Ana Teresa Pugas Carvalho

Immunohistochemical study of intestinal eosinophils in inflammatory bowel disease

Background Eosinophil accumulation and activation are characteristic features of inflammation in allergic diseases and in host defense against parasites. Goals To investigate the involvement of eosinophils in inflamed and noninflamed mucosa of patients with inflammatory bowel disease (IBD). Study Specimens of inflamed colonic mucosa from 15 patients with ulcerative colitis (UC) and inflamed and noninflamed colonic mucosa from 15 patients with Crohns disease (CD) were submitted to histologic and immunohistochemical studies. Twelve patients with irritable bowel syndrome were studied as controls. Sirius red was used to label eosinophils in tissue. EG1, EG2, and anti–hIL-5 were used as primary antibodies in an indirect alkaline phosphatase-labeled immunostaining protocol. Both positive and negative lamina propria cells were assessed by a quantitative grading system and the results expressed as cell numbers per mm2. Results Increased proportions of eosinophils stained with Sirius red, EG1, EG2, and anti–hIL-5+ cells were found in the colon of patients with UC and in inflamed and noninflamed colon of CD patients as compared with controls. Crohns disease patients showed increased proportions of EG1+ and EG2+ cells as compared with those with UC. Increased proportions of IL-5+ cells were detected in UC patients as compared with those with CD. Conclusion Quantitative eosinophil alterations and IL-5+ cells may indicate enhanced cellular activation with degranulation, which is implicated in the pathogenesis of IBD. Increase in IL-5+ cells may reflect a predominant local Th2 response in UC as compared with CD.

Prevalence of periodontitis and DMFT index in patients with Crohn's disease and ulcerative colitis

AIM To compare the prevalence of periodontal disease and the decayed, missing and filled teeth (DMFT) index in patients with Crohns disease (CD) and ulcerative colitis (UC) with those without these diseases. MATERIAL AND METHODS Ninety-nine CD (39.0 SD+/-12.9 years), 80 UC (43.3 SD+/-13.2) and 74 healthy controls (40.3 SD+/-12.9) were compared for DMFT index and presence of periodontitis. Probing pocket depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP), plaque and DMFT index were measured on all subjects. The presence of periodontitis was defined as having CAL > or =3 mm in at least four sites in different teeth. RESULTS Significantly more patients with UC (90.0%; p<0.001) and CD (81.8%; p=0.03) had periodontitis than controls (67.6%). Among smokers, UC patients had significantly more periodontitis. CD had a greater mean DMFT score (18.7 versus 13.9; p=0.031) compared with controls and UC had greater median PPD (2.2 versus 1.7 mm; p<0.0001) than controls. Among non-smokers, CD (2.4 mm; p<0.0001) and UC showed deeper pockets (2.3 mm; p<0.0001) compared with controls (1.5 mm). UC had a greater mean DMFT score (15.3 versus 12.1; p=0.037) compared with controls. CONCLUSIONS CD and UC patients had higher DMFT and prevalence of periodontitis than controls, but smoking was an effect modifier.

Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease: evidence of altered expression of FasL and perforin cytotoxic pathways

Background and aimsAbnormal apoptosis may result in the persistence of activated intestinal T-cells in inflammatory bowel disease (IBD). We investigated apoptosis in distinct mucosal compartments, and the expression of Fas/Fas ligand and perforin in the inflamed and non-inflamed intestinal mucosa of patients with IBD.MethodsColon specimens from 15 patients with ulcerative colitis (UC) and inflamed and non-inflamed mucosa from 15 patients with Crohn’s disease (CD) were analysed for densities and distribution of apoptotic cells determined by the terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick-end labelling (TUNEL) method. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68, by double immunofluorescence with confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser.ResultsColonic lamina propria (LP) and epithelium from patients with UC showed higher rates of apoptosis than controls, but no difference was shown regarding patients with CD. In LP, co-expression of Fas was reduced with T-cells in inflamed CD mucosa, and with macrophages in all patients with IBD. No difference was found in the expression of Fas on B-cells. Rates of FasL-expressing cells in LP were higher in IBD than in controls, with no correlation with the rates of apoptosis. Rates of perforin-expressing cells in LP were greater in UC than in controls, and correlated to the rates of apoptosis. No difference was shown regarding the inflamed and non-inflamed CD mucosa. Rates of FasL and perforin-expressing intra-epithelial lymphocytes showed no difference among groups.ConclusionsIncreased expression of FasL in IBD colonic LP not parallelled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells. Expression of perforin is correlated to the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in UC.

Subgingival microflora in inflammatory bowel disease patients with untreated periodontitis.

Objective To analyze the subgingival microflora composition of inflammatory bowel disease (IBD) patients with untreated chronic periodontitis and compare them with systemically healthy controls also having untreated chronic periodontitis. Method Thirty IBD patients [15 with Crohn’s disease (CD) and 15 with ulcerative colitis (UC)] and 15 control individuals participated in the study. All patients had been diagnosed with untreated chronic periodontitis. From every patient, subgingival plaque was collected from four gingivitis and four periodontitis sites with paper points. Samples from the same category (gingivitis or periodontitis) in each patient were pooled together and stored at −70°C until analysis using a checkerboard DNA–DNA hybridization technique for 74 bacterial species. Results Multiple-comparison analysis showed that the groups differed in bacterial counts for Bacteroides ureolyticus, Campylobacter gracilis, Parvimonas micra, Prevotella melaninogenica, Peptostreptococcus anaerobius, Staphylococcus aureus, Streptococcus anginosus, Streptococcus intermedius, Streptococcus mitis, Streptococcus mutans, and Treponema denticola (P<0.001). CD patients had significantly higher levels of these bacteria than UC patients either in gingivitis or in periodontitis sites (P<0.05). CD patients harbored higher levels of P. melaninogenica, S. aureus, S. anginosus, and S. mutans compared with controls both at gingivitis and at periodontitis sites (P<0.05). UC patients harbored higher levels of S. aureus (P=0.01) and P. anaerobius (P=0.05) than controls only in gingivitis sites. Conclusion Our study showed that even with similar clinical periodontal parameters, IBD patients harbor higher levels of bacteria that are related to opportunistic infections in inflamed subgingival sites that might be harmful for the crucial microbe–host interaction.

Cytokine expression in gingival and intestinal tissues of patients with periodontitis and inflammatory bowel disease: An exploratory study

OBJECTIVE To evaluate the expression of the cytokines IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IL-17A, IL-17F, sCD40L, and TNF-α in gingival tissue and intestinal mucosa of patients having both periodontitis and inflammatory bowel disease (IBD) and assess how they cluster in both tissues. METHODS This cross-sectional study selected 28 patients with periodontitis (18 with Crohns disease and 10 with ulcerative colitis) from the IBD gastroenterology outpatient clinic at the Pedro Ernesto University Hospital. Patients were assessed using questionnaire, medical chart check and periodontal examination. Gingival and intestinal biopsies were collected and homogenized using a cell disruptor. Cytokines expression was evaluated through multiplex technology. Cluster analysis was performed based on cytokinés correlation strength and presented in dendrograms. RESULTS Crohns disease and ulcerative colitis patients exhibited no significant difference between them in cytokine levels (p>0.05), so they were analysed together. Significantly higher levels of IL-17A, IL-17F, IL-22, IL-25, IL-33, IL-10, and INF-γ were found in gingival tissues in comparison with intestinal mucosa (p<0.05). In gingival tissue, cytokines formed the following clusters: IL-25/IL-10/IL-33 (r=0.775), IL-22/IL-23/IL-6 (r=0.681) and IL-6/IL-25/IL-33/IL-10 (r=0.660). In intestinal mucosa, the following clusters were formed: IL-6/IL-21/IL-10 (r=0.880), IL-17A/IL-6/IL-21/IL-10 (r=0.826), IL-I7F/IL-33/IL-25 (r=0.813) and IL-23/IL-2/IL-17A/IL-6/IL-21/IL-10 (r=0.785). CONCLUSION Expression of IL-17A, IL-17F, IL-22, IL-25, IL-33, IL-10, and INF-γ was significantly increased in gingival tissue in comparison with intestinal mucosa of patients with periodontitis and IBD. The cytokine clustering pattern was different in gingival and intestinal tissues.

Thiopurine-methyltransferase variants in inflammatory bowel disease: prevalence and toxicity in Brazilian patients.

AIM To analyze the prevalence of thiopurine-methyltransferase (TPMT) genotypes and their association with drug toxicity in inflammatory bowel disease (IBD) patients from southeastern Brazil. METHODS A total of 219 consecutive patients with IBD, of which 146 had Crohns disease and 73 had ulcerative colitis, regularly seen at the outpatient unit of the Division of Gastroenterology at the University Hospital Pedro Ernesto of the State University of Rio de Janeiro, a tertiary referral center, were enrolled in this study from February 2009 to January 2011. We analyzed the presence of major TPMT genetic variants (TPMT 2, 3A, 3C) in IBD patients by means of a specific allele and RFLP-PCR. Genomic DNA was isolated from peripheral blood leukocytes by proteinase-K/Sodium Dodecyl Sulfate digestion and phenol-chloroform extraction. TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes were detected by real-time polymerase chain reaction followed by direct sequencing with specific primers. Clinical data were systematically recorded, and correlated with the genotype results. RESULTS The distribution of the selected TPMT gene polymorphism TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes was 3.6%, 5.4%, and 7.7% of the patients, respectively. Among the side effects recorded from patients taking azathioprine, 14 patients presented with pancreatitis and/or an elevation of pancreatic enzymes, while 6 patients had liver toxicity, and 2 patients exhibited myelosuppression/neutropenia. TPMT polymorphisms were detected in 37/219 patients (8 heterozygous for 2, 11 heterozygous for 3A, and 18 heterozygous for 3C). No homozygotic polymorphisms were found. Despite the prevalence of the TPMT 3C genotype, no differences among the genotype frequencies were significant. Although no association was detected regarding myelotoxicity or hepatotoxicity, a trend towards the elevation of pancreatic enzymes was observed for TPMT 2 and TPMT 3C genotypes. CONCLUSION The prevalence of TPMT genotypes was high among Brazilian patients. Variants genes 2 and 3C may be associated with azathioprine pancreatic toxicity in a IBD southeastern Brazilian population.

The socio-economic impact of work disability due to inflammatory bowel disease in Brazil

BackgroundInflammatory bowel disease (IBD) might have economic and social impacts in Brazil, where its prevalence has increased recently. This study aimed to assess disability due to IBD in the Brazilian population and demographic factors potentially associated with absence from work.MethodsAnalysis was performed using the computerized Single System of Social Security Benefits Information, with a cross-check for aid pension and disability retirement, for Crohn’s disease (CD) and ulcerative colitis (UC). Additional data were obtained from the platform, including the average values, benefit duration, age, gender and region of the country.ResultsTemporary disability occurred more frequently with UC, whereas permanent disability was more frequent with CD. Temporary disability affected more younger patients with CD than patients with UC. Temporary work absences due to UC and CD were greater in the South, and the lowest absence rates due to CD were noted in the North and Northeast. Absence from work was longer (extending for nearly a year) in patients with CD compared to those with UC. The rates of temporary and permanent disability were greater among women. Permanent disability rates were higher in the South (UC) and Southeast (CD). The value of benefits paid for IBD represented approximately 1% of all social security benefits. The benefits paid for CD were higher than for UC, whereas both tended to decrease from 2010 to 2014.ConclusionsIn Brazil, IBD frequently causes disability for prolonged periods and contributes to early retirement. Reduction trends may reflect improvements in access to health care and medication. Vocational rehabilitation programs may positively impact social security and the patients’ quality of life.

Effects of oral nutritional supplementation on the intestinal mucosa of patients with AIDS

Weight loss is a major component of the clinical syndrome in patients with acquired immunodeficiency syndrome (AIDS). The impact of malnutrition on the outcome of the disease has been unappreciated in many investigations. The authors evaluated the effects of oral nutritional supplementation on the morphology and immunology of the intestinal mucosa of patients with AIDS. Twelve patients with AIDS without diarrhea or opportunistic infections, with at least 10% of body weight loss over 1 year, were submitted to anthropometric measures, peripheral blood T-lymphocyte counts, and peroral jejunal biopsy before and after oral nutritional supplementation. An industrialized peptide-based formula containing omega-3 fatty acids was given for 6 weeks. Jejunal samples were analyzed by histomorphometry, including villous-to-crypt ratio, lamina propria, and intraepithelial lymphocyte count. Immunologic assessment of the intestinal mucosa was made by indirect immunoperoxidase using monoclonal antibodies against CD3, CD4, and CD8. Seven patients with irritable bowel syndrome and two healthy volunteers were selected as a control group for histologic and immunohistochemical comparisons. After 6 weeks the patient group maintained their body weight and increased their tricipital fold. The number of peripheral blood T cells, albumin, transferrin, and the number of CD3+, CD4+, and CD8+ cells in jejunal mucosa as well as the intestinal morphometry remained stable. Oral supplementation contributed to maintaining body weight and may constitute a reasonable adjuvant therapeutic tool against AIDS progression.

Activity of inflammatory bowel disease influences the expression of cytokines in gingival tissue

HighlightsCytokines were assessed in gingiva and intestine from periodontitis patients with IBD.IL‐4, IL‐10 and IL‐21 were higher in gingival tissue from patients with active IBD.An inflammation score was increased in gingiva from patients with IBD activity. Abstract This study assessed the cytokine expression in gingival and intestinal tissues from periodontitis patients with inflammatory bowel disease (IBD) and evaluated if IBD activity is a covariate to the amount of gingival cytokines. Paired gingival and intestinal tissues were collected from 21 patients and homogenised using a cell disruptor. Cytokine expression (IL‐1&bgr;, IL‐4, IL‐6, IL‐10, IL‐21, IL‐22, IL‐23, IL‐25, IL‐31, IL‐33, IL‐17A, IL‐17F, IFN‐&ggr;, sCD40L, and TNF‐&agr;) was evaluated using bead‐based multiplex technology. An inflammation score was developed using the intestinal cytokines that showed good accuracy to discriminate IBD active patients from those in remission and then a similar score was applied to gingival tissue. IL‐4, IL‐10 and IL‐21 expressions were significantly increased in gingival tissue from patients with an active disease as compared to those with a disease in remission. The inflammation score (mean value of IL‐1&bgr;, IL‐6, IL‐21, and sCD40L) was significantly higher in gingival tissue from patients with IBD activity. There was a significant correlation between gingival and intestinal inflammation scores (rho = 0.548; P = 0.01). Significantly higher IL‐23 and IFN‐&ggr; levels and lower IL‐31 and TNF‐&agr; levels were observed in gingival tissues than in intestinal ones. Activity of inflammatory bowel disease influenced the cytokine expression in gingival tissue.

Multidrug resistance 1 gene polymorphisms may determine Crohn's disease behavior in patients from Rio de Janeiro

OBJECTIVES: Conflicting data from studies on the potential role of multidrug resistance 1 gene polymorphisms in inflammatory bowel disease may result from the analysis of genetically and geographically distinct populations. Here, we investigated whether multidrug resistance 1 gene polymorphisms are associated with inflammatory bowel diseases in patients from Rio de Janeiro. METHODS: We analyzed 123 Crohns disease patients and 83 ulcerative colitis patients to determine the presence of the multidrug resistance 1 gene polymorphisms C1236T, G2677T and C3435T. In particular, the genotype frequencies of Crohns disease and ulcerative colitis patients were analyzed. Genotype-phenotype associations with major clinical characteristics were established, and estimated risks were calculated for the mutations. RESULTS: No significant difference was observed in the genotype frequencies of the multidrug resistance 1 G2677T/A and C3435T polymorphisms between Crohns disease and ulcerative colitis patients. In contrast, the C1236T polymorphism was significantly more common in Crohns disease than in ulcerative colitis (p = 0.047). A significant association was also found between the multidrug resistance 1 C3435T polymorphism and the stricturing form of Crohns disease (OR: 4.13; p = 0.009), whereas no association was found with penetrating behavior (OR: 0.33; p = 0.094). In Crohns disease, a positive association was also found between the C3435T polymorphism and corticosteroid resistance/refractoriness (OR: 4.14; p = 0.010). However, no significant association was found between multidrug resistance 1 gene polymorphisms and UC subphenotypic categories. CONCLUSION: The multidrug resistance 1 gene polymorphism C3435T is associated with the stricturing phenotype and an inappropriate response to therapy in Crohns disease. This association with Crohns disease may support additional pathogenic roles for the multidrug resistance 1 gene in regulating gut-microbiota interactions and in mediating fibrosis. Understanding the effects of several drugs associated with multidrug resistance 1 gene variants may aid in the selection of customized therapeutic regimens.