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.     

G-CSF administration is neuroprotective following transient cerebral ischemia even in the absence of a functional NOS-2 gene

Granulocyte colony-stimulating factor (G-CSF) is a candidate neuroprotective factor following cerebral ischemia. To determine whether G-CSF acts partly through the inhibition of nitric oxide synthase (NOS)-2 expression, we administered G-CSF to male NOS-2−/− mice after cerebral ischemia. Although male NOS-2−/− mice exhibit resistance to the gross effects of cerebral ischemia, they display neuronal loss and skilled motor deficits following cerebral ischemia. Administration of G-CSF during reperfusion reduced motor deficit and neuronal loss. Thus, G-CSF is still effective in NOS-2 gene-deficient mice, suggesting that part of the mechanism of action is independent of NOS-2.     

Triple antiplatelet therapy for preventing vascular events: a systematic review and meta-analysis

Background  

Dual antiplatelet therapy is usually superior to mono therapy in preventing recurrent vascular events (VEs). This systematic review assesses the safety and efficacy of triple antiplatelet therapy in comparison with dual therapy in reducing recurrent vascular events.     

Human ovarian granulosa cells and follicular fluid indices: the relationship to oocyte maturity and fertilization in vitro

The study investigates the correlation between oocyte maturity and fertilization and a variety of hormonal parameters in follicular fluid and ovarian granulosa cells. A methodology for purification of granulosa cells from contaminating blood cells is also established. A total of 63 follicular aspirates were collected at oocyte retrieval from 30 women superovulated using the long luteinizing hormone- releasing hormone (LHRH analogue)/human menopausal gonadotrophin regimen. Oestradiol, progesterone, testosterone and human chorionic gonadotrophin (HCG) were quantified in follicular fluid and granulosa cells were immunostained for human chorionic gonadotrophin. Immunopurification of granulosa cells from contaminating blood cells was performed. HCG in follicular fluid was significantly high in follicles yielding immature (grade 3) oocytes (P=0.002); there was no correlation with fertilization. Aspirates from follicles containing mature (grade 1) oocytes and oocytes that subsequently fertilized had significantly more granulosa cells immunobound to HCG (P < 0.001, P=0.02). Moreover, the immunomagnetic purification technique provided >98% pure population of granulosa cells. The data demonstrate that HCG in follicular fluid and on granulosa cells may help to predict oocyte maturity and fertilization. Furthermore, immunomagnetic beads provide a reliable procedure for the purification of ovarian granulosa cells.      

Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus- specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.      

阿片受体拮抗剂在TNF－α所致体温升高中的作用

为探索细胞介素ＴＮＦ－α升体温效应与下丘脑前部、视前区（ＰＯＡＨ）中的阿片受体的关系。应用脑神经核团微量注射方法给自由状态下的雄性ＳＤ大鼠ＰＯＡＨ区微量注射ＴＮＦ－α致热源。给药前３０ｍｉｎ分别用通常阿片受体拮抗剂Ｎａｌ（１０～２０μｇ）和特异性阿片受体μ、δ和κ的拮抗剂ＣＴＡＰ（１．０～２．５μｇ）、ＮＴＩ（０．２５～０．５μｇ）和ｎｏｒ－ＢＮＩ（０．１～３μｇ）对ＰＯＡＨ做预处理。结果：单独给ＴＮＦ－α可致剂量相关的体温升高△Ｔ（１℃～１．４℃）；经Ｎａｌ１０μｇ，ＣＴＡＰ１．０μｇ和ＮＴＩ０．５μｇ处理后使ＴＮＦ－α的升体温效应减弱；用Ｎａｌ２０μｇ，ＣＴＡＰ２．５μｇ和ＮＴＩ０．２５μｇ处理后可完全阻断ＴＮＦ－α所致的体温升高；ｎｏｒ－ＢＮＩ（０．１～３μｇ）对ＴＮＦ－α的升体温效应无影响。     

Response from lieberman and colleagues

Respond on comments on Lieberman's article: Cyclosiloxanes Produce Fatal Liver and Lung Damage in Mice. Environ Health Perspect 107:161-165