Rachele Ciccocioppo

Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease

Objective External fistulas represent a disabling manifestation of Crohns disease with a difficult curability and a high relapse rate despite a large therapeutic armamentarium. Stem cell therapy is a novel and promising approach for treatment of chronic inflammatory conditions. We therefore investigated the feasibility, safety and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in the treatment of fistulising Crohns disease. Patients and methods We enrolled 12 consecutive outpatients (eight males, median age 32 years) refractory to or unsuitable for current available therapies. MSCs were isolated from bone marrow and expanded ex vivo to be used for both therapeutic and experimental purposes. Ten patients (two refused) received intrafistular MSC injections (median 4) scheduled every 4 weeks, and were monitored by surgical, MRI and endoscopic evaluation for 12 months afterwards. The feasibility of obtaining at least 50×106 MSCs from each patient, the appearance of adverse events, and the efficacy in terms of fistula healing and reduction of both Crohns disease and perianal disease activity indexes were evaluated. In addition, the percentage of both mucosal and circulating regulatory T cells expressing FoxP3, and the ability of MSCs to influence mucosal T cell apoptosis were investigated. Results MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohns disease and perianal disease activity indexes (p<0.01 for both), and rectal mucosal healing were induced by treatment without any adverse effects. The percentage of mucosal and circulating regulatory T cells significantly increased during the treatment and remained stable until the end of follow up (p<0.0001 and p<0.01, respectively). Furthermore, MSCs have been proven to affect mucosal T cell apoptotic rate. Conclusions Locally injected MSCs represent a feasible, safe and beneficial therapy in refractory fistulising Crohns disease.

The immune recognition of gluten in coeliac disease

Coeliac disease, the most common intestinal disorder of western populations, is an autoimmune enteropathy caused by an abnormal immune response to dietary gluten peptides that occurs in genetically susceptible individuals carrying the HLA‐DQ2 or ‐DQ8 haplotype. Despite the recent progresses in understanding the molecular mechanisms of mucosal lesions, it remains unknown how increased amounts of gluten peptides can enter the intestinal mucosa to initiate the inflammatory cascade. Current knowledge indicates that different gluten peptides are involved in the disease process in a different manner, some fragments being ‘toxic’ and others ‘immunogenic’. Those defined as ‘toxic’ are able to induce mucosal damage either when added in culture to duodenal endoscopic biopsy or when administered in vivo, while those defined as ‘immunogenic’ are able to specifically stimulate HLA‐DQ2‐ or DQ8‐restricted T cell clones isolated from jejunal mucosa or peripheral blood of coeliac patients. These peptides are able to trigger two immunological pathways: one is thought to be a rapid effect on the epithelium that involves the innate immune response and the other represents the adaptive immune response involving CD4+ T cells in the lamina propria that recognize gluten epitopes processed and presented by antigen presenting cells. These findings are the subject of the present review.

Matrix metalloproteinase pattern in celiac duodenal mucosa.

Matrix metalloproteinases (MMPs) are a family of endopeptidases playing a key role in tissue remodelling in both physiological and pathological conditions. Since little information is available about their role in celiac disease (CD), our aims were to quantify their expression/activity and to investigate their relation to proinflammatory cytokines in this condition. Duodenal biopsies from untreated, treated celiac patients and controls were used to quantify the expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, MMP-14, their inhibitor TIMP-1, IFN-γ and TNF-α by using real-time reverse transcription-polymerase chain reaction and the gelatin/casein/elastin activities by gel zymography, and to isolate lamina propria mononuclear cells (LPMCs). These cells and myofibroblasts isolated from jejunal specimens were incubated in the absence or presence of IFN-γ and TNF-α. MMP-1 and MMP-12 mRNA levels were significantly increased in active CD compared to treated (P<0.01 and P<0.0005, respectively) and normal mucosa (P<0.01 and P<0.0005, respectively), and this was paralleled by an upregulation of caseinolytic and elastolytic activities. Furthermore, MMP-12 levels significantly (P<0.05) correlated with those of IFN-γ and the degree of villous flattening. MMP-2 turned out to be significantly (P<0.05) reduced in untreated and treated celiacs compared to controls. In active CD, transcripts of TIMP-1 were higher than in treated and controls (P<0.005 and P<0.05, respectively), such as those of IFN-γ (P<0.05), whereas TNF-α levels were suppressed (P=0.0001). In physiological condition, myofibroblasts represent the main source of MMP-2, whereas LPMCs produce almost all MMPs only after cytokine stimulation. Conversely, cells isolated from active patients constitutively express MMPs without any increase after cytokine stimulation, while those from treated patients are in a resting condition. In conclusion, our results show the presence of a peculiar MMP pattern in active CD strongly dominated by MMP-12, correlating either with IFN-γ or the degree of mucosal damage.

Altered expression, localization, and phosphorylation of epithelial junctional proteins in celiac disease

We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.

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.

Anemia of chronic disease and defective erythropoietin production in patients with celiac disease

Anemia due to hematinic deficiencies is common in paients with untreated celiac disease. This study shows that, in addition to iron and vitamin deficiencies, anemia of chronic disease has a significant role in some patients with celiac disease. See related perspective article on page 1761. Background Anemia due to hematinic deficiencies is common in patients with untreated celiac disease. Although celiac disease is a chronic condition characterized by an intense inflammatory response of the intestinal mucosa, scant data are available about the prevalence of anemia of chronic disease in celiac disease. Design and Methods One hundred and fifty-two patients with celiac disease at presentation were studied. Anemia was investigated by determining complete blood counts, body iron status, serum levels of the soluble transferrin receptor, erythropoietin, prohepcidin and interferon-γ. Genotyping for HFE mutations associated with hereditary hemochromatosis was performed. Fifty-three anemic patients were re-evaluated for hematologic response after 1 year on a gluten-free diet. Results At the time of diagnosis of celiac disease the prevalence of anemia was 34%. Fifty-three out of 65 anemic patients had either iron and/or vitamin deficiency (folate, vitamin B12). Hereditary hemochromatosis mutations did not affect the prevalence of anemia. In 11 cases iron status parameters were indicative of anemia of chronic disease, sometimes in association with iron deficiency (6 patients). Patients with anemia of chronic disease had low levels of erythropoietin for the degree of anemia and increased serum interferon-γ. In most cases anemia improved following a gluten-free diet, response rates being similar in anemia of chronic disease and in anemia due to hematinic deficiencies. Conclusions Our study shows that, in addition to iron and vitamin deficiencies, anemia of chronic disease has a significant role in some patients with celiac disease. Suppression of intestinal inflammatory changes as a result of a gluten-free diet improves anemia by correcting iron and vitamin malabsorption as well as mechanisms contributing to anemia of chronic disease.

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.

Infliximab downregulates basic fibroblast growth factor and vascular endothelial growth factor in Crohn's disease patients

Background : The use of infliximab in the treatment of Crohns disease patients with symptomatic stenosis is controversial.

Doppler enhancement after intravenous levovist injection in Crohn's disease

Although transabdominal bowel sonography (TABS) has been proposed as a reliable tool to assess increased bowel wall thickness (BWT), the most common sonographic pattern in patients with Crohns disease (CD), its accuracy is limited in the diagnosis of CD. We therefore tried to assess whether color Doppler enhancement with Levovist, a galactose-based intravenous sonographic contrast agent able to enhance the arterial Doppler signal, increases TABS accuracy. Thirty-one patients with ileal CD, diagnosed by endoscopy and enteroclysis, and 20 healthy volunteers were examined with conventional TABS. Color Doppler of the intramural enteric vessels was then performed before and after intravenous injection of Levovist. Twenty-two CD patients had a BWT >4 mm, and 16 of them presented with active disease. Two of the remaining nine CD patients, all with BWT <4 mm, presented with active disease. By means of color Doppler we identified six patients with inactive disease, normal BWT, and normal basal Doppler signal intensity, who showed an enhanced Doppler signal in intramural vessels after contrast agent bolus. Four of these patients, identified only by color Doppler after Levovist injection, relapsed within 6 months. In our experience, sensitivity and specificity of TABS, integrated with additional stimulated acoustic emission mode, were 96.7% and 100%, respectively. The use of Levovist in color Doppler increases the accuracy of TABS in CD diagnosis and follow-up.

Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease

BACKGROUND AIMS Because of their capacity to modulate the immune response and promote tissue repair, mesenchymal stromal cells (MSC) represent a potential novel treatment for autoimmune/inflammatory diseases, including Crohns disease (CD). The aim of the study was in vitro characterization of MSC from active CD patients for future clinical application. METHODS MSC from the bone marrow (BM) of seven CD patients (median age 32 years) were expanded ex vivo in the presence of 5% platelet lysate; cells were investigated for clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential, genetic stability and ability to suppress in vitro proliferation of both autologous and allogeneic lymphocytes to polyclonal mitogens. Results were compared with those of BM MSC of four healthy donors (HD). RESULTS MSC were successfully expanded from all patients. Colony-forming unit-fibroblast (CFU-F) frequency and proliferative capacity were comparable in CD and HD MSC. CD MSC showed typical spindle-shaped morphology and differentiated into osteoblasts, adipocytes and chondrocytes. Surface immunologic markers did not differ between CD and HD MSC, with the only exception of sizeable levels of HLA-DR at early culture passages [12-84% at passage (P)1] in the former. CD MSC ceased their growth at variable passages (from P8 to P25) and entered senescence without any change in morphology/proliferation rate. Array-comparative genomic hybridization demonstrated that CD MSC do not show imbalanced chromosomal rearrangements. Both CD and HD MSC inhibited in vitro proliferation of lymphocytes to mitogens. CONCLUSIONS CD MSC show biologic characteristics similar to HD MSC and can be considered for anti-inflammatory and reparative cell therapy approaches in patients with refractory disease.