P. Cazzola

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Oral butyrate for mildly to moderately active Crohn's disease

Background : Butyrate exerts anti‐inflammatory effects in experimental colitis and on Crohns disease lamina propria mononuclear cells in vitro.

Prevalence and pathogenesis of anemia in inflammatory bowel disease. Influence of anti-tumor necrosis factor-α treatment

Background Anemia is a common complication of inflammatory bowel disease, but its epidemiology may be changing due to earlier diagnosis and improved treatments. We investigated the prevalence and pathogenesis of anemia in patients with inflammatory bowel disease. Design and Methods In a cross-sectional study 263 out-patients with inflammatory bowel disease (165 with Crohn’s disease, 98 with ulcerative colitis) were investigated. The influence of time from diagnosis, disease activity, inflammation and the status of iron and hematinic vitamins on the level of hemoglobin and prevalence of anemia were evaluated. In a second group of 27 patients with Crohn’s disease, undergoing anti-tumor necrosis factor-α treatment with infliximab because of refractory or fistulizing disease, we determined the effects of infliximab on disease activity, hemoglobin, serum erythropoietin levels, iron status and inflammation. Results In all, 104 of the 263 patients with inflammatory bowel disease were anemic. Age, gender and azathioprine treatment had no influence on anemia. The prevalence of anemia was highest at diagnosis (65%), decreased during the first 4 years after disease onset, and was stable thereafter. Active disease was associated with higher rates of anemia. At diagnosis most anemic patients had anemia of chronic disease; during follow-up iron deficiency and multifactorial forms of anemia became more prevalent. Eighteen of 27 patients undergoing treatment with infliximab were anemic; most of them had anemia of chronic disease. Infliximab reduced disease activity and improved anemia in 12 patients. This was mediated by an increased production of erythropoietin for the degree of anemia. In vitro infliximab increased the growth of erythroid progenitors from the peripheral blood of patients with active disease. Conclusions Anemia is a common problem in out-patients with inflammatory bowel disease; the prevalence and severity of anemia are related to the activity of the bowel disorder. The pathogenesis of anemia changes during the course of the disease, with anemia of chronic disease having a major role at diagnosis and iron deficiency and multifactorial forms of anemia during follow-up. In patients requiring anti-tumor necrosis factor-α treatment, response to therapy improves erythropoiesis.

Serum bFGF and VEGF correlate respectively with bowel wall thickness and intramural blood flow in Crohn's disease

Serum levels of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)—two factors known to promote tissue repair, fibroblast proliferation, and angiogenesis—were measured in Crohn’s disease patients and correlated with bowel wall thickness (BWT), measured by conventional grey scale ultrasonography, and with the ileal intramural vessel flow, measured by contrast-enhanced color Doppler imaging. Serum samples were obtained from 25 patients with active Crohn’s disease and 22 healthy volunteers, all sex- and age-matched. Serum bFGF and VEGF levels were measured by ELISA assay. All the patients were examined with conventional transabdominal bowel sonography. Color Doppler of the intramural enteric vessels was then performed after the intravenous injection of Levovist, a galactose-based sonographic contrast agent. In Crohn’s disease patients, serum bFGF and VEGF were significantly higher compared with healthy volunteers. A positive correlation between serum bFGF and BWT and between serum VEGF and color Doppler signal intensity was found. The raised serum bFGF levels in Crohn’s disease patients with intestinal strictures compared with patients with other phenotypes (fistulizing, inflammatory), together with the correlation observed between serum bFGF and BWT, suggests a possible involvement of bFGF in the process of transmural fibrogenesis in Crohn’s disease. The higher levels of VEGF in those patients with increased intramural blood flow suggests that VEGF may be considered a marker of angiogenesis in this condition.

Depletion of immunoglobulin M memory B cells is associated with splenic hypofunction in inflammatory bowel disease.

OBJECTIVES:IgM memory B cells that are responsible for the protection against infections by encapsulated bacteria, require the spleen for their generation and/or survival. Since the association between inflammatory bowel disease and functional hyposplenism is well described, our aim was to verify whether IgM memory B cells mirror the reduced splenic function in Crohns disease and ulcerative colitis patients.METHODS:Peripheral blood samples were obtained from 32 Crohns disease and 29 ulcerative colitis patients, 33 healthy controls, and 27 splenectomized patients. Perendoscopic intestinal biopsies were also collected from 15 of 32 Crohns disease patients, 14 of 29 ulcerative colitis patients and 13 of 33 control subjects. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function and flow cytometry was performed to analyze both peripheral and mucosal B cells.RESULTS:Twelve of 32 Crohns disease patients and 13 of 29 ulcerative colitis patients had pitted red cell values >4% and were considered to be hyposplenic. In inflammatory bowel disease patients circulating IgM memory B cells were significantly lower than in control subjects. We observed a significant inverse correlation between the frequency of circulating IgM memory B cell and the pitted red cell values in inflammatory bowel disease patients with hyposplenism. To exclude the possibility that the reduction of circulating IgM memory B cells reflected their recruitment in the inflamed bowel mucosa, lamina propria B-cell populations were also characterized. We found that the frequency of IgM memory B cells was similar in the blood and in the lamina propria of the same patient.CONCLUSIONS:Our findings show that peripheral IgM memory B cells are reduced in inflammatory bowel disease patients and this defect seems to be related to the impairment of splenic function.

Splenic function and IgM-memory B cells in Crohn's disease patients treated with infliximab

Background: Under experimental chronic inflammation, tumor necrosis factor (TNF)‐&agr; plays a role in damaging spleen marginal zone. This latter has a crucial function in mounting B cell‐dependent immune responses against infections by encapsulated bacteria. In Crohns disease (CD), a chronic inflammatory disorder where TNF‐&agr; is centrally involved, impaired splenic function may increase the susceptibility to bacterial infections. On this basis, we aimed to investigate the influence of anti‐TNF therapy on splenic function in CD patients. Methods: Peripheral blood samples were obtained from 15 CD patients before and after treatment with infliximab administered at weeks 0, 2, and 6 at a dose of 5 mg/kg. Counting of erythrocytes with membrane abnormalities (pitted red cells) was used as an indicator of splenic function. Multicolor flow cytometry was performed to analyze circulating B cells. Results: A substantial clinical improvement in 10 of the 15 CD patients was associated with a significant reduction of pitted red cells (from median 6.0% to 3.6%; P < 0.01) after 10 weeks of treatment. In responder patients the improvement of splenic function was accompanied by a parallel increase of circulating IgM‐memory B cells (from median 6.9% to 13.3%; P < 0.005). Splenic function was not ameliorated in nonresponder patients. Conclusions: Splenic function improved in CD patients who responded to infliximab and was accompanied by a concomitant restoration of the IgM‐memory B cell pool responsible for the protection against encapsulated bacteria. Restoration of splenic function after infliximab treatment is intriguing and requires further investigation.

Increased CD8+ intraepithelial lymphocyte infiltration and reduced surface area to volume ratio in the duodenum of patients with ulcerative colitis.

Abstract Objective. Recent evidence suggests the involvement of the upper gastrointestinal tract in ulcerative colitis (UC). By conducting a prospective controlled study, we explored the immunological abnormalities in the duodenal mucosa of UC patients. Methods. Duodenal and colonic biopsies were collected from 24 corticosteroid-free UC patients and 21 controls. Colonization by Helicobacter pylori and positivity for anti-endomysial antibodies was an exclusion criteria. The severity of duodenal and colonic inflammation was determined by endoscopic and histologic scores. Morphometry was performed to measure the surface area to volume ratio (SV). Duodenal CD3+ and CD8+ intraepithelial lymphocytes (IELs) and lamina propria mononuclear cells (LPMCs) were detected by immunohistochemistry. Results. Fifteen UC patients and 14 controls were Helicobacter pylori and anti-endomysial antibody negative and were thus included in the study. Microscopic duodenitis was reported in 4 of the 15 UC patients (26.6%), and in none of the controls. A significantly higher number of CD3+ and CD8+ IELs and LPMCs was found in UC patients than in controls. A significant positive correlation between the percentage of both CD3+ and CD8+ IELs and disease activity was found in UC patients. SV was significantly reduced in UC patients compared to controls, and inversely correlated with the percentage of CD8+ IELs. Conclusions. The duodenum of UC patients is infiltrated by a higher number of CD8+ IELs which correlates with the degree of villous flattening and disease activity, but not with extent of the colonic lesions. Further studies are needed to clarify whether the duodenum is a target organ in UC.

Splenic Hypofunction in Whipple's Disease

and carcinoids (1) . EUS showing hypoechoic SETs arising from the second echolayer (muscularis mucosa) or from the fourth echolayer (muscularis propria) can represent mesenchymal tumors like GIST (2) . Among these, large (>3 cm) hypoechoic masses with inhomogeneous echogenicity, cystic spaces, and irregular margins that invade into other layers or structures on EUS examinations are more likely to represent malignant processes (3,4) . In addition, a correct recognition among these lesions is very important as all GISTs are considered to have malignant potential, and surgical resection is usually recommended irrespective of symptoms (5) . However, a preoperative pathological diagnosis of GIST or leiomyomas by immunohistochemistry studies on EUS-fi ne needle aspiration-obtained cytology samples has low diagnostic yield, specially for small ( < 3 cm) tumors (6) . Our case represents a very rare occurrence of two SETs arising from diff erent anatomical locations and diff erent wall layers, but both suspicious for mesenchymal tumors, which seems quite unusual. To the best of our knowledge, we have not found any reports of multifocal mesenchymal lesions. As the symptomatic lesion in esophagus causing dysphagia was not amenable to endoscopic resection given deep location, surgery was elected. Surgery proved both lesions to be benign leiomyomas.

Efficacy of butyrate in the treatment of mild to moderate Crohn's disease

Abstract Background Butyrate exerts anti-inflammatory effects in experimental colitis and in lamina propria mononuclear cells of patients with Crohns disease. Aims To assess the safety and efficacy of butyrate in Crohns disease. Patients Thirteen patients with mild to moderate Crohns disease were treated with enteric-release sodium butyrate tablets at a dosage of 4 g/day for eight weeks. Methods Before and after treatment, patients underwent coloscopy with evaluation of the clinical activity of their disease, systemic inflammation index and mucosal expression of interleukin (IL)-1β, IL-6, IL-12, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and nuclear factor (NF)-κB. Results Of the nine patients (69%) responding to treatment, 7 (53%) exhibited complete response and 2 exhibited partial response. Endoscopic and histological evaluation scores were significantly improved ( p p Conclusions Oral administration of butyrate may be effective in inducing clinical improvement or remission in patients with Crohns disease.