Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Myeloid-derived suppressor cells (MDSCs), characterized by the co-expression of CD11b and Gr1, are a heterogeneous population of immature myeloid cells that exhibit strong suppressive functions against T cell responses. In inflammatory conditions like IBD, there is an increase in MDSCs but this is not sufficient to improve intestinal inflammation in IBD. Herein, we investigated the expansion of MDSCs in TNBS-induced acute colitis and whether the adoptive transfer of in vitro generated MDSCs ameliorated intestinal inflammation. We found that CD11b+Gr1+ MDSCs were significantly increased in experimental colitis. Further, this increase correlated to some extent with the severity of the disease. As per our protocol, MDSCs were generated from bone marrow cells co-cultured with hepatic stellate cells (HSCs), an essential cell type to obtain functional MDSCs in vitro. Adoptive transfer of HSC-induced MDSCs improved body weight loss and significantly downregulated inflammatory cytokines TNF, IFN-γ, and IL-17 in colonic tissue. Our results indicate MDSCs are immunoregulatory players in intestinal inflammation and that the adoptive transfer of in vitro generated MDSCs may provide a novel therapeutic approach for inflammatory bowel disease.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Myeloid-derived suppressor cells (MDSCs), characterized by the co-expression of CD11b and Gr1, are a heterogeneous population of immature myeloid cells that exhibit strong suppressive functions against T cell responses. In inflammatory conditions like IBD, there is an increase in MDSCs but this is not sufficient to improve intestinal inflammation in IBD. Herein, we investigated the expansion of MDSCs in TNBS-induced acute colitis and whether the adoptive transfer of in vitro generated MDSCs ameliorated intestinal inflammation. We found that CD11b+Gr1+ MDSCs were significantly increased in experimental colitis. Further, this increase correlated to some extent with the severity of the disease. As per our protocol, MDSCs were generated from bone marrow cells co-cultured with hepatic stellate cells (HSCs), an essential cell type to obtain functional MDSCs in vitro. Adoptive transfer of HSC-induced MDSCs improved body weight loss and significantly downregulated inflammatory cytokines TNF, IFN-γ, and IL-17 in colonic tissue. Our results indicate MDSCs are immunoregulatory players in intestinal inflammation and that the adoptive transfer of in vitro generated MDSCs may provide a novel therapeutic approach for inflammatory bowel disease.

The sympathetic nervous system (SNS) is part of an integrative network that functions to restore homeostasis following injury and infection. The SNS provides negative feedback control over inflammation through the secretion of catecholamines from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Central autonomic structures receive information regarding the inflammatory status of the body and reflexively modulate SNS activity. Evidence suggests that inflammation and infection can also directly regulate ACC function. However, the precise alterations in ACC function that occur in response to regional inflammation, systemic inflammation and exposure to bacterial products have yet to be fully characterized. The present thesis was therefore performed to test the hypothesis that gastrointestinal (GI) and systemic inflammation modulate ACC Ca2+ signaling, and that ACCs possess the ability to directly detect microbe-associated molecular patterns (MAMPs). Ca2+ signaling was assessed in single ACCs isolated from control mice and mice with GI or systemic inflammation using Ca2+ imaging and perforated patch clamp electrophysiology. Acute and chronic GI inflammation consistently reduced high-K+-stimulated Ca2+ transients in ACCs through an inhibition of voltage-gated Ca2+ current. In contrast, systemic inflammation significantly enhanced high-K+-stimulated Ca2+ transients and catecholamine secretion through an increase in Ca2+ release from the endoplasmic reticulum. Incubation of control ACCs in serum obtained from mice with systemic inflammation produced a similar increase in Ca2+ signaling, suggesting that circulating mediators play an important role in this response. To determine whether ACCs can directly detect MAMPs, Ca2+ signaling, excitability and neurotransmitter release were assessed in control ACCs and ACCs incubated in media containing lipopolysaccharide (LPS). Unlike GI and systemic inflammation, LPS did not affect ACC Ca2+ signaling. However, LPS dose- and time-dependently hyperpolarized ACC resting membrane potential and enhanced large conductance Ca2+-activated K+ currents. Consistent with membrane hyperpolarization, LPS reduced ACC excitability and inhibited neuropeptide Y release. These effects were mediated by Toll-like receptor 4 and nuclear factor-κB. In summary, GI and systemic inflammation produce opposite effects on ACC Ca2+ signaling through distinct mechanisms, and ACCs can directly detect MAMPs. These findings extend our knowledge of the complex integration performed by the immune system-nervous system network during health and disease. / Thesis (Ph.D, Physiology) -- Queen's University, 2013-08-20 17:15:23.945

It had long been known that individuals with inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative colitis (UC), have locally reduced vitamin C levels in the intestinal mucosa, which equates to an overall loss of the antioxidant capacity and increase risk of oxidative tissue damage. The aim of the present work was to expand on this concept, through investigating the role of genetic variations in intestinal vitamin C transporters in vulnerability to IBD and further characterizing their functions. The studies focused on a known intestinal transporter for ascorbic acid, SLC23A1, and a novel intestinal transporter of dehydroascorbic acid, SLC2A14 (GLUT14). To investigate any association between the SLC23A1 and SLC2A14 with IBD, genomic DNA of 311 Caucasian individuals with IBD, participated in the Manitoba IBD Cohort Study, and 142 healthy controls were genotyped for tagging single nucleotide polymorphism (SNP) of each gene, using TaqMan Assays. New splice variants of SLC23A1 and SLC2A14 were determined by In silico analyses, followed by sub-cloning of the splice variants to verify their subcellular locations. Substrates and functions were determined, using the Xenopus laevis oocyte system for each transporter. The presence of the SLC23A1 variant rs10063949 G allele elevated the risk for CD by 150% (Odds ratios (OR) = 2.54, 95% CI 1.83-3.53). An allele dosage effect was confirmed; compared to rs10063949-AA homozygotes the 10063949-AG heterozygotes have a 150% elevated risk and the 10063949-GG homozygotes have a 370% elevated CD risk (OR=2.54, 95% CI 1.38-4.66; OR=4.72, 95% CI 2.53-8.81, p<0.001; respectively). No relation was observed between genetic variants in SLC23A1 and UC. Through database search, a novel 5’exon was discovered for SLC23A1 locus which is located 1078 nucleotides upstream of the canonical first exon. The two first exons are not mutually exclusive since they splice together to create a novel SLC23A1 protein isoform, we named it isoform 1A, with a N-terminus that is elongated by 36 amino acid. The novel SLC23A1 isoform located at the plasma membrane, but mediates only 7% of the ascorbic acid transport exhibited by the shorter isoform. The presence of the SLC2A14 SNP rs2889504 A allele elevated the risk for UC by 260% and CD by 468% (OR: 3.60, 95% CI: 1.95-6.64; OR: 4.68, 95% CI: 2.78-8.50, respectively). The rs10846086-G allele elevated the risk of UC and CD approximately 3-fold (OR: 2.91, 95% CI: 1.49-5.68; OR: 3.00, 95% CI: 1.55-5.78, respectively). The variant rs12815313-T increased the risk for CD by 112% (OR: 2.12, 95% CI: 1.33-3.36). All the genetic variations in SLC2A14 gene, associated with IBD, were independent from each other, strengthening the evidence that functional SNPs in the SLC2A14 locus contribute to IBD. It was identified that the two major GLUT14 isoforms locate to the plasmalemma membrane and mediate cellular uptake of dehydroascorbic acid. Significant expression in extra-testicular tissues was confirmed for SLC2A14, notably in intestinal segments, explaining the association with IBD. Re-analysis of genomic showed a dramatically expanded locus of SLC2A14, containing twenty exons which covered 103,477 nucleotides from the first Transcriptional Start Site (TSS) to the termination of the longest transcript. All together, the presented evidence indicate that functional SNPs in the SLC2A14 gene and SLC23A1 could contribute to vitamin C imbalance in mucosal cells which contributes to an elevated risks of IBD. Furthermore, novel information about genetic and functional characteristics of SLC23A1 and GLUT14 transporters was identified. / February 2016

BACKGROUND: Anemia is a common extra-intestinal manifestation of disease in patients with inflammatory bowel disease (IBD), and iron deficiency anemia (IDA) represents the most prevalent form of anemia in this population ((Gasche, Dejaco et al. 1997) (Plantz, Maxwell et al. 2016)). IDA can result from a combination of decreased dietary iron intake, impaired intestinal absorption, and excessive gastrointestinal (GI) bleeding, all of which can lead to decreased iron stores and poor iron utilization. Furthermore, chronic IDA in children with IBD can adversely influence development and cognition, as well as contribute to the increased fatigue and decreased stamina observed in these patients (Laass, Straub et al. 2014). Treatment of IDA in pediatric patients with IBD can be with either oral iron supplementation or parenteral iron infusions, and both can result in an improvement in iron parameters and subsequent improvement in patient overall quality of life. However, there is wide variability in physician practice with respect to the identification and treatment of IDA in pediatric patients with IBD. OBJECTIVE: To assess physician practice at Boston Children’s Hospital with respect to the identification and treatment of IBD-related IDA in a population of patients admitted for management of a flare in their underlying ulcerative colitis. This information will be useful to develop initiatives directed at improving recognition and management of IDA in this population. METHODS: The Institutional Review Board (IRB) at Boston Children's Hospital (BCH) approved the study protocol. Patient electronic medical records were reviewed and abstracted from subjects with ulcerative colitis (UC) that were treated at BCH between January 2005 and January 2013. Each patient was assigned a study identification number to protect private health information, and this data was stored in a computer file behind the hospital’s internet firewall. Inpatient and outpatient office notes, as well as clinical data, were reviewed to track the identification of patients with anemia as well as physician management practices. All of the information collected was stored in the Research Electronic Database Capture (REDCap) and used for analysis. RESULTS: A total of 243 patients met initial inclusion criteria. Subsequent review of the electronic medical records revealed that only 178 patients had complete data available for use in our analysis. 55.5% of the 178 study subjects were anemic at admission, with a mean hemoglobin value of 9.32 g/dL (SD 1.89) and a mean MCV value of 77.49 fl (SD 10.24). None of the patients in this cohort had iron studies (plasma iron, ferritin, total iron binding capacity) included in their admissions laboratory testing. Only 24 patients received oral iron supplementation during their hospital stay while none received IV iron infusions. Over half of the total patient cohort (78.7 %,) were not prescribed oral iron supplementation at the time of discharge. 84% of patients that were discharged anemic remained anemic at the time of their first post-discharge ambulatory visit. Less than half of the total patient cohort (30.34%) had iron studies included in their follow-up ambulatory laboratory testing. Patients that were given iron supplementation at the time of discharge showed increased gains in their hemoglobin levels at follow-up compared to patients with no iron supplementation at the time of discharge (p <0.0001). CONCLUSION: These data suggest that there is a low prevalence with respect to the identification and treatment of IDA and anemia in pediatric patients with ulcerative colitis (UC) at admission, discharge, and follow-up clinic visits. A secondary finding was a statistically significant increase in hemoglobin levels in anemic patients treated with some form of iron supplementation. As a result, our data support both the need to improve recognition of IDA in pediatric patients admitted for a UC flare as well as document the clinical value to this effort. Additional studies are necessary to determine if improved iron surveillance and therapy will improve patient quality of life, linear growth, and cognition in patients with IBD.

Patients with inflammatory bowel disease, particularly those on systemic immunosuppression, have been shown to be at increased risk of herpes zoster infection. Herpes zoster (also known as shingles) is a condition resulting from reactivation of varicella zoster virus (VZV), which causes chickenpox. VZV reactivation is thought to be due to impairment of cell-mediated immunity. Some immunosuppressive agents have been shown to be associated with higher risk for herpes zoster reactivation than others. Until recently, the only vaccine for herpes zoster was a live-attenuated vaccine, which is contraindicated in most immunosuppressed IBD patients due to their immunosuppressive therapy. Recently, an inactivated subunit vaccine has been developed and investigated for use in immunocompetent adults, as well as select groups of immunocompromised individuals. This novel vaccine has not yet been studied in IBD patients but holds promise for use in this population. The proposed study is a single-center prospective pilot study comparing immunogenicity and safety of the inactivated herpes zoster vaccine in patients with IBD (ulcerative colitis or Crohn’s disease) treated with high-level combination immunosuppression (both anti-TNF biologics and immunomodulators) to those not on systemic immunosuppressive therapy (5-aminosalicylates or no treatment). Investigators will compare cell-mediated responses between groups using an intracellular cytokine staining assay with flow cytometry assessed prior to vaccination and at four time points up to 12 months after completion of the immunization sequence. Adverse effects will also be monitored. This study will help to identify whether the novel herpes zoster vaccine is immunogenic and safe for use in IBD patients and whether these parameters are significantly impacted by intensity of immunosuppressive treatment. An additional goal is to provide preliminary data with which to develop future studies of vaccine immunogenicity and efficacy in this target population.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-03Bitstream added on 2014-06-13T20:53:16Z : No. of bitstreams: 1 simoncini_vs_me_botib.pdf: 667182 bytes, checksum: 02774471f02be04fbff31c82acb30112 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A Doença Inflamatória Intestinal (DII) engloba uma série de doenças que afetam o trato intestinal, especialmente o cólon, sendo as principais a retocolite ulcerativa e a doença de Crohn, ambas com alta taxa de morbidade e mortalidade, além de representarem um dos mais importantes fatores de risco para o câncer colorretal, que no Brasil é o quarto tipo de tumor com maior incidência de mortes. Inúmeros medicamentos são usados no controle e no tratamento destas doenças (aminosalicilatos, glicocorticóides, imunomoduladores e antibióticos), no entanto, todos apresentam sérios efeitos colaterais e são de custo extremamente elevado. Além da busca por novas drogas para o tratamento destas doenças ainda sem cura, uma importante estratégia preventiva e curativa se baseia no uso de prebióticos na dieta, os quais poderiam tanto melhorar as condições de vida do paciente como permitir por efeito sinérgico a redução das doses dos fármacos comumente utilizados nestas doenças e conseqüentemente de seus efeitos adversos e tóxicos. Com base nestas informações, o presente trabalho visou 1. Avaliar os efeitos preventivos da dieta enriquecida com banana verde (Banana „Nanica‟ Musa sp AAA) na colite ulcerativa experimental induzida por ácido trinitrobenzenosulfônico em ratos; 2. Avaliar a influência do uso da dieta enriquecida com banana verde sobre a atividade antiinflamatória da prednisolona; 3. Comprovar se a dieta enriquecida com banana verde pode ser considerada um prebiótico. Através da realização deste estudo foi possível concluir que a administração da dieta enriquecida com a banana verde nas concentrações de 10% e 20% protege o cólon de ratos da colite experimental através de mecanismo que envolve a diminuição do estresse oxidativo evidenciado com a manutenção dos níveis de glutationa; além disso, a dieta enriquecida não aumenta a atividade... / The inflammatory bowel disease (IBD) consists in two major diseases that affect the gastrointestinal tract, specially the colon, ulcerative colitis and Crohn‟s disease, both with high mortality and morbity. These diseases represent a major risk for the development of colorectal cancer, which, in Brazil is correlated with a high mortality. Many drugs are used to control these illnesses but most of them present serious side effects and/or are very expensive. The search of new drugs is still a viable method to discovery different treatments for IBD however a new preventive and curative method can be the use of prebiotics. These prebiotics can improve the patients‟ life conditions and possibly permit the decrease of the drugs doses that are used and consequently side and toxics effects. In light of this, the aims of the present study was to evaluate the preventive effects of dietary supplementation with green banana (Banana „Nanica‟ Musa sp AAA) in experimental models of rat colitis induced by trinitrobenzenesulphonic acid (TNBS), evaluate the influence of the dietary supplementation in the dose of a corticoid (prednisolone) and then be able to analyze if the dietary supplementation can be considerate a prebiotic. Our results revealed that the ingestion of the dietary supplementation with green banana in 10% and 20% showed significant anti-inflammatory activities in the experimental model of rat colitis induced by TNBS. This activity was related with the reduction of the oxidative stress indicated with the maintenance of glutathione levels; besides, the anti-inflammatory activity of prednisolone was not altered with the dietary supplementation and the anti inflammatory preventive effects of the dietary supplementation was not correlated with a prebiotic activity

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-28Bitstream added on 2014-06-13T20:53:16Z : No. of bitstreams: 1 almeidajunior_ld_me_botib_parcial.pdf: 353637 bytes, checksum: e154d67d475601f9d66edd862735410a (MD5) Bitstreams deleted on 2015-02-04T11:39:27Z: almeidajunior_ld_me_botib_parcial.pdf,Bitstream added on 2015-02-04T11:40:12Z : No. of bitstreams: 1 000725128.pdf: 1021495 bytes, checksum: 10e5b8e91e721bace643318731693faa (MD5) / A Doença Inflamatória Intestinal (DII) é uma doença com etiologia desconhecida e sem terapêutica curativa disponível, englobando, fundamentalmente, duas doenças distintas: a Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU), ambas caracterizadas por uma inflamação crônica do intestino, com períodos de exacerbação seguidos de intervalos prolongados com remissão dos sintomas, cujo tratamento com os fármacos disponíveis apresentam sérios efeitos colaterais. Portanto, o desenvolvimento de novas estratégias de tratamento que combinem eficácia e segurança é uma importante meta na terapia da DII, sendo que as plantas medicinais são indispensáveis fontes de novos compostos de valor terapêutico. Physalis angulata L. é uma planta nativa brasileira que cresce especialmente nas regiões norte e nordeste do Brasil e em outros países tropicais da África, América e Ásia, sendo amplamente utilizada pela população para o tratamento de uma série de doenças, especialmente aquelas que possuem características inflamatórias. Estudos in vitro em culturas de células realizados por nosso grupo de pesquisa, somados aos dados descritos na literatura mostram que a P. angulata é capaz de modular vários mediadores inflamatórios. Dessa forma, o objetivo deste trabalho foi avaliar a atividade anti-inflamatória intestinal do extrato padronizado em fitoesteróis totais de P. angulata, nas fases aguda e crônica (com recidiva) do processo inflamatório intestinal induzido por acido trinitrobenzenosulfônico (TNBS) em ratos. Os resultados obtidos mostraram, pela primeira vez, que o extrato padronizado produziu uma série de efeitos que melhoraram a resposta dos animais frente à lesão intestinal promovida pelo TNBS. Estes efeitos foram mais pronunciados nos estudos de fase aguda, indicando uma propriedade preventiva importante... / Inflammatory Bowel Disease (IBD) is a disease with unknown etiology and no curative treatment available, embracing essentially two distinct diseases: Crohn's disease (CD) and ulcerative colitis (UC), both characterized by chronic inflammation of the intestine, with periods of exacerbation followed by long intervals with symptom remission, whose treatment with available drugs have serious side effects. Therefore, the development of new treatment strategies that combine effectiveness and safety is an important goal in the treatment of IBD, where medicinal plants are sources of essential novel compounds of therapeutic value. Physalis angulata L. is a native Brazilian plant that grows especially in Northern and Northeastern Brazil and other tropical countries of Africa, America and Asia and is widely used by people to treat a number of diseases, especially those with inflammatory characteristics. In vitro studies in cell cultures conducted by our research group, combined with the previously reported data, show that P. angulata is capable of modulating several inflammatory mediators. Thus, the objective of this study was to evaluate the intestinal anti-inflammatory activity of P. angulata standardized extract in total phytosterols, in the acute and chronic (with relapse) phases of the intestinal inflammatory process induced by trinitrobenzene sulfonic acid (TNBS) in rats. The results showed, for the first time, that the standardized extract produced a series of effects that improved the response of animals against intestinal injury promoted by TNBS. These effects were more pronounced in the acute studies, indicating an important preventive property by reducing the activity of myeloperoxidase and alkaline phosphatase, in addition with the ability of the extract to prevent depletion of glutathione induced by intestinal... (Complete abstract click electronic access below)