RESUMO
BACKGROUND: Four constructs are encompassed by the term "falls-related psychological concerns" (FrPC); "fear of falling" (FOF), "falls-related self-efficacy" (FSe), "balance confidence" (BC) and "outcome expectancy" (OE). FrPC are associated with negative consequences including physical, psychological, and social. Identifying factors associated with FrPC could inform interventions to reduce these concerns. METHODS: Sixty-two empirical papers relating to psychological factors associated with FrPC in community-dwelling older people (CDOP) were reviewed. Four levels of evidence were used when evaluating the literature: good, moderate, tentative, and none. RESULTS: Evidence that anxiety predicted FOF, BC, and OE was tentative. Moderate evidence was found for anxiety predicting FSe. Good evidence was found for depression predicting FSe. Moderate evidence was found for depression predicting both FOF and BC. No evidence was found for depression predicting OE. Tentative evidence was found for FSe predicting depression. Good and moderate evidence was found for quality of life (QoL) being predicted by FOF and BC respectively. Tentative evidence was found for FSe predicting QoL. Moderate evidence was found for QoL predicting both FSe and BC. No evidence was found for QoL predicting FOF. Good and moderate evidence was found for activity avoidance/restriction (AA/AR) being predicted by FOF and FSe respectively. Tentative evidence was found for BC and OE predicting AA/AR, as well as for AA/AR predicting FOF. Moderate evidence for activity level (AL) predicting FOF was identified, however the evidence of this predicting FSe and BC was tentative. Evidence for FOF, FSe, and BC predicting AL was tentative as was evidence to suggest FOF predicted coping. CONCLUSIONS: Mixed evidence has been found for the association of psychological factors in association with FrPCs. Future research should employ theoretically grounded concepts, use multivariate analysis and longitudinal designs.
Assuntos
Acidentes por Quedas/estatística & dados numéricos , Envelhecimento/psicologia , Ansiedade/diagnóstico , Depressão/diagnóstico , Medo/psicologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Avaliação Geriátrica , Humanos , Vida Independente , Masculino , Pessoa de Meia-Idade , Equilíbrio Postural , Escalas de Graduação Psiquiátrica , Psicologia , Qualidade de Vida , AutoeficáciaRESUMO
Conventional methods for studying paracrine signaling in vitro may not be sensitive to short-range effects resulting from signal dilution or decay. We employ a microfabricated culture substrate to maintain two cell populations in microscale proximity. Individual populations can be quickly retrieved for cell-specific readouts by standard high-throughput assays. We show that this platform is sensitive to short-range interactions that are not detectable by common methods such as conditioned media transfer or porous cell culture inserts, as revealed by gene expression changes in a tumor-stromal crosstalk model. In addition, we are able to detect population-specific gene expression changes that would have been masked in mixed co-cultures. We thus demonstrate a tool for investigating an important class of intercellular communication that may be overlooked in conventional biological studies.
Assuntos
Técnicas de Cocultura/métodos , Perfilação da Expressão Gênica/métodos , Comunicação Parácrina/fisiologia , Linhagem Celular Tumoral , Técnicas de Cocultura/instrumentação , Fibroblastos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genéticaAssuntos
Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia/tratamento farmacológico , Nitrofenóis/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Animais , HumanosRESUMO
This study examined the effects of infection with barley and cereal yellow dwarf viruses (BYDVs) on wild grass species in California, a region in which native perennial bunchgrasses have been largely replaced by exotic annual grasses. We sought to determine whether these widespread viruses compromise the fitness of wild hosts and thus have the potential to influence grassland dynamics. Plant viruses have been long overlooked in ecological studies, and their influence on wild hosts has often been assumed to be minimal. We examined the short-term and long-term consequences of infection on field-grown individuals from 18 different populations of wild California grasses (from seven native and one exotic species). Barley yellow dwarf virus infection was aggressive in most hosts and markedly impaired host fitness by reducing growth, survivorship, and fecundity. Previous work indicates that the presence of exotic grasses can more than double BYDV incidence in natives. Given the ubiquity of BYDVs, our results suggest that apparent competition and other virus-mediated processes may influence interactions among native and exotic grasses and potentially contribute to shifts in grassland community composition.
Assuntos
Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Poaceae/virologia , California , Flores/virologia , Poaceae/fisiologia , ReproduçãoRESUMO
Human endothelial cells (EC) costimulate CD4(+) memory T cell activation through CD58-CD2 interactions. In this study we tested the hypothesis that EC activate distinct costimulatory pathways in T cells that target specific transcription factors. AP-1, composed of fos and jun proteins, is a critical effector of TCR signaling and binds several sites in the IL-2 promoter. EC augment c-fos promoter activity in T cells; however, deletion analysis reveals no transcription factor binding sites in the promoter uniquely responsive to EC costimulation. Overexpression of AP-1 proteins in T cells augments the activity of an AP-1-luciferase reporter gene equally in the absence or the presence of EC costimulation. Interestingly, EC stimulate a similar 2- to 3-fold up-regulation of AP-1, NF-AT, NF-kappaB, and NF-IL-2-luciferase reporters. CD2 mAbs completely block EC effects on all of these pathways, as well as costimulation of IL-2 secretion. We conclude that EC costimulation through CD2 does not trigger a single distinct costimulatory pathway in T cells, but rather, it amplifies several pathways downstream of the TCR. Indeed, we find that early EC costimulation acts "upstream" of the TCR by promoting lipid raft aggregation, thus amplifying TCR signaling. Soluble CD2 mAbs block EC-induced raft aggregation, whereas cross-linking CD2 promotes aggregation. These data are consistent with the critical role of CD2 in organizing the T cell-APC contact zone.
Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Endotélio Vascular/imunologia , Microdomínios da Membrana/metabolismo , Linfócitos T/imunologia , Comunicação Celular , Genes fos , Humanos , Junções Intercelulares/metabolismo , Ativação Linfocitária , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Human endothelial cells can be induced to form capillary-like tubular networks in collagen gels. We have used this in vitro model and representational difference analysis to identify genes involved in the formation of new blood vessels. HESR1 (HEY-1/HRT-1/CHF-2/gridlock), a basic helix-loop-helix protein related to the hairy/enhancer of split/HES family, is absent in migrating and proliferating cultures of endothelial cells but is rapidly induced during capillary-like network formation. HESR1 is detectable in all adult tissues and at high levels in well vascularized organs such as heart and brain. Its expression is also enriched in aorta and purified capillaries. Overexpression of HESR1 in endothelial cells down-regulates vascular endothelial cell growth factor receptor-2 (VEGFR2) mRNA levels and blocks proliferation, migration, and network formation. Interestingly, reduction of expression of HESR1 by antisense oligonucleotides also blocks endothelial cell network formation in vitro. Finally, HESR1 expression is altered in several breast, lung, and kidney tumors. These data are consistent with a temporal model for HESR1 action where down-regulation at the initiation of new vessel budding is required to allow VEGFR2-mediated migration and proliferation, but re-expression of HESR1 is necessary for induction of tubular network formation and continued maintenance of the mature, quiescent vessel.
Assuntos
Proteínas de Drosophila , Endotélio Vascular/citologia , Sequências Hélice-Alça-Hélice , Proteínas de Insetos/fisiologia , Fatores de Transcrição , Motivos de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Humanos , Proteínas de Insetos/análise , Proteínas de Insetos/genética , Camundongos , Neovascularização Fisiológica , RNA Mensageiro/análise , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio VascularRESUMO
We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the gel 31 days after implantation. Retroviral-mediated overexpression of a caspase-resistant Bcl-2 protein delays HUVEC apoptosis in vitro for over 7 days. Bcl-2-transduced HUVEC produce an increased density of HUVEC-lined perfused microvessels in vivo compared with untransduced or control-transduced HUVEC. Remarkably, Bcl-2- but not control-transduced HUVEC recruit an ingrowth of perivascular smooth-muscle alpha-actin-expressing mouse cells at 31 days, which organize by 60 days into HUVEC-lined multilayered structures resembling true microvessels. This system provides an in vivo model for dissecting mechanisms of microvascular remodeling by using genetically modified endothelium. Incorporation of such human endothelial-lined microvessels into engineered synthetic skin may improve graft viability, especially in recipients with impaired angiogenesis.
Assuntos
Capilares/citologia , Endotélio Vascular/citologia , Animais , Capilares/ultraestrutura , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/ultraestrutura , Humanos , Camundongos , Camundongos SCID , Microscopia EletrônicaRESUMO
IFN-inducible T cell alpha chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-gamma-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-gamma-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-alpha does not induce I-TAC mRNA alone, but does act synergistically with IFN-gamma. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.
Assuntos
Quimiocinas CXC/biossíntese , Interferon gama/fisiologia , Linfócitos T/imunologia , Capilares/citologia , Capilares/imunologia , Capilares/metabolismo , Adesão Celular/imunologia , Inibição de Migração Celular , Sistema Livre de Células/imunologia , Sistema Livre de Células/metabolismo , Células Cultivadas , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/isolamento & purificação , Quimiotaxia de Leucócito/imunologia , Sinergismo Farmacológico , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , RNA Mensageiro/biossíntese , Pele/citologia , Fator de Necrose Tumoral alfa/fisiologia , Veias UmbilicaisRESUMO
OBJECTIVE: Currently, we lack a theoretical explanation for why squamous cell cervical cancer develops predominantly in specific sites (i.e., along the squamocolumnar junction). We therefore implanted human cervical tissues containing the transformation zone in severe combined immunodeficiency (SCID) mice and studied morphology, steroid effects, gene expression, and human papillomavirus (HPV) factors. METHODS: Normal and dysplastic human cervical tissues (3 x 2 mm) were placed subcutaneously in SCID-beige mice and later assessed by in situ hybridization for HPV 16/18 DNA and by immunohistochemistry for expression of CD31, keratin, proliferating-cell nuclear antigen, HPV 16 E6, p53, and Notch-1 (a binary cell fate determination protein). Some normal tissues were implanted with either a 90-day release 1.7-mg 17beta-estradiol pellet or a 5-mg tamoxifen pellet; others were infected prior to implantation with human recombinant adenovirus 5 vector containing a human cytomegalovirus promoter-driven beta-galactosidase gene and later assessed by X-gal staining. RESULTS: Murine and human vessels formed anastomoses by 3 weeks. For at least 11 weeks, normal tissue retained the transformation zone and normal cell-type-specific keratin expression and exhibited normal proliferation; Notch-1 was present only in the basal cell layer. Dysplastic tissues exhibited koilocytosis, increased levels of cellular proliferation, and aberrant keratin, p53, and Notch-1 expression; HPV 16/18 DNA and HPV 16 E6 protein were detected for at least 6 weeks. Squamous metaplasia of normal cervical epithelium resulted from estrogen exposure, and a predominant columnar differentiation pattern was associated with tamoxifen administration. Through stable adenovirus infection, beta-galactosidase was expressed for at least 6 weeks. CONCLUSIONS: This small manipulatable xenograft model maintains normal and dysplastic human cervical epithelium through neovascularization. Neoplastic tissue retains HPV 16/18 DNA and a premalignant phenotype, including elevated levels of cellular proliferation and aberrant keratin, p53, and Notch-1 expression. These attributes constitute essential features of a biologic model through which one may study HPV-mediated human disease and may be superior to cell culture and transgenic murine systems. Furthermore, this may serve as a model for gene therapy. Finally, we suggest that the normal cervical epithelium is maintained through putative interactions between the Notch locus and cell cycle growth regulators such as p53 and pRb. Neoplastic cervical epithelium may arise through disruption of this pathway. This theory may be testable in our animal model.
Assuntos
Modelos Animais de Doenças , Neovascularização Patológica/patologia , Imunodeficiência Combinada Severa/patologia , Neoplasias do Colo do Útero/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma in Situ/irrigação sanguínea , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma in Situ/virologia , DNA Viral/análise , Estrogênios/farmacologia , Feminino , Humanos , Hibridização In Situ , Camundongos , Camundongos SCID , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/virologia , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/complicações , Imunodeficiência Combinada Severa/metabolismo , Transplante Heterólogo , Neoplasias do Colo do Útero/irrigação sanguínea , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
Human endothelial cells (EC) express MHC class II molecules in vivo and are likely to be involved in presentation of antigens to CD4(+) T cells. We examined, at the single-cell level, EC presentation of superantigens to resting CD4(+) memory T cells. Within 2 h of adherence to class II+ EC early T cell activation is evidenced by translocation of nuclear factor of activated T cells (NFAT), surface expression of CD69, and synthesis of IFN-gamma and IL-2. Naive T cells are not activated. T cell activation is dependent on the prior induction of MHC class II molecules on EC and is blocked by antibodies to LFA-3 (CD58). Our data place EC along a spectrum of antigen-presenting ability. Activated B cells and macrophages trigger more cells to express cytokines than do EC and at lower antigen concentrations; EC are in turn, superior to fibroblasts or smooth muscle cells. Furthermore, the concept of activation thresholds for cytokine synthesis within T cells also extends to earlier activation events: NFAT translocation is relatively easy to trigger, as is CD69 expression; fewer cells can be triggered to express IFN-gamma and fewer still to express IL-2. EC may, therefore, contribute to a graded immune response by inducing qualitatively and quantitatively different responses than professional APC.
Assuntos
Linfócitos B/imunologia , Toxinas Bacterianas , Linfócitos T CD4-Positivos/imunologia , Endotélio Vascular/imunologia , Fibroblastos/imunologia , Memória Imunológica/imunologia , Apresentação de Antígeno/imunologia , Linfócitos B/citologia , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Enterotoxinas/imunologia , Fibroblastos/citologia , Humanos , Antígenos Comuns de Leucócito/imunologia , Macrófagos/imunologia , Modelos Imunológicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/imunologia , Superantígenos/imunologiaRESUMO
The cervical squamocolumnar junction of normal and dysplastic human xenografts was maintained in SCID-beige mice. Dysplastic tissue maintained a dysplastic morphology, irregular pattern of keratin expression, elevated levels of cellular proliferation, and human papillomavirus type 16 and/or type 18 DNA. Hyperplastic changes of normal xenografts occurred via high-dose estrogen exposure, and through recombinant adenovirus infection, the introduction and stable expression of an exogenous gene was accomplished.
Assuntos
Adenoviridae/genética , Colo do Útero/virologia , DNA Viral/análise , Estradiol/farmacologia , Papillomaviridae/genética , Displasia do Colo do Útero/virologia , Animais , Divisão Celular , Feminino , Humanos , Imuno-Histoquímica , Queratinas/análise , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante Heterólogo , beta-Galactosidase/genéticaRESUMO
Academic health centers (AHCs) are experiencing turmoil in all three of their traditional missions of teaching, research, and patient care. The authors examine origins of universities and medical education to place in historical context the stresses affecting AHCs at the end of the 20th century. They describe the cultures of the university to suggest strategies for successful adaptation to these stresses. Clashes of values and norms of the cultures within universities and AHCs can hinder effective adaptation to external change. Administrators, researchers, teachers, and clinicians can have strongly conflicting perspectives. For example, business skill is of increasing importance to the survival of the clinical enterprise, but not typically valued by faculty members. University faculty have often considered accountability as antithetical to academic freedom, and, until recently, accountability was not strongly demanded of AHCs. The authors conclude that AHC faculty must transcend the outdated view that the roles of the scholar, scientist, and healer are in opposition to those of the leader and manager. If AHCs are to survive and prosper through their current cultural transition, their faculty must understand all these roles as part of their intellectual and organizational responsibility.
Assuntos
Centros Médicos Acadêmicos/organização & administração , Docentes de Medicina/organização & administração , Centros Médicos Acadêmicos/história , Centros Médicos Acadêmicos/tendências , História do Século XVIII , História do Século XIX , História do Século XX , História Medieval , Humanos , Liderança , Cultura Organizacional , Inovação Organizacional , Responsabilidade Social , Estados UnidosRESUMO
We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.
Assuntos
Ciclosporina/administração & dosagem , Microcirculação/patologia , Polienos/administração & dosagem , Transplante de Pele/imunologia , Pele/irrigação sanguínea , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimioterapia Combinada , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Genes MHC da Classe II/fisiologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Imunossupressores/administração & dosagem , Queratinócitos/metabolismo , Camundongos , Camundongos SCID , Microcirculação/efeitos dos fármacos , Microcirculação/imunologia , Sirolimo , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Transplante de Pele/patologia , Linfócitos T/imunologia , Transplante Homólogo , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
The aim of this study was to identify molecular mechanisms involved in transcriptional regulation of IL-2 expression following CD2 and CD3 activation in lamina propria (LP) T cells. Studies used T cells from normal, ulcerative colitis, and Crohn's disease mucosa and freshly isolated PBMC, PBMC stimulated with IL-2 alone, and PBMC stimulated with IL-2 and cocultured with B cell lines (LP-like T cells). Electrophoretic mobility shift assays were performed with nuclear extracts from cells activated with either anti-CD2 or anti-CD3 Abs. CD2 signaling in LPMC and LP-like T cells led to a pattern of sustained up-regulation of AP-1-binding complexes, whereas CD3 activation resulted in only transient up-regulation. While the pattern of regulation of AP-1 binding observed in normal, uninflamed, or inflamed Crohn's disease LPMC is similar, differences in intensity of AP-1 binding were observed. Activation of LP-like T cells mimics the up-regulation of AP-1 with a kinetic profile similar to that observed with freshly isolated LPMC from Crohn's disease-inflamed tissue. The AP-1 complex formed following CD2 activation is composed of jun/fos heterodimers. The CD2-enhanced responsiveness is reflected in functional analysis experiments utilizing transfection of both multimeric-TRE or IL-2 promoter-luciferase constructs directly into normal, ulcerative colitis, or Crohn's disease LPMC. Our data suggest that activation of LP T cells from normal, ulcerative colitis, or Crohn's disease mucosa through the CD2 pathway leads to induction of AP-1 complexes that bind to the IL-2 promoter, and may play a pivotal role in modulating IL-2 production in the gut.
Assuntos
Antígenos CD2/fisiologia , Interleucina-2/genética , Ativação Linfocitária , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Linfócitos T/imunologia , Fator de Transcrição AP-1/metabolismo , Animais , Linhagem Celular , Doença de Crohn/imunologia , Interleucina-2/metabolismo , Camundongos , Transcrição Gênica , Regulação para CimaRESUMO
A human skin allograft injury model in immunodeficient mice, engrafted with human peripheral blood mononuclear cells from a different donor, has been used to test whether reagents that block human T cell CD2 interactions with its principal ligand, LFA-3 (CD58), can inhibit immune reactions in vivo. In this model, human skin grafts show a reproducible pattern of progressive human T-cell infiltration and human graft microvascular injury that resembles human first-set skin graft rejection. Murine Mab to human LFA-3 or human LFA-3-IgG1 fusion protein, but not isotype-matched control antibodies, each markedly protected skin grafts from leukocyte infiltration and injury. These data provide the first evidence that LFA-3 functions in vivo and establish the ability of this new model to test human-specific immune modulators.
Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Transplante de Pele/imunologia , Quimeras de Transplante/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Humanos , Imunoglobulina G/metabolismo , Ativação Linfocitária , Camundongos , Camundongos SCID , Linfócitos T/imunologia , Transplante HomólogoRESUMO
The ability to analyze transcriptional regulation in non-transformed T-cells has been hampered by the inability to reproducibly transiently transfect these cells with DNA constructs. We have previously demonstrated that normal human whole mononuclear and CD4 T-cells can be consistently transiently transfected with plasmid DNA. Human cells were most receptive to plasmid DNA uptake between 19.5 and 20 h after prestimulation with a submitogenic dose of the polyclonal T-cell activator, PHA. Here we report an alteration and optimization of this protocol for non-transformed murine splenic T-cells, using concanavalin A instead of PHA as the preactivation stimulus. When coupled with the high sensitivity of luciferase reporter gene constructs, this protocol facilitates the analysis of a variety of T-cell-specific promoters in non-transformed T-cells. In addition, we directly demonstrate that murine T-cells are specifically transiently transfected among a population of whole mononuclear cells by using an expression vector for green fluorescent protein.
Assuntos
DNA/administração & dosagem , DNA/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transfecção/métodos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Concanavalina A/administração & dosagem , Eletroporação , Genes Reporter , Humanos , Técnicas Imunológicas , Técnicas In Vitro , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/genética , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Baço/citologia , Baço/imunologiaRESUMO
The possible clinical use of the methyl xanthine derivative, pentoxifylline (PF), for the treatment of T cell-dependent diseases is being noted with increasing interest. In this paper, we studied the molecular consequences of PF treatment during lymphocyte activation. We found that in T cells, anti-CD3-induced c-Rel expression was blocked by PF, whereas the induction of other NF-kappaB family members was not significantly affected. However, induction of NF-AT, which has the same signaling requirements as c-Rel induction, was not inhibited by PF. Among genes that respond to these transcription factors, IL-2 mRNA induction was suppressed by PF, whereas IL-2R(alpha) chain mRNA induction was not affected. These observations implicated c-Rel as an IL-2 promoter factor, for which experimental support was obtained from transient transfection experiments. In contrast with the observation in T cells, c-Rel induction was not blocked by PF in B cells. The greater selectivity of PF, compared with FK506, at both the molecular and cellular levels may prove advantageous in manipulating T cell responses in vivo.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , Proteínas Nucleares , Pentoxifilina/farmacologia , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/farmacologia , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , Fatores de Transcrição NFATC , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-rel , Tacrolimo/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/farmacologiaRESUMO
Porcine aortic endothelial cells (PAECs), unlike human endothelial cells, express a surface protein recognized by human CTLA4Ig fusion protein that costimulates human T cells through CD28. We have cloned porcine CD86 (pCD86) from an immortalized porcine endothelial cell line, PEC-A, that expresses high levels of this CTLA-4-binding protein. pCD86 mRNA is expressed in PEC-A and PAECs but not in human endothelial cells. Expression of stably transfected pCD86 in CHO cells modestly costimulates human T cell proliferation and IL-2 secretion. Expression of transiently transfected pCD86 in human umbilical vein endothelial cells strongly costimulates IL-2 production by human T cells, comparable to costimulation by PAECs. Costimulation of human T cells by pCD86 in both systems is as effective as costimulation by human CD80 or CD86, and can be blocked by human CTLA4Ig. We conclude that pCD86 contributes to the strong xenoreactivity of porcine endothelium.
Assuntos
Antígenos CD/imunologia , Endotélio Vascular/imunologia , Imunoconjugados , Ativação Linfocitária/fisiologia , Glicoproteínas de Membrana/imunologia , Suínos/imunologia , Linfócitos T/imunologia , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos CD/isolamento & purificação , Antígenos de Diferenciação/imunologia , Aorta , Antígeno B7-2 , Sequência de Bases , Células CHO , Antígeno CTLA-4 , Células Cultivadas , Clonagem Molecular , Cricetinae , Cricetulus , DNA Complementar/genética , Endotélio Vascular/química , Endotélio Vascular/citologia , Humanos , Interleucina-2/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Linfócitos T/metabolismo , Transfecção , Veias UmbilicaisRESUMO
IL-4 and IL-13 each act on human endothelial cells (ECs) to induce expression of vascular cell adhesion molecule-1. On hematopoietic cells. IL-4 responses may be mediated either through a pathway involving gc, the common signaling subunit of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, or through a gc-independent pathway that may be alternatively activated by IL-13. We find that human ECs do not express gc, as detected by indirect immunofluorescence and FACS analysis or by a reverse transcription-PCR method. Like IL-4, IL-13 activates a protein tyrosine kinase that phosphorylates the IL-4R binding protein. In addition, we find that IL-4 and IL-13 each induce tyrosine phosphorylation of the JAK2 tyrosine kinase. Furthermore, both IL-4 and IL-13 induce binding of the Stat6 transcription factor to a consensus sequence oligonucleotide. We conclude that the IL-4 response of human ECs involves the IL-13 shared pathway that is independent of gc, and uses JAK2-Stat6 signaling.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Interleucina-3/farmacologia , Interleucina-4/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores de Interleucina-2/fisiologia , Transativadores/metabolismo , Sequência de Bases , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , Janus Quinase 2 , Dados de Sequência Molecular , Fosforilação , RNA Mensageiro/análise , Fator de Transcrição STAT6 , Tirosina/metabolismo , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Cultured human endothelial cells (EC) increase CD40 ligand expression on polyclonally activated human peripheral blood CD4+ helper T cells compared to T cells activated in the absence of accessory cells or in the presence of peripheral blood adherent cells or B cells. Induction of CD40 ligand expression appears to be biphasic with early induction observable at 6 h and later induction at 24 h. EC cause T cells to increase CD40 ligand expression during the early phase at 6 h after activation. CD40 ligand expression is restricted to the CD4+ helper T cell subset of the peripheral blood T cells, even when EC is present. Blocking monoclonal antibodies to co-stimulatory molecules on EC and T cells indicate that the CD2/LFA-3 pathway, which also contributes to induction of augmented interleukin-2 (IL-2) secretion is involved in EC-induced up-regulation of CD40 ligand. Exogenous IL-2 can also increase CD40 ligand expression. However, increased IL-2 secretion in the presence of EC can not fully account for endothelial-induced CD40 ligand up-regulation as (1) the effect of exogenous IL-2 is greater at 24 h than at 6 h, whereas the opposite is true for EC; (2) the effect of saturating levels of IL-2 is considerably smaller than that of EC; and (3) blocking of IL-2 receptors does not fully inhibit endothelial effects on CD40 ligand expression. We conclude that EC provide unique co-stimulatory signals that effect the phenotype of activated CD4+ T cells.