Mathematical Models Including microRNA Levels of Mesenteric Adipose Tissue May Predict Postoperative Relapse in Crohn's Disease Patients.

Background and Aims: Recent evidence suggests that the mesenteric adipose tissue (MAT) near the affected intestine may play a role in Crohn's disease (CD) pathophysiology. Modulation of several transcripts has already been identified in the MAT of CD in the literature. Therefore, our aim was to validate the microRNA (miRNA) transcript levels and their target genes in the MAT of active CD patients and correlate them with clinical and epidemiological data. Methods: Samples from the MAT of surgical specimens from 25 active CD patients were obtained. The control group comprised fifteen patients who underwent surgery for other diseases, except inflammatory bowel diseases. Transcriptional levels of miRNA and their target genes were assessed by quantitative real-time polymerase chain reaction. The correlation between transcripts and clinical characteristics was obtained using multiple linear regression. The mathematical models (M) underwent a statistical filter to ensure robustness and reliability (P value < .05; adjusted R-squared (Rˆ2)> .99; correct predictions of more than 60%). Results: miRNA-650 and miRNA-29c were upregulated in the MAT of CD compared to the control group (P < .0001 and P = .0032, respectively), besides presenting decreased levels of their target genes. Two were target genes of the miRNA-650: glutamine-fructose-6-phosphate transaminase 2 (P = .012) and aldehyde dehydrogenase 4 family (P = .0035); and 4 were targets of the miRNA-29c: cell death-inducing DFFA-like effector c (P = .001), E2F transcription factor-1 (P = .007), hypoxia-inducible factor 3 subunit alpha (P = .0029), and pyruvate dehydrogenase kinase 4 (P = .0054). We found 2 M with statistical strength and robustness. The performance test identified one model with 100% accuracy for predicting the month of recurrence and determining patients with less risk of early relapse after surgery. Conclusion: We demonstrate that miRNA-650 and miRNA-29c and some of their target genes, besides clinical and epidemiological variables, may be useful in a model to predict when disease relapse may occur in CD patients who underwent surgery. These findings constitute a potential tool to guide postoperative clinical management.

Ferric carboxymaltose for anemia in Crohn's disease patients at a tertiary center: A retrospective observational cohort study.

BACKGROUND: Although the gastrointestinal tract is the most affected by Crohn's disease (CD), the condition triggers other consequent manifestations, and iron deficiency anemia (IDA) is one of the most common. Intravenous (IV) iron replacement is currently available through several drugs, such as ferric hydroxide sucrose and ferric carboxymaltose (FCM). However, the clinical management of these conditions can be challenging. AIM: To elucidate the drug's effectiveness, the present study analyzed, through medical records, the clinical and epidemiological data of a cohort of patients with active CD who received IV FCM for the IDA treatment. METHODS: This retrospective observational study included 25 patients with active CD, severe anemia, and refractory to previous conventional treatments. Patients were evaluated two times: During the last treatment with ferric hydroxide sucrose and treatment with FCM. RESULTS: After treatment with FCM, parameters of IDA assessment significantly improved, serum hemoglobin (Hb) levels increased in 93% of patients (P < 0.0001), and in 44%, there was an increase of ≥ 2 g/dL in a single application. In addition, 86% of the patients showed an increase in serum iron (P < 0.0001) and ferritin (P = 0.0008) and 50% in transferrin saturation (P = 0.01). The serum iron levels at baseline showed a negative association with the ileal and colonic CD and use of biologics and a positive association with patients who developed CD later in life after the age of 40 (A3) and with a stenosing (B2) and fistulizing (B3) phenotype. The values of Hb and hematocrit after ferric hydroxide sucrose treatment remained similar to those found before treatment. CONCLUSION: This study demonstrated that FCM is an important therapeutic strategy for treating IDA in CD patients, achieving satisfactory results in refractory cases.

Pouchitis: insight into the pathogenesis and clinical aspects.

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease and familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease. Both diseases, despite being different, may require the same surgical procedure: proctocolectomy with ileal pouch-anal anastomosis (IPAA). The main complication after this procedure is pouch inflammation (pouchitis). This inflammatory complication can affect up to 60 percent of patients who receive IPAA for UC, and a very small percentage of the FAP patients. The purpose of this review was to determine the current molecular mechanisms in its pathogenesis and detail the risk factors involved in pouchitis, its diagnosis, and treatment.

Endoplasmic Reticulum Stress in Colonic Mucosa of Ulcerative Colitis Patients Is Mediated by PERK and IRE1 Pathway Activation.

Ulcerative colitis (UC) is characterized by a chronic overproduction of proinflammatory cytokines. During an acute phase, the endoplasmic reticulum (ER) is overloaded and the protein folding process is impaired, a condition named ER stress. This state induces a response (unfolded protein response (UPR)), initiated by the activation of IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which has previously been linked to intestinal inflammation in experimental models. ER stress and UPR activation trigger the activation of proinflammatory, autophagy, and apoptosis genes, in addition to promoting protein degradation. Therefore, the goal of this study was to evaluate the activation of ER stress and UPR in colonic mucosa of UC patients. Patient and Methods. Transcriptional analysis of ER stress- and UPR-related genes was performed by qPCR from intestinal mucosa of patients with UC. We also performed in situ hybridization (ISH) and immunohistochemistry (IHQ) of PERK/eIF2α and IRE1/Xbp-1 pathways and UPR-related chaperones. Results. We first evaluated inflammatory genes via qPCR, and we observed that all analyzed proinflammatory transcripts were upregulated in UC patients. ISH and IHQ images showed that ER stress is activated via PERK/eIF2α and IRE1/Xbp-1 pathways not only in intestinal epithelial cells but also in cells of the lamina propria of UC colonic mucosa. Transcriptional analysis confirmed that EIF2AK3 was upregulated in UC patients. UPR-related genes, such as ATF3, STC2, and DDIT3, along with the chaperones and cochaperones DNAJC3, CALR, HSP90B1, and HSPA5, were also upregulated in UC patients. In addition, we observed that proapoptotic and autophagy genes (Bax and ATG6L1, respectively) were also upregulated. Conclusion. Our results suggest that ER stress and UPR are indeed activated in UC patients and this may contribute to the chronic inflammatory process seen in UC. The increased apoptosis and autophagy markers further support the activation of these findings once they are activated to counterbalance tissue damage. These findings provide new insights into the molecular mechanisms that maintain UC activity and open new possibilities to attenuate intestinal inflammation.

New translational and experimental insights into the role of pro-resolving lipid mediators in inflammatory bowel disease.

The resolution of inflammation is an active process, guided by specialized pro-resolution lipid mediators (SPMs). These mediators originate from polyunsaturated fatty acids, such as omega-3. Sufficient evidence suggests that the beneficial effects attributed to omega-3 are, at least in part, the result of the immunomodulatory action of the SPMs, which act systemically by overcoming inflammation and repairing tissue damage, without suppressing the immune response. Recent studies suggest that an imbalance in the synthesis and/or activity of these compounds may be associated with the pathogenesis of several inflammatory conditions, such as inflammatory bowel disease (IBD). Thus, this review highlights the advances made in recent years with regard to the endo-genous synthesis and the biological role of lipoxins, resolvins, protectins, and maresins, as well as their precursors, in the regulation of inflammation; and provides an update on the participation of these mediators in the development and evolution of IBD and the therapeutic approaches that these immunomodulating substances are involved in this context.

The role of chemokines and adipokines as biomarkers of Crohn's disease activity: a systematic review of the literature.

INTRODUCTION: Crohn's disease (CD) is an inflammatory bowel disease (IBD) that affects the gastrointestinal tract and can have a major impact on the patient's quality of life and social/professional activities. Asymptomatic patients, or those with mild symptoms, experience the active disease with subclinical manifestation. Systematic review (SR) was performed to look for evidence for the role of chemokines and adipokines as markers for CD activity. METHODS: This SR was conducted by searching published studies in international and regional databases up till July, 2020. CD patients were adults with the disease in activity or remission. All adipokines and chemokines were considered for the analysis and the Rayyan QCRI system was used. RESULTS: In total, 20 studies were included. Six addressed chemokines and eight adipokines as potential biomarkers of CD activity. CXCL8 was the most studied chemokine (8 studies) and the results were controversial, with 62.5% showing a significant association with CD activity. CXCL10 was investigated by 4 studies and 50% identified it as a potential biomarker. CCL2, CCL11, CCL26 and CXCL1 were examined by 2 articles each. CXCL8 (P=0.002/P=0.001) and CXCL1 (P<0.001) presented the lowest? P value, which qualifies them as potential markers of disease activity. All the adipokines were tested in peripheral blood but 44.4% were also tested in intestinal mucosa, while the percentage in the chemokines' studies was 76.9% in peripheral blood, 46.1% in intestinal mucosa and 7.6% in urine sample respectively. CONCLUSION: The development of disease activity biomarkers for CD is becoming relevant for clinical practice. Chemokines and adipokines have the potential to signalize CD activity, but validation in larger cohorts of patients, preferable multicenter studies are still needed.

Intestinal Microbiota in the SARS-CoV-2 Infection: What Is Known?

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, emerged last year in China and quickly spread to millions of people around the world. This virus infects cells in different tissues and causes pulmonary (e.g., pneumonia and acute respiratory distress syndrome), neurological, cardiovascular, and intestinal manifestations, which can be the result of a direct viral effect or secondary to endothelial, thrombotic, or immunological alterations. In this chapter, we discuss recent studies which highlighted the relevance of the intestinal microbiota for other infectious respiratory diseases. We present the "altered microbiota" (dysbiotic) as a point of connection between conditions that are risk factors for the development of severe forms of COVID-19. In addition, we describe the findings of recent studies reporting alterations of microbiota composition in COVID-19 patients and speculate on how this may impact in development of the disease.

Role of diet and nutrition in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are closely linked to nutrition. The latest research indicates that diet and nutrition are significantly involved in the etiopathogenesis of the disease, although their specific role throughout its clinical course still remains unclear. This study reviewed how diet and nutrition are associated with IBD development and management. Even though specific diets have been shown to bring about positive outcomes, there is currently no scientific consensus regarding an appropriate diet that would benefit all IBD patients. We suggest that individualized dietary recommendations are of the greatest importance and that diets should be planned to provide individual IBD patients with specific nutrient requirements while keeping all the clinical aspects of the patients in mind. Further research is clearly necessary to investigate nutritional factors involved in IBD development and, especially, to evaluate the applications of the diets during the course of the disease.

Microbiota-derived short-chain fatty acids do not interfere with SARS-CoV-2 infection of human colonic samples.

Microbiota-derived molecules called short-chain fatty acids (SCFAs) play a key role in the maintenance of the intestinal barrier and regulation of immune response during infectious conditions. Recent reports indicate that SARS-CoV-2 infection changes microbiota and SCFAs production. However, the relevance of this effect is unknown. In this study, we used human intestinal biopsies and intestinal epithelial cells to investigate the impact of SCFAs in the infection by SARS-CoV-2. SCFAs did not change the entry or replication of SARS-CoV-2 in intestinal cells. These metabolites had no effect on intestinal cells' permeability and presented only minor effects on the production of anti-viral and inflammatory mediators. Together our findings indicate that the changes in microbiota composition of patients with COVID-19 and, particularly, of SCFAs do not interfere with the SARS-CoV-2 infection in the intestine.

Histological grading evaluation of non-alcoholic fatty liver disease after bariatric surgery: a retrospective and longitudinal observational cohort study.

Non-alcoholic fatty liver disease (NAFLD) is a chronic disease with several degrees of histological features which may progress to cirrhosis. Obesity is an important risk factor and although NAFLD has no specific pharmacological treatment, bariatric surgery has been associated with NAFLD regression in severely obese patients. However, few longitudinal histological studies support this finding. Therefore, firstly, a retrospective study was performed including clinical and histological data of 895 obese patients who underwent bariatric surgery. In addition, histological analyses of 30 patient's liver biopsies were evaluated at two timepoints (T1 and T2). The retrospective analysis of the total number of patients revealed that the average body mass index (BMI) was 35.91 ± 2.81 kg/m2. The liver biopsies during bariatric surgery showed that 53.52% did not present NAFLD, 30.16% had NASH, 15.98% isolated steatosis and 0.34% liver cirrhosis. The median BMI of the longitudinal cohort decreased from 37.9 ± 2.21 kg/m2 at the time of bariatric surgery (T1) to 25.69 ± 3.79 kg/m2 after 21 ± 22 months after the procedure (T2). The prevalence of NAFLD in T1 was 50%, and 16.67% in T2. The histological area of collagen fiber was lower in T2 compared to T1 (p = 0.0152) in the majority of patients, which was also illustrated by immunohistochemistry for Kupffer cell and myofibroblast formation markers. These findings confirmed the NAFLD regression after bariatric surgery and, for the first time, showed the amelioration of these features using more accurate histopathological techniques.

Dietary Patterns Associated to Clinical Aspects in Crohn's Disease Patients.

Diet is an important factor in both the pathogenesis and in the clinical course of Crohn's disease (CD). However, data on dietary patterns of CD patients are rather limited in the literature. This cross-sectional study included 60 patients with CD, aged 18-60 years. Dietary intake was assessed using a validated food frequency questionnaire to measure food consumption patterns by principal component analysis (PCA). Multiple regression analysis was performed to investigate the association between dietary patterns and clinical and demographic variables. Three dietary patterns were identified: "Traditional + FODMAP" was associated with symptoms, gender, previous surgeries, and duration of the disease. "Fitness style" was positively associated with physical activity and negatively associated with body mass index and smoking. "Snacks and processed foods" was positively associated with duration of the disease and negatively associated with age. According to the weekly food consumption analysis, patients with active disease consumed less coffee and tea. We found significant associations between the three dietary patterns and the variables, but not with the stage of the disease. Prospective studies are necessary to determine the effects of food consumption patterns on the clinical course of CD.

Whole transcriptional analysis identifies markers of B, T and plasma cell signaling pathways in the mesenteric adipose tissue associated with Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a multifactorial disease characterized by chronic intestinal inflammation. The increased visceral adiposity near the affected intestinal area, of which mesenteric adipose tissue (MAT) is the main component, is a feature of CD. Both protective and pathological roles have been attributed to this disease-associated tissue in CD. To understand the contribution of MAT to CD pathophysiology, a molecular and cellular signature of disease-associated MAT in CD patients was provided. METHODS: We performed an observational study with whole transcriptional analysis by RNA sequencing (RNA-seq) of MAT and ileal mucosa from CD patients with active disease and controls. qPCR and immunohistology were performed for validation analysis. RESULTS: RNA-seq identified 17 significantly regulated genes (|FC| > 1.5; FDR < 0.05) in CD-MAT compared to non-IBD controls, with a marked upregulation of plasma cell genes (i.e., IGLL5, MZB1, CD79A, POU2AF1, FCRL5, JCHAIN, DERL3, SDC1, PIM2). A less strict statistical cutoff value (|FC| > 1.5, nominal p ≤ 0.05) yielded a larger list of 651 genes in CD-MAT compared to controls. CD ileum showed the significant regulation compared to control ileum of 849 genes (|FC| > 1.5; FDR < 0.05) or 2654 genes (|FC| > 1.5, nominal p ≤ 0.05). Ingenuity Pathway Analysis revealed the significant regulation of pathways related to T- and B cell functionality in the MAT of CD patients. Despite the differences between the MAT and ileal signatures of CD patients, we identified a subset of 204 genes significantly modulated in both tissues compared to controls. This common signature included genes related to the plasma cell signature. Genes such as S100A8, S100A9 (calprotectin) and IL1B, which are associated with acute inflammatory response, were exclusively regulated in the ileal mucosa of CD disease. In contrast, some genes encoding for lymphocyte receptors such as MS4A1, CD3D and CD79A were exclusively regulated in CD-MAT, exhibiting a different pattern of immune cell activation compared to the ileal mucosa in CD patients. qPCR and immunohistology confirmed the presence of large infiltrates of CD3+ CD20+ lymphocytes and CD138+ plasma cells in CD-MAT. CONCLUSION: Our data strongly supports the role of CD-associated MAT as a site for T-, B- and plasma cell activation, and suggests that it could also act as a reservoir of memory immune responses.

ER stress activation in the intestinal mucosa but not in mesenteric adipose tissue is associated with inflammation in Crohn's disease patients.

Chronic/abnormal activation of endoplasmic reticulum (ER) stress is linked to the exacerbation of the inflammatory process and has been recently linked to Crohn's disease (CD) pathophysiology. We investigated the intestinal mucosa and the mesenteric adipose tissue (MAT) collected from CD patients with active disease (CD group) and from non-IBD patients (CTR group) to study ER stress activation and to address tissue-specific modulation in CD. The intestinal mucosa of CD patients showed an upregulation in the expression of ER stress related genes, including ATF3, DNAJC3, STC2, DDIT3, CALR, HSPA5 and HSP90B1. Results showed that EIF2AK3 gene was upregulated, along with increased protein expression of p-eIF2α and p-eIF2α/eIF2α ratio. Additionally, ERN1 gene expression was upregulated, along with an increased spliced/activated form sXBP1 protein. Despite the upregulation of ATF6 gene expression in the intestinal mucosa of CD patients, no differences were found in ATF6 protein expression. Lastly, the analysis of MAT revealed unchanged levels of ER stress markers along with no differences in the activation of UPR. However, chaperone gene expression was modulated in the MAT of CD patients. To conclude, our results address tissue-specific differences in UPR activation in CD and point the ER stress as an important pro-inflammatory mechanism in CD, specifically in the intestinal mucosa.

Serum Levels of Infliximab and Anti-Infliximab Antibodies in Brazilian Patients with Crohn's Disease.

OBJECTIVES: The aim of this study was to evaluate the quantitative serum level of infliximab (IFX) as well as the detection of anti-infliximab antibodies (ATIs) in patients with Crohn's disease (CD). METHOD: Forty patients with CD under treatment at a tertiary center in southeastern Brazil were evaluated. Their use of infliximab was continuous and regular. We analyzed and compared the differences in the IFX and ATI levels between the patients with active CD (CDA) and those with CD in remission (CDR). RESULTS: There was no difference in the IFX level between the CDA and CDR groups (p>0.05). Eighty percent of all patients had IFX levels above the therapeutic concentration (6-10 µg/mL). Two (9%) of the 22 patients with active disease and four (22.2%) of the 18 patients in remission had undetectable levels of IFX. Four (66.6%) of the six patients with undetectable levels of IFX had positive ATI levels; three of these patients were in remission, and one had active disease. In addition, the other two patients with undetectable levels of IFX presented ATI levels close to positivity (2.7 and 2.8 AU/ml). None of the patients with therapeutic or supratherapeutic IFX levels had positive ATI levels. CONCLUSIONS: The undetectable levels of IFX correlated with the detection of ATIs, which was independent of disease activity. Immunogenicity was not the main factor for the loss of response to IFX in our study, and the majority of patients in both groups (CDA and CDR) had supratherapeutic levels of IFX.

Serum Levels of Infliximab and Anti-Infliximab Antibodies in Brazilian Patients with Crohn's Disease

OBJECTIVES: The aim of this study was to evaluate the quantitative serum level of infliximab (IFX) as well as the detection of anti-infliximab antibodies (ATIs) in patients with Crohn's disease (CD). METHOD: Forty patients with CD under treatment at a tertiary center in southeastern Brazil were evaluated. Their use of infliximab was continuous and regular. We analyzed and compared the differences in the IFX and ATI levels between the patients with active CD (CDA) and those with CD in remission (CDR). RESULTS: There was no difference in the IFX level between the CDA and CDR groups (p>0.05). Eighty percent of all patients had IFX levels above the therapeutic concentration (6-10 μg/mL). Two (9%) of the 22 patients with active disease and four (22.2%) of the 18 patients in remission had undetectable levels of IFX. Four (66.6%) of the six patients with undetectable levels of IFX had positive ATI levels; three of these patients were in remission, and one had active disease. In addition, the other two patients with undetectable levels of IFX presented ATI levels close to positivity (2.7 and 2.8 AU/ml). None of the patients with therapeutic or supratherapeutic IFX levels had positive ATI levels. CONCLUSIONS: The undetectable levels of IFX correlated with the detection of ATIs, which was independent of disease activity. Immunogenicity was not the main factor for the loss of response to IFX in our study, and the majority of patients in both groups (CDA and CDR) had supratherapeutic levels of IFX.

Ileal pouch of ulcerative colitis and familial adenomatous polyposis patients exhibit modulation of autophagy markers.

Total retocolectomy with ileal pouch-anal anastomosis (IPAA) is the surgery of choice for patients with ulcerative colitis (UC) that are refractory to clinical treatment. Pouchitis is one of the most common complications after this procedure. Defects in autophagy have been reported in inflammatory bowel diseases. However, there are no studies on the IP. Therefore, we studied markers for autophagy in the IP mucosa of UC and FAP patients comparing them to controls with a normal distal ileum. Sixteen patients with IP in "J" shape, asymptomatic and with endoscopically normal IP were evaluated. The control group consisted of eight patients with normal colonoscopy. There was a significant decrease in the transcriptional levels of ATG5, MAP1LC3A and BAX in the FAP group. There was also a decrease in the protein level of Beclin-1 in the UC and FAP compared to the control group. Although the LC3II levels by immunoblot were higher in the UC group, LC3/p62 co-localization were lower in the immunofluorescence analysis in the UC and FAP compared to the control group. Corroborating these results, there was an increase of p62 by immunoblot in the UC group. These findings indicated a modulation of macroautophagy markers in the IP, which may explain the mucosa inflammation predisposition.

Transcriptional and Molecular Pathways Activated in Mesenteric Adipose Tissue and Intestinal Mucosa of Crohn's Disease Patients.

Crohn's disease (CD) is a chronic inflammatory disorder, characterized by cytokine imbalance and transcription signaling pathways activation. In addition, the increase of mesenteric adipose tissue (MAT) near the affected intestinal area is a hallmark of CD. Therefore, we evaluated the transcription signaling pathways and cytokines expression in intestinal mucosa and MAT of active CD patients. Ten patients with ileocecal CD and eight with noninflammatory diseases were studied. The biopsies of intestinal mucosa and MAT were snap-frozen and protein expression was determined by immunoblotting. RNA levels were measured by qPCR. The pIkB/IkB ratio and TNFα level were significantly higher in intestinal mucosa of CD when compared to controls. However, STAT1 expression was similar between intestinal mucosa of CD and controls. Considering the MAT, the pIkB/IkB ratio was significantly lower and the anti-inflammatory cytokine IL10 was significantly higher in CD when compared to controls. Finally, the protein content of pSTAT1 was higher in MAT of CD compared to controls. These findings reinforce the predominance of the proinflammatory NF-kB pathway in CD intestinal mucosa. For the first time, we showed the activation of STAT1 pathway in MAT of CD patients, which may help to understand the physiopathology of this immune mediated disease.