[Nuclear Her2 expression in hepatocytes in liver disease]. / Nukleäre HER2/neu-Expression in Hepatozyten bei Lebererkrankungen.

BACKGROUND: Her2 is a well-known member of the epidermal growth factor receptor (EGFR) superfamily, a group of transmembrane receptors that mediate effects of proliferation and survival and thus play an important role in tumorigenesis. EGFRs can translocate to the nucleus and may mediate DNA repair and cell cycle arrest. OBJECTIVES: The aim of this study was to characterize hepatocellular Her2 expression in different liver diseases. MATERIALS AND METHODS: Her2 expression was analyzed by immunohistochemistry in 674 liver biopsies. RESULTS: Hepatocytes often revealed a nuclear and cytoplasmic Her2 expression in different liver diseases with the strongest association to alcoholic steatohepatitis. The histologic parameters of hepatocellular ballooning and the presence of Mallory-Denk bodies strongly correlated with Her2 positivity. Interestingly, in hepatocellular carcinomas (HCC) nuclear Her2 expression was frequently observed. Furthermore, Her2 positive hepatocytes showed a loss of estrogen receptor expression and increased expression of p21, a cell cycle regulator, and pSTAT3, a downstream effector of nuclear Her2. CONCLUSIONS: Nuclear Her2 expression in hepatocytes with further metabolic and cell cycle alterations may imply a so far unknown mechanism of a stress response. So far, the effects on disease course and a possible role of nuclear Her2 in progression to HCC are unclear and the subject of future research.

Febrile ulceronecrotic Mucha-Habermann disease following suspected hemorrhagic chickenpox infection in a 20-month-old boy.

We present the youngest pediatric patient so far with febrile ulcerative Mucha-Haberman disease (FUMHD) after an admitting clinical picture of hemorrhagic varicella infection. With a time to diagnosis of 25 days, the 20-month-old boy responded to low dose cyclosporine and prednisolone given for 3 months and is free of disease after 4 years of follow up. We describe a polyclonal CD8+ T cell response with elevated pro-inflammatory cytokines and a fivefold upregulation of the high-affinity Fc receptor type I (CD64) on granulocytes. Early consideration of FUMHD in the differential diagnosis of a systemic inflammatory disease combined with a generalized necrotizing rash is important for early and adequate management of children with this rare and challenging disease.

[Molecular and metabolic changes in human clear cell liver foci]. / Molekulare und metabolische Veränderungen in humanen klarzelligen Leberherden.

Activation of the AKT/mTOR and Ras/MAPK pathways and the lipogenic phenotype are evident both in human hepatocellular carcinoma and in the rat model of insulin-induced hepatocarcinogenesis in the earliest preneoplastic lesions, i.e. clear cell foci (CCF) of altered hepatocytes. These CCFs have also been described in the human liver but characterization of molecular and metabolic changes are still pending. In this study, human sporadic CCFs were investigated in a collection of human non-cirrhotic liver specimens using histology, histochemistry, immunohistochemistry, electron microscopy and molecular pathological analysis. Human CCFs occurred in approximately 33 % of non-cirrhotic livers and stored masses of glycogen in the cytoplasm, largely due to reduced activity of glucose-6-phosphatase. Hepatocytes revealed an upregulation of the AKT/mTOR and the Ras/MAPK pathways, the insulin receptor, glucose transporters and enzymes of glycolysis and de novo lipogenesis. Proliferative activity was 2-fold higher than in extrafocal tissue. The CCFs of altered hepatocytes are metabolically and proliferatively active lesions even in humans. They resemble the well-known preneoplastic lesions from experimental models in terms of morphology, glycogen storage, overexpression of protooncogenic signaling pathways and activation of the lipogenic phenotype, which are also known in human hepatocellular carcinoma. This suggests that hepatic CCFs also represent very early lesions of hepatocarcinogenesis in humans.

[Overwhelming postsplenectomy infection syndrome]. / Fulminante Pneumokokkensepsis nach Splenektomie.

The overwhelming postsplenectomy infection syndrome (OPSI) is a fulminant sepsis that is mainly caused by Streptococcus pneumoniae and is characterized by a particular high mortality. Patients whose spleen was removed due to a hematological disease are at special risk. Even after the recommended immunization against Streptococcus pneumoniae 20-30 % of these patients do not develop an adequate level of antibody response. Therefore, this particular group of patients must be trained how to behave in case of fever and need to obtain immediate specific sepsis therapy with antibiotic prophylaxis.

Deregulation of DNA-dependent protein kinase catalytic subunit contributes to human hepatocarcinogenesis development and has a putative prognostic value.

BACKGROUND: The DNA-repair gene DNA-dependent kinase catalytic subunit (DNA-PKcs) favours or inhibits carcinogenesis, depending on the cancer type. Its role in human hepatocellular carcinoma (HCC) is unknown. METHODS: DNA-dependent protein kinase catalytic subunit, H2A histone family member X (H2AFX) and heat shock transcription factor-1 (HSF1) levels were assessed by immunohistochemistry and/or immunoblotting and qRT-PCR in a collection of human HCC. Rates of proliferation, apoptosis, microvessel density and genomic instability were also determined. Heat shock factor-1 cDNA or DNA-PKcs-specific siRNA were used to explore the role of both genes in HCC. Activator protein 1 (AP-1) binding to DNA-PKcs promoter was evaluated by chromatin immunoprecipitation. Kaplan-Meier curves and multivariate Cox model were used to study the impact on clinical outcome. RESULTS: Total and phosphorylated DNA-PKcs and H2AFX were upregulated in HCC. Activated DNA-PKcs positively correlated with HCC proliferation, genomic instability and microvessel density, and negatively with apoptosis and patient's survival. Proliferation decline and massive apoptosis followed DNA-PKcs silencing in HCC cell lines. Total and phosphorylated HSF1 protein, mRNA and activity were upregulated in HCC. Mechanistically, we demonstrated that HSF1 induces DNA-PKcs upregulation through the activation of the MAPK/JNK/AP-1 axis. CONCLUSION: DNA-dependent protein kinase catalytic subunit transduces HSF1 effects in HCC cells, and might represent a novel target and prognostic factor in human HCC.

Repeated exposure of the oral mucosa over 12 months with cold plasma is not carcinogenic in mice.

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

[Hepatocellular carcinoma in the non-cirrhotic liver]. / Hepatozelluläre Karzinome in der nichtzirrhotischen Leber.

Of hepatocellular carcinomas (HCC), 15-20% occur in the non-cirrhotic liver. All factors which cause HCC when liver cirrhosis (LC) is present, can also lead to HCC without LC. On the basis of the relative frequency, HCC can be roughly differentiated into 3 groups: 1) HCC, rarely occurring without cirrhosis (e.g. virus hepatitis, alcohol abuse). 2) HCC, frequently occurring without LC (alpha1-antitrypsin deficiency, hemochromatosis, non-alcoholic fatty liver disease). 3) HCC, consistently occurring without LC (glycogen storage disease type 1, consumption of oral contraceptives/anabolic steroids). In groups 1 and 2 the level of hepatocellular toxicity necessary to reach LC is not yet achieved but the carcinogenic effect is already strong enough to induce HCC, possibly owing to the influence of additional carcinogens or host factors. In group 3, the carcinogenic effect is mediated by a long-standing alteration of the hepatocellular metabolism that is of low toxic effect and does not lead to cell death, but is nevertheless carcinogenic. In these cases, the initial formation of hepatocellular adenomas that subsequently transform into HCC is a common finding (adenoma-carcinoma sequence).

Unique and shared functions of different connexins in mice.

BACKGROUND: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two 'knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. RESULTS: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. CONCLUSIONS: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

Characterization of yellow plaques found in the small bowel during double-balloon enteroscopy.

BACKGROUND AND STUDY AIMS: The aim of this study was to characterize yellow (or whitish) plaques of the small bowel that were found during double-balloon enteroscopy (DBE) performed for small-bowel evaluation. PATIENTS AND METHODS: Patients who were being evaluated for small-bowel pathology at our institution (for a variety of indications) were included in the study. In 16 patients, DBE revealed yellow or whitish submucosal plaques, defined as small, raised, submucosal lesions that were well circumscribed and covered by normal-appearing small-bowel mucosa. Biopsy tissue obtained during the procedures was stained with hematoxylin and eosin and with periodic acid-Schiff stain, and was subjected to immunochemical testing using endothelial markers (anti-CD31 and anti-CD34). RESULTS: These 16 patients were identified out of a total of 150 DBE procedures performed in 120 patients (eight men, eight women; mean age 62, range 33 - 78). The lesions were mostly single (range 1 to > 5 lesions), ranging in size from 2 mm to 15 mm, and were slightly raised (from 1 mm to 2 mm). In four cases the plaques could not be biopsied because the patient had a coagulation disorder or because the DBE was being performed to investigate severe acute bleeding. In the other 12 patients, a characteristic white-yellow liquid exudated from the biopsy site in 80 % of lesions, and these 12 patients were shown to have lymphangiectasias. No association with an infiltrative disorder could be detected. CONCLUSIONS: Yellow and white submucosal plaques are found in up to 13 % of patients undergoing DBE. They are most likely to be lymphangiectasias and are a normal anatomical variant. They do not require further work-up.

Jagged 1 is a major Notch ligand along cholangiocarcinoma development in mice and humans.

Intrahepatic cholangiocarcinoma (ICC) is a rare yet deadly malignancy with limited treatment options. Activation of the Notch signalling cascade has been implicated in cholangiocarcinogenesis. However, while several studies focused on the Notch receptors required for ICC development, little is known about the upstream inducers responsible for their activation. Here, we show that the Jagged 1 (Jag1) ligand is almost ubiquitously upregulated in human ICC samples when compared with corresponding non-tumorous counterparts. Furthermore, we found that while overexpression of Jag1 alone does not lead to liver tumour development, overexpression of Jag1 synergizes with activated AKT signalling to promote liver carcinogenesis in AKT/Jag1 mice. Histologically, tumours consisted exclusively of ICC, with hepatocellular tumours not occurring in AKT/Jag1 mice. Furthermore, tumours from AKT/Jag1 mice exhibited extensive desmoplastic reaction, an important feature of human ICC. At the molecular level, we found that both AKT/mTOR and Notch cascades are activated in AKT/Jag1 ICC tissues, and that the Notch signalling is necessary for ICC development in AKT/Jag1 mice. In human ICC cell lines, silencing of Jag1 via specific small interfering RNA reduces proliferation and increases apoptosis. Finally, combined inhibition of AKT and Notch pathways is highly detrimental for the in vitro growth of ICC cell lines. In summary, our study demonstrates that Jag1 is an important upstream inducer of the Notch signalling in human and mouse ICC. Targeting Jag1 might represent a novel therapeutic strategy for the treatment of this deadly disease.

Evaluation of different models of experimentally induced liver cirrhosis for MRI research with correlation to histopathologic findings.

RATIONALE AND OBJECTIVES: Three models of experimentally induced liver cirrhosis were evaluated for MRI research on chronic liver disease. The influence of different histopathologic changes in liver fibrosis and cirrhosis on relaxation times and signal intensities was studied in vitro and in vivo. METHODS: Liver fibrosis and cirrhosis in rats was induced by oral or subcutaneous administration of carbon tetrachloride (CCl4) or by thioacetamide (TAA) in drinking water. On histology, the degree of liver fibrosis and cirrhosis, fatty infiltration, iron accumulation, and inflammatory changes were measured semiquantitatively. The amount of connective tissue was quantitatively determined by morphometry. The results were correlated with T1 and T2 relaxation times and signal intensities of the liver studied in vitro by relaxometry and in vivo by MRI. RESULTS: In both groups with CCl4 administration, histology revealed different degrees of liver fibrosis and cirrhosis. Subcutaneous injection of CCl4 also resulted in increased fatty infiltration. On the contrary, TAA produced complete liver cirrhosis in all animals. Overall, there was a good correlation between the liver T2 relaxation time and the amount of connective tissue in liver fibrosis and cirrhosis. However, the degree of liver fibrosis and cirrhosis was also strongly correlated with the degree of inflammatory changes. In the group with CCl4 administration, there was a good correlation between the fatty infiltration and the T1 relaxation time, as well as with the liver signal intensity on the T1-weighted gradient echo sequence. An increased iron accumulation was also correlated with the degree of liver fibrosis/cirrhosis; however, there was no significant influence of the iron on relaxation times or signal intensities. CONCLUSIONS: The TAA model is easier to perform and more reliable in liver cirrhosis induction than the CCl4 models. Although there is a positive correlation between the T2 relaxation times and the degree of liver fibrosis/cirrhosis, this probably results from the associated inflammatory changes and is not caused by the increased amount of connective tissue.

Adenovirus-induced liver necrosis in a case of AIDS.

Adenovirus-induced liver necrosis is rare. Because the era of AIDS (acquired immunodeficiency syndrome) this entity was seen predominantly in infants suffering from inborn immunodeficiency syndromes or from iatrogenic immunosuppression because of bone marrow or liver transplantation. Here, we report a case of a 30-year-old woman with AIDS who developed fever and rapidly progressing liver failure. A frozen section from a needle biopsy of the liver allowed a quick diagnosis of viral liver necrosis. The light-microscopic and electron microscopic aspects were typical of adenovirus infection and should be known to the surgical pathologist. The diagnosis was confirmed by immunohistochemistry and DNA hybridization analysis.

Hyperproliferative state of liver acini and pancreatic islets after intraportal transplantation of a small mass of islets of Langerhans in streptozotocininduced diabetic rats.

After the portal-embolic transplantation of a low number of isologous islets of Langerhans into streptozotocin-diabetic rats altered liver acini emerge down-stream to the islet grafts. The hepatocytes of these altered liver acini store high amounts of glycogen and/or fat. The proliferation of the hepatocytes of such altered acini and simultaneously their apoptotic elimination were increased strongly, compared with the surrounding unaltered liver acini. After transplantation of a high number of islets, altered liver acini were not observed. The reason for these focal phenomena was supposed to be the persistent systemic hyperglycemic diabetic state which stimulates the beta cells to produce insulin and to secrete it in maximal amounts. This hypothesis has been supported by electron-microscopical examination of the transplanted islets in a former publication, but the proliferative activity of the transplanted islet epithelial cells has not so far been examined. Furthermore, the period of observation has been only three weeks, and it seemed of interest to determine whether the locally increased hepatocytic proliferation and the stimulation of the beta-cells would persist for a longer time. 450-500 (group I, n = 14) or 1500-2000 (group II, n = 8) isologous pancreatic islets were transplanted to adult male Lewis-rats made diabetic by streptozotocin. Group I animals stayed hyperglycemic after transplantation, group II animals became normoglycemic. 5-bromo-2'-desoxyuridine (BrdU) was applied by osmotic minipumps for 6 days before death (6, 13, 21 and 67-77 days after islet transplantation). In group I animals the mitotic index (number of mitotic figures per 1000 hepatocytic nuclei), the apoptotic index (number of apoptotic bodies per 1000 hepatocytic nuclei) and the immunohistochemical-labeling index for BrdU (BrdU-labeled hepatocytic nuclei per 100 hepatocytic nuclei) of these altered acini (A) were compared with the adjacent non-altered acini (NA). The mitotic index in A was 40-fold to 100-fold increased compared with NA (for the whole period of observation). The apoptotic index in A was 6-fold to 20-fold increased compared with NA. The BrdU-LI in A was 20-fold to 80-fold increased compared with NA. In group II animals altered liver acini did not emerge. Furthermore, BrdU-LI of epithelial cells of group I islets were significantly higher compared with group II islets (28.4 vs. 6.3 at day 6, 28.8 vs. 4.2 at day 13, 23.2 vs. 2.9 at day 21, 15.3 vs. 0.1 at days 67 to 77, respectively). It is supposed that two different types of proliferative stimuli were active in this model of low number islet transplantation: 1) The chronic hyperglycemia induces endocrine epithelial cell proliferation in the grafts. 2) The local chronic hyperinsulinism induces glycogen and fat storage, proliferation and apoptotic elimination of the hepatocytes in the liver acini down-stream to the stimulated islets.

Virus-like particles in a case of acute hepatitis with human GB virus-C viraemia.

BACKGROUND/AIMS: It is still controversial whether the human GB virus-C can cause liver injury, as in situ demonstration of the virus is still inconclusive. METHODS: Here we describe the case report of a patient with two episodes of severe acute hepatitis, who had a meticulous clinical, immunological and microbiological work-up, including human GB virus-C RNA detection by in situ hybridization and electron microscopy. RESULTS: Human GB virus-C viraemia was found as the only potential cause of hepatitis in this patient. Furthermore, virus-like particles could be demonstrated in the cytoplasm of single hepatocytes by electron microscopy and human GB virus-C RNA was detected in the liver by in situ hybridization. CONCLUSION: The presence of human GB virus-C RNA in serum together with the demonstration of human GB virus-C RNA in the liver, favour acute human GB virus-C infection as the cause of liver injury in this patient. Thus virus-like particles and hepatic human GB virus-C RNA should be specifically looked for by electron microscopy and in situ hybridization, especially during the diagnostic work-up of patients with unexplained hepatitis.