Christoph G. Dietrich

Enhanced expression of MMP-7 and MMP-13 in inflammatory bowel disease: a precancerous potential?

&NA; Matrix metalloproteinases (MMPs) are responsible for the turnover and degradation of extracellular matrix. They play a crucial role in the growth and migration of colorectal carcinoma cells. Colorectal carcinomas are characterized by enhanced expression of MMP‐2, MMP‐9, MMP‐7, and MMP‐13. The aim of this study was to determine the expression levels of MMP‐2, MMP‐9, MMP‐7, MMP‐13, and MMP‐14 and their specific inhibitor TIMP‐1 in inflammatory bowel diseases and precancerous lesions of the colon, i.e., Crohns disease and ulcerative colitis, and in adenomatous polyps (APs) for comparison. Biopsy samples of pathological and healthy tissue were obtained from 40 patients with inflammatory bowel disease (ulcerative colitis, n = 17; Crohns disease, n = 23) and from 19 patients with APs. mRNA was measured by quantitative real‐time polymerase chain reaction to study MMP and TIMP‐1 gene expression in both pathological and normal mucosal specimens. For MMP‐2, MMP‐9, and TIMP‐1, protein expression also was quantified with sandwich enzyme‐linked immunosorbent assay. In biopsy specimens of Crohns disease and ulcerative colitis, significantly increased levels of MMP‐2, MMP‐7, and MMP‐13 mRNA were found. MMP‐2 and MMP‐9 showed enhanced secretion on the protein level. AP revealed an increased transcription of MMP‐7 and MMP‐13 genes. MMP‐14 mRNA was decreased in APs. MMPs, especially MMP‐7 and MMP‐13, which are expressed primarily on the tumor cell surface, are elevated in inflammatory bowel disease, which may have more chance to evolve into malignancy than normal tissue. In APs, increased expression of MMP‐7 and MMP‐13 may serve as an early indicator for colorectal carcinogenesis.

Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C

Abstract: u2028Background: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients.

The presence of non-organ-specific autoantibodies is associated with a negative response to combination therapy with interferon and ribavirin for chronic hepatitis C

BackgroundNon-organ-specific autoantibodies are found in a considerable number of anti-HCV positive patients. Previous studies investigated the clinical relevance of these antibodies in patients treated with interferon monotherapy, but not combination therapies.MethodsAnti-nuclear, anti-smooth muscle, anti-mitochondrial, anti-neutrophil-cytoplasmatic and anti-liver/kidney microsomal antibodies were determined in 78 consecutive anti-HCV positive patients by indirect immunofluorescence. The presence of these antibodies was related to demographic variables and to the outcome of antiviral combination therapy with interferon-α and ribavirin in 65 patients.ResultsIn our study, positivity for autoantibodies was associated with higher alanine aminotransferase levels and higher mean values for HCV-RNA (p < 0.01). Furthermore, negativity for non-organ-specific autoantibodies was associated with a favourable treatment outcome of combination therapy with at least one negative RT-PCR for HCV-RNA during treatment (OR 4.65, 95% CI 1.31 to 16.48, p = 0.02). ANA and SMA staining patterns and titers were not correlated to treatment response. With multiple logistic regression analysis, positivity for autoantibodies and HCV genotype were independently associated with outcome of antiviral combination therapy (p = 0.02).ConclusionsThe absence of non-organ-specific autoantibodies might indicate a significantly higher chance for viral clearance in response to combination therapy for chronic hepatitis C infection. Therefore, despite of an overall higher treatment response, the addition of the immunomodulatory drug ribavirin could accentuate immunological differences that affect treatment outcome and might have been less obvious in earlier studies analysing interferon monotherapy.

CC chemokine receptor 5 Δ32 polymorphism in two independent cohorts of hepatitis C virus infected patients without hemophilia

Recently CC chemokine receptor 5 (CCR5) related immune mechanisms and a functional mutation of the CCR5 gene have been implicated in hepatitis C virus (HCV) infection in a cohort of predominantly hemophiliac patients. The present study investigated the frequency and clinical consequences of the CCR5 Δ32 mutation in two genetically homogeneous populations of HCV infected patients with a different risk profile for infection. Genomic DNA samples from 333 German patients with chronic HCV infection were screened by PCR for the presence of the CCR5 Δ32 polymorphism. In-hospital patients admitted for other diseases than viral hepatitis but with a comparable risk for HCV exposure were used as control population (n=125). Allele frequencies of CCR5 Δ32 polymorphism did not differ significantly between the two groups (7.6% and 9.5%, respectively) and control subjects (10.4%), and did not deviate from Hardy-Weinberg equilibrium in any group. Furthermore, there were no major differences between patients with respect to HCV genotypes, viral loads, liver enzymes, or fibrosis scores in relation to the presence or absence of the heterozygous CCR5 Δ32 mutation. Differences in inflammatory scores in liver biopsy samples and response to antiviral therapy in CCR5 Δ32 heterozygotes in one cohort could neither be reproduced in the other group of patients nor when both cohorts were pooled. These results argue against a strong effect of the CCR5 Δ32 deletion regarding these phenotypes. In conclusion, we found no increased frequency of the CCR5 Δ32 polymorphism in two independent cohorts of patients with HCV infection but without hemophilia as the main risk factor for infection. As the major difference to investigations demonstrating an association between CCR5 Δ32 and HCV infection is the selection of cases and controls, our study emphasizes the importance of epidemiological criteria for association studies of HCV infection.

Haplotype‐tagging RANTES gene variants influence response to antiviral therapy in chronic hepatitis C

The response to antiviral therapy for chronic hepatitis C virus (HCV) is complex and is determined by both environmental and genetic factors. Recently, interacting gene polymorphisms of the chemokine RANTES have been shown to affect HIV disease progression. Our aim was to assess if these RANTES variants are associated with response to anti‐HCV therapy. Three linked RANTES single nucleotide polymorphisms (403 G/A, Int1.1 T/C, and 3′ 222 T/C) were determined in 297 Caucasian patients who were treated for chronic HCV infection and 152 control subjects. Characteristic nucleotide combinations on single chromosomes (haplotypes) were reconstructed and tested for disease association. Four common RANTES haplotypes (prevalence 73%) were identified in patients and controls. There was a strong association of RANTES haplotype distribution with outcome of antiviral combination therapy (P = .007). Specifically, RANTES haplotypes carrying Int1.1 C and 3′ 222 C alleles were more frequent in nonresponders than in patients with a sustained response to antiviral therapy (odds ratio 1.9, P = .01). The influence of these RANTES haplotypes on the outcome of therapy was more pronounced in patients infected with HCV genotypes 1 and 4 (odds ratio 2.3, P = .02). Because RANTES haplotypes carrying Int1.1 C are known to down‐regulate RANTES transcriptional activity in vitro, the haplotype analysis fits the hypothesis of a diminished T helper 1 lymphocyte response in patients with a negative response to antiviral therapy. In conclusion, RANTES haplotypes might contribute to the polygenic interaction between HCV and the host immune system and could help to risk stratify patients prior to antiviral therapy. (HEPATOLOGY 2004;40:327–334.)

Cytokine blockade inhibits hepatic tissue inhibitor of metalloproteinase‐1 expression and up‐regulates matrix metalloproteinase‐9 in toxic liver injury

Abstract: Background: Tissue inhibitor of metalloproteinases (TIMP)‐1, the most important endogenous inhibitor of matrix metalloproteinases, plays a pivotal role in the pathogenesis of liver fibrosis and may represent an effective therapeutic target in the design of antifibrotic strategies for chronic liver diseases.

Cytokine-independent repression of rodent Ntcp in obstructive cholestasis.

Cholestatic liver injury is associated not only with accumulation of bile acids but also with activation of proinflammatory cytokines. Common bile duct ligation (CBDL) induces sustained downregulation of the Na+/taurocholate cotransporter (Ntcp) in rodent liver. Although repression of Ntcp during endotoxemia is cytokine mediated, it is unclear whether inflammatory cytokines contribute to this downregulation in obstructive cholestasis. Cytokine inactivation in CBDL rats and mice was either performed directly with tumor necrosis factor alpha (etanercept) or interleukin 1 beta inactivation (anakinra/AMG 719) or indirectly Kupffer cell depletion via intraperitoneal administration of liposome‐encapsulated dichloromethylene bisphosphonate. Protein and messenger RNA (mRNA) expression of Ntcp and short heterodimer partner (SHP) were analyzed via Western and Northern blotting. Key regulators of Ntcp (hepatocyte nuclear factor 1 alpha [HNF‐1α], HNF‐4α, retinoid X receptor alpha [RXRα]:retinoic acid receptor alpha [RARα]) were studied via electrophoretic mobility shift analysis and nuclear Western blot analysis. Both methods of cytokine inactivation failed to maintain Ntcp protein or mRNA expression within 3 days after CBDL in either rats or mice (20%‐40% of sham controls), while SHP mRNA expression increased three‐ to five‐fold. Decreased nuclear HNF‐1α and HNF‐4α protein levels (45% and 60% of sham controls, respectively) and HNF‐1α binding activity (32% of sham controls) were not restored during cytokine inactivation after CBDL, indicating cytokine‐independent mechanisms of Ntcp regulation. RXRα:RARα binding remained unchanged in all experimental conditions. In conclusion, during obstructive cholestasis accumulating bile acids per se, without major contribution of cytokines, leads to downregulation of Ntcp via repression of HNF‐1α and HNF‐4α. (HEPATOLOGY 2005;41:470–477.)

Efficacy of submucosal injection of different solutions inclusive blood components on mucosa elevation for endoscopic resection

Background Endoscopic resection has become the standard treatment for noninvasive gastrointestinal malignancies. In flat mucosal tumors, normal saline is frequently used for submucosal fluid injection in order to reduce the risk of complications during endoscopic resection. Recent studies have demonstrated longer-lasting mucosa elevation by injection of agents such as hyaluronic acid or glyceol, rather than normal saline. We investigated the efficacy of different blood components in comparison with other solutions for use as a submucosal fluid cushion. Methods Normal saline, sodium hyaluronate, glyceol, hydroxyethyl starch, serum, plasma, and whole blood were evaluated for their effectiveness in creating a submucosal cushion. One milliliter of each solution was injected into the submucosa of 5 × 5 cm specimens of resected porcine stomach. Mucosa elevation was measured before and up to 60 minutes after injection. Results The shortest duration of mucosa elevation was observed after injection of normal saline, glyceol, and 0.125% hyaluronic acid. A significantly longer duration was obtained after injection of hydroxyethyl starch, 0.25% and 0.5% hyaluronic acid, serum, and plasma. However, whole blood generated a longer-lasting mucosa elevation than all other agents. Conclusion The results of the current study suggest that whole blood is more effective in generating long-lasting mucosa elevation than any other commonly used solution. Because autologous blood is readily available at almost no cost, this seems to be an optimal agent for creating the mucosa elevation needed for endoscopic resection. Further in vivo studies in humans are needed to clarify the potential role of autologous blood for long-lasting endoscopic mucosa resection or endoscopic submucosal dissection.

Extrahepatic cholestasis downregulates Oatp1 by TNF-α signalling without affecting Oatp2 and Oatp4 expression and sodium-independent bile salt uptake in rat liver

Hepatic uptake of bile salts is mediated by sodium‐dependent and sodium‐independent transport systems. During extrahepatic cholestasis, both the function and the expression of the Na+/taurocholate cotransporting polypeptide (Ntcp) are downregulated. To test whether sodium‐independent organic anion‐transporting polypeptides are also affected by extrahepatic cholestasis, the function and expression of all three Oatps have been determined in common bile duct‐ligated (CBDL) rats. Oatp1/Oatp1a1 protein mass remained unchanged after CBDL for 1 day, but then declined by 75±7% and 90±17%, respectively, after 3 and 7 days. In contrast, Oatp2/Oatp1a4 and Oatp4/Oatp1b2 protein expression was not affected by CBDL as compared with controls. After CBDL, Oatp1 mRNA was rapidly downregulated by 68±21% of untreated controls (P<0.05) within 24u2003h, and remained at similar levels at 3 and 7 days. Cytokine‐inactivation studies with etanercept pretreatment demonstrated that TNF‐α‐dependent signals mediated the down‐regulation of this transporter gene at both protein and mRNA levels during obstructive cholestasis. Sodium‐independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in Km nor Vmax values. These results indicate that sodium‐independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, because its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes.

Downregulation of Breast Cancer Resistance Protein in colon adenomas reduces cellular xenobiotic resistance and leads to accumulation of a food-derived carcinogen

Several molecular changes in colorectal adenomas provide the basis of the adenoma–carcinoma sequence. We investigated the expression of xenobiotic ATP‐binding cassette (ABC) transporters in humans and in ApcMin mice and conducted functional studies estimating the importance of the expression changes. Twenty‐nine adenomas from 21 patients and eight adenomas from four ApcMin mice were analyzed using Western blotting and quantitative Real‐time polymerase chain reaction (RT‐PCR). Adjacent healthy tissue served as control for each polyp. Breast cancer resistance protein (BCRP) was significantly downregulated in human colorectal adenomas (to 28 ± 35% of adjacent healthy tissue). This was in line with data from ApcMin mice adenomas, where downregulation was significant as well (to 58 ± 34%). In parallel, quantitative RT‐PCR showed BCRP mRNA downregulation in human adenomas (to 17 ± 31%). Basal multidrug resistance‐associated protein 2 expression was low and did not change in adenomas; multidrug resistance transporter 1 expression also did not differ between adenomas and healthy tissue. In a functional study, ApcMin mice received radioactively labelled 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐β] pyridine (PhIP), a food colon carcinogen and substrate of BCRP, by oral gavage with analysis of PhIP accumulation and DNA adduct formation 48 hr later. In this setting, we could demonstrate a higher carcinogen concentration in adenomas of ApcMin mice (181 ± 113% of normal tissue) including immunohistochemical detection of PhIP–DNA adducts. We conclude that significant transcriptional downregulation of BCRP/Bcrp leads to higher carcinogen concentrations in colorectal adenomas of mice and men. This might promote the adenoma–carcinoma sequence by higher genotoxic effects. The results indicate a possible role of transporter deficiencies in susceptibility for colon carcinoma.