GMP-based CD133(+) cells isolation maintains progenitor angiogenic properties and enhances standardization in cardiovascular cell therapy.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

pRb2/p130, vascular endothelial growth factor, p27(KIP1), and proliferating cell nuclear antigen expression in hepatocellular carcinoma: their clinical significance.

Hepatocarcinoma (HCC) is the fifth most common cancer, with more than one million fatalities occurring annually worldwide. Multiple risk factors are associated with HCC disease etiology, the highest incidence being in patients with chronic hepatitis B virus and hepatitis C virus, although other factors such as genetic makeup and environmental exposure are involved. Multiple genetic alterations including the activation of oncogenes and inactivation of tumor suppressor genes are required for malignancy in human cancers and are correlated with increased stages of carcinogenesis and further tumor progression. In this study of 21 HCC patients, we analyzed pRb2/p130, vascular endothelial growth factor (VEGF), p27((KIP1)), and proliferating cell nuclear antigen as potential HCC molecular biomarkers. In our sample set, we found that p27((KIP1)) was absent. Univariate survival analysis showed that proliferating cell nuclear antigen expression (diffuse staining >50% of positive cells in tumor) was confirmed as a significant HCC prognostic biomarker for determining patient survival agreeing with previous studies (P = 0.0126, log-rank test). Lower pRb2/p130 expression was associated to a borderline P value of inverse correlation with tumor malignancy and to a positive correlation with respect to the time from HCC diagnosis (Spearman coefficient = 0.568; P < 0.05). Conversely, higher VEGF expression was associated with a poor survival (P = 0.0257, log-rank test). We demonstrate for the first time that pRb2/p130 is inversely correlated with VEGF expression and tumor aggressiveness (P < 0.05) in p27((KIP1))-negative HCC patients. pRb2/p130 and VEGF expression are independent from tumor staging, suggesting their possible role as independent prognostic molecular biomarkers in HCC. Furthermore, we have evidence that VEGF together with pRb2/p130 may act as new HCC biomarkers in a p27((KIP1))-independent manner. Additional studies with larger numbers of patient data would allow the use of multivariable techniques and would be able to further identify patients with poorer survival.

Successful MARS treatment in severe cholestatic patients with acute on chronic liver failure.

Extracorporeal liver devices have gained great attention as a complementary approach to liver transplantation in patients with acute on chronic liver failure. Among others, Molecular Adsorbent Recycling System (MARS) is a hemodiafiltration against albumin able to remove low molecular weight toxins. We aimed to validate the use of MARS in patients presenting with acute on chronic liver failure with severe cholestasis. We enrolled 7 patients with acute on chronic liver failure, presenting with bilirubin >25 mg/dl and hepatorenal syndrome and/or hepatic encephalopathy grade >II. Liver biochemistry, coagulation, blood cell count, electrolytes, ammonia, lactate, blood urea nitrogen, creatinine, bile acids, Fischer ratio, and encephalopathy grade were assessed before and after each MARS treatment. MARS can represent a safe therapeutic choice to achieve a quick improvement of neurological status, a hemodynamic stability, and a better clinical outcome. In particular, our encouraging results suggest that also, patients with severe cholestasis may represent in the future a good indication for MARS treatment.