YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.

YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy.

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.

A feasibility study for the analysis of reparative dentinogenesis in pOBCol3.6GFPtpz transgenic mice.

AIM: To examine the feasibility of using the pOBCol3.6GFPtpz [3.6-green fluorescent protein (GFP)] transgenic mice as an in vivo model for studying the biological sequence of events during pulp healing and reparative dentinogenesis. METHODOLOGY: Pulp exposures were created in the first maxillary molar of 12-16-week-old 3.6-GFP transgenic mice with CD1 and C57/Bl6 genetic background. Direct pulp capping on exposed teeth was performed using mineral trioxide aggregate followed by restoration with a light-cured adhesive system (AS) and composite resin. In control teeth, the AS was placed in direct contact with the pulp. Animals were euthanized at various time points after pulp exposure and capping. The maxillary arch was isolated, fixed and processed for histological and epifluorescence analysis to examine reparative dentinogenesis. RESULTS: Analysis of teeth immediately after pulp exposure revealed absence of odontoblasts expressing 3.6-GFP at the injury site. Evidence of reparative dentinogenesis was apparent at 4 weeks in 3.6-GFP mice in CD1 background and at 8 weeks in 3.6-GFP mice with C57/Bl6 background. The reparative dentine with both groups contained newly formed atubular-mineralized tissue resembling a dentine bridge and/or osteodentine that was lined by cells expressing 3.6-GFP as well as 3.6-GFP expressing cells embedded within the atubular matrix. CONCLUSION: This study was conducted in a few animals and did not allow statistical analysis. The results revealed that the 3.6-GFP transgenic animals provide a unique model for direct analysis of cellular and molecular mechanisms of pulp repair and tertiary dentinogenesis in vivo. The study also shows the effects of the capping material and the genetic background of the mice in the sequence and timing of reparative dentinogenesis.

Phosphoproteomics and cancer research.

Protein phosphorylation plays key roles in the regulation of normal and cancer cells. It is a highly dynamic process. Protein kinases are the targets of several new cancer drugs and drug candidates. However, some of the main issues related to new drugs are how they function and the selection of those patients that will likely respond best to a particular treatment regime. There is an urgent need to understand and monitor kinase signalling pathways. Phosphoproteomics requires the enrichment of phosphorylated proteins or peptides from tissue or bodily fluids, and the application of technologies such as mass spectrometry (MS) to the identification and quantification of protein phosphorylation sites. As the field develops it will provide pharmacodynamic readouts of disease states and cellular drug responses in tumour samples. There have been a number of recent advances, but there are still technical hurdles and bioinformatics challenges that need to be addressed.

The Ca2+-dependent protease Calpain A regulates Cactus/I kappaB levels during Drosophila development in response to maternal Dpp signals.

Regulation of NF kappaB activity is central to many processes during development and disease. Activation of NF kappaB family members depends on degradation of inhibitory I kappaB proteins. In Drosophila, a nuclear gradient of the NF kappaB/c-rel protein Dorsal subdivides the embryonic dorsal-ventral axis, defining the extent and location of mesodermal and ectodermal territories. Activation of the Toll pathway directs Dorsal nuclear translocation by inducing proteosomal degradation of the I kappaB homologue Cactus. Another mechanism that impacts on Dorsal activation involves the Toll-independent pathway, which regulates constitutive Cactus degradation. We have shown that the BMP protein Decapentaplegic (Dpp) inhibits Cactus degradation independent of Toll. Here we report on a novel element of this pathway: the calcium-dependent protease Calpain A. Calpain A knockdowns increase Cactus levels, shifting the Dorsal gradient and dorsal-ventral patterning. As shown for mammalian I kappaB, this effect requires PEST sequences in the Cactus C-terminus, implying a conserved role for calpains. Alteration of Calpain A or dpp results in similar effects on Dorsal target genes. Epistatic analysis confirms Calpain A activity is regulated by Dpp, indicating that Dpp signals increase Cactus levels through Calpain A inhibition, thereby interfering with Dorsal activation. This mechanism may allow coordination of Toll, BMP and Ca(2+) signals, conferring precision to Dorsal-target expression domains.

Nonclassic congenital lipoid adrenal hyperplasia: a new disorder of the steroidogenic acute regulatory protein with very late presentation and normal male genitalia.

CONTEXT: Lipoid congenital adrenal hyperplasia is a severe disorder of adrenal and gonadal steroidogenesis caused by mutations in the steroidogenic acute regulatory protein (StAR). Affected children typically present with life-threatening adrenal insufficiency in early infancy due to a failure of glucocorticoid (cortisol) and mineralocorticoid (aldosterone) biosynthesis, and 46,XY genetic males have complete lack of androgenization and appear phenotypically female due to impaired testicular androgen secretion in utero. OBJECTIVE: The objective of this study was to investigate whether nonclassic forms of this condition exist. PATIENTS AND METHODS: Sequence analysis of the gene encoding StAR was undertaken in three children from two families who presented with primary adrenal insufficiency at 2-4 yr of age; the males had normal genital development. Identified mutants were tested in a series of biochemical assays. RESULTS: DNA sequencing identified homozygous StAR mutations Val187Met and Arg188Cys in these two families. Functional studies of StAR activity in cells and in vitro and cholesterol-binding assays showed these mutants retained approximately 20% of wild-type activity. CONCLUSIONS: These patients define a new disorder, nonclassic lipoid congenital adrenal hyperplasia, and represent a new cause of nonautoimmune Addison disease (primary adrenal failure).

Exercise improves insulin and leptin sensitivity in hypothalamus of Wistar rats.

Prolonged exercise of medium to high intensity is known to promote a substantial effect on the energy balance of rats. In male rats, moderately to severely intense programs lead to a reduction in food intake. However, the exact causes for the appetite-suppressive effects of exercise are not known. Here, we show that intracerebroventricular insulin or leptin infusion reduced food intake in exercised rats to a greater extent than that observed in control animals. Exercise was associated with a markedly increased phosphorylation/activity of several proteins involved in leptin and insulin signal transduction in the hypothalamus. The regulatory role of interleukin (IL)-6 in mediating the increase in leptin and insulin sensitivity in hypothalamus was also investigated. Treatment with insulin or leptin markedly reduced food intake in exercised rats that were pretreated with vehicle, although no increase in sensitivity to leptin- and insulin-induced anorexia after pretreatment with anti-IL-6 antibody was detected. The current study provides direct measurements of leptin and insulin signaling in the hypothalamus and documents increased sensitivity to these hormones in the hypothalamus of exercised rats in an IL-6-dependent manner. These findings provide support for the hypothesis that the appetite-suppressive actions of exercise may be mediated by the hypothalamus.

Leptin inhibits apoptosis in thymus through a janus kinase-2-independent, insulin receptor substrate-1/phosphatidylinositol-3 kinase-dependent pathway.

The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we used Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, whereas signal transduction was evaluated by immunoprecipitation, immunoblot, and confocal microscopy. The Ob receptor (ObR) was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was colocalized with Janus kinase (JAK)-2 and signal transducer and activator of transcription-3, and an acute, in vivo, injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of signal transducer and activator of transcription-3. The treatment with leptin also led to the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG(490) but was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY(294002) and an antisense oligonucleotide to IRS-1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS-1/phosphatidylinositol 3-kinase pathway.

p40phox: the last NADPH oxidase subunit.

The phagocytic NADPH-oxidase is a multiprotein system activated during the inflammatory response to produce superoxide anion (O2-), which is the substrate for formation of additional reactive oxygen species (ROS). The importance of this system for innate immunity is established by chronic granulomatous disease (CGD), a primary immunodeficiency caused by defects in the NADPH oxidase. In this review, we present and discuss recent knowledge about p40phox, the last NADPH oxidase component to be identified. Furthermore, its interaction with cellular pathways outside of the NADPH oxidase is discussed. Described in this review is evidence that p40phox participates in NADPH oxidase dynamics within cells, what is known about its role in the oxidase, the possibility that p40phox participates in non-NADPH oxidase processes in phagocytic and non-phagocytic cells and whether p40phox could mediate a similar function in other NADPH oxidases. An improved understanding of p40phox should provide new insights about NADPH oxidase, the physiology of phagocytic cells and the innate immune system.

Cross-talk between the insulin and leptin signaling systems in rat hypothalamus.

OBJECTIVE: To investigate whether insulin and leptin share common intracellular signal transduction pathways and to determine whether these hormonal signaling systems modulate each other's action in rat hypothalamus. RESEARCH METHODS AND PROCEDURES: Male Wistar rats were studied after chronic implantation of an intracerebroventricular catheter into the third ventricle. Immunoprecipitation and immunoblotting were used to examine the activation of insulin and leptin signaling molecules in the rat hypothalamus. RESULTS: Insulin alone is able to produce molecular activation of insulin receptor substrates (IRSs)/phosphatidylinositol 3-kinase (PI 3-kinase)/Akt and mitogen-activated protein (MAP) kinase signaling pathways in hypothalamus, whereas leptin alone activates MAP kinase and IRSs/PI 3-kinase signaling with no effect on Akt. Combined infusion of leptin and insulin provokes a dual action. There was no quantitative potentialization of any single hormone's action on the elements of the insulin signaling pathway, IRSs/PI 3-kinase/Akt, and MAP kinase. Conversely, leptin plus insulin leads to quantitative potentialization of molecular signaling through the Janus kinase/signal transducer and activator of transcription pathway. DISCUSSION: We provide evidence for a convergence of leptin and insulin signaling at the level of IRSs-PI 3-kinase and a divergence at the level of Akt. Moreover, our results indicate a direct and positive cross-talk between insulin and leptin at the level of Janus kinase 2 and signal transducer and activator of transcription 3 tyrosine phosphorylation. This mechanism may serve to potentiate the activity of both insulin and leptin pathways and to increase stimulation in physiological processes such as the control of food intake and body weight, which are under the combined control of insulin and leptin.

Macrophages and mast cells control the neutrophil migration induced by dentin proteins.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1beta, TNF-alpha, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP-treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1beta, TNF-alpha, and MIP-2 by macrophages. This inhibitory activity of the DSP-stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.

The maternal JAK/STAT pathway of Drosophila regulates embryonic dorsal-ventral patterning

Activation of NFkappaB plays a pivotal role in many cellular processes such as inflammation, proliferation and apoptosis. In Drosophila, nuclear translocation of the NFkappaB-related transcription factor Dorsal is spatially regulated in order to subdivide the embryo into three primary dorsal-ventral (DV) domains: the ventral presumptive mesoderm, the lateral neuroectoderm and the dorsal ectoderm. Ventral activation of the Toll receptor induces degradation of the IkappaB-related inhibitor Cactus, liberating Dorsal for nuclear translocation. In addition, other pathways have been suggested to regulate Dorsal. Signaling through the maternal BMP member Decapentaplegic (Dpp) inhibits Dorsal translocation along a pathway parallel to and independent of Toll. In the present study, we show for the first time that the maternal JAK/STAT pathway also regulates embryonic DV patterning. Null alleles of loci coding for elements of the JAK/STAT pathway, hopscotch (hop), marelle (mrl) and zimp (zimp), modify zygotic expression along the DV axis. Genetic analysis suggests that the JAK kinase Hop, most similar to vertebrate JAK2, may modify signals downstream of Dpp. In addition, an activated form of Hop results in increased levels of Cactus and Dorsal proteins, modifying the Dorsal/Cactus ratio and consequently DV patterning. These results indicate that different maternal signals mediated by the Toll, BMP and JAK/STAT pathways may converge to regulate NFkappaB activity in Drosophila.

The maternal JAK/STAT pathway of Drosophila regulates embryonic dorsal-ventral patterning.

Activation of NFkappaB plays a pivotal role in many cellular processes such as inflammation, proliferation and apoptosis. In Drosophila, nuclear translocation of the NFkappaB-related transcription factor Dorsal is spatially regulated in order to subdivide the embryo into three primary dorsal-ventral (DV) domains: the ventral presumptive mesoderm, the lateral neuroectoderm and the dorsal ectoderm. Ventral activation of the Toll receptor induces degradation of the IkappaB-related inhibitor Cactus, liberating Dorsal for nuclear translocation. In addition, other pathways have been suggested to regulate Dorsal. Signaling through the maternal BMP member Decapentaplegic (Dpp) inhibits Dorsal translocation along a pathway parallel to and independent of Toll. In the present study, we show for the first time that the maternal JAK/STAT pathway also regulates embryonic DV patterning. Null alleles of loci coding for elements of the JAK/STAT pathway, hopscotch (hop), marelle (mrl) and zimp (zimp), modify zygotic expression along the DV axis. Genetic analysis suggests that the JAK kinase Hop, most similar to vertebrate JAK2, may modify signals downstream of Dpp. In addition, an activated form of Hop results in increased levels of Cactus and Dorsal proteins, modifying the Dorsal/Cactus ratio and consequently DV patterning. These results indicate that different maternal signals mediated by the Toll, BMP and JAK/STAT pathways may converge to regulate NFkappaB activity in Drosophila.

Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver.

Chronic leptin treatment markedly enhances the effect of insulin on hepatic glucose production unproportionally with respect to body weight loss and increased insulin sensitivity. In the present study the cross-talk between insulin and leptin was evaluated in rat liver. Upon stimulation of JAK2 tyrosine phosphorylation, leptin induced JAK2 co-immunoprecipitation with STAT3, STAT5b, IRS-1 and IRS-2. This phenomenon parallels the leptin-induced tyrosine phosphorylation of STAT3, STAT5b, IRS-1 and IRS-2. Acutely injected insulin stimulated a mild increase in tyrosine phosphorylation of JAK2, STAT3 and STAT5b. Leptin was less effective than insulin in stimulating IRS phosphorylation and their association with PI 3-kinase. Simultaneous treatment with both hormones yielded no change in maximal phosphorylation of STAT3, IRS-1, IRS-2 and Akt, but led to a marked increase in tyrosine phosphorylation of JAK2 and STAT5b when compared with isolated administration of insulin or leptin. This indicates that there is a positive cross-talk between insulin and leptin signaling pathways at the level of JAK2 and STAT5b in rat liver.

Control of human luteal steroidogenesis.

The human corpus luteum (CL) undergoes a dynamic cycle of differentiation, steroid hormone production and regression during the course of non-fertile cycles. In humans and other primates, luteal steroidogenesis is absolutely dependent on pituitary-derived LH. However, changes in LH and LH receptor expression do not explain the marked decline in progesterone production at the end of the luteal phase. Changes in the level of the steroidogenic acute regulatory protein (StAR), a gene whose expression is controlled by LH most likely account for the cyclic pattern of progesterone production. During the mid-to-late luteal phase of a fertile cycle, chorionic gonadotropin (hCG) rescues the CL, overcoming the actions of the factors inducing luteolysis. Although the agents causing regression of the CL in a non-fertile cycle are not yet known, intra-luteal growth factors and cytokines that modify the action of LH probably contribute to the reduction of StAR expression and the subsequent fall in progesterone production.

Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon and requirement of interferon regulatory factor 1.

The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection with Toxoplasma gondii was investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected with T. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-gamma (IFN-gamma(-/-)) or IFN regulatory factor -1 (IRF-1(-/-)). These findings indicate the important role of endogenous IFN-gamma and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection with T. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected with T. gondii. Together with the results showing the extreme susceptibility of the IFN-gamma(-/-) and the IRF-1(-/-) mice to infection with T. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.

Prolactin and autoimmunity.

Prolactin (PRL) is a versatile hormone that is produced by the anterior pituitary gland and various extrapituitary sites including immune cells. Furthermore, PRL has widespread influences on proliferation and differentiation of a variety of cells in the immune system and is, in effect, a cytokine. PRL-receptors (PRL-R) are distributed throughout the immune system and are included as members of the cytokine receptor superfamily. PRL-R signal transduction is mediated by a complex array of signaling molecules of which JAK2, Stat1 and Stat5 pathway have been well studied. In PRL-stimulated T cells, the transcription factor gene, interferon regulatory factor-1 provides a mechanism whereby PRL can regulate the immune response. The human PRL gene is situated on the short arm of chromosome 6 close to the major histocompatibility complex. Polymorphisms of the human PRL gene have implications for production of lymphocyte PRL in SLE. Mild and moderate hyperprolactinemia (HPRL) has been demonstrated in 20-30% of SLE patients and is associated with active disease. HPRL may have a role in lupus nephritis and central nervous system involvement of SLE patients. HPRL stimulated the production of autoantibodies. These evidences support the important role of PRL in autoimmunity and autoimmune diseases, mainly SLE.

MAGUK proteins: structure and role in the tight junction.

ZO-1, ZO-2 and ZO-3 are tight junction (TJ)-associated proteins that belong to the MAGUK family. In addition to the presence of the characteristic MAGUK modules (PDZ, SH3 and GK), ZOs have a distinctive carboxyl terminal with splicing domains, acidic- and proline-rich regions. The modular organization of these proteins allows them to function as scaffolds, which associate to transmembrane TJ proteins, the cytoskeleton and signal transduction molecules. ZOs shuttle between the TJ and the nucleus, where they may regulate gene expression.

Role of interferon and interferon regulatory factors in early protection against Venezuelan equine encephalitis virus infection.

To investigate the role of type I interferon (IFN) and its regulatory transacting proteins, interferon regulatory factors (IRF-1 and IRF-2), in early protection against infection with virulent Venezuelan equine encephalitis virus (VEE), we utilized mice with targeted mutations in the IFN-alpha/beta receptor, IRF-1, or IRF-2 genes. IFN-alpha/beta-receptor knockout mice are highly susceptible to peripheral infection with virulent or attenuated VEE, resulting in their death within 24 and 48 h, respectively. Treatment of normal macrophages with anti-IFN-alpha/beta antibody prior to and during infection with molecularly cloned virulent VEE resulted in increased VEE replication. However, treatment with high doses of IFN or IFN-inducing agents failed to alter percentage mortality or average survival times in mice challenged with a low dose of virulent VEE. In IRF-1 and IRF-2 knockout mice (IRF-1(-/-) and IRF-2(-/-)), the 100% protection against virulent VEE that is conferred by attenuated VEE within 24 h in control C57BL/6 mice was completely absent in IRF-2(-/-) mice, whereas 50% of IRF-1(-/-) mice were protected. IRF-2(-/-) mice were deficient in clearing VEE virus from the spleen and the brain compared to the heterozygous IRF-2(+/-) knockout or C57BL/6 (+/+) mice. Furthermore, a distinct pattern of histopathological changes was observed in brains of IRF-2(-/-) mice after VEE exposure. Taken together, these findings imply that the altered immune response in IRF-1 and IRF-2 knockout mice results in altered virus dissemination, altered virus clearance, and altered virus-induced pathology. Thus, type I interferon, as well as IRF-1 and IRF-2, appears to play an important and necessary role in the pathogenesis of, and protection against, VEE infection.