MHC-II-independent CD4+ T cells induce colitis in immunodeficient RAG-/- hosts.

CD4(+) alpha beta T cells from either normal C57BL/6 (B6) or MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice engrafted into congenic immunodeficient RAG1(-/-) B6 hosts induced an aggressive inflammatory bowel disease (IBD). Furthermore, CD4(+) T cells from CD1d(-/-) knockout (KO) B6 donor mice but not those from MHC-I(-/-) (homozygous transgenic mice deficient for beta(2)-microglobulin) KO B6 mice induced a colitis in RAG(-/-) hosts. Abundant numbers of in vivo activated (CD69(high)CD44(high)CD28(high)) NK1(+) and NK1(-) CD4(+) T cells were isolated from the inflamed colonic lamina propria (cLP) of transplanted mice with IBD that produced large amounts of TNF-alpha and IFN-gamma but low amounts of IL-4 and IL-10. IBD-associated cLP Th1 CD4(+) T cell populations were polyclonal and MHC-II-restricted when derived from normal B6 donor mice, but oligoclonal and apparently MHC-I-restricted when derived from MHC-II-deficient (A alpha(-/-) or A beta(-/-)) B6 donor mice. cLP CD4(+) T cell populations from homozygous transgenic mice deficient for beta(2)-microglobulin KO B6 donor mice engrafted into RAG(-/-) hosts were Th2 and MHC-II restricted. These data indicate that MHC-II-dependent as well as MHC-II-independent CD4(+) T cells can induce a severe and lethal IBD in congenic, immunodeficient hosts, but that the former need the latter to express its IBD-inducing potential.

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.

Estudi dels factors mol•leculars i cel•lulars implicats en les alteracions corticals de la Síndrome de down

Investigación producida a partir de una estancia en en el Instituto de Neurociencias de la Universidad Miguel Hernández entre enero y mayo del 2007. El SD o trisomía del cromosoma 21 es la aneuploidía cromosómica más frecuente y constituye la principal causa de retraso mental. Las cuestiones que aún son objeto de debate en el SD son: 1) si pueden existir, entre los genes triplicados, algunos que contribuyan de forma más importante a algunos de los fenotipos observables en SD y, 2) hasta qué punto los fenotipos observados derivan de alteraciones del neurodesarrollo o de alteraciones funcionales en el adulto. Con el fin de abordar esta cuestión nos hemos centrado en las alteraciones cognitivas del SD y hemos realizado la caracterización del papel de Dyrk1A en el desarrollo de una estructura clave para esta función: la corteza cerebral. Los resultados obtenidos muestran que la sobrexpresión de Dyrk1A produce un desajuste proliferativo dando lugar a un retraso en la formación de la subplaca, con consecuencias en la laminación de la placa cortical. Las alteraciones en la corticogénesis van a tener consecuencias en el establecimiento de la conectividad tálamo-cortical que se encuentra marcadamente retrasada. En el hipocampo, los ratones transgénicos mostraron una reducción del grosor de las capas. Estos resultados pueden ser relevantes para el SD, puesto que es similar a lo observado en fetos SD.

Papel de la Interleucina 6 (IL-6) en la regulación de la expresión de Osteopontina (OPN) y CD44 tras axotomía del nervio facial

Investigación producida a partir de una estancia en la University of Sidney, Australia, entre octubre del 2008 y enero del 2009. Se ha desarrollado el proyecto titulado "Papel de la interleucina 6 (IL6) en la regulación de la expresión de Osteopontina (OPN) y de CD44 tras axotomía del nervio facial". Tras efectuar una transección del nervio facial, se indujo una reactividad glial en el núcleo facial (NF) localizado en el tronco cerebral, utilizando ratones transgénicos que sobrexpresan IL6 bajo promotor GFAP (tg GFAP-IL6), es decir selectivamente en astrocitos. Se han utilizado técnicas histoquímicas e inmunohistoquímicas, así como también se ha completado el estudio utilizando análisis de RPA, western blotting y citometría de flujo para la identificación de poblaciones celulares. Los resultados obtenidos indican que la OPN se expresa constitutivamente en las neuronas del NF. Tras axotomía del nervio facial, la expresión de OPN y CD44 incrementa en los ratones WT, mientras que en los tg GFAP-IL6 disminuye significativamente, sugiriendo que la IL6 podría estar involucrada en la modulación de la expresión de ambas moléculas. Sin embargo, no se ha visto diferencias en otros receptores de OPN como la integrina Alpha-5. La ctometría de flujo corroboró algunos de los resultados histológicos sobre la reactividad microglial y permitió concluir que la proporción de microglía activada (CD11b+/CD45+mid) y macrófagos (CD11b+/CD45+high) que expresan CD44 incrementa en in los tg GFAP-IL6 versus WT donde la mayor parte de microglia activada mostraba un perfil CD11b+/CD45+low.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

Notch1 control of oligodendrocyte differentiation in the spinal cord.

We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.

Participation of connexin-based channels in the control of vascular function

SUMMARYIntercellular communication is achieved at specialized regions of the plasma membrane by gap junctions. The proteins constituting the gap junctions are called connexins and are encoded by a family of genes highly conserved during evolution. In adult mouse, four connexins (Cxs) are known to be expressed in the vasculature: Cx37, Cx40, Cx43 and Cx45. Several recent studies have provided evidences that vascular connexins expression and blood pressure regulation are closely linked, suggesting a role for connexins in the control of blood pressure. However, the precise function that each vascular connexin plays under physiological and pathophysiological conditions is still not elucidated. In this context, this work was dedicated to evaluate the contribution of each of the four vascular connexins in the control of the vascular function and in the blood pressure regulation.In the present work, we first demonstrated that vascular connexins are differently regulated by hypertension in the mouse aorta. We also observed that endothelial connexins play a regulatory role on eNOS expression levels and function in the aorta, therefore in the control of vascular tone. Then, we demonstrated that Cx40 plays a pivotal role in the kidney by regulating the renal levels of COX-2 and nNOS, two key enzymes of the macula densa known to participate in the control of renin secreting cells. We also found that Cx43 forms the functional gap junction involved in intercellular Ca2+ wave propagation between vascular smooth muscle cells. Finally, we have started to generate transgenic mice expressing specifically Cx40 in the endothelium to investigate the involvement of Cx40 in the vasomotor tone, or in the renin secreting cells to evaluate the role of Cx40 in the control of renin secretion.In conclusion, this work has allowed us to identify new roles for connexins in the vasculature. Our results suggest that vascular connexins could be interesting targets for new therapies caring hypertension and vascular diseases.

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent.

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

Connexin implication in the control of the murine beta-cell mass.

Diabetes develops when the insulin needs of peripheral cells exceed the availability or action of the hormone. This situation results from the death of most beta-cells in type 1 diabetes, and from an inability of the beta-cell mass to adapt to increasing insulin needs in type 2 and gestational diabetes. We analyzed several lines of transgenic mice and showed that connexins (Cxs), the transmembrane proteins that form gap junctions, are implicated in the modulation of the beta-cell mass. Specifically, we found that the native Cx36 does not alter islet size or insulin content, whereas the Cx43 isoform increases both parameters, and Cx32 has a similar effect only when combined with GH. These findings open interesting perspectives for the in vitro and in vivo regulation of the beta-cell mass.

Cardiac Lineage Protein-1 (CLP-1) Regulates Cardiac Remodeling via Transcriptional Modulation of Diverse Hypertrophic and Fibrotic Responses and Angiotensin II-transforming Growth Factor β (TGF-β1) Signaling Axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

Mast cells are dispensable for normal and activin-promoted wound healing and skin carcinogenesis.

The growth and differentiation factor activin A is a key regulator of tissue repair, inflammation, fibrosis, and tumorigenesis. However, the cellular targets, which mediate the different activin functions, are still largely unknown. In this study, we show that activin increases the number of mature mast cells in mouse skin in vivo. To determine the relevance of this finding for wound healing and skin carcinogenesis, we mated activin transgenic mice with CreMaster mice, which are characterized by Cre recombinase-mediated mast cell eradication. Using single- and double-mutant mice, we show that loss of mast cells neither affected the stimulatory effect of overexpressed activin on granulation tissue formation and reepithelialization of skin wounds nor its protumorigenic activity in a model of chemically induced skin carcinogenesis. Furthermore, mast cell deficiency did not alter wounding-induced inflammation and new tissue formation or chemically induced angiogenesis and tumorigenesis in mice with normal activin levels. These findings reveal that mast cells are not major targets of activin during wound healing and skin cancer development and also argue against nonredundant functions of mast cells in wound healing and skin carcinogenesis in general.

Mechanisms of B cell survival induced by BAFF, APRIL and their receptors

RESUME : BAFF est un membre de 1a famille du TNF qui contrôle l'homéostasie des lymphocytes B. BAFF lie les récepteurs TACI, BCMA et BAFF-R sur les cellules B, tandis qu'APRIL, son proche homologue, lie seulement TACI et BCMA. BAFF et APRIL sont des protéines transmembranaires pouvant -être relâchées sous forme de cytokines trimériques solubles suite à un clivage protéolytique. Le BAFF soluble peut s'assembler en 60-mère. Les rôles physiologiques des BAFF membranaires et solubles sont inconnnus. Nous avons étudié la capacité de diverses formes de BAFF et APRIL à activer différents récepteurs. BAFF-R répond à toutes les formes dé BAFF, tandis que TACI nécessite du BAFF ou de l'APRIL membranaire ou oligomérisé pour être activé et pour transmettre des signaux de survie dans les lymphocytes B primaires. TACI ne répond pas aux ligands trimériques bien qu'il puisse les lier. TACI est essentiel pour la réponse humorale aux antigènes présentant des épitoges répétitifs, une réponse qui est indépendante des lymphocytes T (réponse TI-2). Des souris exprimant moins de BAFF ont un pourcentage modérément réduit de lymphocytes B et leur réponse TI-2 est atténuée. Par contre, des souris qui n'expriment que du BAFF membranaire ont encore moins de cellules B mais répondent efficacement aux antigènes TI-2. Ces résultats suggèrent que le BAFF soluble est impliqué dans le maintien de la population des lymphocytes B, alors que le BAFF membranaire peut activer TACI lors d'are réponse TI-2. Le BAFF 60-mère est un autre activateur potentiel de TACI in vivo. Le BAFF 60-mère existe dans des surnageants de cellules productrices de BAFF mais n'est pas détecté dans le plasma de souris saines, même lorsqu'elles présentent des niveaux élevés de BAFF. BAFF 60-mère est néanmoins présent dans le plasma de souris transgéniques pour BAFF et de souris déficientes en TACI. Comme ces deux lignées présentent des signes d'autoimmunité, ces résultats suggèrent que la présence de BAFF 60-mère pourrait être liée à des conditions pathologiques. Summary : The TNF family ligand BAFF is essential for B cell homeostasis. BAFF binds to the receptors TACI, BCMA and BAFF-R on B cells, whereas its close homolog APRIL binds to TACI and BCMA only. BAFF and APRIL are transmembrane proteins, which can be proteolytically processed to release trimeric soluble cytokines. Soluble BAFF 3-mer can further assemble in a 60-mer. The physiological roles of membrane-bound and soluble BAFF are unknown. We studied the ability of various forms of BAFF and APRIL to signal through different receptors. BAFF-R responded to all forms of BAFF, but TACI required membrane-bound, cross-licked or oligomeric BAFF or APRIL in order to transmit productive signals in primary B cells. TACI was unresponsive to trimeric ligands, although it could bind them. TACI is essential for T-cell independent antibody responses to antigens with repetitive epitopes (TI-2 responses). Mice expressing lower than normal levels of BAFF displayed a moderate B cell reduction and impaired TI-2 responses, whereas mice expressing membrane-bound BAFF displayed severe B cell reduction, but unimpaired TI-2 responses. These results suggest that processed BAFF is involved in the maintenance of the B cell pool and that membrane-bound BAFF can activate TACI during T-cell independent humoral responses. BAFF 60-mer is another potential activator of TACI in vivo. BAFF 60-mer was detected in the supernatant of BAFF-producing cells, but not in the plasma of healthy mice with either norma1 or elevated BAFF levels. It was however present in sera of BAFF transgenic mice and TACI-/- mice, both of which suffer from autoimmunity, suggesting that GAFF 60-mer may be linked to pathogenic conditions.

SCAP is required for timely and proper myelin membrane synthesis.

Myelination requires a massive increase in glial cell membrane synthesis. Here, we demonstrate that the acute phase of myelin lipid synthesis is regulated by sterol regulatory element-binding protein (SREBP) cleavage activation protein (SCAP), an activator of SREBPs. Deletion of SCAP in Schwann cells led to a loss of SREBP-mediated gene expression involving cholesterol and fatty acid synthesis. Schwann cell SCAP mutant mice show congenital hypomyelination and abnormal gait. Interestingly, aging SCAP mutant mice showed partial regain of function; they exhibited improved gait and produced small amounts of myelin indicating a slow SCAP-independent uptake of external lipids. Accordingly, extracellular lipoproteins partially rescued myelination by SCAP mutant Schwann cells. However, SCAP mutant myelin never reached normal thickness and had biophysical abnormalities concordant with abnormal lipid composition. These data demonstrate that SCAP-mediated regulation of glial lipogenesis is key to the proper synthesis of myelin membrane, and provide insight into abnormal Schwann cell function under conditions affecting lipid metabolism.

Increased efflux of amyloid-β peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.

The formation and accumulation of toxic amyloid-β peptides (Aβ) in the brain may drive the pathogenesis of Alzheimer's disease. Accordingly, disease-modifying therapies for Alzheimer's disease and related disorders could result from treatments regulating Aβ homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aβ or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aβ burden in AβPPPS1, hAβPPSL, and AβPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aβ peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aβ in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aβ in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aβ peptide in pre-plaque mhAβPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aβ peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aβ transport across the Blood Brain Brain (BBB). Thus increased Aβ clearance through the BBB may contribute to reduced Aβ burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects.

Regulation of gammadelta versus alphabeta T lymphocyte differentiation by the transcription factor SOX13.

alphabeta and gammadelta T cells originate from a common, multipotential precursor population in the thymus, but the molecular mechanisms regulating this lineage-fate decision are unknown. We have identified Sox13 as a gammadelta-specific gene in the immune system. Using Sox13 transgenic mice, we showed that this transcription factor promotes gammadelta T cell development while opposing alphabeta T cell differentiation. Conversely, mice deficient in Sox13 expression exhibited impaired development of gammadelta T cells but not alphabeta T cells. One mechanism of SOX13 function is the inhibition of signaling by the developmentally important Wnt/T cell factor (TCF) pathway. Our data thus reveal a dominant pathway regulating the developmental fate of these two lineages of T lymphocytes.