Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.     

Administration of S-nitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact

Background  

Traumatic brain injury (TBI) is a major cause of preventable death and serious morbidity in young adults. This complex pathological condition is characterized by significant blood brain barrier (BBB) leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. Following other brain injuries, nitric oxide modulators such as S-nitrosoglutathione (GSNO) maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether GSNO shows efficacy in a rat model of experimental TBI.     

Ongoing normal pregnancy after transfer of zona-free blastocysts: implications for embryo transfer in the human

Recent reports suggest that transfer of day 5 blastocysts improves implantation rates in in-vitro fertilization programmes. This paper reports a successful ongoing pregnancy after the transfer of zona-free day 6 expanded and hatching blastocysts. The patient was 37 years old and had undergone six stimulated and two thaw cycles previously, all of which had failed. Stimulation was by down-regulation and oocytes recovered transvaginally by ultrasound guidance. Two pronuclear embryos were co-cultured on Vero cells to day 6. The zonae of two hatching and two fully expanded blastocysts were removed using 0.5% pronase, and the zona-free blastocysts were then transferred. Pregnancy was confirmed on day 18 with a positive human chorionic gonadotrophin (HCG) and ultrasound at 6 weeks showed a single healthy fetal heart inside a clear sac. At 14 weeks a triple test (oestriol, J-HCG and alpha- fetoprotein) was normal and at 22 weeks a detailed ultrasound scan showed no congenital anomalies. This is the first report in the human of a normal ongoing pregnancy after the transfer of zona-free day 6 embryos.      

Prevalence and clinical profile of autosomal dominant type 2 diabetes from a diabetes centre in India

AimsTo study the prevalence, and clinical profile of autosomal dominant (AD) inherited forms of type 2 diabetes mellitus (T2DM).MethodsDetailed pedigree charts were drawn on 510 consecutive T2DM subjects attending a tertiary care diabetes centre in South India. Clinical and biochemical features of T2DM subjects with and without AD inheritance were compared.ResultsOverall, 36.1% of T2DM had one parent with diabetes, in 10.6%, both parents had diabetes and 10.2% had features of AD. Age at diagnosis of diabetes was the lowest among AD group compared to other groups (p for trend <0.001). Only 22.6% of T2DM with AD inheritance had age at diagnosis of diabetes below 25 years and in 26.4%, it was diagnosed above 45 years. There were no significant differences in the clinical features, including prevalence of diabetic complications, between T2DM with and without AD inheritance.ConclusionsIn this clinic-based study, 10.2% of T2DM subjects had evidence of AD inheritance. While the AD cases occurred at younger age, older age at diagnosis was not uncommon. Clinical features, including complications, did not differ between the T2DM patients with or without AD.     

The Feasibility of Early Second Trimester Pregnancy (12 to 14 weeks) Termination with Single Intra-Amniotic Injection of 15-Methyl PGF2α

Summary: Prostaglandin analogues have proven to be safe and effective for second trimester pregnancy terminations when given by various routes. Of these, the intra-amniotic (IA) route has proven to be safer, especially in women with medical disorders, as it causes the least occurrence of systemic side-effects. Previously, blind transabdominal IA instillation of the analogue 15-methyl PGF₂a was routinely possible only after 16 weeks' gestation. With the advent of ultrasound-guided techniques, it has now become possible to use the IA route for very early second trimester pregnancies. This study examines the use of single 1.5 mg 15-methyl PGF₂a IA injection for termination of 12–14 weeks pregnancies. Single injection resulted in successful abortion in 76.5% of women and, in the unsuccessful cases, additional prostaglandin injection resulted in successful abortion in all women within 48 hours without problems. It is therefore feasible to use ultrasound-guided IA prostaglandin injection for successful termination of early second trimester pregnancies.     

Modulatory effect of hesperidin on benzo(a)pyrene induced experimental lung carcinogenesis with reference to COX-2, MMP-2 and MMP-9

Hesperidin is a naturally occurring flavonoid that has been reported to possess anticancer effects. The purpose of this study is to evaluate the effect of hesperidin in modulating the expressions of cyclooxygenase-2 (COX-2), mast cells (MCs) and matrix metalloproteinases (MMPs) during benzo(a)pyrene (B(a)P) induced lung carcinogenesis in mice. B(a)P (50 mg/kg body weight) induced animals showed increased mast cell density (MCD) as revealed by toluidine blue staining and severe expression of COX-2 along with upregulated expression of MMP-2 and MMP-9 as revealed by Western blotting and immunohistochemistry. Supplementation of hesperidin (25 mg/kg body weight) to lung cancer bearing mice attenuated MCD and downregulated the expressions of COX-2, MMP-2 and MMP-9. These observations show that hesperidin exerts its anti-carcinogenic activity against lung cancer by altering the expressions of COX-2, MMP-2 and MMP-9.     

The effects of quercetin on antioxidant status and tumor markers in the lung and serum of mice treated with benzo(a)pyrene

Chemoprevention has emerged as a very effective preventive measure against carcinogenesis. Several bioactive compounds present in fruits and vegetables have revealed their cancer curative potential on benzo(a)pyrene (B(a)P) induced carcinogenesis. In the present study, the efficacy of quercetin on the level of lipid peroxides, activities of antioxidant enzymes and tumor marker enzymes in B(a)P induced experimental lung carcinogenesis in Swiss albino mice was assessed. In lung cancer bearing animals there was an increase in lung weight, lipid peroxidation and marker enzymes such as aryl hydrocarbon hydroxylase, gamma glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase and adenosine deaminase with subsequent decrease in body weight and antioxidant enzymes-superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, reduced glutathione, vitamin E and vitamin C. Quercetin supplementation (25 mg/kg body weight) attenuated all these alterations, which indicates the anticancer effect that was further confirmed by histopathological analysis. Overall, the above data shows that the anticancer effect of quercetin is more pronounced when used as an chemopreventive agent rather than as a chemotherapeutic agent against B(a)P induced lung carcinogenesis.