A doença Inflamatória Intestinal (DII) é uma desordem caracterizada pela inflamação crônica do trato gastrointestinal. Os dois principais tipos de DII são a Retocolite Ulcerativa (RCU) e a Doença de Crohn (DC) e ambas cursam com importantes alterações no estado nutricional (EN). O objetivo deste estudo foi identificar as diferenças na composição corporal entre pacientes com DC, RCU e indivíduos saudáveis, além de comparar o estado nutricional dos três grupos de pacientes, ajustando para fatores que podem interferir no EN, como o uso atual de corticosteróides, a atividade física, a atividade de doença, a idade e o sexo. Foi realizado um estudo transversal que incluiu 101 pacientes com DII (50 com DC e 51 com RCU) e 35 indivíduos saudáveis, selecionados no Ambulatório do Hospital Universitário Pedro Ernesto (HUPE) da Universidade do Estado do Rio de Janeiro (UERJ). Foram colhidas informações sócio-demográficas e pessoais, tais como: prática de atividade física, tabagismo, doenças pregressas e procedimentos cirúrgicos prévios. Outras informações necessárias à pesquisa foram coletadas em prontuário médico. A avaliação antropométrica foi realizada por meio das seguintes medidas: peso corporal; altura; circunferências do braço, da cintura (CC) e do quadril; dobras cutâneas do tríceps, subescápula, supra-ilíaca e da coxa; e circunferência muscular do braço (CMB). A análise da composição corporal foi realizada por meio da bioimpedância elétrica (BIA), utilizando-se o aparelho Biodynamics modelo 450. As variáveis laboratoriais analisadas foram: glicose, hemograma completo, perfil lipídico, proteínas totais, albumina, globulina, velocidade de hemossedimentação e proteína C reativa. O peso, o índice de massa corporal, a CC e o percentual de gordura corporal calculado a partir da aferição das dobras cutâneas, foram menores nos pacientes com DC, quando comparados aos indivíduos saudáveis e/ou aos pacientes com RCU. A CMB foi menor nos pacientes com DC e RCU quando comparados aos indivíduos saudáveis, porém sem apresentar diferenças entre os dois grupos de pacientes. Por BIA, verificou-se que os pacientes com DC apresentaram valores de massa magra, massa celular corpórea, massa extracelular, água corporal total e água extracelular menores quando comparados aos indivíduos saudáveis. Os níveis séricos de colesterol total, proteínas totais e albumina, e a contagem total de hemácias foram menores nos indivíduos com DC quando comparados aos indivíduos do grupo controle e/ou aos indivíduos do grupo da RCU. Os pacientes com RCU exibem composição corporal semelhante à da população saudável. Em contraposição, os pacientes com DC apresentam EN amplamente comprometido com depleção de gordura corporal e massa magra em relação aos demais indivíduos / Inflammatory Bowel Disease (IBD) is a disorder characterized by diffuse inflammation of the gastrointestinal tract. The two main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD), both coursing with important changes in nutritional status (NS). The objective of this study was to identify differences in body composition between patients with CD, UC, and healthy subjects and to compare the NS of these three groups of patients, adjusting for factors that can interfere in NS such as current use of corticosteroids, physical activity, disease activity, age and sex. It was conducted a cross-sectional study which included 101 patients with IBD (50 with CD and 51 with UC) and 35 healthy subjects, selected at the Ambulatory of Pedro Ernesto University Hospital (HUPE) of the Rio de Janeiro State University (UERJ). Socio-demographic and personal information such as physical activity, smoking, prior diseases and previous surgical procedures were collected. Other necessary information for the research were collected from medical records. The anthropometric evaluation was carried out through the following measures: body weight; height; mid-arm, waist and hip circumferences; skinfold thickness of the triceps, subscapular, suprailiac, and thigh; and mid-arm muscle circumference (MAMC). The body composition analysis was performed by bioelectrical impedance (BIA) using the equipment Biodynamics model 450. The laboratory variables analyzed, included: glucose, complete blood count, lipid profile, total protein, albumin, globulin, erythrocyte sedimentation rate, and C-reactive protein. Weight, body mass index, waist circumference, and percentage body fat calculated from skinfold measurements were lower in CD patients compared to healthy subjects and/or the patients with UC. The MAMC was lower in patients with CD and UC compared to healthy subjects, but without showing differences between the two groups of patients. Through BIA, it was found that CD patients had values of lean body mass, body cell mass, extracellular mass, total body water, and extracellular water smaller when compared to healthy subjects. Seric levels of total cholesterol, total protein, and albumin, as well as red blood cell count and relative count of lymphocytes were lower in the individuals with CD than the individuals of the control group and/or the patients with UC. Patients with UC exhibit body composition similar to that of the healthy population. In contrast, CD patients have widely NS committed with depletion of body fat and lean mass in relation to other individuals.

Abstract The function of the transmembrane protein FasL is to complex with the Fas receptor in a target cell and induce target cell apoptosis. Fas/FasL-mediated apoptosis plays important role in immunoregulation. FasL expression is mostly seen in activated lymphocytes. We have characterized endothelial FasL expression in different functional compartments of lymph nodes and gut-associated lymphoid tissue. Furthermore, we have explored the functional role of endothelial FasL expression by analyzing correlation with apoptosis of lymphocyte subpopulations in lymph nodes and by assessing endothelial expression under different conditions by activation of immune functions in gastrointestinal mucosa. Immunohistochemical stainings (Fas, FasL, CD3, CD20, CD19, CD23, CD56, FVIII) were performed on 20 reactive lymph node tissues (I and II), 60 pediatric endoscopy biopsy samples (III) or 60 samples from gut resections (IV). A double-staining method combining apoptosis detection with the TUNEL-method and lymphocyte classification with FasL, Fas and cell lineage markers was optimized. Patient groups included non-pathological lymph nodes, pediatric cow’s milk-sensitive enteropathy, pediatric celiac disease, appendicitis, ulcerative colitis and Crohn’s disease. Control groups included normal biopsy samples from pediatric patients and non-pathological resecate samples from the appendix, colon or ileum to correspond to patient groups. Quantitative analysis (positive vessels or cells per mm2) was performed thoroughly for each anatomical region. In a subset of patients, soluble FasL in the serum was quantified with standard enzyme-linked immunosorbent assay. In reactive lymph nodes FasL expression was predominantly present in high endothelial venules located in the paracortical area, where apoptotic T and B lymphocytes, some expressing Fas, were subsequently found. In the gut wall vascular FasL expression was seen in high endothelial vessels near lymphoid follicles. Serum FasL was elevated in children with an abundance of mucosal lymphoid follicles. In IBD, vascular FasL was upregulated in ulcers and in the submucosa of colons affected by Crohn’s disease. The results indicate that endothelial FasL is characteristically present in high endothelial venules of lymphoid tissues. Detection of apoptotic Fas expressing lymphocytes adjacent to such vessels supports the idea that endothelial FasL functions as a selective gatekeeper by inducing apoptosis of Fas+ lymphocytes entering from the blood stream. / Tiivistelmä Solukalvon läpäisevän proteiinin, FasL:n, tehtävä on sitoutua kohdesolun Fas-reseptoriin ja indusoida kohdesolun apoptoosi. Fas/FasL-välitteinen apoptoosi on merkittävä tekijä immunologisessa säätelyssä. FasL ilmentyy pääsääntöisesti aktivoituneissa lymfosyyteissä. Olemme kuvanneet tutkimuksessamme FasL:n endoteelistä ilmentymistä imukudoksen eri toiminnallisissa alueissa ja suoliston lymfaattisessa kudoksessa. Lisäksi kartoitimme endoteelin FasL:n toiminnallista merkitystä analysoimalla sen yhteyttä lymfosyyttien alaryhmien apoptoosiin imusolmukkeissa ja arvioimalla FasL:n endoteelistä ilmentymistä suoliston limakalvon immunologisesti erilaisissa sairauksissa. Teimme immunohistokemiallisia värjäyksiä (Fas, FasL, CD3, CD20, CD19, CD23, CD56 ja FVIII) 20 reaktiiviselle imusolmukkeelle (I ja II), 60 lapsen endoskooppiselle biopsianäytteelle (III) sekä 60 suoliresekaattinäytteelle (IV). Optimoimme kaksoisvärjäysmenetelmän, missä yhdistettiin apoptoosin havainnointimenetelmä TUNEL ja FasL-, Fas- tai solulinjamarkkeri. Potilasryhmiin kuului potilaita, joilla oli normaalit imusolmukkeet, sekä potilaita, jotka sairastivat lasten viivästynyttä lehmänmaitoallergiaa, lasten keliakiaa, umpilisäketulehdusta, haavaista paksusuolitulehdusta tai Crohnin tautia. Verrokkiryhmiin kuului normaaleja biopsianäytteitä lapsipotilailta sekä terveitä resekaattinäytteitä umpilisäkkeestä sekä paksu- tai sykkyräsuolesta potilasryhmien mukaisesti. Jokaiselle anatomiselle alueelle suoritimme perusteellisen määrällisen analyysin (positiivista suonta tai solua per mm2). Osalle ryhmistä suoritimme seerumin liukoisen FasL:n määrityksen entsyymivälitteisellä immunosorbenttimäärityksellä. Reaktiivisissa imusolmukkeissa FasL:n ilmentyminen näkyi pääsääntöisesti parakortikaalialueen korkeaendoteelisissä venuleissa, missä myös apoptoottiset T- ja B-lymfosyytit (joista osa ilmensi Fasia) sittemmin näkyivät. Suoliston seinämässä havaitsimme verisuoniperäistä FasL:n ilmentymistä korkeaendoteelisissä suonissa lymfaattisten itukeskusten lähettyvillä. Niillä lapsipotilailla, joilla havaitsimme limakalvon lymfaattisten itukeskuksien lisääntymistä, oli myös seerumin FasL-pitoisuus koholla. Tulehduksellisissa suolistosairauksissa verisuoniperäinen FasL oli lisääntynyt limakalvon haavaumissa sekä Crohnin tautia sairastavien potilaiden submukoosassa. Tulokset osoittavat verisuoniperäisen FasL:n tyypillisesti ilmentyvän imukudoksen korkeaendoteelisissa suonissa. Apoptoosin havaitseminen Fasia ilmentävissä lymfosyyteissä näiden suonien läheisyydessä tukee ajatusta siitä, kuinka verisuoniperäinen FasL toimii valikoivana portinvartijana ja aiheuttaa Fas-positiivisten lymfosyyttien apoptoosin estämällä niiden pääsyn verenkierrosta.

Magister Chirurgiae Dentium - MChD / Purpose: To assess the oral health status of 4-15-year-old schoolchildren in the Western Region of the Eastern Cape Province, by determining the prevalence of dental caries, periodontal disease, dental fluorosis, malocclusion and oral mucosal lesions and to create baseline data for planning oral health services in this region. Materials and Methods: The prevalent analytic survey was undertaken on 822 schoolchildren between the ages of 4-15 years in 27 different schools. The sample size selected for this region by the Department of Statistics, University of Pretoria, for the National Oral Health Survey of 1999/2000 was used. The data on 209, 210, 209 and 194 scholars in the 4-5, 6, 12 and 15-year-old age groups respectively were collected. The World Health Organization's (1997) criteria were used to determine the prevalence of caries, periodontal treatment needs, the dental fluorosis, malocclusion and prevalence of oral mucosal lesions. Six calibrated and trained examiners recorded the data on a survey form. The WHO survey forms were used for data collection. The data were analysed and processed by the author on Microsoft Excel 2000. Results: The sample size consisted of 1.35 % Asian, 51.89 % Black, 26.07 % Coloured and 20.06 % White scholars. Nearly equal amounts of males (48.3 %) and females (51.7%) were examined. The dental caries prevalence in the primary dentition of 4-5-year-olds with an average age of 4.43 years was 40.67%, with a mean dmft of 3,56. The dental caries prevalence in the permanent dentition of6-, 12- and 15-year-oldage groups was 67.62%, 46.05% and 67.92% respectively. The mean DMFT for 6-, 12- and 15 year olds were 0.05, 1.19 and 2.02 respectively. Gender differences and differences in prevalence of dental caries between the populations groups were recorded. The Coloured children experienced the highest dental caries prevalence. In the 4-5- and 15-year-old groups the males were affected more than the females by dental caries. Prevalent differences were recorded between rural and urban areas in the same population group. The prevalence of periodontal disease in 373, 12-15-year-olds was 79.09%. Only 4.02 % were in need of dental hygiene instructions and polishing. Dental education, dental hygiene instructions, dental scaling and polishing were needed by 75.07% of the children. Dental fluorosis prevalence in 450, 6-15-year-olds was 13.03%. Dental fluorosis did not affect 86.97% of the scholars; 10.63% had mild fluorosis and 2.05% was severely affected. The mean fluoride concentration in the drinking water in this region were 1.07 mgIL, ranging between 0.3-3 mgIL. Definite malocclusion was recorded in only 0.48% of the 12-year-old group, whom needed elective treatment. In the 12-year-olds, 99.52% needed no or slight treatment for no or minor malocclusions. The majority of children had no oral mucosal lesions (91.55%). The prevalence of oral mucosal lesions was 8.5% in this survey. The most frequent conditions were traumatic lesions (1.96%), dento-alveolar abscess (1.22%) and herpes labialis (1.22%). The intra-examiner reliability for caries, dental fluorosis, periodontal treatment needs and prevalence of malocclusions was 97.66%, 100%, 88.89% and 86.67% respectively. The inter-examiner reliability was 98.75%, 98.34%, 100%, 100% and 100% respectively for the five examiners. Conclusion: This survey indicated a higher prevalence of dental caries in the primary dentition (63.48%), than the permanent dentition (41.3%). The mean dmft was 3.65 and the DMFT 1.2, which indicated a low mean caries experience for these children. The WHO goal for the year 2000 for the 6-year-olds of 50% being caries free has not reached for only 32.56% were caries free. The mean DMFT of 1.2 for 12-year-olds in this survey is below the WHO goal of a mean DMFT of 1.5 or less. The lack of available preventative service provision, were ) indicated by the low percentage of fissure sealants (2.8%). The need for dental treatment was highlighted by the need mostly for one-surface restorations. Preventative treatment, two-surface fillings and extractions were also needed. Periodontal disease was a major public oral health concern for this region for a prevalence of 79.09% was recorded for the 12-15-year-olds. The majority of the children affected by periodontal disease (75.07%) were in need of professional cleaning and calculus removal. The prevalence of dental fluorosis was 8.2%, 19.87% and 13.05% for 6-, 12- and IS-year age groups respectively. The prevalence of malocclusion was 0.48% for the 12-year-old group. The prevalence of oral mucosa lesions was 8.5% for 4-15-year-old schoolchildren. Therefore, the conclusion can be made that dental fluorosis; malocclusions and oral mucosal lesions were no public concern in this region. The major public oral health concerns were dental caries in the primary dentition and periodontal disease in all age groups. Recommendations: Dental services in this region should be directed towards prevention of dental caries in the primary dentition and periodontal disease in all age groups. The focus of dental services should be primarily on preventative programs and treatments. The public dental treatments needs indicated in this survey, namely scaling and polishing, restorations and extractions, consists of the primary health care package delivery. Currently, no evaluation tool exists to monitor the efficiency of oral health programs, no baseline data for the Province exists. Therefore, the urgent need for dental research and oral health service planning and delivery are recommended.

P-glycoprotein (P-gp) protects the mammalian intestinal epithelium by effluxing toxins from the epithelial cells as well as release of human endocannabinoids that inhibit neutrophil infiltration. Diminished or dysfunctional P-gp is associated with intestinal inflammation including ulcerative colitis (UC). Due to the microbiome dysbiosis associated with UC, we hypothesize that the healthy microbiota promote colonic P-gp expression. Utilizing mouse models of antibiotic treatment, microbiota reconstitution, and metabolite perturbation, we have shown butyrate and secondary bile acids, dependent on vancomycin-sensitive bacteria, induce P-gp expression in vivo. We have shown these metabolites together potentiate induction of P-gp in intestinal epithelial cell lines in vitro, which is sufficient to inhibit primary human neutrophil transmigration. Furthermore, in UC patients we find diminished P-gp expression is coupled to reduction of anti-inflammatory endocannabinoids and luminal content with reduced capability to induce P-gp expression. Additionally, we have found butyrate contributes to P-gp expression via histone deacetylase inhibition, and secondary bile acids regulate P-gp expression via nuclear receptors pregnane X receptor and vitamin D receptor. Employing RNA sequencing (RNAseq) in IECs uncovered signaling networks that are uniquely triggered with the combination of butyrate and secondary bile acids, suggesting additional pathways required for maximal P-gp expression in the colon. Together we identify a mechanistic link between cooperative functional outputs of the complex microbial community and suppression of intestinal inflammation. These data emphasize the importance of the intestinal microbiome in driving the P-gp axis to suppress aberrant neutrophil infiltration and identify potential therapeutic targets for promoting P-gp expression in an inflamed colon to reset homeostasis.

Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies. / <p>QC 20180412</p>

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.