Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.     

Thrombosis in inflammatory bowel disease: clinical setting, procoagulant profile and factor V Leiden

Patients with inflammatory bowel disease have an increased frequency of thromboembolism, and microvascular thrombosis has been proposed as a contributory pathogenic factor. The mechanism of enhanced procoagulant activity is not understood. We examined the clinical setting of thromboembolic events in 52 patients with Crohn's disease or ulcerative colitis, and assessed the procoagulant laboratory profile, including Factor V Leiden, in a subset of 20 patients to identify procoagulant risk factors. Patients who developed thrombosis tended to be young; 60% of thrombotic events occurred in patients under 50 years. Multiple thromboembolic episodes occurred in 13% and unusual sites of thrombosis (e.g. intracardiac, cerebral, inominate veins) in 11%. No risk factor was identifiable in 52% of cases and two-thirds of thromboses occurred in an out-patient setting. The mortality rate was 8%. Evidence for inflammatory disease activity was found in only 45% of patients with ulcerative colitis at the time of the thromboembolic event, in contrast to 89% of those with Crohn's disease. Assays for specific coagulation defects were negative in all cases tested (protein S, C were normal in 17/17; anti-thrombin III, anti-phospholipid antibodies and activated protein C resistance were negative in 20/20, and only 1/20 patients was found to be heterozygous for Factor V leiden. Thrombosis in inflammatory bowel disease is important because it occurs in a young population, often in unusual sites, and has a high mortality. The development of thrombosis is related to active inflammatory disease in most patients with Crohn's disease but apparently not in those with ulcerative colitis. Since approximately half of the patients had no other identifiable risk factor, there remains a substantial group of patients with IBD who develop thrombosis for unknown reasons.      

Comparison of the subacute toxicity and efficacy of 3-hydroxypyridin-4- one iron chelators in overloaded and nonoverloaded mice

Five orally effective iron chelators of the 3-hydroxypyridin-4-one series have been administered intraperitoneally to iron-overloaded and nonoverloaded male mice at a dose of 200 mg/kg/24 h for a total of 60 days to investigate the effect on iron loading and toxicity. There was a significant reduction in hepatic iron at the end of the study in the iron-overloaded mice with all compounds studied using chemical iron quantitation (P less than .001) and with Perls' stain (P less than .01). Liver iron removal with the hydroxypyridinones ranged from 37% with CP20 to 63% with CP51, compared with 46% removal for desferrioxamine (DFO). There was no significant reduction in splenic or cardiac iron with any chelator. There were no deaths in iron-overloaded animals receiving any of the hydroxypyridin-4-ones, but significantly more deaths in the nonoverloaded groups as a whole (P less than .03). No weight loss was observed with any chelator. Significant reductions in hemoglobin and white cell count were observed with CP20(L1). No histologic abnormalities of kidney, spleen, bone marrow, or stifle joints were observed. Intracytoplasmic inclusion bodies were observed in the centrilobular hepatocytes of animals administered each of the hydroxypyridin-4-ones, while the DFO-treated and control groups showed no such changes.     

Iron,HCV and the liver

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Novel method of intraoperative liver tumour localisation with indocyanine green and near-infrared imaging

INTRODUCTIONFluorescence imaging (FI) with indocyanine green (ICG) is increasingly implemented as an intraoperative navigation tool in hepatobiliary surgery to identify hepatic tumours. This is useful in minimally invasive hepatectomy, where gross inspection and palpation are limited. This study aimed to evaluate the feasibility, safety and optimal timing of using ICG for tumour localisation in patients undergoing hepatic resection.METHODSFrom 2015 to 2018, a prospective multicentre study was conducted to evaluate feasibility and safety of ICG in tumour localisation following preoperative administration of ICG either on Day 0–3 or Day 4–7.RESULTSAmong 32 patients, a total of 46 lesions were resected: 23 were hepatocellular carcinomas (HCCs), 12 were colorectal liver metastases (CRLM) and 11 were benign lesions. ICG FI identified 38 (82.6%) lesions prior to resection. The majority of HCCs were homogeneous fluorescing lesions (56.6%), while CLRM were homogeneous (41.7%) or rim-enhancing (33.3%). The majority (75.0%) of the lesions not detected by ICG FI were in cirrhotic livers. Most (84.1%) of ICG-positive lesions detected were < 1 cm deep, and half of the lesions ≥ 1 cm in depth were not detected. In cirrhotic patients with malignant lesions, those given ICG on preoperative Day 0−3 and Day 4−7 had detection rates of 66.7% and 91.7%, respectively. There were no adverse events.CONCLUSIONICG FI is a safe and feasible method to assist tumour localisation in liver surgery. Different tumours appear to display characteristic fluorescent patterns. There may be no disadvantage of administering ICG closer to the operative date if it is more convenient, except in patients with liver cirrhosis.