Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease

Intestinal fibrosis and strictures frequently occur in Crohn's disease but not ulcerative colitis. We have recently shown that, compared to myofibroblasts obtained from normal and ulcerative colitis tissue, myofibroblasts isolated from fibrotic Crohn's disease mucosal samples express significantly lower amounts of transforming growth factor (TGF)-beta 3, but the expression of TGF-beta 2 was significantly greater. We now report that in myofibroblast cultures established from fibrotic Crohn's disease mucosal samples there is significantly higher constitutive expression of tissue inhibitor of metalloproteinase (TIMP)-1 compared to similar cells isolated from normal or ulcerative colitis tissue. Myofibroblasts derived from normal mucosa and from mucosa affected by ulcerative colitis or Crohn's disease also expressed matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 but did not express MMP-9. Recombinant (r) TGF-beta 1 and rTGF-beta 2, but not rTGF-beta 3, induced expression of TIMP-1 in normal intestinal myofibroblasts. These studies illustrate a potential mechanism by which differential expression of isoforms of TGF-beta may lead to excessive deposition of extracellular matrix and stricture formation via TIMP-1-mediated inhibition of MMP activity.     

A PIGH mutation leading to GPI deficiency is associated with developmental delay and autism

We identified an individual with a homozygous missense variant (p.Ser103Pro) in a conserved residue of the glycosylphosphatidylinositol (GPI) biosynthesis gene PIGH. This gene encodes an essential component of the phosphatidylinositol N‐acetylglucosaminyltransferase complex, in the first step of the biosynthesis of GPI, a glycolipid anchor added to more than one hundred human proteins, several being critical for embryogenesis and neurological functions. The affected individual had hypotonia, moderate developmental delay, and autism. Unlike other reported individuals with GPI deficiency, the proband did not have epilepsy; however, he did have two episodes of febrile seizures. He had normal alkaline phosphatase and no brachytelephalangy. Upon analysis of the surface expression of GPI‐anchored proteins on granulocytes, he was demonstrated to have GPI deficiency. This suggests that PIGH mutations may cause a syndrome with developmental delay and autism, but without an epileptic encephalopathy, and should increase the awareness of the potentially deleterious nature of biallelic variants in this gene.     

Expression of antimicrobial neutrophil defensins in epithelial cells of active inflammatory bowel disease mucosa

BACKGROUND/AIMS: The normal intestinal epithelium is increasingly being recognised as an important component of the mucosal innate protection against microorganisms. Human neutrophil defensins 1-3 (HNP 1-3) and lysozyme are components of the systemic innate immunity. The aim of this study was to investigate the expression of HNP 1-3 and lysozyme in normal and active inflammatory bowel disease (IBD) mucosa. METHODS: Mucosal tissue sections were studied by immunohistochemistry using antibodies to neutrophil defensins 1-3 and lysozyme. Extracts of purified intestinal epithelial cells were used for immunoblotting studies and antimicrobial activity against the phoP negative strain of Salmonella typhimurium. RESULTS: Surface epithelial cells strongly immunoreactive for neutrophil defensins and lysozyme were seen in active ulcerative colitis and Crohn's disease (but not normal or inactive IBD) mucosal samples. Many of these cells coexpressed both of the antimicrobial proteins. Immunoblotting studies confirmed the expression of neutrophil defensins in extracts of purified ulcerative colitis epithelial cells, which also demonstrated antimicrobial activity. CONCLUSION: HNP 1-3 and lysozyme are expressed in surface enterocytes of mucosa with active IBD and they may play an important role in intestinal host defence against luminal microorganisms.     

Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor^® 488 (toxin A⁴⁸⁸) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A⁴⁸⁸‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A⁴⁸⁸ in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A⁴⁸⁸‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A⁴⁸⁸‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.     

Slow-release 5-amino-salicylic acid (Pentasa) for the treatment of active Crohn's disease

A double-blind, placebo-controlled trial of a slow-release preparation of 5-amino-salicylic acid (Pentasa) has been performed in 40 patients with active Crohn's disease. Over a 6-week period, Pentasa (1.5 g daily) was no different to the dummy tablet in terms of clinical activity (Harvey-Bradshaw Index) or laboratory indicators of inflammation. No serious adverse reactions occurred.     

Ante-mortem characterization of sudden deaths as first-manifestation in Italy

Journal of Interventional Cardiac Electrophysiology - There is a relative paucity of data on ante-mortem clinical characteristics of young (age 1 to 35 years) sudden death (SD) victims....     

Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment.

Clostridium difficile is the aetiological agent of pseudomembranous colitis, and animal studies suggest the essential role of secreted toxin A in inducing disease. This study examined the biological responses to toxin A by human intestinal epithelial cells. Confluent monolayers of Caco2, HT29, and T84 cells and primary epithelial cells in organ cultures of human colonic biopsy specimens and after detachment with EDTA were studied. Interleukin 8 was assayed using enzyme linked immunosorbent assay (ELISA). Purified C difficile toxin A induced cell rounding and detachment of monolayers of the epithelial cell lines. Cells in detached monolayers initially remained viable while adherent to each other. Subsequently, an increasing number of apoptotic cells appeared in suspension. Exposure to toxin A for 24 hours induced interleukin 8 production in T84 and HT29 cells. Toxin A also induced epithelial cell rounding, detachment, and apoptosis in organ cultures of human colonic biopsy specimens. During culture (in medium only), EDTA detached colonic epithelial cells produced interleukin 8 and cell death occurred by apoptosis. Colonic disease by C difficile may be initiated by toxin A mediated induction of epithelial cell interleukin 8 production and apoptosis after cell detachment from the basement membrane. Studies on isolated (toxin untreated) colonic epithelial cells suggest that interleukin 8 production and apoptosis occur as a consequence of cell injury and detachment.     

Macrophage subpopulations in lamina propria of normal and inflamed colon and terminal ileum.

The aim of this study was to characterise human intestinal macrophages in normal and inflamed ileum and colon. Immunoperoxidase staining with a panel of monoclonal antibodies and histochemical staining for acid phosphatase and non-specific esterase was performed. In the superficial lamina propria, normal colonic macrophages were larger and more strongly positive for acid phosphatase and non-specific esterase than those in normal terminal ileum. There were more macrophages staining with monoclonal antibody RFD1 in the superficial lamina propria of the latter. Studies in inflammatory bowel disease tissue showed the presence of macrophages staining with monoclonal antibodies RFD9 and 3G8 which were rarely present in normal tissue and represented a different pattern from that seen in infectious colitis. Studies on isolated intestinal macrophages confirmed the findings in tissue sections. Subpopulations of intestinal macrophages are likely to have different functional roles. Phenotypic changes during inflammation may be induced by mediators of inflammation or may represent a recently recruited population of cells.     

Comparative modes of action of lactitol and lactulose in the treatment of hepatic encephalopathy.

Lactitol, an unabsorbed sugar with defined laxative threshold and superior taste properties has been suggested as an alternative to lactulose in the treatment of hepatic encephalopathy. In the present study we have compared the colonic metabolism of the two sugars using an in vitro faecal incubation system. Both sugars were readily metabolised by faecal bacteria producing volatile fatty acids and the metabolism was inhibited by neomycin. The effect of lactitol and lactulose on terminal ileal and colonic pH was monitored in six normal subjects using a radiotelemetry technique. Both sugars significantly lowered right colonic pH (basal -6.51 +/- 0.48 vs lactitol -5.63 +/- 0.50; lactulose -5.18 +/- 0.82, p less than 0.05). The pH of rest of the colon and terminal ileum was unaffected. Neomycin given concurrently with lactulose abolished acidification of right colon. As lactitol and lactulose have similar effects within the colon, lactitol would appear to have a role in the treatment of hepatic encephalopathy. As neomycin antagonises the effect of lactulose in the colon, its concurrent use may be less effective in the treatment of hepatic encephalopathy.