 is mediated via a particular signaling pathway, we compared early signaling events initiated in resting T cells by the stimulatory mAb BW 828 with signals triggered by the nonstimulating CD28 mAb 9.3. Stimulation of T cells with BW 828 induced an increase in intracellular Ca2+, but did not lead to detectable activation of the protein kinases p56(lck) and c-Raf-1. This pathway resulted in the induction of the transcription factors NF-kappa B, NF-AT, and proteins binding to the CD28 response element of the IL-2 promoter. On the other hand, stimulation of T cells with mAb 9.3 increased the level of intracellular Ca2+ and triggered the activation of p56(lck) and c-Raf-1, but was unable to induce the binding of transcription factors to the IL-2 promoter. In contrast to the differential signaling of BW 828 and 9.3 in resting T cells, the two mAbs exhibited a similar pattern of early signaling events in activated T cells and Jurkat cells (p56(lck) activation, association of phosphatidylinositol 3-kinase with CD28), indicating that the signaling capacity of CD28 changes with activation. These data support the view that stimulation through CD28 can induce some effector functions in T cells and suggest that this capacity is associated with a particular pattern of early signaling events.
Assuntos
Antígenos CD28/fisiologia , Citocinas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Transcrição Gênica/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD28/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Interfase/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Transcrição/biossínteseRESUMO
The c-Raf-1 kinase is a downstream effector of Ras signaling. Both proteins are highly oncogenic when they are mutationally activated, but only the Ras GTPase is frequently mutated in naturally occurring tumors. Although the c-Raf-1 protein was found to be amplified in different lung cancer cell lines, overexpression of the wild-type c-Raf-1 protein was shown to be insufficient to transform cultured cells. Here we have addressed the question of whether overexpression of the wild-type c-Raf-1 kinase can induce lung cancer in mice. We show that lung-targeted expression of oncogenically activated or wild-type c-Raf-1 proteins induces morphologically indistinguishable lung adenomas in transgenic mice. Compared with mice transgenic for the activated c-Raf-1-BxB, tumor development is delayed and occurs at a lower incidence in wild-type c-Raf-1 transgenic mice. Our studies show that the c-Raf-1 expression level is a critical parameter in tumor development and should be analyzed in more detail to evaluate its potential in the induction of cancer.