Development of a liver-specific Tet-off AAV8 vector for improved safety of insulin gene therapy for diabetes.

BACKGROUND: Diabetes mellitus is caused by a partial or complete lack of insulin production in the body. We have previously shown that a single injection of an adeno-associated virus serotype 8 (AAV8) vector carrying a modified and codon optimized human insulin gene induced hepatic production of insulin and corrected streptozotocin (STZ)-induced diabetes in mice for more than 1 year. Insulin production was constitutive, analogous to long-acting insulin therapy. METHODS: We have developed a single AAV8 vector with a Tet-Off regulatable system as a safety mechanism to turn off insulin secretion should hypoglycaemia develop in vector-treated diabetic mice. We first transfected HepG2 cells or freshly isolated rat hepatocytes in vitro with the Tet-Off system (pAAV-Tetoffbidir -Alb-luc) regulating a luciferase reporter gene. We subsequently incorporated a furin-cleavable codon-optimised human proinsulin cDNA into pAAV-Tetoffbidir backbone to form the doxycycline inducible pAAV-Tetoffbidir -Alb-hINSco. RESULTS: Using STZ-induced diabetic mice, we were able to switch off insulin secretion repeatedly with doxycycline administration, and showed full restoration of insulin secretion on withdrawing doxycycline. CONCLUSIONS: The present study provides proof of concept that, under circumstances when inappropriate basal insulin secretion is a safety concern, insulin secretion from AAV8 gene therapy can be turned off reversibly with doxycycline.

Effects of malnutrition on economic productivity in China as estimated by PROFILES.

OBJECTIVE: To calculate the effects of malnutrition on economic productivity in China. METHODS: PROFILES was used to quantify the function consequences of malnutrition in term of protein energy malnutrition, iron deficiency and iodine deficiency. RESULTS: Productivity gained due to improved iodine nutrition. The reduction in the TGR in 1992 to 2001 increased the net present value of further economic productivity by yen 142 billion. Reduction of the TGR rate to 5% over next 10 years would result in future productivity gains with value of yen 40 billion. Productivity gain due to reductions in child stunting would result in future economic productivity gains with the value of yen 101 billion. Reducing stunting further over the next 10 years would gain yen 20 billion. Productivity gain due to reduction of iron deficiency anemia reduced by 30% over the next 10 years would gain worth yen 107 billion and if childhood anemia reduced by 30% over next 10 years would gain yen 348 billion. CONCLUSION: These interventions have huge economic payoff. That is likely to exceed their costs many times over.

Effects of malnutrition on child survival in China as estimated by PROFILES.

OBJECTIVE: To estimate the benefits of reductions in underweight and Vitamin A deficiency for child survival in China that might be expected as a result of lowering the prevalence of these conditions. METHODS: Profiles, a process of nutrition policy analysis was used to quantify the functional consequences of malnutrition in terms of child survival. RESULTS: Underweight: The actual reduction in underweight between 1992 and 2001 (from 15.7% to the current 10.1%) resulted in saving of 176,000 child lives. As estimated, without improvements, 612,000 children will die due to underweight between 2001 and 2010, 281,000 (46%) of them living in western provinces. Reducing underweight prevalence from 10.1% to 8% could overall save 62,000 lives. The reduction of underweight prevalence in the west alone might save 56,000 lives. Vitamin A in China as a whole, vitamin A deficiency accounts, as estimated, for 7.5% of deaths of children 6-59 months old, representing 206,000 deaths over the past ten years. Halving the prevalence over the period would save 49,000 child lives. The higher prevalence and higher mortality rates in western provinces mean that even with only 28% of the Chinese population, over half of child deaths there are related to vitamin A.

[Multiple factors analysis on malnutrition of children and under five in different patterns in China in 2000].

In order to analyze the major factors contributing to malnutrition of children aged under five at 40 inspective spots, a cluster method was used and four patterns were set up in this study. Multiple logistic regression was applied in each pattern to identify the most important factor. The results showed that the problem of child malnutrition in urban areas has been solved, but the problem in rural is still severe. The attributable risk of education level of mother, breast feeding and water supply were the major factors contributing to the nutrition status of children and therefore, the synthetical measures should be take for improving the nutrition status of children.

[Epidemiological survey of chronic vascular complications of type 2 diabetic in-patients in four municipalities].

OBJECTIVE: To provide an epidemiological description and risk factors of chronic vascular complications of type 2 diabetic in-patients in four municipalities including Beijing, Shanghai, Tianjin, and Chongqing. METHODS: Data of the study came from 3,469 Type 2 diabetic in-patients from 1991 to 2000 in 10 medical centers of Beijing, Shanghai, Tianjin, and Chongqing. A variety of parameters of in-patients were evaluated retrospectively to know the prevalence and risk factors of chronic vascular complications in the study patients. RESULTS: Overall, the detailed prevalence of chronic vascular complications is listed as follows: diabetic retinopathy 31.5%, diabetic nephropathy 39.7%, diabetic neuropathy 51.1%, hypertension 41.8%, coronary heart disease (CHD) 25.1%, cerebral vascular disease (CVD) 17.3%, vessel complication of lower limbs 9.3%. Multivariate logistic regression analysis shows that diabetes family history, duration of diabetes (> 5 years), and systolic blood pressure (> 125 mmHg) are the risk factors for diabetic retinopathy; duration of diabetes (> 5 years), systolic blood pressure (> 125 mmHg), LDL-C (> 3.12 mmol/L), and triglyceride (> 1.70 mmol/L) are the risk factors for diabetic nephropathy; age (> 45 years), duration of diabetes (> 5 years), HbA1C (> 7.0%), systolic blood pressure (> 125 mmHg), and LDL-C (> 3.12 mmol/L), are the risk factors for CHD; age (> 45 years), duration of diabetes (> 5 years), systolic blood pressure (> 125 mmHg), and triglyceride (> 1.70 mmol/L) are the risk factors for CVD. CONCLUSION: In order to improve patients' outcome, multiple metabolic controls in type 2 diabetic patients are urgently needed, which include decreasing glycemia, reducing hypertension and improving lipid levels.

The role of R-spondin2 in keratinocyte proliferation and epidermal thickening in keloid scarring.

Keloids are found only in humans and the underlying biochemical mechanisms of their pathogenesis remain unknown. R-spondins (Rspos) are a relatively new group of secreted proteins known to be Wnt/ß-catenin signaling agonists, but their role in keloids has yet to be elucidated. We investigated the expression levels of R-spondin2 (Rspo2) in cell lysates and conditioned media of monocultures and co-cultures of fibroblasts and keratinocytes derived from keloids and normal skin. In this study we found increased protein expression and secretion of Rspo2 in respective monocultures of keloid fibroblasts and keratinocytes when compared with their normal counterparts. Double-chamber co-culture experiments implicated the role of keloid keratinocytes (KKs) in the induction of Rspo2 secretion from fibroblasts because of epithelial-mesenchymal interactions. Addition of recombinant human Rspo2 in culture increased the proliferation of keratinocytes and it acted synergistically with Wnt3a through the canonical Wnt/ß-catenin pathway. Overexpression of Rspo2 in normal fibroblasts brought about thicker epidermis when compared with control fibroblasts in a skin organotypic culture model. This observation coincides with the hyperproliferative phenotype of thickened epidermis seen in keloids. Taken together, the results suggest the possible double paracrine action of KKs in inducing higher expression of Rspo2 in fibroblasts that promotes keratinocyte proliferation and epidermal thickening.

Keloid fibroblasts are more sensitive to Wnt3a treatment in terms of elevated cellular growth and fibronectin expression.

BACKGROUND: Current evidence suggests the potential role of Wnt signalling in keloids pathogenesis but such literature remains scanty. We hypothesize that Wnt signalling is upregulated in keloid fibroblasts (KFs) and this promotes cellular growth, migration and extracellular matrix (ECM) production in such fibroblasts. OBJECTIVES: To verify the downregulation of secreted frizzled-related protein 1 (SFRP1), a Wnt inhibitor and test KFs sensitivity to Wnt3a treatment compared to NFs in terms of activation of Wnt/ß-catenin, cellular growth, migration and ECM expressions. Next, to investigate if ectopic expression of SFRP1 and treatment of quercetin in KFs can reverse their phenotypes. METHODS: Quantitative Real-time PCR and western blotting were used to verify SFRP1 expression in NFs and KFs. The fibroblasts were tested with Wnt3a conditioned media and its effects were tested for (1) the cells' sensitivity to direct Wnt signalling via the activation of TCF reporter assay and protein expression of ß-catenin, (2) cellular growth, (3) cell migration and (4) expressions of ECM components. Finally KFs were stably transduced with SFRP1 and treated with 2 doses of quercetin. RESULTS: Lower levels of SFRP1 were confirmed at mRNA and protein levels in KFs which partly explained their sensitivity to Wnt3a treatment in terms of higher Wnt activation, cellular growth and fibronectin expression. Interestingly, Wnt3a did not promote higher cell migration rate and increase in collagen I expression. Ectopic expression of SFRP1 and quercetin treatment was able to mitigate Wnt3a-mediated phenotype of KFs. CONCLUSIONS: Using SFRP1 or inhibitors of Wnt signalling might be one of the therapeutic solutions to treat keloid scarring.

[A discussion on utility and purposed value of obesity and abdomen obesity when body mass index, waist circumference, waist to hip ratio used as indexes predicting hypertension and hyper-blood glucose].

OBJECTIVE: Discussion on utility and purposed value of obesity and abdomen obesity when body mass index (BMI), waist circumference (WC), waist to hip ratio (WHR) used as indexes predicting hypertension, hyper-blood glucose, and both clusters, to provide scientific basis for the decision on the indexes and their cut-off points of obesity and abdomen obesity in Chinese people. METHODS: Using the data of diabetes mellitus (DM) from epidemiological studies carried out in 11 provinces/autonomous regions/municipalities of China from July 1995 to June 1997. Partial relative analysis, logistic multi-factors regression analysis, interaction analysis were used. Relative risk (RR), attributable risk proportion (ARP) and population attributable risk proportion (PARP) of hypertension, hyper-blood glucose, and the both cluster as BMI, WC, WHR with the different cut off points were analysed. RESULTS: 1) The correlations between BMI, WC and blood pressure, blood glucose were better than the WHR. 2) After adjusted by age, sex, occupation leisure physical activity, education degree and the family history of DM, the results suggested that BMI, WC, WHR were important predictive factors, with relative importance as BMI > WC > WHR. 3) There were augment interactions on BMI, WC and WHR with hypertension, hyper-blood glucose, with the interaction of BMI and WC in particular. Their pure attributable interaction proportion were from 5.95% to 29.34%. 4) The values of RR were about 2.5 when BMI >/= 23, >/= 24 and >/= 25, suggesting the relationship with exposure factors and diseases were with medium and high maleficent extent. Their ARP were from 0.580 to 0.623 with PARP from 0.259 to 0.425. The values of RR were from 2.06 to 3.08 as WC >/= 85 cm in males, WC >/= 80 cm in females while WC >/= 90 cm in males, WC >/= 80 cm in females, which suggested that the relationship with exposure factors and diseases were in medium and high maleficent extent. Their ARP were from 0.515 to 0.676 while PARP from 0.241 to 0.431. CONCLUSIONS: Since the maleficent extent of exposure factors to diseases, the acceptability for overweight and obesity in population, and the prevention and care for overweight and obesity were just in the introduction stage in China. The utility value of predicted hypertension, hyper-blood glucose in BMI and WC seemed to be better then in WHR. We suggested that BMI used as the obesity index, with the diagnostic cut-off point BMI >/= 24. WC as the abdomen obesity index. The diagnostic cut-off points are suggested to be WC >/= 85 cm in males, and WC >/= 80 cm in females.