RESUMEN
Osteoarthritis (OA) is the most prevalent musculoskeletal disease in humans, causing pain, loss of joint motility and function, and severely reducing the standard of living of patients. Cartilage tissue engineering attempts to repair the damaged tissue of individuals suffering from OA by providing mechanical support to the joint as new tissue regenerates. The aim of this study was to create composite three dimensional scaffolds comprised of electrospun poly(D,L-lactide)/poly(L-lactide) (PDLA/PLLA) or poly(D,L-lactide)/polycaprolactone (PDLA/PCL) with salt leached pores and an embedded chitosan hydrogel to determine the potential of these scaffolds for cartilage tissue engineering. PDLA/PLLA-hydrogel scaffolds displayed the largest compressive moduli followed by PDLA/PCL-hydrogel scaffolds. Dynamic mechanical tests showed that the PDLA/PLLA scaffolds had no appreciable recovery while PDLA/PCL scaffolds did exhibit some recovery. Primary canine chondrocytes produced both collagen type II and proteoglycans (primary components of extracellular matrix in cartilage) while being cultured on scaffolds composed of electrospun PDLA/PCL. As a result, a composite electrospun embedded hydrogel scaffold shows promise for treating individuals suffering from OA.
Asunto(s)
Cartílago/química , Quitosano/química , Hidrogeles , Nanofibras , Polímeros/química , Ingeniería de Tejidos , Andamios del Tejido , Ensayo de MaterialesAsunto(s)
Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas c-vav/fisiología , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rac1/genética , Animales , Biomarcadores/metabolismo , Western Blotting , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Skeletal muscle repair after injury usually results in scar tissue and decreased functionality. In this study, we coaxially electrospun poly(ε-caprolactone), multiwalled carbon nanotubes, and a hydrogel consisting of polyvinyl alcohol and polyacrylic acid (PCL-MWCNT-H) to create a self-contained nanoactuating scaffold for skeletal muscle tissue replacement. This was then compared to electrospun PCL and PCL-MWCNT scaffolds. All scaffolds displayed some conductivity; however, MWCNT incorporation increased the conductivity. Only the PCL-MWCNT-H actuated when stimulated with 15 and 20 V. The PCL, PCL-MWCNT, and hydrogel only scaffolds demonstrated no reaction when 5, 8, 10, 15, and 20 V were applied. Thus, all components of the PCL-MWCNT-H scaffold are essential for movement. All three PCL-containing scaffolds were biocompatible, but the PCL-MWCNT-H scaffolds displayed more multinucleated cells with actin interaction. After tensile testing, the MWCNT-containing scaffolds had higher strength than the rat and pig skeletal muscle. Although the mechanical properties were higher than muscle, the PCL-MWCNT-H scaffold shows promise as a potential bioartificial nanoactuator for skeletal muscle.
Asunto(s)
Resinas Acrílicas/farmacología , Músculo Esquelético/fisiología , Nanotubos de Carbono/química , Poliésteres/farmacología , Alcohol Polivinílico/farmacología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Proliferación Celular/efectos de los fármacos , Módulo de Elasticidad/efectos de los fármacos , Conductividad Eléctrica , Microscopía Fluorescente , Células Musculares/citología , Células Musculares/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Nanotubos de Carbono/ultraestructura , Ratas , Estrés Mecánico , Sus scrofaRESUMEN
Traumatic injuries can interrupt muscle contraction by damaging the skeletal muscle and/or the peripheral nerves. The healing process results in scar tissue formation that impedes muscle function. Electrospinning and metal nanoparticles (Nps) can create a scaffold that will trigger muscle cell elongation, orientation, fusion, and striation. Poly(L-lactic acid) (PLLA) and gold (Au) Nps were electrospun to create three composite scaffolds, 7% Au-PLLA, 13% Au-PLLA and 21% Au-PLLA, and compared to PLLA alone. The scaffolds had a conductivity of 0.008 ± 0.003 S/cm for PLLA, 0.053 ± 0.015 S/cm for 7% Au-PLLA, 0.076 ± 0.004 S/cm for 13% Au-PLLA and 0.094 ± 0.037 S/cm for 21% Au-PLLA. Next, a cell study was conducted with rat primary muscle cells and all three Au-PLLA scaffolds. The first cell study showed low cell proliferation on all three of the Au-PLLA scaffolds; however, the second cell study showed that this was not due to Au Nps toxicity. Instead, low cell proliferation may be a marker for myotube differentiation and fusion. Values for the elastic modulus and yield stress for the Au-PLLA scaffolds on days 0, 7, 14, 21 and 28 were much higher than those for skeletal muscle tissue. Therefore, lower amounts of Au Nps may be utilized to create a biodegradable, biocompatible and conductive scaffold for skeletal muscle repair.
Asunto(s)
Oro , Nanopartículas del Metal , Músculo Esquelético/citología , Poliésteres/química , Ingeniería de Tejidos , Animales , Microscopía Electrónica de Rastreo , RatasRESUMEN
A number of surgical interventions in ancient veterinary medicine were modelled on the same procedures in human medicine. This is discussed in some detail for the prolapse of the uterus and the couching of the cataract in horses. In the introductory section, the importance of Switzerland and neighbouring areas for the transmission of ancient veterinary medicine is highlighted.
Asunto(s)
Catarata/historia , Enfermedades de los Caballos/historia , Prolapso Uterino/historia , Medicina Veterinaria/historia , Animales , Catarata/veterinaria , Extracción de Catarata/historia , Extracción de Catarata/veterinaria , Femenino , Antigua Grecia , Historia Antigua , Enfermedades de los Caballos/cirugía , Caballos , Humanos , Ciudad de Roma , Prolapso Uterino/cirugía , Prolapso Uterino/veterinariaRESUMEN
Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.
Asunto(s)
Linfocitos B/inmunología , Quimiotaxis de Leucocito , Factores de Intercambio de Guanina Nucleótido , Proteínas Proto-Oncogénicas/fisiología , Animales , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Marcación de Gen , Células Madre Hematopoyéticas/inmunología , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Inmunoglobulinas/biosíntesis , Activación de Linfocitos , Subgrupos Linfocitarios/clasificación , Tejido Linfoide/inmunología , Ratones , Ratones Noqueados , Agregación Plaquetaria , Proteínas Proto-Oncogénicas/genética , Recombinación Genética , Factores de Intercambio de Guanina Nucleótido Rho , Distribución TisularRESUMEN
The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK natural cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to produce IL-4 in response to in vivo CD3 stimulation. These data highlight the important role of Vav-1 in NK T cell development and NK cell function.
Asunto(s)
Proteínas de Ciclo Celular , Diferenciación Celular , Citotoxicidad Inmunológica/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Anticuerpos/inmunología , Células Cultivadas , Activación Enzimática , Eliminación de Gen , Interleucina-2/inmunología , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/metabolismo , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Receptores de IgG/inmunologíaRESUMEN
Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor-induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1(-/-)Vav-2(-/-) B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-independent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1(-/-)Vav-2(-/-) B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Proteínas de Ciclo Celular , Proteínas Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/inmunología , Animales , Secuencia de Bases , Señalización del Calcio , Diferenciación Celular , Cartilla de ADN/genética , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
The transcriptional coactivator BOB.1/OBF.1 confers B-cell specificity on the transcription factors Oct1 and Oct2 at octamer site-containing promoters. A hallmark of the BOB.1/OBF.1 mutation in the mouse is the absence of germinal center development in secondary lymphoid organs, demonstrating the requirement for BOB.1/OBF.1 in antigen-dependent stages of B-cell differentiation. Here we analyzed earlier stages of B lymphopoiesis in BOB.1/OBF.1-deficient mice. Examination of B-cell development in the bone marrow revealed that the numbers of transitional immature (B220(+) IgM(hi)) B cells were reduced and that B-cell apoptosis was increased. When in competition with wild-type cells, BOB.1/OBF.1(-/-) bone marrow cells exhibited defects in repopulating the bone marrow B-cell compartment and were unable to establish a presence in the periphery of host mice. The defective bone marrow populations in BOB.1/OBF.1(-/-) mice were rescued by conditional expression of a BOB.1/OBF.1 transgene controlled by the tetracycline gene expression system. However, the restored populations did not restore the numbers of IgD(hi) B cells in the periphery, where the BOB.1/OBF.1 transgene was not expressed. These results show that BOB.1/OBF.1(-/-) B cells exhibit multistage defects in B-cell development, including impaired production of transitional B cells and defective maturation of recirculating B cells.
Asunto(s)
Linfocitos B/metabolismo , Transactivadores/genética , Transactivadores/fisiología , Animales , Antibacterianos/farmacología , Apoptosis , Western Blotting , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Bromodesoxiuridina/metabolismo , Diferenciación Celular , Separación Celular , Doxiciclina/farmacología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Genotipo , Inmunoglobulina M/metabolismo , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Mutación , Fenotipo , Regiones Promotoras Genéticas , Bazo/metabolismo , TransgenesRESUMEN
ADP-ribosylation factor 1 (Arf1) plays an important role in early and intra-Golgi protein trafficking. During this process, Arf1 interacts with many different proteins and other molecules that regulate its state of activation or are involved in its intracellular function. To determine which of these proteins interact directly with Arf1 during coat protein type I (COPI) vesicle biogenesis, we probed the molecular environment of Arf1 by use of site-specific photocrosslinking. This method was first used successfully in the field of protein trafficking to study the mechanisms involved in protein translocation across the endoplasmic reticulum during protein synthesis. In such a hydrophobic environment, crosslink yields of up to 30% have been observed. We have now applied this method to study the mechanism of vesicle budding from the cytosolic face of the Golgi apparatus, an aqueous environment. Although the crosslink yield is significantly lower under these conditions, due to predominant reaction of the photolabile probes with water, a specific interaction of Arf1 with subunits of coatomer, the major coat protein of COPI vesicles, could readily be identified.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Proteína Coat de Complejo I/metabolismo , Vesículas Cubiertas/metabolismo , Reactivos de Enlaces Cruzados , Aparato de Golgi/metabolismo , Ingeniería de Proteínas/métodos , Azirinas , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteína Coatómero , Fenilalanina/análogos & derivados , Pruebas de Precipitina , Unión Proteica , ARN de Transferencia/genética , Receptores de Superficie Celular/metabolismo , Supresión Genética , Rayos UltravioletaRESUMEN
The Liber passionalis is an early and hitherto mostly unexplored example of a composite medical work on diagnosis and therapy, similar to the better-known compilations circulating under the titles Petroncellus and Gariopontus (Passionarius Galieni). It shows the efforts made to provide comprehensive coverage of morbid conditions drawn from a choice of the best sources available to the compiler, sources which in some instances complement or enhance our knowledge of ancient medicine in a way overlooked by specialists in the field for a long time and which provide the best clue when trying to assess medical expertise at the turn of the first millennium. The paper explores the transmission, structure and sources of the Liber passionalis, touching on the body of medical literature available in the early Middle Ages.
Asunto(s)
Libros/historia , Diagnóstico , Religión y Medicina , Terapéutica/historia , Historia MedievalRESUMEN
The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav-/- B cells. In vivo, vav-/- mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav-/- B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav-/- mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Ciclo Celular , Proteínas Proto-Oncogénicas/fisiología , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Anticuerpos Antivirales/biosíntesis , Antígenos/administración & dosificación , Antígenos Virales/administración & dosificación , Antígenos Virales/inmunología , Linfocitos B/virología , Nucleótidos de Guanina/fisiología , Haptenos/administración & dosificación , Haptenos/inmunología , Inmunoglobulina M/biosíntesis , Inyecciones Intravenosas , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitrofenoles/administración & dosificación , Nitrofenoles/inmunología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Fenilacetatos , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
The guanine nucleotide-exchange factor Vav is a regulator of antigen-mediated cytoskeletal reorganization required for receptor clustering, proliferation and thymic selection. Moreover, Vav has been identified as a major substrate in the CD28 signal transduction pathway and overexpression of Vav enhances TCR-mediated IL-2 secretion in T cells. Here we show that CD3- plus CD28-mediated proliferation and IL-2 production were reduced in vav gene-deficient T cells. However, Vav had no apparent role in phorbol 12-myristate 13-acetate-plus CD28-mediated proliferation and IL-2 production, suggesting that Vav acts downstream of the TCR/CD3 complex. In vivo, Vav expression was crucial to generate primary vesicular stomatitis virus (VSV)-specific cytotoxic T cell responses. In contrast, vav-/- mice exhibited a reduced but significant footpad swelling after lymphocytic choriomeningitis virus (LCMV) infections and mounted a measurable primary cytotoxic T cell response to LCMV. Upon in vitro restimulation, cytotoxic T cell responses of both VSV- and LCMV-infected mice reached near normal levels. Our data provide the first genetic evidence that Vav is an important effector molecule that relays antigen receptor signaling to IL-2 production and activation of cytotoxic T cells.
Asunto(s)
Antígenos CD28/inmunología , Proteínas Oncogénicas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Interleucina-2/biosíntesis , Ratones , Ratones Noqueados , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunologíaRESUMEN
The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.
Asunto(s)
Apoptosis , Hígado/embriología , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Animales , Daño del ADN , Marcación de Gen , Hematopoyesis/genética , Hígado/citología , Ratones , Ratones Noqueados , Mutagénesis , Neovascularización Fisiológica/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genéticaRESUMEN
The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav-/- thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28-mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav-/- thymocytes. Vav was found to bind constitutively to PKC-theta in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.
Asunto(s)
Apoptosis/genética , Apoptosis/inmunología , Proteínas de Ciclo Celular , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/genética , Linfocitos T/inmunología , Linfocitos T/patología , Animales , Antígenos CD28/inmunología , Complejo CD3/inmunología , Citocinas/inmunología , Citoesqueleto/inmunología , Citoesqueleto/patología , Ratones , Ratones Transgénicos , Péptidos , Proteínas Proto-Oncogénicas c-vav , Transducción de Señal/inmunologíaRESUMEN
The haematopoietic-specific Rho-family guanine-nucleotide exchange factor Vav is a regulator of lymphocyte antigen receptor signaling leading to proliferation of B and T cells, generation of the B1 cell lineage and IL-2 production and maturation in T cells. The specific role it plays in these events, however, has not yet been resolved. Recent findings suggest that Vav is recruited to activated antigen receptors and requires both tyrosine phosphorylation and the presence of activating phospholipids for catalytic activity towards Rho-family GTPases. Studies form vav-deficient mice show that in response to antigen receptor activation, Vav is not essential for activation of JNK kinase pathways, but is required for actin polymerisation and T cell capping. We discuss Vav function in the light of these new findings.
Asunto(s)
Proteínas de Ciclo Celular , Citoesqueleto/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Actinas/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Factores de Intercambio de Guanina Nucleótido , Activación de Linfocitos , Ratones , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-vav , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Vav is a guanine-nucleotide exchange factor for the Rho-like small GTPases RhoA, Rac1 and Cdc42, which regulate cytoskeletal reorganization and activation of stress-activated protein kinases (SAPK/JNKs). Vav is expressed in hematopoietic cells and is phosphorylated in T and B cells following activation of various growth factor or antigen receptors. Vav interacts with several signaling molecules in T cells, but the functional relevance of these interactions is established only for Slp76: they cooperate to induce activity of the transcription factor NF-AT and interleukin-2 expression. We have investigated the role of Vav in T cells by generating vav-/- mice. RESULTS: Mice deficient for vav were viable and healthy, but had impaired T-cell development. In vav-/- T cells, in response to activation of the T-cell receptor (TCR), cell cycle progression, induction of NF-ATc1 activity, downregulation of the cell-cycle inhibitor p27Kip1, interleukin-2 production, actin polymerization and the clustering of TCRs into patches and caps--a cytoskeletal reorganization process--were defective. TCR-mediated activation of mitogen-activated protein kinase and SAPK/JNK was unaffected. Ca2+ mobilization was impaired in vav-/- thymocytes and T cells. In wild-type cells, Vav constitutively associated with the cytoskeletal membrane anchors talin and vinculin. In the absence of Vav, phosphorylation of Slp76, Slp76-talin interactions, and recruitment of the actin cytoskeleton to the CD3 zeta chain of the TCR co-receptor were impaired. CONCLUSIONS: Vav is a crucial regulator of TCR-mediated Ca2+ flux, cytoskeletal reorganization and TCR clustering, and these are required for T-cell maturation, interleukin-2 production and cell cycle progression.