Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

Chondrogenic differentiation within magnetic-responsive multilayered liquified capsules containing collagen II/TFG-β3 coated microparticles

The use of stem cells is a promising therapeutic approach for the substantial challenge to regenerate cartilage. Considering the two prerequisites, namely the use of a 3D system to enable the chondrogenic differentiation and growth factors to avoid dedifferentiation, the diffusion efficiency of essential biomolecules is an intrinsic issue. We already proposed a liquified bioencapsulation system containing solid microparticles as cell adhesion sites1. Here, we intend to use the optimized system towards chondrogenic differentiation by encapsulating stem cells and collagenII-TGF-β3 PLLA microparticles. As a proof-of-concept, magnetite-nanoparticles were incorporated into the multilayered membrane. This can be a great advantage after implantation procedures to fixate the capsules in situ with the held of an external magnetic patch and for the follow-up through imaging. Results showed that the production of glycosaminoglycans and the expression of cartilage-relevant markers (collagen II, Sox9, aggrecan, and COMP) increased up to 28 days, while hypertrophic (collagen X) and fibrotic (collagen I) markers were downregulated. The presence of nanofibers in the newly deposited ECM was visualized by SEM, which resembles the collagen fibrils of native cartilage. The presence of the major constituent of cartilage, collagen II, was detected by immunocytochemistry and afranin-O and alcian blue stainings revealed a basophilic ECM deposition, which is characteristic of neocartilage. These findings suggest that the proposed system may provide a suitable environment for chondrogenic differentiation.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Infección de macrófagos con el Virus Encefalitis Saint Louis: efecto sobre el fenotipo celular y la apoptosis.

El virus Encefalitis Saint Louis (VESL) (género Flavivirus) experimenta una re-emergencia en la región central del país, con la ocurrencia de un brote en Córdoba y el aislamiento de cepas de distintos genotipos. Está demostrado que los Flavivirus neurotrópicos, como VESL, replican en macrófagos y células dendríticas, tanto en el tejido local como en nódulos linfáticos satélites, para luego llegar a torrente sanguíneo y ser transportados a sistema nervioso central. Es así que el nivel de viremia inicial es regulado por la depuración del virus que realizan los macrófagos. Estas células reconocen a los virus por medio de receptores de reconocimiento de patrones moleculares asociados a patógenos, que incluyen a los receptores Toll-like (TLR). La relación entre los TLR y los virus, se fundamenta en tres aspectos: 1) los TLR al ser estimulados por moléculas derivadas de virus activan vías de señalización que inducen la producción de citoquinas pro-inflamatorias, como TNF- , IL-1, 6, 8 y 18, INF-  y , que median la respuesta inmune antiviral; 2) las señales que dependen de los TLR median efectos inmunopatogénicos, como la apoptosis y la patogénesis del virus; 3) algunas estrategias terapéuticas o profilácticas antivirales se basan en la estimulación de los TLR mediante los respectivos agonistas. Como parte de la respuesta del macrófago a la infección viral, hay proliferación, diferenciación y muerte celular. A la hora de morir, estas células pueden seguir el camino que lleva a la necrosis o el de la apoptosis. Durante la activación de la respuesta inmune frente a antígenos extraños, la apoptosis es requerida para eliminar las células efectoras, una vez que han ejecutado su función y así evitar el desarrollo de procesos deletéreos para el huésped. Estudios realizados con distintos Flavivirus documentan el incremento de apoptosis de macrófagos durante la progresión de la infección y también su relación con la severidad de la patología. De acuerdo a los antecedentes expuestos, se formulan las siguientes hipótesis de estudio: 1-El fenotipo de activación del macrófago infectado con VESL está relacionado con el genotipo viral. 2-La clase de inmunomoduladores liberados y el grado de apoptosis de los macrófagos infectados con el VESL dependen del receptor de reconocimiento utilizado por el virus.El objetivo principal es caracterizar la respuesta inmune inducida en macrófagos infectados in vitro con diferentes genotipos de VESL. Para ello se plantean los siguientes objetivos específicos: 1-Determinar la capacidad de replicación de VESL en macrófagos.2-Evaluar la expresión de molécula de superficie, receptores y la producción de inmunomoduladores en macrófagos infectados con VESL.3-Analizar el impacto de la infección con VESL sobre la apoptosis de macrófagos.4-Correlacionar la expresión de antígenos de superficie, receptores, producción de inmunomoduladores, apoptosis y carga viral con el genotipo viral que infecta al macrófago.Se utilizará una línea línea celular mieloide U937 y cepas del VESL genotipo III, V y VII. Se estudiará la infección de las mismas y determinará la expresión de: CD14, CD16, CD54/ICAM-1, HLA-DR, Fas, R-TNF, CD86, IL4R, TLR2, TLR3, TLR4 y TLR7 por Citometría de Flujo. En el sobrenadante de los cultivos infectados se cuantificarán las concentraciones de IFN-, IFN-, TNF-, IL-1, IL-6, IL-8, IL-10, IL-12, IL-18 y TGF- por técnica de ELISA.Se determinará la apoptosis en los macrófagos infectados mediante marcación con Anexina V-Ficoeritrina y análisis de fragmentación del ADN.La emergencia de esta virosis en nuestro medio amerita abordar distintos aspectos de la respuesta inmune en esta infección. El conocimiento de las características de la activación del macrófago cuando se infecta con VESL, los inmunomoduladores liberados y el impacto de la infección sobre la apoptosis de ésta célula, aportaría posibles blancos para el diseño futuro de estrategias terapéuticas o profilácticas contra esta infección.

Modulación de la activación de células dendríticas por antígenos de Fasciola hepatica y ligandos para receptores Toll: Potencial terapéutico en respuestas anti-inflamatorias. Fasciola hepatica antigens and TLR ligands modulate dendritic cells activation: therapeutic potential in anti-inflammatory responses.

Antecedentes: En nuestro laboratorio hemos demostrado que antígenos (Ags) de Fasciola hepatica inducen en células dendríticas murinas (CD), diferentes propiedades tolerogénicas como la incapacidad por si mismos de inducir la maduración de las células, la resistencia a la maduración por ligandos de TLR, el incremento en la producción de IDO y también la capacidad de esta estas células de dirigir la respuesta inmune hacia un perfil Th2 y T reg. Por otra parte ha sido bien documentado que CD con características tolerogénicas, ya sea inmaduras o semimaduras, son útiles para reducir respuestas inflamatorias excesivas tales como las que ocurren en enfermedades autoinmunes. Además hemos demostrado que CD tratadas con Ags del parásito en conjunto con un ligando Toll (CpG-ODN) producen altos niveles de citoquinas anti-inflamatorias (IL-10 y TGF-) bajos de citoquinas proinflamatorias (TNF, IL-6, IL-12). Hipótesis: El fenotipo semimaduro alcanzado en las CDpodría ser utilizado para reducir la inflamación en un modelo de enfermedad autoinmune en donde existe una exacerbada respuesta Th1 y Th17, ya que la producción elevada de IL-10 y TGF- podría inhibir o controlar estas respuestas de manera directa o a través de la inducción de células T regulatorias. Objetivos: En este proyecto nosotros proponemos la inmunización de animales susceptibles (ratones DBA1/j), al desarrollo de artritis inducida por colágeno (AIC) con CD tratadas con Ags de F. hepatica en conjunto con CpG-ODN para reducir los síntomas clínicos de la enfermedad. Materiales a utilizar: En nuestro laboratorio hemos desarrollado un modelo de artritis inducida por colágeno (AIC) mediante dos inmunizaciones de ratones DBA1/j con colágeno tipo II bovino y adyuvante de Freund. El modelo permitió establecer un índice clínico mediante la hinchazón en las patas de los animales. Doce días posteriores a la primera inmunización los animales serán inyectados con CD tratadas con: 1. PBS, 2.Extracto total de F.hepatica (TE) + CII, 3. CpG + CII, 4. TE+CpG+CII Se realizará la observación macroscópica diaria, a partir de los 7 días de la 2a inmunización Luego del sacrificio las articulaciones de las patas se prepararán para realizar un análisis histológico. Se detectará en suero los niveles de anticuerpos IgG1 (perfil Th2) y de IgG2a (perfil Th1) mediante la técnica de ELISA. Se detectará también el perfil de citoquinas en los nódulos drenantes por la técnica de ELISA y adicionalmente la poblaciónes celulares de células T regulatorias (Treg) CD4+CD25+Foxp3 o células Tr1. Resultados esperados: Pensamos que el tratamiento de los animales que desarrollan AIC con CD semimaduras (por el tratamiento con TE y CpG), serán capaces de migrar a los órganos linfaticos y secretar TGF-be(inductora de células T reg), IL-10 (inductoras de células Tr1), IDO inhibitoria de la respuesta de Li T y promotor de células T reg, también podría generarse una respuesta Th2 (por la presencia de antígenos del parásito), y estas respuestas aisladas o en forma sinérgica podrían inhibir las respuestas de tipo Th17 y Th1 asociadas a la patología en esta enfermedad. Importancia del proyecto: En el desarrollo de la artritis existe un aumento de la inmunidad mediada por células, asi como de la respuesta inmune humoral hacia componentes de la matriz del cartílago. El tratamiento convencional de la artritis recae en general en el uso de inmunosupresores no-específicos, los cuales poseen una variedad de efectos adversos y la inhibición de la respuesta inflamatoria no es específica. En este proyecto proponemos el uso de CD tratadas con antígenos del helminto F. hepatica y CpG ligando Tol que capacita a estas células para generar una respuesta adaptativa de tipo regulatoria, útil en la inhibición de las respuestas inflamatorias como la que ocurre durante la progresión de artritis reumatoidea en un modelo experimental en ratones. We have shown that F. hepatica Ags-treated dendritic cells (DC) together with a TLRl ligand (CpG-ODN) produce high levels of anti-inflammatory cytokines (IL-10 and TGF-Beta) and low of proinflammatory cytokines (TNF, IL-6, IL -12). Hypothesis: The semimature phenotype achieved by DC, could be used to reduce inflammation in a model of autoimmune disease. The high production of IL-10 and TGF-Beta by these cells could directly or through the induction of T reg cells inhibit the inflammatory response. Objective: In this project we propose the immunization of DBA1 / j mice, susceptible to the development of collagen-induced arthritis (CIA) with F. hepatica-treated DC in conjunction with CpG-ODN to reduce clinical signs of disease. Materials: In our laboratory, we developed the CIA model by two immunizations of DBA1 / j mice with bovine type II collagen and Freund's adjuvant. The model allowed to stablish a clinical index by swelling in the legs of animals. Twelve days after the first immunization the animals are injected with DC treated with: 1. PBS 2. F.hepatica Extract (TE) + CII, 3. CpG + CII, 4. TE + CpG + CII Macroscopic observation will take place daily from 7 days of the 2nd immunization. After sacrifice the joints of the legs will be prepared for histological analysis. Serum levels of IgG1 antibodies (Th2 profile) and IgG2a (Th1 profile) will be detected by ELISA. It will also detected the cytokine profile in draining lymph nodes by ELISA and additionally the cell populations of regulatory T cells (Treg) CD4 + CD25 + Foxp3 or Tr1 cells. Expected results: We believe that the treatment of animals that had developed CIA with DC will be able to migrate to lymphatic organs and secrete TGF-B (T reg cell-inducing), IL-10 (inducing Tr1 cells), IDO (inhibitory of T cells and inducing of T reg cells) could alone or in synergy inhibit Th17-type responses and Th1 associated with the pathology in this disease.

Anemia, disfunção renal e desnutrição associadas à insuficiência cardíaca em pacientes valvopatas

FUNDAMENTO: A doença valvar pode cursar com insuficiência cardíaca (IC), anemia e disfunção renal (DR), aumentando o risco nutricional e piorando o prognóstico dos pacientes. OBJETIVO: Avaliar a prevalência de anemia e DR em pacientes portadores de valvopatias com ou sem IC, bem como estabelecer correlação com o estado nutricional. MÉTODOS: Foram avaliados 104 pacientes internados na enfermaria de valvopatias do PROCAPE/UPE, no período de ago-out/2008. Os dados foram obtidos das fichas de acompanhamento nutricional e dos prontuários. As variáveis coletadas foram: sexo, idade, estado nutricional segundo o índice de massa corpórea (IMC), presença de IC, anemia, DR. Considerou-se como anemia valores de hemoglobina < 13 g/dl nos homens e < 12 g/dl nas mulheres. A DR foi estabelecida de acordo com a taxa de filtração glomerular (TGF), sendo calculada pela fórmula proposta por Cockcroft e Gault. RESULTADOS: A prevalência de anemia e DR em pacientes com IC foi de 71,1% e de 68,8%, e nos pacientes sem IC foi de 48,1% e de 60,0%, respectivamente, com diferença estatisticamente significativa para anemia (p = 0,022). Dos pacientes, 48,1% estavam eutróficos, 26,9% com excesso de peso e 25,0% com algum grau de desnutrição. Os pacientes com IC apresentaram uma frequência maior de baixo peso (p = 0,020). O estado nutricional não apresentou associação com anemia (p = 0,117), mas apresentou associação com DR, sendo a função renal diminuída mais frequente nos pacientes com baixo peso (p = 0,000). CONCLUSÃO: Houve significância estatística quando comparamos as prevalências de desnutrição, anemia e DR entre pacientes com e sem IC.

Fator de diferenciação de crescimento 15: um novo biomarcador em pacientes com disfunção diastólica?

FUNDAMENTO: O fator de diferenciação de crescimento-15 ou GDF-15, uma citocina de resposta ao estresse relacionada ao fator transformador de crescimento beta (TGF-ß), está elevado e independentemente relacionado à prognóstico adverso na insuficiência cardíaca sistólica. OBJETIVO: O objetivo do presente estudo é investigar os níveis plasmáticos de GDF-15 em pacientes com disfunção diastólica pré-clínica ou insuficiência cardíaca com fração de ejeção normal (ICFEN). MÉTODOS: Avaliamos 119 pacientes com fração de ejeção (FE) normal, encaminhados à angiografia coronariana eletiva, dos quais 75 (63%) tinham doença arterial coronariana (DAC). Os indivíduos foram classificados como tendo disfunção diastólica ventricular esquerda leve (DDVE grau I, n = 61), ICFEN (DDVE grau II ou III, n = 38) ou função diastólica normal (controles, n = 20). Em um subgrupo de 20 indivíduos, alterações no débito cardíaco (DC) foram medidas através de reinalação de gás inerte (Innocor®) em resposta a um teste hemodinâmico ortostático. RESULTADOS: Os níveis de GDF-15 na ICFEN [mediana 1,08, variação interquartil (0,88-1,30) ng/ml] eram significantemente mais altos do que nos controles [0,60 (0,50-0,71) ng/ml, p = 0,003] e em pacientes com DDVE grau I [0,78 (0,62-1,04) ng/ml, p < 0.001]. Além disso, os níveis de GDF-15 estavam significantemente elevados em pacientes com DDVE grau I, em comparação aos controles (p = 0,003). Adicionalmente, GDF-15 estava correlacionado com os marcadores ecocardiográficos de disfunção diastólica e estava correlacionado com a magnitude da resposta do CO à alteração na posição do corpo de ereta para supina (r = -0,67, p = 0,005). CONCLUSÃO: Os níveis de GDF-15 estão elevados em indivíduos com ICFEN e podem diferenciar função diastólica normal de DDVE. Além disso, os níveis de GDF-15 estão associados com uma redução na resposta do DC no teste hemodinâmico ortostático.

O polimorfismo do gene AKL7 está associado ao risco de síndrome metabólica e à remodelação cardiovascular

FUNDAMENTO: Quinase Tipo Receptor de Ativina 7 (ALK7) é um tipo de receptor I para a superfamília TGF-β e recentemente apresentou ter uma função importante na manutenção de homeostase metabólica. OBJETIVO: Investigar a associação do polimorfismo do gene ALK7 à síndrome metabólica (SMet) e remodelação cardiovascular em pacientes com SMet. MÉTODOS: O polimorfismo de nucleotídeo único rs13010956 no gene ALK7 foi genotipado em 351 indivíduos chineses submetidos à ultrassonografia cardíaca e das carótidas. As associações do polimorfismo do gene ALK7 ao fenótipo e aos parâmetros da síndrome metabólica e características ultrassônicas cardiovasculares foram analisadas. RESULTADOS: O polimorfismo de rs13010956 no gene ALK7 foi considerado significativamente relacionado ao fenótipo de SMet em mulheres (p < 0,05) e significativamente associado à pressão sanguínea em populações totais (p < 0,05) e femininas (p < 0,01). Outras análises revelaram que rs13010956 estava associado à média da espessura íntima-média de artérias carótidas em mulheres (p < 0,05). Após o controle do índice de massa corporal, pressão arterial, glicemia em jejum e triglicérides, o rs13010956 também foi considerado significativamente associado ao índice de massa do ventrículo esquerdo em populações totais (p < 0,05) e femininas (p < 0,05). CONCLUSÃO: Nossos achados sugeriram que o polimorfismo de rs13010956 do gene ALK7 estava significativamente vinculado ao risco de SMet em mulheres e pode estar envolvido na remodelação cardiovascular em pacientes com SMet.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Genome-wide patterns of latitudinal differentiation among populations of Drosophila melanogaster from North America.

Understanding the genetic underpinnings of adaptive change is a fundamental but largely unresolved problem in evolutionary biology. Drosophila melanogaster, an ancestrally tropical insect that has spread to temperate regions and become cosmopolitan, offers a powerful opportunity for identifying the molecular polymorphisms underlying clinal adaptation. Here, we use genome-wide next-generation sequencing of DNA pools ('pool-seq') from three populations collected along the North American east coast to examine patterns of latitudinal differentiation. Comparing the genomes of these populations is particularly interesting since they exhibit clinal variation in a number of important life history traits. We find extensive latitudinal differentiation, with many of the most strongly differentiated genes involved in major functional pathways such as the insulin/TOR, ecdysone, torso, EGFR, TGFβ/BMP, JAK/STAT, immunity and circadian rhythm pathways. We observe particularly strong differentiation on chromosome 3R, especially within the cosmopolitan inversion In(3R)Payne, which contains a large number of clinally varying genes. While much of the differentiation might be driven by clinal differences in the frequency of In(3R)P, we also identify genes that are likely independent of this inversion. Our results provide genome-wide evidence consistent with pervasive spatially variable selection acting on numerous loci and pathways along the well-known North American cline, with many candidates implicated in life history regulation and exhibiting parallel differentiation along the previously investigated Australian cline.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND:: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18. METHODS:: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS:: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS:: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations. (Inflamm Bowel Dis 2010).

A corneal dystrophy associated with transforming growth factor beta-induced Gly623Asp mutation an amyloidogenic phenotype.

PURPOSE: To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset. DESIGN: Experimental study. PARTICIPANTS: Four affected and 6 nonaffected family members. METHODS: Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15. MAIN OUTCOME MEASURES: Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. RESULTS: The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein. CONCLUSIONS: In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects.

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-β. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

Multiple expression control mechanisms of peroxisome proliferator-activated receptors and their target genes.

The peroxisome proliferator-activated receptors (PPAR) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. As ligand-activated receptors, they form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs). PPARs are activated by fatty acids and their derivatives, whereas RXR is activated by 9-cis retinoic acid. This heterodimer binds to peroxisome proliferator response elements (PPRE) residing in target genes and stimulates their expression. Recent reports now indicate that PPARs and RXRs can function independently, in the absence of a hetero-partner, to modulate gene expression. Of importance, these non-canonical mechanisms underscore the impact of both cofactors and DNA on gene expression. Furthermore, these different mechanisms reveal the increasing repertoire of PPAR 'target' genes that now encompasses non-PPREs containing genes. It is also becoming apparent that understanding the regulation of PPAR expression and activity, can itself have a significant influence on how the expression of subgroups of target genes is studied and integrated in current knowledge.