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.

[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.

The IGF axis and hepatocarcinogenesis.

Deregulation of the insulin-like growth factor (IGF) axis, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and the expression of the IGF receptors, has been identified in the development of hepatocellular carcinoma (HCC). Characteristic alterations detected in HCC and hepatoma cell lines comprise the increased expression of IGF-II and the IGF-I receptor (IGF-IR), which have emerged as crucial events in malignant transformation and the growth of tumours. Alterations of IGFBP production and the proteolytic degradation of IGFBPs resulting in an excess of bioactive IGFs, as well as the defective function of the IGF degrading IGF-II/mannose 6-phosphate receptor (IGF-II/M6PR), may further potentiate the mitogenic effects of IGFs in the development of HCC.

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.

Fatal pneumonia in an AIDS patient coinfected with adenovirus and Pneumocystis carinii.

BACKGROUND: Adenovirus infections are common in immunocompromised hosts. However, pulmonary adenovirus infections rarely cause significant morbidity in HIV-infected patients. PATIENT: Here we describe a 27-year-old man with AIDS who presented with tachypnea, hypoxemia and an infiltrate in the upper left lobe on chest X-ray. Bronchoscopy was performed and Pneumocystis carinii was detected in bronchoalveolar lavage (BAL) fluid. Treatment with cotrimoxazole and prednisone initially resulted in improvement, but after 10 days the patient's clinical condition deteriorated rapidly and he died after 23 days due to respiratory failure. RESULTS: On autopsy histopathologic examination showed abundant "smudge cells," suggestive of adenoviral infection. Electron microscopy revealed adenovirus-like particles arrayed in a paracrystalline manner. Subsequent immunohistochemistry confirmed the extensive presence of adenovirus in addition to P. carinii. CONCLUSION: This case demonstrates a pathogenetic role for adenovirus coinfection in P. carinii pneumonia (PCP). Earlier diagnosis, e.g. by PCR analysis of the BAL fluid or transbronchial biopsy, might have led to the consideration of ribavirin treatment.

Analysis of the IGF axis in preneoplastic hepatic foci and hepatocellular neoplasms developing after low-number pancreatic islet transplantation into the livers of streptozotocin diabetic rats.

Preneoplastic hepatic foci have been demonstrated in liver acini, which drain the blood from intraportally transplanted pancreatic islets in streptozotocin-induced diabetic rats with mild persisting diabetes. In long-term studies of this animal model, hepatocellular adenomas and carcinomas (HCC) developed after a sequence of characteristic preneoplastic hepatic foci. In this experimental model, the local hyperinsulinism is thought to have a causative role. Because insulin and the insulin-like growth factor (IGF) axis are closely linked, an altered gene expression of the IGF axis components is likely. Therefore, preneoplastic hepatic foci and HCC were studied for the expression of IGF axis components. Glycogen-storing "early" preneoplastic hepatic foci were detectable several days after pancreatic islet transplantation. Northern blot analysis, in-situ hybridization, and immunohistochemical studies of these "early" lesions demonstrated increased expressions of IGF-I and IGF binding protein-4 (IGFBP-4) in altered parenchymal cells, and a decreased expression of IGFBP-1. IGF-II was not detected in these preneoplastic foci. HCC arising in this model had decreased expressions of IGF-I and IGFBP-4 but IGFBP-1 expression was not significantly altered. Some HCC showed a more than 100-fold overexpression of IGF-II, whereas other tumors were completely negative for IGF-II expression. Low IGF-I receptor expression was detected in preneoplastic foci and adjacent nonaltered liver tissue. However, HCC tissue consistently showed an increased IGF-I receptor expression, rendering these tissues susceptible to the mitogenic effects of IGF. The altered gene expression in glycogen-storing preneoplastic hepatic foci, especially the up-regulation of IGF-I and IGFBP-4 with the down-regulation of IGFBP-1, resemble the insulin-dependent regulation of these components in normal rat hepatocytes. These data agree with previous studies demonstrating a correspondence of the focal character, morphology, and enzyme pattern of preneoplastic hepatic foci with insulin effects on hepatocytes. The development from preneoplastic foci to HCC may be driven by insulin itself and/or an altered IGF axis component or yet unidentified factors.