Gut microbiota, metabolome and immune signatures in patients with uncomplicated diverticular disease.

OBJECTIVE: The engagement of the gut microbiota in the development of symptoms and complications of diverticular disease has been frequently hypothesised. Our aim was to explore colonic immunocytes, gut microbiota and the metabolome in patients with diverticular disease in a descriptive, cross-sectional, pilot study. DESIGN: Following colonoscopy with biopsy and questionnaire phenotyping, patients were classified into diverticulosis or symptomatic uncomplicated diverticular disease; asymptomatic subjects served as controls. Mucosal immunocytes, in the diverticular region and in unaffected sites, were quantified with immunohistochemistry. Mucosa and faecal microbiota were analysed by the phylogenetic platform high taxonomic fingerprint (HTF)-Microbi.Array, while the metabolome was assessed by 1H nuclear magnetic resonance. RESULTS: Compared with controls, patients with diverticula, regardless of symptoms, had a >70% increase in colonic macrophages. Their faecal microbiota showed depletion of Clostridium cluster IV. Clostridium cluster IX, Fusobacterium and Lactobacillaceae were reduced in symptomatic versus asymptomatic patients. A negative correlation was found between macrophages and mucosal Clostridium cluster IV and Akkermansia. Urinary and faecal metabolome changes in diverticular disease involved the hippurate and kynurenine pathways. Six urinary molecules allowed to discriminate diverticular disease and control groups with >95% accuracy. CONCLUSIONS: Patients with colonic diverticular disease show depletion of microbiota members with anti-inflammatory activity associated with mucosal macrophage infiltration. Metabolome profiles were linked to inflammatory pathways and gut neuromotor dysfunction and showed the ability to discriminate diverticular subgroups and controls. These data pave the way for further large-scale studies specifically aimed at identifying microbiota signatures with a potential diagnostic value in patients with diverticular disease.

Moderate to severe and prolonged left lower-abdominal pain is the best symptom characterizing symptomatic uncomplicated diverticular disease of the colon: a comparison with fecal calprotectin in clinical setting.

BACKGROUND: Left lower-abdominal pain is considered the best symptom to differentiate between symptomatic uncomplicated diverticular disease (SUDD) and irritable bowel syndrome (IBS). However, this statement has not been validated yet. GOALS: The aim of this study was to assess whether prolonged left lower-quadrant pain is the best symptom characterizing SUDD and be able to differentiate SUDD from IBS-like symptoms in diverticulosis, and to compare the location of abdominal pain with fecal calprotectin (FC) expression. STUDY: Seventy-two patients suffering from abdominal pain and having diverticula at colonoscopy were enrolled. Patients were classified according to SUDD definition (abdominal pain for at least 24 consecutive hours in left lower abdomen) (42 patients) and IBS-like symptoms fulfilling Rome III criteria (30 patients). Abdominal pain was assessed using a 10-point visual scale, assigning numerical values from 0 (absence of pain) to 10 (severe pain). FC expression was assessed by a rapid test in all patients enrolled. RESULTS: FC test was positive in 27 (64.3%) patients in the SUDD group and in no patient in the IBS-like group (P<0.0001). In patients with SUDD, there was a significant correlation between the severity of the abdominal pain and the FC score (P=0.0015). Extension of diverticulosis correlated with FC score (P=0.022) and the severity of diverticulosis (P=0.005). CONCLUSIONS: Severe and prolonged left lower-abdominal pain seems to be the best symptom characterizing SUDD, and it can differentiate these patients from those harboring diverticula but suffering from IBS-like according to Rome III criteria.

From diverticulosis to complicated diverticular disease: Progression of myogenic alterations and oxidative imbalance.

BACKGROUND: The natural history and pathophysiology of diverticular disease (DD) are still uncertain. An ex-vivo human complicated DD (cDD) model has recently shown a predominant transmural oxidative imbalance. The present study aims to evaluate whether the previously described alterations may precede the symptomatic form of the disease. METHODS: Colonic surgical samples obtained from patients with asymptomatic diverticulosis (DIV), complicated DD, and controls were systematically and detailed morphologically and molecularly analyzed. Therefore, histologic, histomorphometric, immunohistochemical evaluation, and gene and protein expression analysis were performed to characterize colonic muscle changes and evaluate chronic inflammation, oxidative imbalance, and hypoxia. Functional muscle activity was tested on strips and isolated cells in response to contractile and relaxant agents. KEY RESULTS: Compared with controls, DD showed a marketed increase in muscle layer thickness, smooth muscle cell syncytium disarray, and increased interstitial fibrosis; moreover, the observed features were more evident in the cDD group. These changes mainly affected longitudinal muscle and were associated with altered contraction-relaxation dynamics and fibrogenic switch of smooth muscle cells. Chronic lymphoplasmacytic inflammation was primarily evident in the mucosa and spared the muscle. A transmural increase in carbonylated and nitrated proteins, with loss of antioxidant molecules, characterized both stages of DD, suggesting early oxidative stress probably triggered by recurrent ischemic events, more pronounced in cDD, where HIF-1 was detected in both muscle and mucosa. CONCLUSION & INFERENCES: The different DD clinical scenarios are part of a progressive process, with oxidative imbalance representing a new target in the management of DD.

Musosal tumour necrosis factor α in diverticular disease of the colon is overexpressed with disease severity.

AIM: Inflammation occurs in diverticular disease (DD), but there is little information on inflammatory cytokines such as tumour necrosis factor α (TNF-α). The aim of this study was to assess TNF-α expression in DD and to see whether it is related to the severity of the disease. METHOD: Twenty-four patients with symptomatic DD were divided into those with acute uncomplicated diverticulitis (AUD) (12 patients) and those with symptomatic uncomplicated diverticular disease (SUDD) (12 patients). Twelve further patients with asymptomatic diverticulosis (AD), six with segmental colitis associated with diverticulosis (SCAD), with ulcerative colitis (UC) and six healthy individuals (HC) were enrolled as controls. TNF-α expression in the colonic mucosa was assessed by the amount of mRNA codifying for the synthesis of TNF-α. RESULTS: TNF-α expression was significantly higher in AUD than in HC (P=0.0007), in AD (P=0.0001) and in SUDD (P=0.0179). It was significantly higher also in SUDD than in HC (P=0.0007) and in AD (P=0.0001). TNF-α expression in AUD did not differ significantly from that in UC (P=0.0678) and SCAD (P=0.0610). It was significantly higher in UC, SCAD and AUD than in SUDD (P=0.0007, P=0.0001, P=0.0179). CONCLUSION: TNF-α expression in DD seems to be related to the severity of the disease. In particular, it appears to be overexpressed in DD with inflammation (AUD and SUDD) compared with DD without (AD).

Absence of mucosal inflammation in uncomplicated diverticular disease.

BACKGROUND: Uncomplicated diverticular disease is a common condition in patients older than 50 years. Symptoms are aspecific and overlapping with those of irritable bowel syndrome. Nowadays, patients are often treated with antinflammatory drugs (5-aminosalicilic acid). AIM: Our purpose was to evaluate the presence of inflammation in the colonic mucosa of patients with symptomatic uncomplicated diverticular disease compared with subjects without diverticula. METHODS: Endoscopic biopsies of colon from 10 patients with symptomatic uncomplicated diverticular disease and 10 from subjects without diverticula (controls) were taken. Specimens were homogenised and IL2, IL4, IL5, IL8, IL10, IL12p70, IL13, IFN gamma, TNF alfa (searchlight multiplex technique), TGF beta, transglutaminase type 2 and caspase 9 were measured. Histochemistry for transglutaminase type 2 and TUNEL were performed on the histological sections, in addition to morphologic evaluation, as markers of tissue remodelling and apoptosis. For statistical analysis Student's t test and Spearman correlation test were used. RESULTS: No histological differences were detected between the patients with an uncomplicated diverticular disease and controls. Mean values of mucosal cytokines and of the other tested parameters did not show statistically significant differences between patients with uncomplicated diverticular disease and controls. CONCLUSIONS: Even if based on a small number of patients, the study demonstrates the absence of inflammation in the mucosa of subjects affected by uncomplicated diverticular disease.

Alterations in enteric calcitonin gene-related peptide in patients with colonic diverticular disease: CGRP in diverticular disease.

Diverticular disease (DD) is one of the most prevalent diseases of the large bowel. Lately, imbalance of neuro-muscular transmission has been recognized as a major etiological factor for DD. Neuronal calcitonin gene-related peptide (CGRP) is a potent gastrointestinal smooth muscle relaxant shown to have a widespread effect within the alimentary tract. Nevertheless, CGRPergic innervation of the enteric ganglia has never been considered in the context of motility impairment observed in DD patients. Changes in CGRP and calcitonin receptor-like receptor (CRLR) abundance within enteric ganglia were investigated in sigmoid samples from symptomatic and asymptomatic DD patients using quantitative fluorescence microscopy. CGRP effect on gastrointestinal smooth muscle was investigated using organ bath technique. We found CGRP levels within the enteric ganglia to be declined by up to 52% in symptomatic DD patients. Conversely, CRLR within the enteric ganglia was upregulated by 41% in symptomatic DD. Longitudinal smooth muscle displayed an elevated (+10.5%) relaxant effect to the exogenous application of CGRP in colonic strips from symptomatic DD patients. Samples from asymptomatic DD patients consistently showed intermediate values across different experiments. In conclusion, the present study demonstrates that CGRPergic signaling is subject to alteration in DD. Our results suggest that a hypersensitization mechanism to gradually decreasing levels of CGRP-IR nerve fibers takes place during DD progression. Alterations to CGRPergic signaling in DD disease may have implications for physiological abnormalities associated with colonic DD.

Interstitial cells of Cajal, enteric nerves, and glial cells in colonic diverticular disease.

BACKGROUND: Colonic diverticular disease (diverticulosis) is a common disorder in Western countries. Although its pathogenesis is probably multifactorial, motor abnormalities of the large bowel are thought to play an important role. However, little is known about the basic mechanism that may underlie abnormal colon motility in diverticulosis. AIMS: To investigate the interstitial cells of Cajal (the gut pacemaker cells), together with myenteric and submucosal ganglion and glial cells, in patients with diverticulosis. PATIENTS: Full thickness colonic samples were obtained from 39 patients undergoing surgery for diverticulosis. Specimens from tumour free areas of the colon in 10 age matched subjects undergoing surgery for colorectal cancer served as controls. METHODS: Interstitial cells of Cajal were assessed using anti-Kit antibodies; submucosal and myenteric plexus neurones and glial cells were assessed by means of anti-PGP 9.5 and anti-S-100 monoclonal antibodies, respectively. RESULTS: Patients with diverticulosis had normal numbers of myenteric and submucosal plexus neurones compared with controls (p = 0.103 and p = 0.516, respectively). All subtypes of interstitial cells of Cajal were significantly (p = 0.0003) reduced compared with controls, as were glial cells (p = 0.0041). CONCLUSIONS: Interstitial cells of Cajal and glial cells are decreased in colonic diverticular disease, whereas enteric neurones appear to be normally represented. This finding might explain some of the large bowel motor abnormalities reported to occur in this condition.

Visceral hypersensitivity in symptomatic diverticular disease and the role of neuropeptides and low grade inflammation.

BACKGROUND: Recurrent abdominal pain is reported by a third of patients with diverticulosis, particularly those with previous episodes of acute diverticulitis. The current understanding of the etiology of this pain is poor. Our aim was to assess visceral sensitivity in patients with diverticular disease and its association with markers of previous inflammation and neuropeptides. METHODS: Patients with asymptomatic and symptomatic diverticular disease underwent a flexible sigmoidoscopy and biopsy followed 5-10 days later by visceral sensitivity testing with barostat-mediated rectal distension. Inflammation was assessed by staining of serotonin (5HT) and CD3 positive cells. mRNA levels of tumor necrosis factor alpha (TNF α) and interleukin-6 (IL-6) were quantitated using RT-PCR. Neuropeptide expression was assessed from percentage area staining with substance P (SP) and mRNA levels of the neurokinin 1 & 2 receptors (NK1 & NK2), and galanin 1 receptor (GALR1). KEY RESULTS: Thirteen asymptomatic and 12 symptomatic patients were recruited. The symptomatic patients had a lower first reported threshold to pain (28.4 mmHg i.q.r 25.0-36.0) than the asymptomatic patients (47 mmHg i.q.r 36.0-52.5, P < 0.001). Symptomatic patients had a higher median overall pain rating for the stimuli than the asymptomatic patients (P < 0.02). Symptomatic patients had greater median relative expression of NK1 and TNF alpha mRNA compared with asymptomatic patients. There was a significant correlation between barostat VAS pain scores and NK 1 expression (Figure 4, r(2) 0.54, P < 0.02). CONCLUSIONS & INFERENCES: Patients with symptomatic diverticular disease exhibit visceral hypersensitivity, and this may be mediated by ongoing low grade inflammation and upregulation of tachykinins.

Mucosal expression of basic fibroblastic growth factor, Syndecan 1 and tumor necrosis factor-alpha in diverticular disease of the colon: a case-control study.

BACKGROUND: Inflammation may be detected in diverticular disease (DD), and fibrosis may also develop. We assessed the mucosal expression of bFGF, SD1, and TNF-α in DD according to the severity of the disease. Moreover, we assessed the response to therapy of these cytokines in acute uncomplicated diverticulitis (AUD). METHODS: Fifteen patients affected by AUD and seven patients affected by symptomatic uncomplicated diverticular disease (SUDD) were enrolled. Patients with asymptomatic diverticulosis (AD), segmental colitis associated with diverticulosis (SCAD), ulcerative colitis (UC), and healthy subjects (HC) served as control groups. KEY RESULTS: The expression of bFGF, SD1, and TNF-α was significantly higher in diverticulitis than in healthy controls, in diverticulosis, and in uncomplicated diverticular disease. Cytokines were significantly higher in uncomplicated diverticular disease than in healthy controls. Cytokine expression in diverticulitis did not differ significantly from that of ulcerative colitis. After treatment, TNF-α expression dropped significantly. CONCLUSIONS & INFERENCES: Mucosal TNF-α is overexpressed only in symptomatic DD, while SD1 and bFGF are already overexpressed in AD. Finally, TNF-α but not SD1 or bFGF expression seems to be influenced by the treatment in AUD.

Tumour necrosis factor-alpha expression in segmental colitis associated with diverticulosis is related to the severity of the endoscopic damage.

BACKGROUND: Tumour necrosis factor-α expression may be increased in segmental colitis associated with diverticulosis. AIMS: To assess tumour necrosis factor-α expression in segmental colitis associated with diverticulosis in relation with the severity of the endoscopic damage. METHODS: 21 patients affected by segmental colitis associated with diverticulosis were studied (15 M, 6 F, mean age 58.87 years, range 43-85 years). Segmental colitis associated with diverticulosis was graduated as mild-moderate (patterns A and C) and severe (patterns B and D). Ten patients with moderate-to-severe ulcerative colitis, 10 patients with moderate-to-severe Crohn's disease, and 10 patients with irritable bowel syndrome served as control groups. RESULTS: Tumour necrosis factor-α expression was significantly higher in segmental colitis associated with diverticulosis B (42.7%) and segmental colitis associated with diverticulosis D (40%) than in segmental colitis associated with diverticulosis A (19.1%) and segmental colitis associated with diverticulosis C (21.1%).Tumour necrosis factor-α expression was lower in segmental colitis associated with diverticulosis A and C than in ulcerative colitis and Crohn's disease, whilst no different tumour necrosis factor-α expression was found between segmental colitis associated with diverticulosis B and D and both ulcerative colitis and Crohn's disease.Finally, tumour necrosis factor-α expression was significantly lower in irritable bowel syndrome (8%±4) than in every type of segmental colitis associated with diverticulosis. CONCLUSIONS: Tumour necrosis factor-α expression in segmental colitis associated with diverticulosis seems to be related to the severity of the endoscopic damage. This behaviour, similar to that of the inflammatory bowel diseases (IBD), confirms that this disease should be considered as a subtype of IBD.

Appearance of epithelial and stromal genomic instability in background colorectal mucosa of sporadic colorectal cancer patients: relation to age and gender.

BACKGROUND: We have previously demonstrated that not only epithelial but also stromal genetic instability possibly contributes to colorectal tumorigenesis. To assess the increasing risk of carcinogenesis in the colorectum with aging, we examined genomic instability in both epithelia and stroma in the background noncancerous mucosa of patients with colorectal carcinomas. METHODS: In 213 noncancerous colorectal mucosa samples from colorectal cancer cases and 51 normal mucosa specimens of diverticulosis cases, epithelial and stromal genomic instability was analyzed with National Cancer Institute standard microsatellite markers, chromosome 17 (Chr.17) markers and tumor suppressor gene-related markers, using a combination of laser-capture microdissection and GeneScan approaches. Results were compared with immunohistochemically demonstrated expression of FHIT, Rb, WT1, hMLH1 and hMSH2. RESULTS: Genomic instability (MSI and LOH) in both epithelia and stroma appeared after around 40 years of age and remained relatively constant thereafter at relatively low frequencies (4.8-30.4%). The Epithelial LOH tended to show a stepwise increase in people in their 40s and 50s along with aging, especially in males. Overall frequencies of both epithelial MSI and LOH in left-side colon and LOH in right-side colon were significantly higher in males than in females. Epithelial hMLH1 expression in MSI (-) cases tended to be reduced with aging. CONCLUSIONS: Genomic instability of both MSI and LOH in noncancerous colonic mucosa, and more particularly epithelial and stromal LOH, appears relatively early in adults, suggesting age-related changes which increase the risk of cancer development, particularly in males.

Faecal calprotectin in colonic diverticular disease: a case-control study.

BACKGROUND AND AIMS: Information about faecal calprotectin (FC) in colonic diverticular disease (DD) are lacking. We assessed FC in colonic DD, comparing it with irritable bowel syndrome (IBS) patients and healthy controls. Moreover, we compared FC levels in different degrees of DD and assessed FC in symptomatic DD before and after treatment. MATERIALS AND METHODS: Forty-eight consecutive patients with a new endoscopic diagnosis of DD (16 with asymptomatic diverticulosis, 16 with symptomatic uncomplicated DD, 16 with acute uncomplicated diverticulitis), 16 healthy controls, and 16 IBS patients were studied. FC was assessed by semi-quantitative method and compared with histological inflammation. Moreover, FC was reassessed in symptomatic DD after 8 weeks of treatment. RESULTS/FINDINGS: FC was not increased in healthy controls and IBS patients. No difference was found between asymptomatic diverticulosis, healthy controls, and IBS patients (p = n.s.). We found higher FC values in acute uncomplicated diverticulitis (p < 0.0005) and in symptomatic uncomplicated DD (p < 0.005) than in healthy controls and in IBS patients. FC values correlated with inflammatory infiltrate (p < 0.0005). FC decreased after treatment to normal values both in acute uncomplicated diverticulitis (p < 0.0005) and in symptomatic uncomplicated DD (p < 0.005) after treatment. INTERPRETATIONS/CONCLUSIONS: FC may be useful to detect colonic inflammation in DD and in distinguishing symptomatic DD from IBS, as well as in assessing response to therapy in DD.

Different matrix micro-environments in colon cancer and diverticular disease.

BACKGROUND AND AIMS: The extracellular matrix and the interactive signalling between its components are thought to play a pivotal role for tumour development and metastasis formation. An altered matrix composition as potential underlying pathology for the development of colorectal cancer was hypothesized. METHODS: In a retrospective study of patients with colon cancer, the extracellular matrix in tumour-free bowel specimen was investigated in comparison with non-infected bowel specimen from patients operated on for colonic diverticulosis. The following matrix parameters with known associations to tumour formation, cell proliferation, invasion and metastasis were analysed by immunohistochemistry and quantified by a scoring system: VEGF, TGF-beta, ESDN, CD117, c-erb-2, cyclin D1, p53, p27, COX-2, YB-1, collagen I/III, MMP-13, PAI and uPAR. Expression profiles and correlations were calculated. RESULTS: The comparison of the two groups revealed a significantly decreased immunostaining for CD117 and TGF-beta in the cancer group (8.5+/-2.6 vs 10.3+/-2,1 and 4.9+/-1.5 vs 8.1+/-3, respectively), whereas PAI scores were significantly higher than in patients with diverticular disease (8.1+/-1.6 vs 6.2+/-0.9). Overall correlation patterns of matrix parameters indicated pronounced differences between tumour-free tissue in cancer patients compared with patients with diverticular disease. CONCLUSIONS: Our results indicate distinct differences in the colonic tissue architecture between cancer patients and patients with diverticulitis that support the notion of an altered matrix composition predisposing to the development of colon cancer.

Case closed? Diverticulitis: epidemiology and fiber.

A major advance in understanding diverticular disease occurred decades ago with the epidemiologic association between fiber intake and the development of diverticular disease. This association has been well documented with investigations into the emergence of diverticular disease in underdeveloped countries where the disease had been virtually unknown before the adoption of a westernized diet, low in fiber. The high frequency of right-sided diverticular disease in Asian countries diverges from what is seen in the West. The physiologic effects of insoluble fiber has been well examined as well, increasing bulk and decreasing transit time, with a deficiency contributing to the high pressures implicated in the physiology which leads to diverticular disease. However, at most, 10% to 25% of individuals with diverticular disease will develop diverticulitis. Risk factors for symptomatic diverticular have been increasingly described in recent years with obesity and red meat intake being of particular importance, in addition to age. However, the known factors poorly identify those at increased risk and the predisposing pathophysiology is incompletely understood as well. Insoluble fiber, but not soluble fiber, has been viewed as the principal component which has been deficient in western diets and is the culprit which leads to the establishment of diverticular disease and in turn, diverticulitis. Soluble fiber and its effect on the intestinal flora is proposed as having significant influence on the development of diverticulitis. This understanding, if demonstrated, would have important implications for the primary and secondary prevention of diverticulitis.

Use of mesalazine in diverticular disease.

Diverticular disease includes a spectrum of conditions sharing the underlying pathology of acquired diverticula of the colon: symptomatic uncomplicated diverticular disease, recurrent symptomatic uncomplicated diverticular disease, and complicated diverticular disease. Goals of therapy in diverticular disease should be to improve symptoms and to prevent recurrent attacks in symptomatic uncomplicated diverticular disease, and to prevent the complications of disease such as diverticulitis. Inflammation seems to play a key role in all forms of the disease. This is the rationale for the use of anti-inflammatory drugs such as mesalazine. Inflammation in such diseases seems to be generated by a heightened production of proinflammatory cytokines, reduced anti-inflammatory cytokines, and enhanced intramucosal synthesis of nitric oxide. The mechanisms of action of mesalazine are not yet well understood. It is an anti-inflammatory drug that inhibits factors of the inflammatory cascade (such as cyclooxygenase) and free radicals, and has an intrinsic antioxidant effect. Some recent studies confirm the efficacy of mesalazine in diverticular disease both in relief of symptoms in symptomatic uncomplicated forms and in prevention of recurrence of symptoms and main complications.

Up-regulation of collagen and tissue inhibitors of matrix metalloproteinase in colonic diverticular disease.

PURPOSE: Thickening of the muscularis propria is a key pathologic feature of colonic diverticulosis but its cause is unknown. This study was designed to investigate the role of collagens, matrix metalloproteinases, and tissue inhibitor of metalloproteinases in colonic diverticulosis. METHODS: Collagen content was determined by Sircol Collagen Assay and standard van Gieson staining. Messenger-RNA expression for matrix metalloproteinases and tissue inhibitor of metalloproteinase was analyzed by quantitative competitive reverse transcription polymerase chain reaction. Immunohistochemical staining was performed to localize tissue inhibitor of metalloproteinases in sections. RESULTS: In mucosa and submucosal layer, complicated diverticular disease samples had a higher collagen content than uncomplicated disease, which in turn had higher levels than controls. There was an 18-fold increase in tissue inhibitor of metalloproteinase-1 mRNA, and a threefold increase in tissue inhibitor of metalloproteinase-2 mRNA in complicated diverticulosis compared with controls. In the muscularis propria, the amount of total soluble collagen also was higher in both uncomplicated and complicated diverticulosis samples than in the controls. Tissue inhibitor of metalloproteinase-1 and metalloproteinase-2 mRNA was significantly increased in diverticulosis compared with controls. Macrophage-like and fibroblast-like cells stained strongly positive for tissue inhibitor of metalloproteinases in the submucosa, serosa, and muscularis propria and in areas around the blood vessels. CONCLUSIONS: Colonic diverticulosis is associated with altered collagen content and tissue inhibitor of metalloproteinases expression. These factors may play a role in remodeling the gut wall in this condition.