Colite microscópica : uma causa rara de diarreia crónica em pediatria

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Reflexiones anticolicas : experimentos medico-practicos chymico-galenicos, quinta esencia de los remedios contra la colica epidemica endemica y respuesta a la Breve reflexión, y crisis medica, que sobre el dolor colico sacò à luz ... don Vicente Boyvia .../

Mode of access: Internet.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH(2)O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; p = 1.18 g CM-3) and a low-density fraction (LDMV; rho=1.12 g cm(-3)). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Increased potency of a novel complement factor 5a receptor antagonist in a rat model of inflammatory bowel disease

We have previously shown that complement factor 5a(C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective, orally active C5a antagonist AcF-[OP(D-Cha) WR]. This study tested the efficacy and potency of a new C5a antagonist, hydrocinnamate (HC)-[OP(D-Cha) WR], which has limited intestinal lumenal metabolism, in this model of colitis. Analogs of AcF-[OP(D-Cha) WR] were examined for their susceptibility to alimentary metabolism in the rat using intestinal mucosal washings. One metabolically stable analog, HC-[OP(D-Cha)WR], was then evaluated pharmacokinetically and investigated at a range of doses (0.03 - 10 mg/kg/ day p.o.) in the 8-day rat TNBS- colitis model, against the comparator drug AcF-[OP(D-Cha) WR]. Using various amino acid substitutions, it was determined that the AcF moiety of AcF-[OP(D-Cha) WR] was responsible for the metabolic instability of the compound in intestinal mucosal washings. The analog HC-[OP( D-Cha) WR], equiactive in vitro to AcF-[OP(D-Cha) WR], was resistant to intestinal metabolism, but it displayed similar oral bioavailability to AcF-[OP(D-Cha) WR]. However, in the rat TNBS- colitis model, HC-[OP(D-Cha) WR] was effective at reducing mortality, colon edema, colon macroscopic scores, and increasing food consumption and body weights, at 10- to 30- fold lower oral doses than AcF-[OP( D-Cha) WR]. These studies suggest that resistance to intestinal metabolism by HC-[OP(D-Cha) WR] may result in increased local concentrations of the drug in the colon, thus affording efficacy with markedly lower oral doses than AcF-[OP(D-Cha) WR] against TNBS-colitis. This large increase in potency and high efficacy of this compound makes it a potential candidate for clinical development against intestinal diseases such as inflammatory bowel disease.

Bone loss in Crohn's disease: Exercise as a potential countermeasure

Crohn's disease (CD) is associated with a number of secondary conditions including osteoporosis, which increases the risk of bone fracture. The cause of metabolic bone disease in this Population is believed to be multifactorial and may include the disease itself and associated inflammation, high-close corticosteroid use, weight loss and malabsorption, a lack of exercise and physical activity, and all underlying genetic predisposition to bone loss. Reduced bone mineral density has been reported in between 5% to 80% of CD sufferers, although it is generally believed that approximately 40% of patients suffer from osteopenia and 15% from osteoporosis. Recent studies Suggest a small but significantly increased risk of fracture compared with healthy controls and, perhaps, sufferers of other gastrointestinal disorders Such as ulcerative colitis. The role of physical activity and exercise in the prevention and treatment of CD-related bone loss has received little attention, despite the benefits of specific exercises being well documented in healthy populations. This article reviews the prevalence of and risk factors for low bone mass in CD patients and examines various treatments for osteoporosis in these patients, with a particular focus on physical activity.

Avaliação do efeito do extrato hidroalcoólico das folhas de Anacardium occidentale L. em ratos submetidos à colite ulcerativa

Ulcerative colitis is a chronic disease characterized by inflammation in the intestinal mucosa, in most cases affects the colon and rectum. The therapeutic drugs are used as aminosalicylates and glucocorticosteroids, but due to the low response and the various side effects caused by them, reveals the need to search for new sources of useful compounds in the treatment of this disease.The species Anacardium occidentale popularly known as cashew, has been used for centuries in folk medicine in the healing aid of skin and mucosa lesions.Recent studies show its expressive antiulcerogenic effect, what we instigated to assess the effect of the extract of A. occidentaleleaves in rats with acute ulcerative colitis, therefore, 42 rats were used male Wistar, divided into 06 groups, and Negative Control (C) Positive Control (C +), treated with Sulfasalazine (Sz500) and treated with Extract A. occidentale at doses of 50 (Ao50), 100 (Ao100) and 200 mg / kg (Ao200).All groups were submitted to experimental colitis Ulcerative except C-, moreover, C- and C + received saline via gavage for 7 consecutive days while the other groups received their respective treatments.Euthanasia of animals took place on the 8th day in which it was collected intestinal colon sample for later analysis macroscopic, histopathological, morphometric and biochemistry, as well as complementary collection of blood and liver tissue. The extract is rich in saponins and phenolic compounds such as flavonoids (quercetin and kaempferol) and tannins.When the Sz500 groups and 100 showed significant protection to damage to lipids and proteins, among the groups subjected to experimental ulcerative colitis, the animals Ao100 group obtained the lowest score in all parameters analyzed.Treatment with 100 mg / kg of A. occidentale extract seems to have a combination of antiinflammatory, antioxidant, bactericidal and anabolic promoted by the bioactive compounds present in the extract.However, it is necessary to investigate harder treating dose of 100mg / kg to higher doses compared to elucidate more properly the best therapeutic dosage ulcerative colitis.

Efeito anti-inflamatório e antioxidante do extrato hidroalcóolico de Turnera subulata na colite ulcerativa induzida por ácido acético em ratos

Inflammatory bowel diseases is composed by a set of chronic and inflammatory disorders, among them is ulcerative colitis (UC). UC treatment is based on anti-inflammatory administration; however, this group of drugs clearly leads to development of undesirable side effects, what stimulate the search for new therapies alternatives. The aim of this study was to evaluate the effect of hydroalcholic Turnera subulata extract on acetic acid-induced acute UC in rats. UC was induced by 1 mL injection of 4% acetic acid via rectal in Wistar mouse. 42 animals were distributed among 6 experimental groups: Control, UC, Sulfasalazine 500 mg/Kg/day (SSZ), T. subulata 50mg/Kg/day (TS 50), T. subulata 100mg/Kg/day (TS 100), T. subulata 200mg/Kg/day (TS 200). Throughout the experiment, body weight, food and water ingestion was daily evaluated. At the end of the experiment, the animals were euthanized and a colon fragment was observed by macroscopic analysis. Colon fragments were also collected for microscopic analysis and oxidative stress evaluation. The means from each group was compared by ANOVA test with a significance level of 5% (p<0.05) using GraphPad Prism Software. As results, we can clearly observe that SSZ group had the greater body weight decrease among the groups throughout the experiments, 14.78%, as well as, the lowest food intake, 6.23 g of food/day. The animals treated with T. subulata extracts showed no important body weight loss when compared to control. UC group showed the highest tissue damage macroscope score, 6.5, while TS 50 showed the lowest tissue damage score: 1. Microscope evaluation showed the presence of edema, haemorraghia and ulceration in all group of animals, except for Control. Nevertheless, TS 50 showed the lowest inflammatory damage among all groups. Oxidative stress analysis revealed that T. subulata treatment modulate catalase and superoxide dismutase activity, we also observed a decrease in protein and lipid peroxidation in response to extract administration. Taken together, these results shows that T. subulata extract exerts anti-inflammatory and anti-oxidant effects on experimental UC.

Efeito anti-inflamatório e antioxidante do extrato hidroalcóolico de Turnera subulata na colite ulcerativa induzida por ácido acético em ratos

Inflammatory bowel diseases is composed by a set of chronic and inflammatory disorders, among them is ulcerative colitis (UC). UC treatment is based on anti-inflammatory administration; however, this group of drugs clearly leads to development of undesirable side effects, what stimulate the search for new therapies alternatives. The aim of this study was to evaluate the effect of hydroalcholic Turnera subulata extract on acetic acid-induced acute UC in rats. UC was induced by 1 mL injection of 4% acetic acid via rectal in Wistar mouse. 42 animals were distributed among 6 experimental groups: Control, UC, Sulfasalazine 500 mg/Kg/day (SSZ), T. subulata 50mg/Kg/day (TS 50), T. subulata 100mg/Kg/day (TS 100), T. subulata 200mg/Kg/day (TS 200). Throughout the experiment, body weight, food and water ingestion was daily evaluated. At the end of the experiment, the animals were euthanized and a colon fragment was observed by macroscopic analysis. Colon fragments were also collected for microscopic analysis and oxidative stress evaluation. The means from each group was compared by ANOVA test with a significance level of 5% (p<0.05) using GraphPad Prism Software. As results, we can clearly observe that SSZ group had the greater body weight decrease among the groups throughout the experiments, 14.78%, as well as, the lowest food intake, 6.23 g of food/day. The animals treated with T. subulata extracts showed no important body weight loss when compared to control. UC group showed the highest tissue damage macroscope score, 6.5, while TS 50 showed the lowest tissue damage score: 1. Microscope evaluation showed the presence of edema, haemorraghia and ulceration in all group of animals, except for Control. Nevertheless, TS 50 showed the lowest inflammatory damage among all groups. Oxidative stress analysis revealed that T. subulata treatment modulate catalase and superoxide dismutase activity, we also observed a decrease in protein and lipid peroxidation in response to extract administration. Taken together, these results shows that T. subulata extract exerts anti-inflammatory and anti-oxidant effects on experimental UC.

Estudio de la infección "Clostridium difficile": incidencia, epidemiología, características clínicas, factores de riesgo de gravedad y recurrencia

Clostridium difficile causes a broad range of diseases in humans, from mild colitis to pseudomembranous colitis and disease refractory to treatment, fulminant and fatal. It is an infection whose frequency, seriousness and related morbidity and mortality have increased in recent years [1-4]. Nowadays it is regarded as an emerging public health problem, and prevention and monitoring are particularly recommended. In recent years, different authors have described a change in its epidemiology, which affects not only the populations traditionally involved, but also children and patients from the community [2, 5]. Moreover, the Spanish situation has proven to be different, in terms of the ribotypes present, to other countries in Europe, Canada and the USA. Thus, the performance of an in-depth study in this type of patients in Spain, as well as the source of the acquisition of Clostridium difficile infection (CDI), is of major relevance. The main predisposing factor to acquiring CDI is the use of antibiotics in the previous 8 weeks (90% cases in some series), even with a single prophylactic dose. Other risk factors are a previous stay in health-care centers, particularly hospitals, being old and immunodepression, including transplantations and HIV [6]. The severity of CDI has been associated both with host factors and microorganism-specific factors...

The use of PET-CT in the assessment of patients with colorectal carcinoma.

Colorectal cancer is the third most commonly diagnosed cancer, accounting for 53,219 deaths in 2007 and an estimated 146,970 new cases in the USA during 2009. The combination of FDG PET and CT has proven to be of great benefit for the assessment of colorectal cancer. This is most evident in the detection of occult metastases, particularly intra- or extrahepatic sites of disease, that would preclude a curative procedure or in the detection of local recurrence. FDG PET is generally not used for the diagnosis of colorectal cancer although there are circumstances where PET-CT may make the initial diagnosis, particularly with its more widespread use. In addition, precancerous adenomatous polyps can also be detected incidentally on whole-body images performed for other indications; sensitivity increases with increasing polyp size. False-negative FDG PET findings have been reported with mucinous adenocarcinoma, and false-positive findings have been reported due to inflammatory conditions such as diverticulitis, colitis, and postoperative scarring. Therefore, detailed evaluation of the CT component of a PET/CT exam, including assessment of the entire colon, is essential.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.