Viral expression and molecular profiling in liver tissue versus microdissected hepatocytes in hepatitis B virus - associated hepatocellular carcinoma

Abstract Background The molecular mechanisms whereby hepatitis B virus (HBV) induces hepatocellular carcinoma (HCC) remain elusive. We used genomic and molecular techniques to investigate host-virus interactions by studying multiple areas of the same liver from patients with HCC. Methods We compared the gene signature of whole liver tissue (WLT) versus laser capture-microdissected (LCM) hepatocytes along with the intrahepatic expression of HBV. Gene expression profiling was performed on up to 17 WLT specimens obtained at various distances from the tumor center from individual livers of 11 patients with HCC and on selected LCM samples. HBV markers in liver and serum were determined by real-time polymerase chain reaction (PCR) and confocal immunofluorescence. Results Analysis of 5 areas of the liver showed a sharp change in gene expression between the immediate perilesional area and tumor periphery that correlated with a significant decrease in the intrahepatic expression of HB surface antigen (HBsAg). The tumor was characterized by a large preponderance of down-regulated genes, mostly involved in the metabolism of lipids and fatty acids, glucose, amino acids and drugs, with down-regulation of pathways involved in the activation of PXR/RXR and PPARα/RXRα nuclear receptors, comprising PGC-1α and FOXO1, two key regulators critically involved not only in the metabolic functions of the liver but also in the life cycle of HBV, acting as essential transcription factors for viral gene expression. These findings were confirmed by gene expression of microdissected hepatocytes. Moreover, LCM of malignant hepatocytes also revealed up-regulation of unique genes associated with cancer and signaling pathways, including two novel HCC-associated cancer testis antigen genes, NUF2 and TTK. Conclusions Integrated gene expression profiling of whole liver tissue with that of microdissected hepatocytes demonstrated that HBV-associated HCC is characterized by a metabolism switch-off and by a significant reduction in HBsAg. LCM proved to be a critical tool to validate gene signatures associated with HCC and to identify genes that may play a role in hepatocarcinogenesis, opening new perspectives for the discovery of novel diagnostic markers and therapeutic targets

Sequential therapy for first-line helicobacter pylori eradication: 10- or 14-day regimen?

Background & Aim: Standard 10-day sequential therapy is advised as first-line therapy for Helicobacter pylori (H. pylori) eradication by current Italian guidelines. Some data suggested that a 14-day regimen may achieve higher eradication rates. This study compared the efficacy of sequential therapy administered for either 10- or 14-days. Methods: This prospective, multicenter, open-label study enrolled patients with H. pylori infection without previous treatment. Patients were receiving a sequential therapy for either 10 or 14 days with esomeprazole 40 mg and amoxicillin 1 g (5 or 7 days) followed by esomeprazole 40 mg, clarithromycin 500 mg and tinidazole 500 mg (5 or 7 days), all given twice daily. Bacterial eradication was checked using 13 C-urea breath test. Eradication cure rates were calculated at both Intention-to-treat (ITT) and per-protocol (PP) analyses. Results: A total of 291 patients were enrolled, including 146 patients in 10-day and 145 in the 14-day regimen. The eradication rates were 87% (95% CI = 81.5-92.4) and 90.3% (95% CI = 85.5-95.1) at ITT analysis with the 10- and 14-day regimen, respectively, and 92.7% (95% CI = 88.3-97) and 97% (95% CI = 94.2-99.9) at PP analysis (p =0.37). Among patients, who earlier had interrupted therapy, bacterial eradication was achieved in 8 out of 9 who completed the first therapy phase and performed at least ≥3 days of triple therapy in the second phase. Conclusion: This study found that both 10- and 14-day sequential therapies achieved a high eradication rate for first-line H. pylori therapy in clinical practice

Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence

Candida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38–mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]–MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render C. albicans incapable of causing epithelial damage and markedly reduce mucosal infection in vivo. Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for C. albicans pathogenicity at mucosal surfaces