Disturbances in apoptosis of lamina propria lymphocytes in Crohn's disease

Introduction

The aim of this study was to assess the potential mechanisms providing resistance to apoptosis of lamina propria lymphocytes (LPL) directlyin intestinal tissues from patients with Crohn''s disease (CD).

Material and methods

Fifty CD patients were enrolled in the study. The control group consisted of healthy patients who underwent surveillance colonoscopy after endoscopic polypectomy. Each CD patient underwent colonoscopy with tissue sampling from inflamed areas of the colon with the assessment of immunohistochemical expression of active caspase 3, Fas, tumour necrosis factor receptor 1 (TNFR1), Bcl-2, Bax, CD4 and CD8. This was compared with healthy intestinal mucosa.

Results

The expression of active caspase 3 was significantly lower in LPL in CD (0.4 ±0.3 vs. 2.8 ±1.5; p = 0.0002). A statistically significant increase of CD4 and CD8 positive cells was noted in CD (2.3 ±0.5 vs. 1.2 ±0.2, p < 0.0001; 2.1 ±0.3 vs. 1.1 ±0.3, p < 0.0001, respectively). It was associated with a significant increase of the Bcl-2 (6.7 ±2.7 vs. 2.9 ±0.8; p < 0.0001) and a decrease of the Bax protein expression (3.4 ±2.1 vs. 5.5 ±1.8; p < 0.0001) in CD. The expression of Fas and TNFR1 did not differ between the study groups.

Conclusions

LPL in CD are resistant to apoptosis when compared with physiological conditions. This is probably due to an imbalance in Bcl-2 family proteins. TNFR1-related pathway is probably not involved in disturbances of LPL apoptosis in CD.     

Commensal and probiotic bacteria may prevent NEC by maturing intestinal host defenses

Necrotizing enterocolitis (NEC) is a devastating disease of prematurity with significant morbidity and mortality. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. The presence of probiotic and commensal bacteria within the gut has been shown to mature the intestinal defense system through a variety of mechanisms. We have shown that commensal and probiotic bacteria can promote intestinal host defenses by reducing apoptotic signaling, blocking inflammatory signaling, and maturing barrier function in immature intestinal epithelia. Future studies aimed at elucidating the mechanisms by which probiotic and commensal bacteria exert their effects will be critical to developing effective preventive therapies for NEC.     

Ultrastructure of intestinal lymphatics in Crohn's disease

The fine structure of intestinal lymphatics in four patients with Crohn's disease and in two control subjects is described. Although obstructed lacteals are considered to be of major importance in the pathogenesis of regional enteritis, no detailed electron microscopic studies of lymphatic capillaries in this disease could be found. Even though both open and closed intercellular junctions were observed in the normal intestinal lymphatics, only closed junctions were noted in the mucosal and submucosal lymphatic capillaries in patients with regional enteritis. A heavy accumulation of protein rich lymph at the abluminal surface of lymphatic capillaries was consistently seen. None of the control lymphatics showed a similar alteration. The described fine structural changes indicate a decreased permeability of the lymphatic wall. Reduced lymphatic permeability could be a contributing element in the development of submucosal edema, a major microscopic feature of Crohn's disease.     

Molecular crosstalk of probiotic bacteria with the intestinal immune system: Clinical relevance in the context of inflammatory bowel disease

It is current knowledge that the intestinal microbiota plays a major role in the development and maintenance of intestinal health. Intestinal epithelial cells (IEC) constitute the interface between the gut lumen and the innate and adaptive immune system. To maintain intestinal homeostasis, the organized and diffuse compartments of the gut-associated lymphoid tissue have to process the continuously varying information at the interface between the luminal side and the host. Dysregulated intestinal immune responses towards commensal bacteria are an important factor in the pathogenesis of inflammatory bowel diseases (IBD). In contrast to the colitogenic effects of enteric bacteria, clinical and experimental studies showed that specific probiotic strains are protective in the context of chronic intestinal inflammation. Although the molecular understanding of bacteria-host interaction is improving, the anti-inflammatory mechanisms induced by these probiotic bacteria are just starting to be unraveled. The present review is meant to summarize and discuss the clinical relevance of probiotics, but it also seeks to give an overview about currently known probiotic mechanisms in the context of chronic intestinal inflammation with a focus on IEC.     

Role of intestinal epithelial cells in immune effects mediated by gram-positive probiotic bacteria: involvement of toll-like receptors

The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4(+) T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens.     

Adherence of probiotic bacteria to human intestinal mucus in healthy infants and during rotavirus infection

The concentration of fecal mucin and the adhesion of specific probiotics and their combinations in the intestinal mucus of infants during and after rotavirus diarrhea and in healthy children were determined. Mucus was prepared from fecal samples from 20 infants during and after rotavirus diarrhea and from 10 healthy age-matched children. Mucin concentration was determined, and the adhesion of five probiotics-Lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Lactobacillus paracasei F19, Lactobacillus acidophilus LA5, and Bifidobacterium lactis Bb12-and their combinations was tested in vitro. The mean concentrations of fecal mucin during and after rotavirus diarrhea, 15.2 and 14.1 mg/g, were comparable to that in healthy children, 14.9 mg/g. The adherence of probiotics ranged from 1 to 34% in healthy subjects as indicated for the following strains: L. rhamnosus GG, 34%; B. lactis Bb12, 31%; L. acidophilus LA5, 4%; L. paracasei F19, 3%; and L. casei Shirota, 1% (P = 0.0001). The distinctive pattern of probiotic adherence was not influenced by rotavirus diarrhea. The adhesion of Bb12 in the presence of GG increased from 31 to 39% in healthy infants (P = 0.018) and in episodes of diarrhea increased from 26 to 44% (P = 0.001). Rotavirus diarrhea does not decrease the production of fecal mucin or with respect to the adhesion of probiotic bacteria tested in vitro. Combination of specific probiotic strains may enhance adherence in a synergistic manner. Optimal clinical application of these interactions may offer novel therapeutic guidelines for the treatment and prevention of gastrointestinal infections.     

Flow cytometry of intestinal intraepithelial lymphocytes in celiac disease

This article reviews the multiple uses of flow cytometry in the diagnosis, monitoring and research of celiac disease, the most prevalent chronic autoimmune gastrointestinal disease. The phenotyping of intraepithelial lymphocytes (IELs) is of clinical relevance in the diagnosis of the disease given the characteristic features of elevated CD3+ IELs (αβ and γδ TcR) and the decrease in CD3− IELs. IEL biomarkers are also useful in the assessment of the response to the gluten-free diet and, importantly, in the diagnosis of the severe complications of celiac disease: refractory celiac disease and enteropathy-associated T-cell lymphoma. Novel applications of flow cytometry for the detection of anti-transglutaminase antibodies (a validated biomarker of celiac disease) and of gluten (the triggering antigen of the autoimmune process) are also discussed. The assessment of diagnostic and prognostic biomarkers by flow cytometry in celiac disease is performed routinely in a growing number of centers and it is an example of the versatility of this technique and its applicability to the research and clinical study of solid tissues.     

miR-200b is involved in intestinal fibrosis of Crohn's disease

Intestinal fibrosis is one of the major serious complications of Crohn's disease (CD). However, there are no effective antifibrotic drugs to treat intestinal fibrosis in CD. Therefore, it is important to understand the pathogenesis of fibrosis in CD. It has been reported that members of the miR-200 family are essential in the regulation of renal fibrogenesis. In this study, we analyzed the function of miR-200a and miR-200b in intestinal fibrosis, which was induced by transforming growth factor β1 (TGF-β1) in vitro. Furthermore, we detected the expression of miR-200a and miR-200b in CD specimens, which were divided into groups of fibrosis and no-fibrosis. The results of this study showed that administration of miR-200b could partially protect intestinal epithelial cells from fibrogenesis in vitro. Furthermore, we found that miR-200b was overexpressed in the serum of the fibrosis group. The results suggest that miR-200b has potential value for diagnostic and therapeutic applications for CD patients with fibrosis complications.     

A case of intestinal Behcet's disease similar to Crohn's colitis

Behcet's disease is a multi-systemic vasculitis and characterized by systemic organ involvement. Although the gastrointestinal and systemic features of Behcet's disease and inflammatory bowel disease overlap to a considerable extent, they are generally viewed as two distinct diseases. A 39-yr-old female was diagnosed as having Behcet's disease. She was admitted to our hospital because of oral and genital ulcer, lower abdominal pain, and frequent diarrhea. Colonoscopy showed diffuse involvement of multiple longitudinal ulcers with inflammatory pseudopolyps with a cobblestone appearance and ano-rectal fistula was suspected. These findings are extremely rare in Behcet's disease. However, there were no granulomas, the hallmark of Crohn's colitis. Microscopically, perivasculitis and multiple lymph follicles compatible with Behcet's disease were seen. Although being rarely encountered, multiple longitudinal ulcers, cobblestone appearance, and ano-rectal fistula can develop in Behcet's disease, as in Crohn's colitis. Therefore, Behcet's disease and Crohn's disease may be closely related and part of a spectrum of disease.     

Mucosal inflammatory cytokine production by intestinal biopsies in patients with ulcerative colitis and Crohn's disease

Chronic inflammation in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis) may be attributed partly to increased secretion of inflammatory cytokines. The aim of this study was to investigate simultaneously the spontaneous release patterns of tumor necrosis factor-alpha (TNF-), interleukin-1-beta (IL-1), and interleukin-6 (IL-6) by organ cultures of inflamed mucosa from IBD patients. Organ cultures of involved IBD mucosa spontaneously produced increased amounts of TNF-, IL-1, and IL-6 compared to normal mucosa. The patterns of cytokine release between Crohn's disease and ulcerative colitis organ cultures were not significantly different. Increased inflammatory cytokine production by lamina propria mononuclear cells (LPMCs) and mucosa treated with EDTA suggests that these cytokines originate mainly from LPMCs. These results confirm the role of inflammatory cytokines in IBD and shed a new light on the role of TNF- in IBD.     

Gliadin antibody production by small intestinal lymphocytes from patients with coeliac disease

Small intestinal lymphocytes (SIL) were isolated from jejunal biopsies from 9 non-coeliac controls, 6 treated and 5 untreated coeliac patients. Six-day cell culture supernatants were assayed for total IgG, IgM and IgA and specific anti-casein and anti-gliadin antibodies by ELISA. SIL from the untreated patients secreted more total IgM and IgA than SIL from the treated patients and more total IgG, IgM and IgA than SIL from the controls. The untreated patients' cells secreted more specific anti-gliadin IgA than those from the treated patients and more anti-gliadin IgG, IgM and IgA than the controls. SIL from the untreated group released more anti-casein IgM than those from either the treated or control groups. There were no differences in total immunoglobulin, or specific anti-casein or anti-gliadin antibody secretion by SIL from the treated and control groups. Comparison of immunoglobulin release from cells lysed prior to culture and that secreted both after 30-min and 6-day cultures showed that the majority of immunoglobulin had been synthesised in vitro and could not be accounted for by carry-over.     

Role of colonoscopic biopsy in distinguishing between Crohn's disease and intestinal tuberculosis

BACKGROUND: The histological differential diagnosis of Crohn's disease and intestinal tuberculosis can be very challenging, as both are chronic granulomatous disorders with overlapping histological features. AIM: To evaluate selected clinical and histological parameters in colonic biopsy specimens for their ability to discriminate between Crohn's disease and intestinal tuberculosis. METHODS: 25 patients with Crohn's disease and 18 patients with intestinal tuberculosis were selected for this study on the basis of established clinical, radiological and histological criteria. Clinical data and selected histological parameters in colonoscopic biopsy specimens were assessed retrospectively. A total of 103 and 41 biopsy sites were evaluated in patients with Crohn's disease and intestinal tuberculosis, respectively. RESULTS: Clinical parameters helpful in differentiating intestinal tuberculosis from Crohn's disease included chest radiographic features of tuberculosis (56% v 0%), perianal fistulae (0% v 40%) and extraintestinal manifestations of Crohn's disease (0% v 40%). Histopathological features that seemed to reliably differentiate between intestinal tuberculosis and Crohn's disease included confluent granulomas, > or =10 granulomas per biopsy site and caseous necrosis (in biopsy samples of 50%, 33% and 22% of patients with intestinal tuberculosis, respectively, v 0% of patients with Crohn's disease). Features that were observed more often in patients with intestinal tuberculosis than in those with Crohn's disease included granulomas exceeding 0.05 mm(2) (67% v 8%), ulcers lined by conglomerate epithelioid histiocytes (61% v 8%) and disproportionate submucosal inflammation (67% v 10%). CONCLUSION: Clinical features and selected histological parameters in colonoscopic biopsy specimens can help in differentiating between Crohn's disease and intestinal tuberculosis.     

Modulation of inflammation by vasoactive intestinal peptide and bombesin: lack of effects on neutrophil apoptosis

Inhibition of neutrophil apoptosis has been identified as a prominent feature in chronic inflammation, parenchymal damage, and unresolved organ dysfunction. Lung injury animal models suggest that the neuropeptides vasoactive intestinal peptide and bombesin are protective. Therefore, in vitro effects of VIP and bombesin on apoptosis of normal human neutrophils were tested. For measuring effects on cell survival and apoptosis, trypan dye exclusion, colorimetric MTT assay to assess cell survival, and caspase-3 assay and annexin-V binding for analysing apoptosis rates were used. Foetal calf serum, Fas ligand, and tumour necrosis factor-alpha served as modulatory control agents; survival-promoting and apoptosis-inducing activities of the respective agents were confirmed. Vasoactive intestinal peptide and bombesin, however, failed to significantly affect cell death in neutrophils. Data suggest that direct regulation of neutrophil apoptosis is unlikely to be among the mechanisms of lung-protective actions of VIP and bombesin.     

Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33(+) cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and 'virtual' Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0.9 +/- 0.16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1.80 +/- 0.8 (P = 0.004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.     

Kinetics of intestinal intraepithelial lymphocytes during acute graft-versus-host disease in mice

We examined the kinetics of intestinal intraepithelial lymphocytes (IEL) and the incidence of apoptosis at villus or crypt sites during the development of non-irradiated acute graft-versus-host disease (GVHD). The first IEL to increase were host-origin on day 3 and the donor-derived IEL appeared first on day 12 after GVHD induction. Unique CD3⁺CD4⁺CD8α/α⁺ IEL were significantly increased on day 6 and an appreciable number of IEL bearing T cell receptor Vβ capable of recognizing self-superantigen were detected on day 9. The sudden appearance of apoptosis and reduction of mitotic activity occurred on day 12, accompanied by a dramatic decrease of CD3⁺CD4^?CD8α/α⁺ IEL of host origin. CD8α/α⁺ IEL of host origin, which expand and then decrease by apoptosis at the early stage of acute GVHD, may be associated with pathogenesis of the enteropathy occurring during acute GVHD.