Nod2 Deficiency in mice is Associated with Microbiota Variation Favouring the Expansion of mucosal CD4+ LAP+ Regulatory Cells

Nucleotide-binding Oligomerization Domain-2 (NOD2) mutations are associated with an increased risk to develop Crohn's Disease. In previous studies, we have shown that Nod2-/- mice manifest increased proportion of Lamina Propria (LP) CD4+ LAP+ Foxp3- regulatory cells, when compared with Nod2+/+ mice, while CD4+ Foxp3 + regulatory cells were not affected. Here, we investigated the Nod2 gut microbiota, by 16S rRNA pyrosequencing, at steady state and after TNBS-colitis induction in mice reared separately or in cohousing, correlating the microbial profiles with LP regulatory T cells proportion and tissue cytokines content. We found that enrichment of Rikenella and Alistipes (Rikenellaceae) in Nod2-/- mice at 8 weeks of age reared separately was associated with increased proportion of CD4+ LAP+ Foxp3- cells and less severe TNBS-colitis. In co-housed mice the acquisition of Rickenellaceae by Nod2+/+ mice was associated with increased CD4+ LAP+ Foxp3- proportion and less severe colitis. Severe colitis was associated with enrichment of gram-negative pathobionts (Escherichia and Enterococcus), while less severe colitis with protective bacteria (Barnesiella, Odoribacter and Clostridium IV). Environmental factors acting on genetic background with different outcomes according to their impact on microbiota, predispose in different ways to inflammation. These results open a new scenario for therapeutic attempt to re-establish eubiosis in Inflammatory Bowel Disease patients with NOD2 polymorphisms

Endoluminal calprotectin measurement in assessment of pouchitis and a new index of disease activity. A pilot study

Pouchitis is the most common complication following proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC). To provide a standardized definition of pouchitis clinical, endoscopic and histological markers were grouped and weighted in the pouch disease activity index (PDAI). However, the delay in the assessment of the final score due to the time requested for histological analysis remains the main obstacle to the index implementation in clinical practice so that the use of modified-PDAI (mPDAI) with exclusion of histologic subscore has been proposed. We tested the ability of calprotectin measurement in the pouch endoluminal content to mimic the histologic score as defined in the PDAI, the index that we adopted as gold standard for pouchitis diagnosis. Calprotectin was measured by ELISA in the pouch endoluminal content collected during endoscopy in 40 consecutive patients with J-pouch. In each patient PDAI and mPDAI were calculated and 15% of patients were erroneously classified by mPDAI. ROC analysis of calprotectin values vs. acute histological subscore 3 identified different calprotectin cut-off values with corresponding sensitivity and specificity allowing the definition and scoring of different range of calprotectin subscores. We incorporated the calprotectin score in the mPDAI obtaining a new score that shows the same specificity as PDAI for diagnosis of pouchitis and higher sensitivity when compared with mPDAI. The use of the proposed new score, once validated in a larger series of patients, might be useful in the early management of patients with symptoms of pouchitis

Investigación de mercados sobre la gestión del manejo del cliente en CORDIALSACosta Rica S.A

Proyecto de Graduación (Bachillerato en Administración de Empresas) Instituto Tecnológico de Costa Rica, 2007.CORDIALSA Costa Rica S.A. es una empresa que se dedica a la comercialización de galletas, pastelería, chocolates y modificadoresde la leche. Esta corporación se instaló en el país a finales del año 2004, al adquirir las plantas de galletas y chocolates de Nestlé. La compañía se dedica a comercializar los productos previamente mencionados en el país, Centroamérica y Panamá. Para el desarrollo de sus productos en CORDIALSA se aplican políticas de calidad integrada al estar comprometidos con sistemas de gestión de calidad y ambientales ya que se tiene como objetivos alimentar y deleitar a los consumidores al ofrecer un servicio que asegure la preferencia de los clientes, además de velar por el uso racional de los recursos naturales prevenir y controlar los aspectos ambientales para reducir los impactos sobre el medio ambiente, su objetivo es lograr un destacado retorno de las inversiones, superior al costo del capital empleado. Con respecto al negocio de alimentos, se busca siempre mejorar la calidad de vida del consumidor y el progreso de sus trabajadores, además del crecimiento rentable con marcas líderes, servicio superior y una excelente distribución nacional e internacional, sin olvidar el desarrollo de actividades con el mejor talento humano, innovación sobresaliente y un comportamiento corporativo ejemplar. La empresa manufactura en el país galletas, chocolates, modificadores de leche y pastelería industrial para distribuir en Costa Rica y Centroamérica. Para ello cuenta con dos plantas: la de galletas y la de chocolates. El presente trabajo es una investigación de mercados para conocer la percepción que tienen los actuales clientes tipo “A” de CORDIALSA con respecto a la gestión del manejo del cliente, dentro del territorio nacional, su propósito, es tener un mejor desempeño a la hora de relacionarse con los clientes tipo “A”, o en otras palabras, aquellos clientes que por sus niveles de compras son los que le resultan más rentables a la compañía (tomando en cuenta cadenas de supermercados, almacenes mayoristas y distribuidores especializados de territorio). Evaluar los niveles de satisfacción, la calidad del servicio y del producto, así como tomar en cuenta inquietudes, sugerencias y preferencias de los clientes son algunos de los aspectos a determinar a lo largo de esta investigación. gestión del manejo del cliente es una parte de la estrategia de negocios centrada en el cliente. Se fundamenta en recopilar información sobre los clientes, para poder dar valor a la oferta. La empresa debe trabajar para conocer las necesidades de los mismos y así poder adelantar una oferta y mejorar la calidad en la atención, es decir, la gestión del manejo del cliente es el proceso de remodelación de las empresas para adaptarse a las necesidades del mismo, es cuando se detecta la necesidad de replantear los conceptos "tradicionales" del marketing y emplear los conceptos del marketing relacional. En la actualidad, mantener buenas relaciones con los clientes es muy importante, y para ello, se debe seguir un proceso que conlleva varios pasos, siendo uno de estos investigar entre los clientes su modo de trabajo, sus necesidades, la percepción que tienen de la compañía, y los medios para mejorar el servicio que se les brinda. De esta manera el cliente se sentirá apoyado y tendrá una mejor disposición para que el negocio se mantenga y desarrolle. Con la estrategia se definen programas que, en primera instancia, reconocen y bonifican los mejores clientes con los mejores desempeños, es decir, aquellos cuyos volúmenes de compra, frecuencia de compra, monto de la inversión, moralidad comercial y antigüedad en la relación, se tornan más valiosos para la organización y quienes normalmente generan los mayores volúmenes de ingreso con que cuenta la empresa. En el caso de la comercializadora CORDIAL S.A. el proyecto se enfocará en sus clientes de primer nivel o sea el canal intermediario y de entre éstos los tipos “A” (los que le generan más ganancias), que son los encargados de que el producto llegue al consumidor final, es por esto que fortalecer las relaciones que se tiene con ellos es importante ya que así se podrá abarcar un mercado más amplio obteniendo ambos los beneficios de un esfuerzo conjunto por mejorar los niveles de venta y el proceso para realizarlas.Instituto Tecnológico de Costa Rica. Escuela de Administración de Empresas

CD3+CD4+LAP+Foxp3-regulatory cells of the colonic lamina propria limit disease extension in ulcerative colitis

Background and Aims: In ulcerative colitis (UC), inflammation begins in the rectum and can extend proximally throughout the entire colon. The extension of inflammation is an important determinant of disease course, and may be limited by the action of regulatory T cells (Tregs). In this cross-sectional study, we evaluated the relationship between UC extension and the proportions of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3- Tregs in the colonic lamina propria (LP) of 79 UC patients and 29 controls. The role of these cells in UC extension was also investigated in the murine oxazolone-induced colitis model. Methods: Patients: Disease extension was classified according to the Montreal classification. Where possible, endoscopic biopsies of involved and uninvolved tissue were obtained from UC patients. Mouse model: Colitis was induced by intrarectal oxazolone administration. Lamina propria mononuclear cells were isolated from patient biopsies and mouse colon tissue using enzymatic method and the percentage of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-cells evaluated by immunofluorescence. Confocal microscopy was applied for the visualization and quantification of CD4+LAP+ cells on tissue histological sections. Results: In UC patients with distal colitis the proportion of LP CD3+CD4+Foxp3+ Tregs was significantly higher in inflamed tissue than uninvolved tissue. As opposite, the proportion of LP CD3+CD4+LAP+ Tregs was significantly higher in uninvolved tissue than involved tissue. Both LP CD3+CD4+Foxp3+ and LP CD3+CD4+LAP+ Tregs proportion in involved tissue was significantly higher than in controls irrespective of the extension of inflammation. In mice with oxazolone-induced distal colitis, treatment with LAP-depleting antibody was associated with the development of extensive colitis. Conclusions: Our findings suggest that CD3+CD4+LAP+Foxp3-Tregs limit the extension of inflammatory lesions in UC patients

Probiotic administration in patients with ileal pouch-anal anastomosis for ulcerative colitis is associated with expansion of mucosal regulatory cells.

BACKGROUND: Probiotics have anti-inflammatory effects in patients with inflammatory bowel disease and appear to regulate mucosal immune response through reductions in proinflammatory cytokines. The probiotic VSL#3 prevents pouchitis if started within a week of ileostomy closure and maintains remission following antibacterial treatment in patients with refractory or recurrent pouchitis. However, the efficacy of probiotics and their effects on regulatory cells if started at a greater time after surgery in patients undergoing ileal pouch anal anastomosis (IPAA) for ulcerative colitis are unknown. METHODS: We conducted an open-label study in which 31 patients at different periods from surgery without signs and symptoms of pouchitis were randomized to 2 sachets of VSL#3 once daily or no treatment for 12 months. Pouchitis disease activity index (PDAI) was evaluated at baseline and after 3, 6, and 12 months. The percentage of CD4+ T lymphocytes expressing CD25 and the inactive form of transforming growth factor-beta [latency-associated peptide (LAP)] were evaluated at baseline and after 3 and 6 months in peripheral-blood mononuclear cells and mucosal biopsies. Variation in tissue interleukin-1beta and Foxp3 mRNA expression was also evaluated. RESULTS: During the study period, VSL#3-treated patients showed a significant reduction in PDAI score and a significant increase in the percentage of mucosal CD4+CD25(high) and CD4+ LAP-positive cells compared with baseline values. Tissue samples at different points showed a significant reduction in IL-1beta mRNA expression, and a significant increase in Foxp3 mRNA expression. CONCLUSIONS: We conclude that VSL#3 administration in patients with IPAA modulates the PDAI and expands the number of mucosal regulatory T cells

IL-13 mRNA tissue content identifies two subsets of adult ulcerative colitis patients with different clinical and mucosa-associated microbiota profiles

BACKGROUND AND AIM: A personalized approach to therapy has great promise to improve disease outcomes. To this end, the identification of different subsets of patients according with the prevalent pathogenic process might guide in the choice of therapeutic strategy. We hypothesize that UC patients might be stratified according to distinctive cytokine profiles and/or to a specific mucosa-associated microbiota. METHODS: In a cohort of clinically and endoscopic active UC patients and controls, we analyzed by qPCR the mucosal cytokine mRNA content and the mucosa-associated microbiota composition assessed by the 16SrRNA gene sequencing. RESULTS: We demonstrate, by means of data-driven approach, the existence of a specific UC patient subgroup characterized by elevated IL-13mRNA tissue content separated by patients with low IL-13 mRNA tissue content. The two subsets differ in clinical-pathological characteristics. High IL-13mRNA patients are younger at diagnosis and show higher prevalence of extensive colitis than low IL-13mRNA ones. They also show a more frequent use of steroid/immunosuppressant/anti-TNFα therapy during a one-year follow-up. The two subgroups show a differential enrichment of mucosa associated microbiota genera with prevalence of Prevotella in patients with high IL-13mRNA tissue content and Sutterella and Acidaminococcus in patients with low IL-13mRNA tissue content. CONCLUSION: Assessment of mucosal IL-13mRNA might help in the identification of the patients' subgroup that might benefit from a therapeutic approach modulating IL-13

Overexpression of CIITA in T Cells Aggravates Th2-Mediated Colitis in Mice

The MHC class II transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC class II restricted antigen presentation. Previously we suggested another role of CIITA in Th1/Th2 balance by demonstrating that forced expression of CIITA in murine T cells repressed Th1 immunity both in vitro and in vivo. However, the results were contradictory to the report that CIITA functioned to suppress the production of Th2 cytokine by CD4+ T cells in CIITA deficient mice. In this study, we investigated the influence of constitutive expression of CIITA in T cells on Th2 immune response in vivo using murine experimental colitis model. In the dextran sodium sulfate-induced acute colitis, a disease involving innate immunity, CIITA transgenic mice and wild type control mice showed similar progression of the disease. However, the development of oxazolone-induced colitis, a colitis mediated by predominantly Th2 immune response, was aggravated in CIITA-transgenic mice. And, CD4+ T cells from the mesenteric lymph node of CIITA-transgenic mice treated with oxazolone exhibited a high level of IL-4 secretion. Together, these data demonstrate that constitutive expression of CIITA in T cells skews immune response to Th2, resulting in aggravation of Th2-mediated colitis in vivo

Probiotic VSL#3-induced TGF-β ameliorates food allergy inflammation in a mouse model of peanut sensitization through the induction of regulatory T cells in the gut mucosa.

SCOPE: Among food allergies, peanut allergy is frequently associated with severe anaphylactic reactions. In the need for safe and effective therapeutic strategies, probiotics may be considered on the basis of their immunomodulatory properties. The aim of the present study was to investigate the immunological mediators involved in the effects of probiotic VSL#3 oral supplementation on Th2 inflammation and anaphylaxis in a mouse model of peanut allergy. METHODS AND RESULTS: VSL#3 supplementation to peanut-sensitized mice was effective in ameliorating anaphylaxis and Th2-mediated inflammation, by promoting regulatory responses in the jejunum mucosa and in the mesenteric lymph node, as evaluated by ELISA, real-time PCR, histologic, and immunohistochemical analysis. Probiotic-induced TGF-β mediates its protective effects through the induction of regulatory T cells expressing FOXP3 and/or latency-associated peptide, as proven by in vivo blockade of TGF-β in VSL#3-treated mice with a neutralizing monoclonal antibody one day before challenge. CONCLUSION: TGF-β, induced in the gut by VSL#3 supplementation, is capable of reducing the Th2 inflammation associated with food anaphylaxis in a mouse model of peanut sensitization. TGF-β acts through the induction/maintenance of regulatory T cells expressing FOXP3 and/or latency-associated peptide. Probiotics supplementation may represent an effective and safe strategy for treating food allergies in adult population