Strictures in Crohn's disease are characterised by an accumulation of mast cells colocalised with laminin but not with fibronectin or vitronectin.

BACKGROUND/AIMS: Intestinal fibrosis and stricture formation is an unresolved problem in Crohn's disease. The aim of this study was to investigate whether mast cells accumulate in these tissues and whether their localisation is associated with extracellular matrix components. METHODS: Mast cells were visualised by immunohistochemical staining of the mast cell specific proteases chymase and tryptase. Their localisation in relation to extracellular matrix components was shown by immunohistochemical double labelling. RESULTS: In strictures in Crohn's disease, a striking accumulation of mast cells was seen particularly in the hypertrophied and fibrotic muscularis propria, with a mean (SEM) mast cell number of 81.3 (14.9) v 1.5 (0.9)/mm(2) in normal bowel (p<0.0005). All mast cells in the muscularis propria were colocalised with patches of laminin. In contrast, in the submucosa, laminin was exclusively found in the basal lamina of blood vessels where many adherent mast cells were seen. No colocalisation of mast cells was found with fibronectin or vitronectin. CONCLUSIONS: The large accumulation of mast cells in the muscle layer of strictured bowel suggests a functional role for these cells in the hypertrophic and fibrotic response of the smooth muscle cells. The colocalisation with laminin indicates a mechanism of interaction between smooth muscle cells and mast cells that may be important in the role of mast cells in the process of fibrosis.

Spontaneous rupture of liver in a patient with Ehlers Danlos disease type IV.

Ehlers-Danlos syndrome (EDS) type IV is an autosomal dominant connective tissue disease caused by mutations in the type III collagen gene resulting in extreme tissue fragility. Affected individuals are at risk of dramatic and often fatal complications, mostly spontaneous arterial, uterine, or colonic ruptures. Phenotypic expression of EDS type IV is variable and clinical signs are generally quite subtle, thus making a prompt diagnosis difficult. The case of a 33-year-old woman is described who presented with a wide range of clinical features and sequelae that eventually led to the diagnosis of EDS type IV. She presented with spontaneous liver rupture, renal infarction, and pneumothorax, all representing rare complications of EDS type IV. Prior history revealed a uterine rupture in advanced pregnancy associated with ischemic necrosis of the descending and sigmoid colon. EDS type IV should be suspected in young individuals who present with such unusual complications. Early diagnosis is essential if severe or even lethal complications are to be avoided in the diagnostic and therapeutic management of such patients.

In situ detection of lipid peroxidation by-products in chronic liver diseases.

Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes. The deleterious consequences of this mechanism are related in part to the formation of reactive aldehydic products that bind to intra- or extracellular molecules to form adducts. Specific antibodies directed against malondialdehyde (MDA) and 4-hydroxynonenal (HNE) adducts, major aldehydic metabolites of lipid peroxidation, allowed us to investigate in situ, with an immunohistochemical procedure, the occurrence of lipid peroxidation in a panel of different chronic liver diseases. Intracellular HNE and MDA adducts were detected respectively in 24 of 39 cases (62%) and in 12 of 34 cases investigated (35%). They were localized mainly in the cytoplasm of hepatocytes, with the strongest staining observed in hemochromatosis, Wilson's disease, and in areas of acute alcoholic hepatitis in cases of alcoholic liver diseases. A peculiar pattern of immunostaining was observed in primary biliary cirrhosis where biliary cells of destroyed but also intact bile ducts strongly expressed HNE adducts. The liver extracellular matrix also displayed MDA adducts (30 of 34 cases, 88%) and HNE adducts (23 of 39 cases, 59%). While HNE adducts were specifically localized on large bundles of collagen fibers, MDA adducts were detected in a thin reticular network and in sinusoidal cells around portal tracts or fibrous septa. In conclusion, lipid peroxidation by-products are detectable in chronic liver diseases. Immunohistochemical results suggest that this mechanism is implicated very early in the pathogenesis of some of these diseases.

In situ detection of lipid peroxidation in chronic hepatitis C: correlation with pathological features.

AIMS: To assess the occurrence of lipid peroxidation in chronic hepatitis C and to evaluate its relation to pathological features and liver iron concentrations. METHODS: Liver biopsy samples of 43 patients with untreated chronic hepatitis C were studied by immunohistochemistry using specific antibodies directed against two major aldehyde metabolites of lipid peroxidation, malondialdehyde (MDA), and 4-hydroxynonenal (HNE). RESULTS: MDA and HNE adducts (aldehydes covalently linked to another molecule) were detected in the liver samples in 77% and 30% of cases, respectively. MDA adducts were detected both in the extracellular matrix and sinusoidal cells localised in areas of periportal and lobular necrosis. HNE adducts appeared in the cytoplasm of only a few hepatocytes. Comparison of the semiquantitative assessment of adducts (MDA and HNE indexes) with the grading and the staging of chronic hepatitis showed that the MDA index was correlated with fibrosis score (p < 0.001) and the grade of activity (p < 0.01). There was also a tendency to correlation with liver iron concentration (p = 0.09). No correlation was observed between the HNE index and pathological features or liver iron concentration. CONCLUSION: Lipid peroxidation products are detectable in the liver of chronic hepatitis C patients. The presence of MDA adducts in areas of active fibrogenesis and the correlation between the MDA index and fibrosis score suggest a role for lipid peroxidation in liver fibrosis.

Imbalance of the interleukin 1 system in colonic mucosa--association with intestinal inflammation and interleukin 1 receptor antagonist [corrected] genotype 2.

BACKGROUND: Interleukin 1 (IL-1) alpha and beta are potent cytokines which play key roles in inflammation. They are controlled by IL-1 receptor antagonist (IL-1ra). AIMS: To investigate the influence of mucosal inflammation and IL-1ra genotype on the IL-1ra:IL-1 balance. PATIENTS AND METHODS: IL-1 alpha, IL-1 beta, and IL-1ra were measured by enzyme linked immunosorbent assay (ELISA) in biopsy specimens taken from inflamed and non-inflamed colon of 60 patients with Crohn's disease (CD), 34 with ulcerative colitis (UC), 15 inflammatory controls, and 103 non-inflamed controls. IL-1ra genotype was determined by polymerase chain reaction and gel electrophoresis. RESULTS: IL-1 alpha and IL-1 beta were significantly increased in inflamed mucosa in inflammatory bowel disease (IBD) (CD: 53.5 (22.4) and 409.9 (118.7) pg/mg protein, respectively; UC: 18.9 (6.8) and 214.5 (78.2) pg/mg, respectively) and non-IBD patients (19.2 (7.4) and 281.4 (121.0) pg/mg, respectively; p < 0.0001) compared with normal controls (2.8 (0.6) and 30.6 (5.6) pg/mg, respectively). In CD IL-1 alpha and beta were also significantly increased in non-inflamed mucosa (6.1 (1.3) pg/mg and 88.7 (17.4) pg/mg, respectively; p < 0.0012). IL-1ra:(IL-1 alpha+beta) ratios were significantly decreased in inflamed mucosa of patients with CD (182 (45); p < 0.0001), UC (425 (136); p = 0.0018) and without IBD (221 (76); p < 0.0001), and in non-inflamed mucosa in CD (369 (149); p < 0.0001) compared with normal controls (1307 (245); p < 0.0001). Patients with IL-1ra genotype 2 had slightly but significantly reduced mucosal IL-1ra concentrations (p = 0.003). The greatest difference was seen in colonic biopsy specimens from patients with inflamed Crohn's disease. CONCLUSION: Mucosal inflammation can modulate the balance of the IL-1:IL-1ra system in colonic mucosa.

Cellular and subcellular localization of acetaldehyde-protein adducts in liver biopsies from alcoholic patients.

Acetaldehyde, the first product of ethanol in hepatocytes, can react with protein to form acetaldehyde-protein adducts (APAs). Because it has been suggested that these adducts could be involved in the pathogenesis of ethanol-induced hepatic lesions and in fibrogenesis, we performed an ultrastructural immunohistochemical study to precisely define the cellular and subcellular localization of APAs. A preembedding technique of indirect immunoperoxidase was performed in liver biopsy specimens from eight patients with alcoholic liver disease, using a specific antiserum against APAs. In all specimens, APAs were detected in the rough endoplasmic reticulum, in some peroxisomes, and in the cytosol of hepatocytes. In four patients with steatofibrosis or cirrhosis, labeling of Ito cells was also observed. In these cases, the same staining pattern was observed in the cytoplasmic processes of myofibroblasts in areas of fibrogenesis. When isolated rat Ito cells were incubated in the presence of acetaldehyde, APAs were also detected in the cytoplasm. These results show that APA formation occurs in hepatocytes at the sites of acetaldehyde production. Detection of APAs in human and rat Ito cells strongly suggests that acetaldehyde can diffuse into Ito cells and bind to cytoplasmic proteins to form local APAs. Because Ito cells are the main effector cells of liver fibrosis, detection of APAs in these cells points to their possible involvement in liver fibrogenesis.

Conservative treatment of acute hepatic failure.

Treatment of patients with acute liver failure has considerably improved in recent years. Effective treatment of these devastating situations requires early assessment of prognosis and early referral of these patients to specialized centers. The conservative management of extrahepatic complications in intensive care units appears to contribute to a better survival of patients whether or not they are subsequently submitted to a transplantation procedure. Specific hepatocyte-targeted treatment by prostaglandin E2 or similar drugs can be an option in the future. Dramatic progress has been made in the temporary substitution of liver function bridging the time period between liver failure and resumption of hepatocellular function due to liver regeneration. Presently, artificial liver assist devices as well as the extracorporeal perfusion of human or pig livers are evaluated in clinical trials. Initial results indicate that these measures allow to bridge the time until an appropriate donor liver is available. Permanent liver transplantation has been shown to save the lives of many patients suffering from acute liver failure. Selection of patients requiring urgent liver transplantation has been facilitated by the use of prognostic scores specifically adapted to the etiology of underlying liver disease. A temporary auxiliary liver transplantation can be a better treatment option preventing the patient from a life-long dependence upon medical surveillance and drug-induced immunosuppression.

A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae.

Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.

Acetaldehyde-modified epitopes in liver biopsy specimens of alcoholic and nonalcoholic patients: localization and association with progression of liver fibrosis.

Acetaldehyde, the first product of ethanol oxidation, has been shown to stimulate collagen gene expression and to form protein-acetaldehyde adducts. Because little is known about these adducts in human liver tissue, we assessed, with an immunohistochemical procedure, the presence and location of acetaldehyde-protein adducts in liver biopsy specimens of alcoholic patients. In addition, we correlated the presence of adducts with the progression or subsequent occurrence of liver fibrosis. The group included 106 patients with high alcohol consumption (> 90 gm ethanol/day for the last 5 yr), 10 nonalcoholic patients with normal livers and 23 patients with other liver diseases. Sixty-four of the 106 alcoholic patients had a second liver biopsy, whose specimen was used to assess the progression of liver fibrosis. Polyclonal antibodies were produced against homologous low-density lipoprotein purified from rabbit serum and modified in vitro in the presence of acetaldehyde. Protein-acetaldehyde adducts could be detected by immunohistochemistry in biopsy specimens of 90 alcoholic patients (85%), in none of the 10 nonalcoholic patients with normal livers and in 65% of the patients with nonalcoholic liver disease. Acetaldehyde-modified epitopes were detected in the intracellular and extracellular compartment. Intracellular protein-acetaldehyde adducts were localized in the cytoplasm of hepatocytes with a more intense staining in zone 3. No correlation existed between the intensity of intracellular staining and the histologically assessed severity of liver disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunobiology of the serine protease allergens from house dust mites.

Extracts of Dermatophagoides pteronyssinus and D. farinae were shown to contain a variety of 30 kDa serine proteases, including trypsin, chymotrypsin, and an elastase-like enzyme. The mite trypsin, unlike chymotrypsin and the elastase enzyme, was heterogeneous with regard to charge. The enzymes were shown to be present at higher concentration in fecally enriched extracts than in whole mite extracts. The proteases were shown to induce vascular permeability and to detach cells in tissue culture. Further study showed that the mite elastase induced non-IgE mediated rat mast cell degranulation. Such properties may contribute to immunogenicity.

[Raynaud syndrome. A case report]. / Raynaud-Syndrom. Eine Fallbeschreibung.

Symptomatology, diagnosis, treatment, and clinical course of a Raynaud syndrome in a sixteen-year old patient beginning in the second year of his life are described. He lost all distal phalanges of both hands and big toes at the age of nine. Remarkable was the laboratory finding of an extremely high serum IgE concentration. A combined therapy of plasmapheresis, immunosuppression and corticosteroids improved the clinical and laboratory findings.

Transport systems for amphipathic compounds in normal and neoplastic hepatocytes.

Photoaffinity labeling of plasma membrane subfractions from liver and of intact liver tissue with a photolabile bile salt derivative, the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid, revealed that the hepatobiliary transport of bile salts is accomplished by transport systems different for sinusoidal uptake and canalicular secretion. Polypeptides with apparent Mr values 54,000 and 48,000 interact with bile salts at sinusoidal membrane, whereas a polypeptide with an apparent Mr of 100,000 is involved in bile salt secretion through the canalicular membrane. Photoaffinity labeling with photolabile derivatives of uncharged and cationic compounds provided evidence that the sinusoidal membrane polypeptides exhibit a broad binding specificity. Photoaffinity labeling studies and kinetic studies suggest that hepatic uptake of different amphipathic anions, uncharged compounds and even of cations is mediated by the sinusoidal transport systems which are involved in the uptake of bile salts. Relatively little is known about the specificity of the canalicular bile salt transport system. The fluorescent bile salt derivative, the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid, is readily taken up into the hepatocytes of all acinar zones and may be used for the evaluation of the functional state of bile salt transport by fluorescence microscopy. Fluorescent microscopic studies with the fluorescent bile salt derivative showed that ascites hepatoma AS 30D cells do not have the ability to take up bile salts and demonstrated the absence of hepatobiliary bile salt transport in the solid Morris hepatoma 7777. Photoaffinity labeling studies revealed that in both tumor cell models, in hepatoma AS 30D and in Morris hepatoma 7777, the plasma membranes were devoid of the polypeptides having affinities to bile salts and amphipathic cations. A slight labeling of bile salt binding membrane polypeptides in plasma membranes from Morris hepatomas 9618A and TC 5123 opens the possibility to study transport in neoplastic hepatocytes.