Improved production of adenovirus vectors expressing apoptotic transgenes

Adenovirus vectors expressing gene products that can induce apoptosis have potential utility in gene therapy applications ranging from the treatment of proliferative diseases to transplantation. However, adenovirus vectors carrying proapoptotic gene products are difficult to produce, as the apoptotic environment is not conducive to adenovirus gene expression and replication. Production of AdFasL/G, an adenovirus vector that expresses high levels of Fas ligand, was severely reduced in the 293 packaging cell line. Increased yields of AdFasL/G were achieved by inclusion of peptide-based caspase inhibitors in the growth medium. However, use of these inhibitors for large-scale production would be difficult and expensive. A screen for gene products that increase the yield of AdFasL/G in 293 cells revealed that the poxvirus serpin CrmA and the adenovirus 14.7K product were able to increase virus yields significantly. Apoptosis induced by AdFasL/G was attenuated in 293CrmA cell lines and virus titers were increased dramatically. However, serial passage of AdFasL/G on 293CrmA cells resulted in the generation of replication-competent adenovirus. To resolve this problem, the CrmA gene was introduced into AE25 cells, an E1-complementing cell line that has limited sequence identity with the vectors. AdFasL/G titers were increased 100-fold on AE25CrmA cells relative to the AE25 cells and RCA contamination was not detectable. In addition, adenovirus vectors that express FADD, caspase 8, and Fas/APO1 were produced efficiently in AE25CrmA and 293CrmA.     

Women with unexplained recurrent pregnancy loss do not have evidence of an underlying prothrombotic state: Experience with calibrated automated thrombography and rotational thromboelastometry

Introduction

Where unexplained recurrent pregnancy loss (RPL) is attributed to an underlying maternal prothrombotic state, empirical prophylactic anticoagulation may be recommended.

Materials and Methods

In the present study we used calibrated automated thrombography and rotational thromboelastometry to determine the procoagulant potential of these women as a rationale for anticoagulation. Fifty women with ≥ three consecutive unexplained losses prior to 14 weeks’ gestation or one loss after this time were compared with forty-one parous women with no miscarriages. Exclusion criteria included antiphospholipid syndrome, inherited thrombophilia and prior venous thromboembolism. Thrombin generation in platelet poor plasma and whole blood thromboelastometry was performed outside pregnancy to determine the presence or not of an underlying prothrombotic state.

Results

Peak thrombin and endogenous thrombin potential were not significantly increased in subjects relative to controls. The use of low tissue factor (1 pM) to better reflect physiological conditions and assay modification to better assess the protein C pathway (5 pM in the presence of thrombomodulin) provided no additional discrimination. Consistent results were shown with thromboelastometry; mean parameters were equivalent between subjects and controls.

Conclusions

These data demonstrate that global coagulation assays provide no evidence of an underlying hypercoagulable state in women with unexplained RPL; this is in keeping with the results of recent randomised controlled trials and strengthens the evidence base against use of anticoagulants in this setting.     

Efficacy and safety of ruzasvir 60 mg and uprifosbuvir 450 mg for 12 weeks in adults with chronic hepatitis C virus genotype 1, 2, 3, 4 or 6 infection

In clinical trials, the three‐drug regimen of ruzasvir (RZR) 60 mg, uprifosbuvir (UPR) 450 mg and grazoprevir 100 mg, with or without ribavirin, has demonstrated promising efficacy and excellent tolerability across a wide range of hepatitis C virus (HCV)‐infected individuals. The present study assessed the efficacy and safety of the two‐drug combination of RZR 60 mg plus UPR 450 mg administered for 12 weeks in participants with HCV genotype (GT) 1‐6 infection. In this open‐label clinical trial, treatment‐naive or ‐experienced and cirrhotic or noncirrhotic participants with chronic HCV GT1‐6 infection received RZR 60 mg plus UPR 450 mg orally once daily for 12 weeks (NCT02759315/protocol PN035). The primary efficacy endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). One hundred and sixty participants were enrolled. SVR12 rates were 96% (52 of 54) in participants with GT1a infection; 100% (15 of 15) in those with GT1b infection; 97% (28 of 29) in those with GT2 infection; 77% (30 of 39) in those with GT3 infection; 90% (18 of 20) in those with GT4 infection; and 67% (2 of 3) in those with GT6 infection. Drug‐related adverse events (AEs) reported by >5% of participants were fatigue (n = 10, 6.3%) and diarrhoea (n = 9, 5.6%). Five participants reported a total of 11 serious AEs, none considered drug‐related. One participant experienced on‐treatment alanine aminotransferase/aspartate aminotransferase elevations that resolved without intervention. Data from the present study indicate that the combination of RZR 60 mg plus UPR 450 mg once daily for 12 weeks was well tolerated overall but was effective only for certain genotypes.     

Ideal cardiovascular health among Ghanaian populations in three European countries and rural and urban Ghana: the RODAM study

Cardiovascular health (CVH) is a construct defined by the American Heart Association (AHA) as part of its 2020 Impact Goal definition. CVH has, until now, not been evaluated in Sub-Saharan African populations. The aim of this study was to investigate differences in the prevalence of ideal CVH and its constituent metrics among Ghanaians living in rural and urban Ghana and Ghanaian migrants living in three European countries. The AHA definition of CVH is based on 7 metrics: smoking, body mass index, diet, physical activity, blood pressure, total cholesterol, and fasting plasma glucose. These were evaluated among 3510 Ghanaian adults (aged 25–70 years) residing in rural and urban Ghana and three European cities (Amsterdam, London and Berlin) in the multi-centre RODAM study. Differences between groups were assessed using logistic regression with adjustments for gender, age, and education. Only 0.3% of all participants met all 7 metrics of the AHA’s definition of ideal CVH. Compared to rural Ghana (25.7%), the proportions and adjusted odds ratio (OR) of individuals who had 6–7 CVH metrics in the ideal category were substantially lower in urban Ghana, (7.5%; OR 0.204, 95% CI 0.15–0.29), Amsterdam (4.4%; 0.13, 0.08–0.19), Berlin (2.7%; 0.06, 0.03–0.11), and London (1.7%; 0.04, 0.02–0.09), respectively. The proportion of ideal CVH for the various metrics ranged from 96% for all sites in the smoking metric to below 6% in the diet metric. The proportion of ideal CVH is extremely low in Ghanaians, especially among those living in urban Ghana and Ghanaian migrants in Europe.     

Hb Adana (HBA2 or HBA1: c.179G > A) and alpha thalassemia: Genotype–phenotype correlation

Alpha thalassemia due to nondeletional mutations usually leads to more severe disease than that caused by deletional mutations. Devastating outcomes such as hydrops fetalis can occur with two nondeletional mutations, therefore warranting DNA‐based workup for suspected carriers with subtle hematological abnormalities for family counseling purposes. We describe three cases with hemoglobin (Hb) Adana, a nondeletional alpha chain mutation, compounded with an alpha globin gene deletion resulting in thalassemia intermedia. We review the literature, draw genotype–phenotype correlations from published cases of Hb Adana, and propose that this correlation can be used by clinicians to help direct diagnostic studies and urge hematologists to thoroughly workup high‐risk patients.     

Antihyperglycaemic and Antioxidant Effects of Adenia Lobata engl. (Passifloraceae) in Streptozotocin-Induced Diabetic Rats

The antihyperglycaemic and antioxidant activities of a Ghanaian medicinal plant namely Adenia lobata Engl (Passifloraceae), used to treat diabetes mellitus in traditional medicine, was investigated. The dried stem powder of A. lobata was successively extracted by Soxhlet with petroleum ether and 70% ethanol to obtain the crude petroleum ether (PEAL: yield =1.1w/w %) and ethanol (EEAL: yield = 5.4 w/w %) extracts. The extracts were assessed for their antihyperglycaemic and antioxidant activities. The antihyperglycaemic activity of PEAL and EEAL were determined in streptozotocin-induced diabetic rats (70 mg/kg body weight). Five groups of diabetic rats were given 150, 300 and 600 mg/kg body weight of PEAL and EEAL orally once daily for 20 days. Glibenclamide (5 mg/kg body weight) was used as positive control while distilled water (5 ml) acted as the normal diabetic control. The blood glucose levels were monitored initially for 6 hours and subsequently over 24 days. Both extracts exhibited statistically significant (p< 0.001) antihyperglycaemic activity throughout the study period, with EEAL showing the greatest activity. The antioxidant properties of the petroleum ether and ethanol extracts of A. lobata (PEAL and EEAL) were evaluated using five assays; total phenolic content, total antioxidant capacity, reducing power, DPPH scavenging effect and lipid peroxidation activity. In all these assays, the antioxidant properties increased with increasing concentration of the extracts.     

Physician prescribing practices: What do we know? Where do we go? How do we get there?

Although drug prescribing is one of the most important components of medical care, little is known about how prescribing practices are determined and how they can be influenced. Enhancing the quality and effectiveness of drug prescribing requires research and better dissemination of information to physicians and other decision-makers. This requires a collaborative effort and a coordinated action plan. Participants at the Physician Prescribing Practices Workshop, held in Ottawa in October 1995, addressed issues and made recommendations in three areas: current knowledge and issues for research in the field of prescribing practices, and the capacity of Canadian databases to study these issues, strategies for disseminating and implementing knowledge and research findings to enhance the quality of prescribing; and the formation of a network to foster collaboration among stakeholders.