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.

[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.

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.

[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.

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.

[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.

[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).

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.

Renal carcinogenesis in models of diabetes in rats: metabolic changes are closely related to neoplastic development.

AIMS/HYPOTHESIS: There is an increased risk of renal cell carcinoma (RCC) in human diabetes mellitus. We therefore examined the influence of hyperglycaemia and glucose-lowering treatment on nephrocarcinogenesis in rats. METHODS: Rats (n = 850), which were either spontaneously diabetic, streptozotocin-diabetic or normoglycaemic, were examined with special reference to Armanni-Ebstein lesions (AEL). RESULTS: Irrespective of the cause of diabetes, diabetic but not normoglycaemic rats developed typical glycogenotic clear-cell AEL. AEL showed strong proliferative activity, which was nearly completely inhibited by EGF receptor blockade (Gefitinib treatment). Many findings suggested a stepwise development of RCCs from AEL. Whereas the number and size of RCCs gradually increased in all diabetic groups, beginning at 6 months after onset of diabetes, normoglycaemic controls did not developed RCC. After 28 months, up to 82% of diabetic animals had at least one RCC. In contrast to the proximal tubules, the distal tubular system, including glycogenotic AEL, had the same levels of enzyme activities as RCC (e.g. high glycogen phosphorylase and synthase activity, lack of glucose 6-phosphatase activity) and the same expression patterns of cytokeratin 7 and several growth factors, along with their receptors and signal transduction proteins (TGF-alpha, EGF receptor, IGF-I, IGF-I receptor, IGF-II receptor, insulin receptor substrate 1, v-raf-1 murine leukemia viral oncogene homologue 1 and mitogen activated protein kinase kinase 1). In addition, direct morphological transitions between distal tubules, AEL and RCCs were frequently observed. All these findings indicate a common origin and a precursor-product relationship of AEL and RCCs. CONCLUSIONS/INTERPRETATION: Nephrocarcinogenesis in diabetic rats results from sustained hyperglycaemia, resulting in an adaptive metabolic response, altered growth factor signalling and subsequent neoplastic transformation of the tubular epithelial cells.

Endoplasmic and vascular surface activation during organ preservation: refining upon the benefits of machine perfusion.

The endoplasmic reticulum (ER) represents a subcellular target reactive to various cytosolic impairments. The involvement of ER-stress in organ preservation was investigated, comparing machine preservation, cold storage (CS) and a novel concept of only temporary perfusion after procurement. Rat livers were retrieved 30 min after cardiac arrest and preserved for 18 h by CS, oxygenated machine perfusion for 18 h (18 h MP) or for 2 h with subsequent CS for 16 h (2 h MP + 16 h CS). Upon reperfusion, 18 h MP significantly improved enzyme leakage (ALT, LDH) and promoted a 2-fold increase of metabolic recovery compared to CS. However, vascular stress, evaluated by endothelin-release, was significantly elevated after 18 h MP. Interestingly, better viability was obtained using the short-term perfusion protocol (2 h MP + 16 h CS), which further reduced enzyme leakage, maintained energetic recovery and mitigated endothelin-release compared to 18 h MP. Caspase 12-mRNA was upregulated in the 18 h MP-group but unchanged after CS or 2 h MP + 16 h CS. Activation/cleavage of caspase 12 protein was significantly enhanced after 18 h MP and very low in the 2 h MP + 16 h CS-group. Correspondingly, electron microscopy showed ultrastructural alterations of ER after CS and especially after 18 h MP but not after 2 h MP + 16 h CS. At this time mitochondrial appearance was unaffected in all groups, suggesting the ER to be an early subcellular target of preservation injury. In our model, ER and vascular endothelium were best protected by only temporary machine perfusion, which also maintained overall graft viability.

Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver.

Ursodeoxycholic acid (UDCA) exerts anticholestatic effects by undefined mechanisms. Previous work suggested that UDCA stimulates biliary exocytosis via Ca(++)- and protein kinase C (PKC)-dependent mechanisms. Therefore, the effect of taurine-conjugated UDCA (TUDCA) was studied in the experimental model of taurolithocholic acid (TLCA)-induced cholestasis on bile flow, hepatobiliary exocytosis, distribution of PKC isoforms, and density of the apical conjugate export pump, Mrp2, in canalicular membranes. Isolated perfused rat livers were preloaded with horseradish peroxidase (HRP), a marker of vesicular exocytosis, and were perfused with bile acids or dimethylsulfoxide (control) only. PKC isoform distribution and membrane density of Mrp2 were studied using immunoblotting and immunoelectron-microscopic techniques. Biliary secretion of the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione (GS-DNP), was studied in the presence or absence of the PKC inhibitor, bisindolylmaleimide I (BIM-I; 1 micromol/L). TLCA (10 micromol/L) impaired bile flow by 51%; biliary secretion of HRP and GS-DNP by 46% and 95%, respectively; membrane binding of the Ca(++)-sensitive alpha-isoform of PKC by 32%; and density of Mrp2 in the canalicular membrane by 79%. TUDCA (25 micromol/L) reversed the effects of TLCA on bile flow, secretion of HRP and GS-DNP, and distribution of alpha-PKC. TUDCA reduced membrane binding of epsilon-PKC and increased Mrp2 density 4-fold in canalicular membranes of cholestatic hepatocytes. BIM-I inhibited the effect of TUDCA on GS-DNP secretion in cholestatic livers by 49% without affecting secretion in controls. In conclusion, TUDCA may enhance the secretory capacity of cholestatic hepatocytes by stimulation of exocytosis and insertion of transport proteins into apical membranes via PKC-dependent mechanisms.