Intestinal Inflammation Levels Research Articles

Application of enzyme-linked immunosorbent assay to detect antimicrobial peptides in human intestinal lumen

Intestinal transplantation is the definitive treatment for intestinal failure. However, tissue rejection and graft-versus-host disease are relatively common complications, necessitating aggressive immunosuppression that can itself pose further complications. Tracking intraluminal markers in ileal effluent from standard ileostomies may present a noninvasive and sensitive way to detect developing pathology within the intestinal graft. This would be an improvement compared to current assessments, which are limited by poor sensitivity and specificity, contributing to under or over-immunosuppression, respectively, and by the need for invasive biopsies. Herein, we report an approach to reproducibly analyze ileal fluid obtained through stoma sampling for antimicrobial peptide/protein concentrations, reasoning that these molecules may provide an assessment of intestinal homeostasis and levels of intestinal inflammation over time. Concentrations of lysozyme (LYZ), myeloperoxidase (MPO), calprotectin (S100A8/A9) and β-defensin 2 (DEFB2) were assessed using adaptations of commercially available enzyme-linked immunosorbent assays (ELISAs). The concentration of α-defensin 5 (DEFA5) was assessed using a newly developed sandwich ELISA. Our data support that with proper preparation of ileal effluent specimens, precise and replicable determination of antimicrobial peptide/protein concentrations can be achieved for each of these target molecules via ELISA. This approach may prove to be reliable as a clinically useful assessment of intestinal homeostasis over time for patients with ileostomies.

Proteomic Analysis Identifies Three Reliable Biomarkers of Intestinal Inflammation in the Stools of Patients With Inflammatory Bowel Disease.

Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC]. To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach. Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls. 2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls. We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.

A200 CROHN’S-ASSOCIATED E. COLI WORSEN COLITIS SEVERITY IN MICE COLONIZED WITH DEFINED MICROBIOTA

Abstract Background Bacterial infections have been postulated to drive the dysregulated inflammation found in inflammatory bowel disease (IBD). In particular, adherent-invasive Escherichia coli (AIEC) isolated from patients with IBD have pathobiont characteristics and have been implicated in IBD pathogenesis. Aims Our aim was to characterize and compare the level of intestinal inflammation and potential microbiota shifts induced by E. coli clinical isolates using a gnotobiotic mouse model of colitis. Methods Adult germ-free C57BL/6 mice were transferred to ISO positive cages in a gnotobiotic facility and colonized with altered Schaedler flora-like (ASF) microbiota and one of three clinical E. coli isolates: E. coli C0004 (n = 5), E. coli LF82 (n = 9), E. coli NRG857c (n = 6), or ASF alone (n = 6). Three weeks later, mice were treated for 5 days with low dose dextran sodium sulphate in drinking water (2%; DSS), followed by 2 days of water. Mice were monitored daily for clinical symptoms (weight, stool consistency, and occult blood). At sacrifice, colon tissue was collected for histological analysis. Cecum contents were cultured to determine bacterial load. Fecal samples were collected for 16S rRNA gene sequencing analysis before and after DSS treatment. Results All mice colonized with an E. coli isolate displayed significantly greater clinical and microscopic scores of colitis compared to ASF alone, but the severity was dependent on the colonized E. coli strain. E. coli NRG857c-colonized mice exhibited more severe symptoms (p &amp;lt; 0.001) two days earlier than mice colonized with other E. coli isolates. Mice colonized with E. coli LF82 or E. coli NRG857c had higher histological scores of colitis compared to mice colonized with E. coli C0004, which were also significantly greater than ASF alone (p &amp;lt; 0.0001). 16S rRNA gene sequencing revealed that ASF-alone-colonized mice lacked Proteobacteria. All E. coli-colonized mice had comparable bacterial loads, which were verified by 16S rRNA analysis. Following DSS, E. coli LF82 and E. coli NRG857c relative abundance remained stable, whereas the relative abundance of Roseburia/ASF 492 declined significantly in all E. coli colonized mice. Conclusions The presence of E. coli pathobionts in mice drive the severity of chemically induced colitis, with AIEC NRG857c inducing the greatest severity. This gnotobiotic mouse model enables us to control the severity of colitis in a well-defined microbiota that is dependent on the colonized E. coli isolate. Using this model, we will be able to assess therapeutic candidates that aim to treat colitis at varying stages of its disease progression. Funding Agencies CCC, CIHRNSERC

Characterization of Skeletal Phenotype and Associated Mechanisms With Chronic Intestinal Inflammation in theWinnieMouse Model of Spontaneous Chronic Colitis

Osteoporosis is a common extraintestinal manifestation of inflammatory bowel disease (IBD). However, studies have been scarce, mainly because of the lack of an appropriate animal model of colitis-associated bone loss. In this study, we aimed to decipher skeletal manifestations in the Winnie mouse model of spontaneous chronic colitis, which carries a MUC2 gene mutation and closely replicates ulcerative colitis. In our study, Winnie mice, prior to the colitis onset at 6 weeks old and progression at 14 and 24 weeks old, were compared with age-matched C57BL/6 controls. We studied several possible mechanisms involved in colitis-associated bone loss. We assessed for bone quality (eg, microcomputed tomography [micro-CT], static and dynamic histomorphometry, 3-point bending, and ex vivo bone marrow analysis) and associated mechanisms (eg, electrochemical recordings for gut-derived serotonin levels, real-time polymerase chain reaction [qRT-PCR], double immunofluorescence microscopy, intestinal inflammation levels by lipocalin-2 assay, serum levels of calcium, phosphorus, and vitamin D) from Winnie (6-24 weeks) and age-matched C57BL6 mice. Deterioration in trabecular and cortical bone microarchitecture, reductions in bone formation, mineral apposition rate, bone volume/total volume, osteoid volume/bone surface, and bone strength were observed in Winnie mice compared with controls. Decreased osteoblast and increased osteoclast numbers were prominent in Winnie mice compared with controls. Upregulation of 5-HTR1B gene and increased association of FOXO1 with ATF4 complex were identified as associated mechanisms concomitant to overt inflammation and high levels of gut-derived serotonin in 14-week and 24-week Winnie mice. Skeletal phenotype of the Winnie mouse model of spontaneous chronic colitis closely represents manifestations of IBD-associated osteoporosis/osteopenia. The onset and progression of intestinal inflammation are associated with increased gut-derived serotonin level, increased bone resorption, and decreased bone formation.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

Levels of Intestinal Inflammation and Fibrosis in Resection Specimens after Preoperative Anti-Tumor Necrosis Factor Alpha Treatment in Patients with Crohn's Disease: A Comparative Pilot Study.

Background Strictures are a common complication in Crohn's disease (CD), found in more than 50% of patients. They are characterized by the excessive deposition of extracellular proteins in the tissue as a result of the chronic inflammatory process. The effect of anti-tumor necrosis factor alpha (TNF-α) therapy on the development of fibrosis is not yet fully understood. Aim To investigate whether the degree of intestinal inflammation and fibrosis is correlated with preoperative anti-TNF-α) therapy on the development of fibrosis is not yet fully understood. Methods This unblinded, prospective, single tertiary center, pilot cohort study included all adult patients with CD who underwent elective, laparoscopic, or open intestinal resection. Preoperative investigations included measurement of blood TNF-α) therapy on the development of fibrosis is not yet fully understood. Results Histopathological specimens from 10 patients with CD who underwent ileocecal or ileocolic resections were retrieved. Four of those patients were on anti-TNF-α) therapy on the development of fibrosis is not yet fully understood. α) therapy on the development of fibrosis is not yet fully understood. α) therapy on the development of fibrosis is not yet fully understood. p=0.01). Anti-TNF-α) therapy on the development of fibrosis is not yet fully understood. α) therapy on the development of fibrosis is not yet fully understood. α) therapy on the development of fibrosis is not yet fully understood. Conclusions Patients who underwent preoperative anti-TNF-α treatment had a higher fibrosis score than controls.α) therapy on the development of fibrosis is not yet fully understood.

Molecular mechanisms of interleukin-38 inhibiting inflammatory bowel disease in children by regulating nuclear factor-κB and signal transduction and activator of transcription 3 pathway

Objective To explore the role of interleukin (IL)-38 in inhibiting inflammatory bowel disease (IBD) in children and to investigate the potential molecular mechanisms. Methods From January 2014 to October 2017, 67 patients with ulcerative colitis (UC) and 115 patients with Crohn′s disease (CD) admitted to Wuhan Children′s Hospital were recruited, and 40 individuals with normal endoscopic findings were selected as control. Serum levels of IL-38 of IBD patients and healthy control were determined by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining (IHC) was used to detect the expression level of IL-38, nuclear factor κB (NF-κB), phosphorylated signal transduction and activator of transcription 3 (p-STAT3), C-reaction protein (CRP) and erythrocyte sedimentation rate (ESR) in the intestinal mucosa of IBD patients and healthy controls. The extent of disease, therapeutic agents and disease activity scores (Mayo score system for UC patients, Crohn′s disease activity index (CDAI) for CD patients) were evaluated. IL-38-C57BL/6 transgenic mice model was established, and dextran sulfate sodium was used to induce IBD mice model. The intestinal inflammation levels were compared between the wild type IBD mice and IL-38 transgenic IBD mice. The levels of IL-38, NF-κB and p-STAT3 in intestinal mucosa of mice of different groups were determined by IHC. The ratio of CD4+ IL-17+ T helper (Th) 17 cells in peripheral blood of mice of different groups was detected by flow cytometry. Independent sample t test, chi square test and Pearson correlation were performed for statistical analysis. Results The results of ELISA showed that the serum levels of IL-38 of UC and CD patients were (6.1±1.9) ng/L and (9.8±2.1) ng/L, respectively, which both were lower than that of healthy controls ((16.4±2.7) ng/L), and the differences were statistically significant (t=23.107 and 15.853, both P<0.05). The results of IHC indicated that the levels of IL-38 in the intestinal mucosal tissues of UC and CD patients were 0.04±0.01 and 0.03±0.01, respectively, which were both lower than that of healthy controls (0.18±0.02), and the differences were statistically significant (t=48.186 and 69.443, both P<0.05). The levels of NF-κB and p-STAT3 of UC and CD patients were 0.150±0.030, 0.160±0.040 and 0.130±0.030, 0.110±0.010, which were all higher than those of healthy controls (0.020±0.003 and 0.010±0.002), and the differences were statistically significant (tUC=27.273 and 23.078, tCD=23.657 and 62.684; all P<0.05). The number of patients with disease at active phase, CRP level, ESR and disease activity scores of UC and CD patients with low IL-38 expression were all significantly higher than those of patients with high IL-38 expression (χUC2=11.552, tUC=7.118, 8.991 and 7.086; χCD2=5.675, tCD=9.559, 9.358 and 11.268; all P<0.05). The results of Pearson correlation analysis demonstrated that the level of IL-38 in the intestinal mucosal tissue of UC patients was negatively correlated with CRP, ESR and Mayo scores (r=-0.291, -0.672 and -0.639; all P<0.05). And the level of IL-38 in the intestinal mucosal tissue of CD patients was negatively correlated with CRP, ESR and CDAI (r=-0.559, -0.471 and -0.353; all P<0.05). The IHC results showed that the levels of NF-κB and p-STAT3 of IL-38 transgenic IBD mice were lower than those of wild type IBD mice (0.14±0.02 vs. 0.32±0.06, 0.12±0.02 vs. 0.44±0.07), and the differences were statistically significant (t=6.971 and 10.767, both P<0.05). The results of flow cytometry showed that the ratio of CD4+ IL-17+ Th17 cells in the peripheral blood of IL-38 transgenic IBD mice was lower than that of wild type IBD mice (0.030±0.006 vs. 0.280±0.050), and the difference was statistically significant (t=12.160, P<0.05). Conclusions The expression level of IL-38 significantly decreases in the intestinal mucosal tissues of IBD patients, while the level of NF-κB and p-STAT3 significantly increases. IL-38 may inhibit IBD by regulating NF-κB and p-STAT3 signaling pathway to alleviate intestinal immune reaction. Key words: Inflammatory bowel diseases; NF-kappa B; interleukin-38; STAT3

A randomized double-blind placebo-controlled trial of a multi-strain probiotic in treatment of symptomatic uncomplicated diverticular disease.

Diverticular disease is a significant burden on healthcare systems that is managed, surgically or medically, mainly as an emergency or acute condition. There are no standardized treatment recommendations for symptomatic uncomplicated disease. We hypothesized that a probiotic would reduce abdominal pain in such patients. We conducted a single-center, double-blind, placebo-controlled trial of probiotic treatment (Symprove) in adult patients with moderate-to-severe chronic, non-acute symptomatic diverticular disease. 143 patients were randomized to receive 1mL/kg/day of probiotic liquid (N=72) or placebo (N=71) daily for 3 months. The primary endpoint was abdominal pain severity. Secondary endpoints consisted of the change in the frequency of eight abdominal symptoms and the level of intestinal inflammation (fecal calprotectin). 120 patients completed the trial. Abdominal pain score, the primary end point, decreased in both groups, but no significant difference between the groups was found (P=0.11). In relation to placebo, the probiotic significantly decreased the frequency of four of the eight secondary endpoints: constipation, diarrhea, mucorrhea, and back pain (P<0.04). No significant differences were found in frequency of abdominal pain, PR bleeding, dysuria, and bloating. Multi-strain liquid probiotic did not improve abdominal pain scores significantly, but significantly improved the frequency of four other symptoms associated with chronic, non-acute symptomatic diverticular disease.

Entamoeba histolytica-Encoded Homolog of Macrophage Migration Inhibitory Factor Contributes to Mucosal Inflammation during Amebic Colitis.

Understanding the mechanisms by which Entamoeba histolytica drives gut inflammation is critical for the development of improved preventive and therapeutic strategies. E. histolytica encodes a homolog of the human cytokine macrophage migration inhibitory factor (MIF). Here, we investigated the role of E. histolytica MIF (EhMIF) during infection. We found that the concentration of fecal EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. Mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx, and mucosal damage. Together, these results uncover a specific parasite protein that increases mucosal inflammation, expands our knowledge of host-parasite interaction during amebic colitis, and highlights a potential immunomodulatory target.

Defect in TLR5 Expression Enhances Spontaneous Visceral Hypersensitivity

Irritable Bowel Syndrome (IBS), such as Crohn’s disease (CD), is an intestinal disease that is associated with colonic hypersensitivity and abdominal pain associated with other symptoms (bloating and bowel dysfunction). Moreover, alterations in innate immune function genes and activities have been described in IBS and CD patients. Among them, Toll-Like Receptor 5 (TLR5) is a primordial innate immune receptor, responsible for the primary defense of the organism against microbial intruders. This receptor acts as a sentinel by detecting the presence of potentially virulent invasive bacteria expressing flagellin, the microbial TLR5 ligand. Mice deficient in TLR5 (T5KO) have been described to uniformly exhibit increased levels of inflammatory markers, resulting in a “sub-clinical” inflammation. Consequently, we hypothesized that, depending on the level of intestinal inflammation, T5KO mice will spontaneously develop a visceral hypersensitivity and thus provide a practical means to study mechanisms underlying inflammation and visceral pain. Two distinct strains of T5KO mice (knockout mouse strains on the C57BL/6 background deficient in TLR5 from Drs. Akira and Flavell) were assayed for intestinal inflammation level and visceral hypersensitivity. Inflammation was monitored using clinical anatomical indicators of colitis such as spleen and colon weight, and assessed by determination of the colonic myeloperoxidase (MPO) activity and expression of systemic pro-inflammatory cytokines. In parallel, visceral hypersensitivity was assessed using a technique based on measuring electromyographic abdominal contractions induced by colorectal distension. This test is to induce, by progressive inflation of a balloon inserted into the colon, stomach cramps whose amplitude is proportional to the colon sensitivity. Mice deficient for TLR5 uniformly exhibited increased levels of inflammatory markers and greater visceral hypersensitivity in comparison to their housekeeping WT mice. Direct involvement of TLR5 in colonic hypersensitivity has been demonstrated. Such impact on colonic sensitivity could be related to its role in the modulation of the intestinal microbiota and its role on micro-colonic inflammations. Thus, approaches focused on TLR5-dependent pathway could be used to initiate new pharmacological strategies based on the use of specific molecules modulating the activity of this protein.

Serum infliximab concentrations in pediatric inflammatory bowel disease

Objective. To study serum infliximab (s-IFX) levels in pediatric patients with inflammatory bowel disease (IBD). Subjects and methods. s-IFX trough levels were measured in a total of 133 blood samples of 37 pediatric IBD patients (Crohn's disease, 23): 48 during the induction phase (weeks 2 and 6) and 85 during maintenance treatment. Antibodies to infliximab (ATI) were determined in 93 samples (30 patients). s-IFX values were related to fecal calprotectin (FC) and serum markers of inflammation. Results. During induction (5 mg/kg) and maintenance therapy, the median s-IFX levels were 17.6 μg/ml (range 0–48 μg/ml) and 3.55 μg/ml (range 0–40 μg/ml), respectively. The IFX levels were similar in ulcerative colitis and Crohn's disease (e.g. during maintenance median 3.2 vs. 2.8 μg/ml, p = 0.718), thus the data are pooled. During induction, the s-IFX level was associated with the total dose of IFX, that is, young children with lower body weight had lower levels (p < 0.001 at week 2 and p < 0.05 at week 6). Shorter administration interval resulted in higher trough levels (p < 0.005). All samples with undetectable s-IFX (6.8%) levels presented ATI. High inflammation (FC >1000 µg/g) during induction was associated with lower s-IFX levels (median 4.0 μg/ml, range 0.47–25 compared to median 20 μg/ml, range 0–48 when FC <1000 µg/g, p < 0.005). There was no significant association between the ESR or values of C-reactive protein and s-IFX levels during induction. Conclusions. In pediatric IBD, lower body weight and higher level of intestinal inflammation are associated to s-IFX levels during induction but relation to therapeutic response is unclear.

Correlation of MRI-determined small bowel Crohn’s disease categories with medical response and surgical pathology

To determine whether magnetic resonance imaging (MRI) can be used to categorize small bowel Crohn's disease (SB CD) into groups that correlate with response to medical therapy and surgical pathology. Data was collected from all patients with MRI evidence of SB CD without significant colonic disease over a 32-mo period. Two radiologists, blinded to clinical findings, evaluated each MRI and grouped them based on bowel wall thickness and wall enhancement. These categories were: (1) "fibrosis", (2) "mild segmental hyper-enhancement and mild wall thickening", (3) "mild segmental hyper-enhancement and marked wall thickening", (4) "marked segmental transmural hyper-enhancement". Patient response to additional medical therapy post-MRI was prospectively determined at 8-wk. Non-responders underwent endoscopy and were offered therapeutic endoscopy or surgery. Surgical pathology was assessed against the MRI category. Fifty-five patients were included. Females and category "2" patients were more likely, and patients with luminal narrowing and hold-up less likely, to respond to medical therapy (P < 0.05). Seventeen patients underwent surgery. The surgical pathological findings of fibrosis and the severity of inflammation correlated with the MRI category in all cases. Our findings suggest that SB CD can be grouped by the MRI findings and that these groups are associated with patients more likely to respond to continued medical therapy. The MRI categories also correlated with the presence and level of intestinal inflammation and fibrosis on surgical pathology, and may be of prognostic use in the management of CD patients.

Saponin-Containing Subfractions of Soybean Molasses Induce Enteritis in the Distal Intestine of Atlantic Salmon

The current work aimed at tracing the causative components for soybean-induced enteritis in Atlantic salmon (Salmo salar L.). Soybean molasses was subjected to phase separation using n-butanol. Three subfractions were obtained as follows: butanol phase, precipitate, and water phase. The biochemical composition of soybean molasses and the obtained subfractions were analyzed in detail: Protein, fat, and ash were quantified according to standard methods. Sucrose, raffinose, and stachyose were quantified using high-performance anion-exchange chromatography. Soyasaponins were quantified using reverse-phase high-performance liquid chromatography. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to evaluate the size distribution of the proteins present in each fraction. Molasses and the different subfractions were thereafter fed to Atlantic salmon in two successive fish trials. The level of intestinal inflammation was evaluated by light microscopy using a semiquantitative scoring system. Histological assessments revealed that Atlantic salmon fed a combination of butanol phase and precipitate displayed significant enteritis. Atlantic salmon fed the water phase displayed normal intestinal morphology. The causative components for soybean-induced enteritis withstand butanol treatment and prolonged heating at 70 degrees C. Sucrose, raffinose, stachyose, nor soybean proteins larger than 10 kDa induce enteritis alone. Soyasaponins, or components that follow the same solubility pattern, trigger the inflammatory reaction. We therefore suggest that soybean-induced enteritis in Atlantic salmon is induced by soyasaponins alone or by soyasaponins in combination with other factors, e.g., antigenic soybean proteins or the intestinal microflora.