Association of polymorphous light eruption with NOD-2 and TLR-5 gene polymorphisms.

BACKGROUND: Polymorphous light eruption (PLE) is a common, immunologically mediated, photosensitive skin disease. After ultraviolet-B (UV-B) irradiation, patients with PLE show reduced Langerhans cell (LC) depletion in the epidermis, which results in a non-suppressive microenvironment in the skin. Interestingly, severe acute graft-versus-host disease (aGvHD) occurred in stem cell transplanted patients that showed no or incomplete depletion of LCs after UVB irradiation. Genetic variation in nucleotide-binding oligomerization domain 2 (NOD-2) and toll-like receptor 5 (TLR-5) genes also confers susceptibility to aGvHD. OBJECTIVES: We hypothesized that PLE is associated with genetic variation in the NOD-2 and TLR-5 genes. METHODS: We investigated single-nucleotid polymorphisms (SNPs) of NOD-2 (R702W, G908R, 3020Cins) and TLR-5 (A592S, P616L, N392STOP) in skin biopsies of patients with PLE (n = 143) and in healthy controls (n = 104) using restriction fragment length polymorphism analysis. RESULTS: The frequency of NOD-2 alleles with the SNP R702W was significantly higher in PLE than in controls (31.8% vs. 6.3%; P < 0.0001), and homozygous carriers of this mutation were more common in PLE (27.9% vs. 0%; P < 0.0001). For SNP 3020Cins, the allele frequency (7.3% vs. 0.7%; P = 0.0025) and the number of heterozygotes (14.7% vs. 1.3%; P = 0.0019) were higher in PLE. The frequency of alleles with the N392STOP SNP of the TLR5 gene, which is associated with a truncated, non-functional receptor, was significantly higher in PLE (21% vs. 5%; 7% vs. 1% homozygotes, 28% vs. 8% heterozygotes; P < 0.0001). The other SNPs did not differ significantly. CONCLUSIONS: This study yielded a high frequency of functional SNPs in the NOD-2 and TLR-5 genes in PLE. The same SNPs are associated with aGvHD and there are similarities in the reaction of LCs after UVB irradiation between aGvHD and PLE. This leads to the hypothesis that patients with PLE may be more susceptible to developing GvHD after stem cell transplantation, an assumption that needs to be investigated further.

Wild-type KRAS is a novel therapeutic target for melanoma contributing to primary and acquired resistance to BRAF inhibition.

Malignant melanoma reveals rapidly increasing incidence and mortality rates worldwide. By now, BRAF inhibition is the standard therapy for advanced melanoma in patients carrying BRAF mutations. However, only approximately 50% of melanoma patients harbor therapeutically attackable BRAF mutations, and overall survival after treatment with BRAF inhibitors is modest. KRAS (Kirsten Rat sarcoma) proteins are acting upstream of BRAF and have a major role in human cancer. Recent approaches awaken the hope to use KRAS inhibition (KRASi) as a clinical tool. In this study, we identified wild-type KRAS as a novel therapeutic target in melanoma. KRASi functions synergistically with BRAF inhibition to reduce melanoma proliferation and to induce apoptosis independently of BRAF mutational status. Moreover, acquired resistance to BRAF inhibitors in melanoma is dependent on dynamic regulation of KRAS expression with subsequent AKT and extracellular-signal regulated kinase activation and can be overcome by KRASi. This suggests KRASi as novel approach in melanoma-alone or in combination with other therapeutic regimes.

mTOR inhibition improves fibroblast growth factor receptor targeting in hepatocellular carcinoma.

BACKGROUND: Systemic therapy has proven only marginal effects in hepatocellular carcinoma (HCC) so far. The aim of this study was to evaluate the effect of targeting fibroblast growth factor receptor (FGFR) on tumour and stromal cells in HCC models. METHODS: Human and murine HCC cells, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), hepatic stellate cells (HSCs), human HCC samples, FGFR inhibitor BGJ398 and mammalian target of rapamycin (mTOR) inhibitor rapamycin were used. Effects on growth, motility, signalling and angiogenic markers were determined. In vivo subcutaneous and syngeneic orthotopic tumour models were used. RESULTS: In tumour cells and ECs, targeting FGFR showed significant inhibitory effects on signalling and motility. Minor effects of FGFR inhibition were observed on VSMCs and HSCs, which were significantly enhanced by combining FGFR and mTOR blockade. In vivo daily (5 mg kg(-1)) treatment with BGJ398 led to a significant growth inhibition in subcutaneous tumour models, but only a combination of FGFR and mTOR blockade impaired tumour growth in the orthotopic model. This was paralleled by reduced tumour cell proliferation, vascularisation, pericytes and increased apoptosis. CONCLUSIONS: Targeting FGFR with BGJ398 affects tumour cells and ECs, whereas only a combination with mTOR inhibition impairs recruitment of VSMCs and HSCs. Therefore, this study provides evidence for combined FGFR/mTOR inhibition in HCC.

Olive oil attenuates the cholesterol-induced development of nonalcoholic steatohepatitis despite increased insulin resistance in a rodent model.

It is indefinite whether nonalcoholic steatohepatitis (NASH) results as by-product from general metabolic perturbations and adipokine dysregulations or whether defined dietary factors also play a pathogenetic role. Here, we examine the effects of a modification of dietary lipids in a NASH inducing diet on metabolic changes as well as hepatic steatosis, inflammation, and fibrosis in rats. Male Wistar rats were fed with variations of the atherogenic diet (AD), which induces pathophysiological changes resembling human NASH. Dietary variants (AD without cholesterol, cholate, or choline; change of neutral fat to olive oil or coconut oil) were fed for 8 weeks. Insulin resistance, adipokine profile, liver histology, and lipid content as well as expression of proinflammatory and profibrogenic genes were examined. AD led to clear signs of hepatic steatosis and inflammation together with an increase in TNF and collagen type 1 expression. AD without cholesterol showed markedly less liver damage without changes of insulin action and adipokine profile. AD with olive oil and AD without cholate clearly attenuated hepatic inflammation, whereas fat deposition and features of the metabolic syndrome were increased in these animals. Insulin resistance and hepatic fat deposition per se do not cause significant hepatic inflammation in this rodent model. However, dietary cholesterol is an important causal agent for the development of NASH. Olive oil plays a protective role in this respect, which might be due to the high content of monounsaturated fatty acids.

Dysbalance in sympathetic neurotransmitter release and action in cirrhotic rats: impact of exogenous neuropeptide Y.

BACKGROUND & AIMS: Splanchnic vasodilation is an essential disturbance in portal hypertension. Increased systemic sympathetic nerve activity is well known, but potential corresponding vascular desensitization is incompletely characterized. Release of splanchnic sympathetic neurotransmitters noradrenaline (NA) and co-transmitter neuropeptide Y (NPY) remains to be elucidated. Finally, the effects of exogenous NPY on these mechanisms are unexplored. METHODS: Portal vein ligated cirrhotic, and control rats were used for in vitro perfusion of mesenteric arteries. Depletion of vascular pressure response was induced by repetitive electric sympathetic perivascular nerve stimulation (PNS) and performed in the absence and presence of exogenous NPY. Additionally, PNS-induced release of NA and NPY was measured. RESULTS: Mesenteric PNS-induced pressure response was lower in portal hypertension. Depletion of the pressure response to PNS, representing the degree of desensitization, was enhanced in portal hypertension. NA release was elevated, whereas NPY release was attenuated in cirrhosis. Administration of exogenous NPY led to marked recovery from desensitization and vasoconstrictive improvement in cirrhotic rats, being associated with more pronounced decrease of NA release. CONCLUSIONS: Pronounced depletion of splanchnic arterial pressure-response to repetitive sympathetic nerve stimulation in cirrhosis is partly attributable to altered NA release as well as to deficient NPY release. External NPY restores vascular contractility and attenuates pathologically elevated NA release in the portal hypertensive mesenteric vasculature, revealing post-, and prejunctional effects at the vascular smooth muscle motor endplate; therefore outlining encouraging therapeutic strategies.

Dietary folic acid activates AMPK and improves insulin resistance and hepatic inflammation in dietary rodent models of the metabolic syndrome.

The AMP activated kinase plays an important role in metabolic control, and pharmacologic enhancement of AMPK activity is used to improve insulin resistance. We hypothesized that high dose of folic acid supplementation might improve insulin sensitivity and hepatic inflammation and examined this by a dietary intervention in (a) the high fat fed rat model of the metabolic syndrome, which shows sole hepatic steatosis as well as (b) in rats fed with a high cholesterol, high cholate diet inducing nonalcoholic steatohepatitis (NASH). Male Wistar rats were fed with folic acid supplemented (40 mg/kg) high fat diet [based on lard, fat content 25% (wt/wt)] or NASH inducing diet (containing 15% fat, 1.25% cholesterol, 0.5% sodium cholate). Metabolic profiling was performed by measuring the animals' visceral fat pads, fasting plasma glucose, insulin, and adipokines as well as in vivo insulin tolerance tests. Hepatic steatosis and inflammation were analyzed semiquantitatively by histological analysis. Folic acid supplementation reduced visceral obesity and improved plasma adiponectin levels. In vivo insulin sensitivity was improved, and in HF-FA rats folic acid increased activation of hepatic AMPK. Further, folic acid supplementation improved hepatic inflammation in animals fed with NASH-inducing diet. Dietary folic acid improved parameters of insulin resistance and hepatic inflammation in rodent models. This might be due to an increased AMK activation.

Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells.

Hepatic stellate cells (HSCs) are pericytes of liver sinusoidal endothelial cells (LSECs) and activation of HSC into a myofibroblast-like phenotype (called transdifferentiation) is involved in several hepatic disease processes including neovascularization during liver metastasis, chronic and acute liver injury. While early smooth muscle cell (SMC) differentiation markers including SM alpha-actin and SM22alpha are expressed in a variety of non-SMC, expression of late-stage markers is far more restricted. Here, we found that in addition to early SMC markers, activated rat HSC express a large panel of characteristic late vascular SMC markers including SM myosin heavy chain, h1-calponin and h-caldesmon. Furthermore, myocardin, which is present exclusively in SMCs and cardiomyocytes and controls the transcription of a subset of early and late SMC markers, is highly expressed in activated HSC. We further studied activated HSC in a functional three-dimensional spheroidal co-culture system together with endothelial cells (EC). Co-culture spheroids of EC and SMC differentiate spontaneously and organize into a core of SMC and a surface layer of EC representing an inside-outside model of the physiological assembly of blood vessels. Replacing SMC by in vitro activated HSC resulted in a similar organized spheroid with differentiated, von-Willebrand factor producing, surface lining quiescent human umbilical vein endothelial cell and a core of HSC. In an in vitro angiogenesis assay, activated HSC induced quiescence in vascular EC-the hallmark of vascular SMC function. Co-spheroids of LSEC and activated HSC formed capillary-like sprouts in gel angiogenesis assays expressing the vascular EC marker VE-cadherin. Our findings indicate that activated HSC are capable to adapt a functional SMC phenotype and to induce formation of tubular sprouts by LSEC and vascular endothelial cells. Since tumors and tumor metastasis induce HSC activation, HSC may take part in tumor-induced neoangiogenesis by adapting SMC-like functions.

The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma.

BACKGROUND: Melanoma inhibitory activity 2 (MIA2) is a novel gene of the MIA gene family. The selective expression of MIA2 in hepatocytes is controlled by hepatocyte nuclear factor (HNF) 1 binding sites in the MIA2 promotor. In contrast, in most hepatocellular carcinomas (HCC) MIA2 expression is down-regulated or lost. AIM: In this study we examined the regulation and functional role of MIA2 in hepatocancerogenesis. METHODS AND RESULTS: In HCC cell lines and tissues HNF-1 expression was lower than in primary human hepatocytes (PHH) and corresponding non-tumorous tissue, respectively, and correlated significantly with the down-regulation of MIA2 expression. Re-expression of HNF-1 in HCC cells reinduced MIA2 in HCC cells to similar levels as found in PHH. Further, MIA2 was re-expressed in HCC cell lines by stable transfection, and the generated cell clones revealed a strongly reduced invasive potential and proliferation rate in vitro. In line with these findings treatment of HCC cells with recombinant MIA2 inhibited proliferation and invasion. In nude mice MIA2 re-expressing HCC cells grew significantly slower and revealed a less invasive growth pattern. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding non-cancerous liver tissue of 85 patients confirmed reduced MIA2 expression in HCC. Furthermore, MIA2 negative HCC tissue showed a significantly higher Ki67 labelling index and loss of MIA2 expression correlated significantly with more advanced tumour stages. CONCLUSION: This study presents MIA2 as an inhibitor of HCC growth and invasion both in vitro and in vivo, and consequently, as a tumour suppressor of HCC. Further, our findings indicate a novel mechanism, how loss of HNF-1 expression in HCC affects tumorigenicity via down-regulation of MIA2.

Hepatitis C - contamination of toothbrushes: myth or reality?

Chronic hepatitis C patients are advised not to share toothbrushes, razors, nail-scissors or other personal articles that potentially may have been in contact with blood, with others. This study examines the contamination of toothbrushes in patients with chronic hepatitis C as a model for a possible unconventional way of transmission. In 30 patients with chronic hepatitis C, 2 mL of saliva (before and after toothbrushing) and the toothbrush rinsing water after toothbrushing were tested for HCV-RNA. Saliva before and after toothbrushing was positive for HCV-RNA in nine (30%) and 11 patients (36.7%), respectively. Twelve of the toothbrush rinsing water specimens (40%) tested HCV-RNA-positive. In six of these 12 patients, the 'native' saliva had been negative for HCV-RNA. Patients with HCV-RNA-positive toothbrush rinsing water showed no significant differences from those with negative rinsing water with respect to certain clinical, biochemical and virological parameters. In conclusion, our study demonstrates a contamination with HCV-RNA of a considerable portion of toothbrushes used by hepatitis C patients, suggesting at least a theoretical risk of infection by sharing these objects and strengthening the recommendations to take care of a clear separation of these personal care objects between patients and their household members.

Association between serum ferritin and the insulin resistance syndrome in a representative population.

OBJECTIVE: Unexplained hepatic iron overload with increased serum ferritin (SF) values has been associated with the insulin resistance syndrome (IRS), defined by the presence of one or more of the following criteria: increased body mass index (BMI), diabetes, hyperlipidemia or hypertension. However, as yet the association between IRS and SF in a representative population has not been investigated. METHODS: The study subjects participated in a nationwide epidemiological survey on metabolic disorders in the adult German population. The 1200 probands included in this study are representative of the German population. To eliminate major causes of secondary iron overload, 114 (9.5%) subjects with excessive alcohol consumption and 16 (1.5%) subjects with serological evidence for hepatitis B or C were excluded. For all remaining 1070 probands, complete clinical data of SF, HbA1c, known diabetes, BMI, cholesterol, high-density lipoprotein-cholesterol and blood pressure were available. RESULTS: SF values were significantly increased in men and women with high BMI (> 25 kg/m2), increased cholesterol (> 200 mg/dl), and increased systolic (> 160 mmHg) blood pressure, in women with diabetes, and in men with increased diastolic (> 95 mmHg) blood pressure. Furthermore, there was a significant correlation between the number of IRS criteria and SF. CONCLUSIONS: This study shows a significant correlation between SF and the presence of IRS criteria in a large representative population. Interestingly, the severity of the IRS seems to be associated with increased SF levels suggesting a causal connection. Further studies are required to investigate the pathophysiological mechanism and consequences of increased SF levels in patients with IRS.

Different effects of a CD14 gene polymorphism on disease outcome in patients with alcoholic liver disease and chronic hepatitis C infection.

AIM: Clinical and experimental data suggest that gut-derived endotoxins are an important pathogenic factors for progression of chronic liver disease. Recently, a C-T (-159) polymorphism in the promoter region of the CD14 gene was detected and found to confer increased CD14 expression and to be associated with advanced alcoholic liver damage. Here, we investigated this polymorphism in patients with less advanced alcoholic liver disease (ALD) and chronic hepatitis C virus (HCV) infection. METHODS: CD14 genotyping was performed by PCR-RFLP analysis in (a) 121 HCV patients, (b) 62 patients with alcohol-associated cirrhosis (Alc-Ci), (c) 118 individuals with heavy alcohol abuse without evidence of advanced liver damage (Alc-w/o Ci), and (d) 247 healthy controls. Furthermore, serum levels of soluble CD14 (sCD14) and transaminases were determined. RESULTS: The TT genotype was significantly more frequent in Alc-Ci compared to Alc-w/o Ci or controls (40.3% vs 23.7% or 24.0%, respectively). In Alc-w/o Ci, serum levels of transaminases did not differ significantly between patients with different CD14 genotypes. In HCV patients, TT-homozygotes had significantly higher sCD14 levels and sCD14 serum levels were significantly higher in patients with advanced fibrosis or cirrhosis. However, no association was found between CD14 genotypes and histological staging or grading. CONCLUSION: Considering serum transaminases as surrogate markers for alcoholic liver damage, the CD14 polymorphism seems to exhibit different effects during the course of ALD. Differences in genotype distribution between cirrhotic HCV patients and alcoholics and the known functional impact of this polymorphism on CD14 expression levels further indicate differences in the pathophysiological role of CD14 and CD14-mediated lipopolysaccharides signal transduction with regard to the stage as well as the type of the underlying liver disease.

Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma.

AIMS: To determine the expression of a protein termed augmenter of liver regeneration (ALR), recently found to have a specific and beneficial effect on the process of liver regeneration in normal and diseased human liver. METHODS AND RESULTS: ALR expression in normal and cirrhotic human livers with various underlying diseases as well as in tissue samples of hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) was analysed by immunohistochemistry and quantitative reverse transciptase-polymerase chain reaction (RT-PCR). Expression analysis of ALR in total liver protein extracts by Western blotting showed mainly dimeric ALR protein. Immunohistochemically, cytosolic and perinuclear immunosignals were found in hepatocytes and cholangiocytes in normal, cirrhotic or cancerous liver tissue and only weak signals in some endothelial cells in normal livers. Quantitative mRNA analysis revealed significantly increased ALR expression in cirrhosis compared with normal liver tissue. In HCC and CCC ALR mRNA expression was also significantly enhanced compared with normal liver tissue, but expression levels did not differ from the matching non-neoplastic tissue in the same patient. CONCLUSIONS: The findings suggest an important role for ALR in hepatocellular regeneration in liver cirrhosis as well as in hepatocarcinogenesis and therefore its potential value in the clinical diagnosis of hepatic cirrhosis and cancer.

Loss of E-cadherin leads to upregulation of NFkappaB activity in malignant melanoma.

Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here, we show that loss of E-cadherin leads to induction of nuclear factor kappa B (NFkappaB) activity in melanoma cell lines. Melanoma cells show constitutively active NFkappaB, whereas no activity is found in primary melanocytes. After re-expression of E-cadherin in melanoma cells, strong downregulation of NFkappaB activity was found. Consistently, NFkappaB activity was induced in primary human melanocytes after inhibition of E-cadherin activity by functionally blocking anti-E-cadherin antibodies. Interestingly, re-expression of E-cadherin-blocked p38 MAPK activity and the p38 MAPK inhibitors SB203580 and SB202190 almost completely prevented NFkappaB activation in melanoma cells. Furthermore, cytoplasmatic beta-catenin induced p38 and NFkappaB activation in malignant melanoma. To our knowledge, this is the first report suggesting a correlation between E-cadherin and NFkappaB activity in melanocytes and melanoma cells. In summary, we conclude that loss of E-cadherin and cytoplasmatic beta-catenin induces p38-mediated NFkappaB activation, potentially revealing an important mechanism of tumorigenesis in malignant melanomas.

Differential effects of deoxycholic acid and taurodeoxycholic acid on NF-kappa B signal transduction and IL-8 gene expression in colonic epithelial cells.

Several effects of bile acids (BAs) on colonic epithelial cells (CECs) have been described, including induction of proliferation and apoptosis. Some of these effects are mediated through activation of the NF-kappa B transcriptional system. In this study, we investigated the molecular mechanisms underlying the BA-induced gene expression in CECs. The human CEC line HT-29 and primary human CECs were treated with dilutions of salts of deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). NF-kappa B binding activity was analyzed with EMSA, RelA translocation with immunofluorescence, and I kappa B alpha- and RelA-phosphorylation with Western blot analysis. IL-8 mRNA and protein expression were assessed by quantitative PCR and ELISA. Functional impact of NF-kappa B activation was determined by blocking the proteasome activity with MG132 or by preventing IKK activity with a dominant-negative IKK beta delivered by adenoviral dominant-negative (dn) IKK beta (Ad5dnIKK beta). DCA and TDCA induced IL-8 expression in a dose- and time-dependent manner. It is interesting that DCA but not TDCA induced I kappa B alpha-phosphorylation, RelA translocation, and NF-kappa B binding activity. Accordingly, the proteasome inhibitor MG132 blocked DCA- but not TDCA-induced IL-8 gene expression. In contrast, TDCA-induced IL-8 gene expression correlated with enhanced RelA phosphorylation, which was blocked by Ad5dnIKK beta. Our data suggest that DCA-induced signal transduction mainly utilized the I kappa B degradation and RelA nuclear translocation pathway, whereas TDCA primarily induced IL-8 gene expression through RelA phosphorylation. These differences may have implications for the understanding of the pathophysiology of inflammation and carcinogenesis in the gut.

Mutation analysis of the HFE gene in German hemochromatosis patients and controls using automated SSCP-based capillary electrophoresis and a new PCR-ELISA technique.

BACKGROUND: The diagnosis of hereditary hemochromatosis (HH) before the onset of iron-overload has been difficult in the past. However, a convincing candidate gene for HH: HFE has been described recently. The aims of this study were: 1) To determine the prevalence of the hemochromatosis associated mutations C282Y and H63D of the HFE gene in patients from Southern Germany with hemochromatosis phenotype; and 2) to test two new, time- and cost-saving methods: automated SSCP-based capillary electrophoresis (SSCP-CE) and a PCR-ELISA technique for the analysis of HFE mutations. METHODS: HFE genotype was studied in 36 unrelated HH patients and 126 controls from Southern Germany. In addition, family screening was performed in 76 relatives. The C282Y and H63D mutations were detected using SSCP-CE and restriction length polymorphism (RFLP). The C282Y mutation was additionally analysed by a PCR-ELISA. RESULTS: Twenty-six (72%) HH patients were homozygous for mutation C282Y, and three compound heterozygous for C282Y and H63D. One patient was homozygous for H63D. By performing family screening, six additional patients with the +/+ C282Y mutation were identified. The results of the SSCP-CE and the PCR-ELISA analysis agreed completely with data obtained by RFLP. CONCLUSIONS: SSCP-CE and PCR-ELISA analysis proved to be reliable methods for HFE genotyping and therefore represent cost- and time-effective alternative methods to the widely used restriction analysis allowing large populations to be screened for HH associated with HFE mutations. Surprisingly, only 72% of our HH patients had the C282Y +/+ genotype. This indicates that hemochromatosis in Southern Germany is genetically more heterogeneous than in other regions. A challenge for the future will be to define the genetic or environmental factors responsible for iron-overload in HH patients who do not show typical alterations of the HFE gene.

[Clinical and genetic aspects of hereditary hemochromatosis]. / Klinische und genetische Aspekte der hereditären Hämochromatose.

Hereditary hemochromatosis (HH) is an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. As laboratory parameters for measuring HH lack specificity, and HH remains asymptomatic for a long time, methods for genetic screening are highly valuable. Mutations in two genes, hfe and TfR2, have recently been found to be responsible for HH. The mutation C282Y in the hfe gene is detected in 70-95% of German and Austrian HH patients. Mutations in the TfR2 gene have been detected only very recently, and results of larger epidemiological studies are not yet available. Molecular methods permit molecular diagnosis and genetic screening.

Hepatocellular carcinoma in southern Germany: epidemiological and clinicopathological characteristics and risk factors.

UNLABELLED: The aetiology of chronic liver disease leading to hepatocellular carcinoma (HCC) and the clinical characteristics of patients with HCC vary considerably internationally and intranationally. This study analyses the characteristics of HCC patients in southern Germany, a low endemic area of HCC. METHODS: The files of 118 consecutive patients with HCC observed in a single tertiary care hospital between 1994 and 2000 have been reviewed. Epidemiological and clinicopathological characteristics such as age at presentation, ethanol consumption, serological hepatitis virus markers, and fibrosis were studied. Additionally, serum levels of alpha-fetoprotein (AFP) were analysed at the time of diagnosis in 77 patients. RESULTS: The male:female ratio was 4:1 and the mean age at presentation was 61.8 years. Alcohol abuse (49.2%) and chronic hepatitis C infection (17.8%) were the most frequent risk factors. Histologically proven liver cirrhosis in the surrounding non-tumorous tissue was present in only 59.0% of cases. AFP levels were elevated in 78% of cases, but only 34% reached >500 ng/ml, a value considered to be significant for the diagnosis of HCC. AFP levels correlated with the stage of fibrosis. SUMMARY AND CONCLUSIONS: The sensitivity of AFP serum levels as a tumour marker is poor but might help to detect at least a minority of cases. As in other populations within Europe, chronic alcohol abuse is frequently associated with HCC in southern Germany, confirming that alcohol is still the most important risk factor for hepatocarcinogenesis in areas with low hepatitis virus prevalence. Considering the poor prognosis of HCC, prevention is of pivotal importance, particularly for patients with chronic liver disease and other risk factors for the development of HCC.

Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview.

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.