Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy

AIM To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA.METHODS Crystal-binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography. Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)-conjugated anti-lgA at 37C. Samples were examined under polarizing and fluorescence light microscopy with digital image processing.RESULTS Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC-conjugated anti-lgA antibodies. Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence.CONCLUSION Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystalbinding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Recurrent thrombotic occlusion of a transjugular intrahepatic portosystemic stent-shunt due to activated protein C resistance

The transjugular intrahepatic portosystemic stent-shunt (TIPS) has successfully been used in the management of refractory variceal bleeding and ascites in patients with portal hypertension. Major drawbacks are the induction of hepatic encephalopathy and shunt dysfunction. We present a 59-year-old woman with alcoholic liver cirrhosis who received a TIPS because of recurrent bleeding from esophageal varices. Stent occlusion occurred 4 mo after placement of the TIPS. Laboratory testing revealed resistance to activated protein C (APC). Combination therapy with low-dose enoxaparin and clopidogrel could not prevent her recurrent stent occlusion. Finally, therapy with high-dose enoxaparin was sufficient to prevent further shunt complications up to now (follow-up period of 1 year). In conclusion, early occlusion of a TIPS warrants testing for thrombophilia. If risk factors are confirmed,anticoagulation should be intensified. There are currently no evidence-based recommendations regarding the best available anticoagulant therapy and surveillance protocol for patients with TIPS.

Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases: Relevance to intracellular signaling pathways

AIM: To study the effect of gelatinases (especially MMP-9)on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells.METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases.RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P＜0.05)and the cells overexpressing MMP-9-H401A showed 62% migration (P＜0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly.Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1deactivates cell signaling pathways of MMP-2 and MMP-9involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1.CONCLUSION: Overexpressing functional TIMP-1-enhanced migration of HepG2-TIMP-1 cells depends on enhanced MMP-activity, especially MMP-9.

Side effects of budesonide in liver cirrhosis due to chronic autoimmune hepatitis: Influence of hepatic metabolism versus portosystemic shunts on a patient complicated with HCC

AIM: To investigate the systemic availability of budesonide in a patient with Child A drrhosis due to autoimmune hepatitis (AIH) and primary hepatocellular carcinoma, who developed serious side effects.METHODS: Serum levels of budesonide, 6β-OH-budesonide and 16α-OH-prednisolon were measured by HPLC/MS/MS;portosysternic shunt-index (SI) was determined by 99mTc nuclear imaging. All values were compared with a matched control patient without side effects.RESULTS: Serum levels of budesonide were 13-fold increased in the index patient. The ratio between serum levels of the metabolites 6β-OH-budesonide and 16α-OH-prednisolone, respectively, and serum levels of budesonide was diminished (1.0 vs. 4.0 for 6β-OH-budesonide, 4.2 vs.10.7 for 16α-OH-prednisolone). Both patients had portosystemic SI (5.7% and 3.1%) within the range of healthy subjects.CONCLUSION: Serum levels of budesonide vary up to 13-fold in AIH patients with Child A cirrhosis in the absence of relevant portosysternic shunting. Reduced hepatic metabolism, as indicated by reduced metabolite-to-drug ratio, rather than portosystemic shunting may explain systemic side effects of this drug in cirrhosis.

Occult celiac disease prevents penetrance of hemochromatosis

AIM: To report a patient with C282Y homozygocity, depleted body iron and intestinal atrophy caused by celiac disease (CD) who experienced resolution of the enteropathy with subsequent normalization of iron metabolism upon glutenfree diet.METHODS: To obtain information on the tissue distribution and quantitative expression of proteins involved in duodenal iron trafficking, we determined the expression of divalent-metal transporter 1 (DMT1), ferroportin 1 (FP1) and transferrin receptor (TfR1) by means of immunohistochemistry and real-time PCR in duodenal biopsies of this patient.RESULTS: Whereas in hereditary hemochromatosis patients without CD, DMT1 expression was up-regulated leading to excessive uptake of iron, we identified a significant reduction in protein and mRNA expression of DMT1 as acompensatory mechanism in this patient with HH and CD.CONCLUSION: Occult CD may compensate tot increased DMT1 expression in a specific subset of individuals withhomozygous C282Y mutations in the hemochromatosis(HFE) gene, thus contributing to the low penetrance of HH.