Inclusion of Jaagsiekte sheep retrovirus proviral elements markedly increases lentivirus vector pseudotyping efficiency.

Retroviral pseudotyping for gene transfer applications endeavors to alter vector tropism and maintain a suitable titer. We investigated the compatibility of the Jaagsiekte sheep retrovirus (JSRV) envelope glycoprotein with the feline immunodeficiency virus (FIV) vector. A construct consisting of the minimal JSRV env coding region expressed from a standard mammalian expression plasmid generated FIV vector titers of approximately 10(4) TU/ml following standard triple transfection, collection of supernatants, and concentration by centrifuge. Interestingly, retention of the native proviral 5' and 3' flanking regions surrounding the JSRV env resulted in exceptional titers of approximately 10(8) TU/ml following the same viral preparation. To discern the regions necessary to achieve this 10,000-fold increase in titer, additional constructs were designed and tested. Our results indicate that the enhanced vector titer correlates with an increase in steady-state levels of envelope RNA that results from a combination of RNA splicing and stability, leading to increased envelope protein production. Expression of four other glycoproteins in an expression plasmid retaining the enhancing elements from the JSRV proviral sequence increased FIV vector titers from 0- to 100-fold. These novel data demonstrate that optimization of the envelope expression construct can profoundly influence titers for lentivirus vectors.     

Know your cholesterol: population screening

To facilitate the goal that all adult Americans know their total serum cholesterol levels, our specific aim was to evaluate the effectiveness, practicality, and cost of total cholesterol sampling in nonfasting self-referred subjects with use of venous blood and mailed results (n = 3844), and to compare these results with capillary blood total cholesterol levels (n = 1167), with immediate turnaround. We used consensus cut points of total cholesterol levels greater than 200, greater than 220, and greater than 240 mg/dl for moderate risk of coronary heart disease and greater than 220, greater than 240, and greater than 260 for high risk for aged 20 through 29, 30 through 39, and greater than 40 years. Total cholesterol level was in the moderate- or high-risk range in 45% and 37% of the venous and capillary cohorts, respectively. Median venous and capillary total cholesterol values were approximately 20 and 10 mg/dl greater than the total cholesterol values in the Lipid Research Clinics Prevalence Study, a difference contributed to by nonfasting versus fasting conditions, use of serum versus plasma, and self-referral bias for subjects with a family history of premature coronary heart disease. The cost per subject in the venous and capillary studies was $5.09 and $7.12 respectively, and $11.40 and $ 18.63 for each subject in the moderate- to high-risk range. Resampling with the subject fasting and follow-up were stressed, and were made available for moderate- and high-risk subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

“As real as it gets”: A Grounded Theory Study of a Reading Intervention in a Juvenile Correctional School

Background

The well-documented statistics regarding the academic struggles of incarcerated youth are disconcerting, and efforts to improve reading performance among this population are greatly needed. There is a dearth of research that provides rich and detailed accounts of reading intervention implementation in the juvenile corrections setting.

Objective

The present study attempted to address this gap in the research base by developing a grounded theory of literacy intervention implementation in one juvenile correctional school.

Methods

Qualitative methods were used for data collection (i.e., individual and focus group interviews) and analysis (i.e., grounded theory). Study participants included representatives from all facets of the facility, including education (both students and adults), security, and administration, to allow for a comprehensive examination of the context.

Results

The context affected the faculty, staff, and students in different ways and influenced the commitment to and implementation of the intervention. Additionally, teachers’ experience and background seemed to influence their perception of the program. Individual student characteristics affected their motivation for participation.

Conclusions

There were many contextual factors, some that contributed to the success of the intervention, and others that impeded its success. It is important that interventions being considered for implementation in challenging or atypical settings account for the contextual variables that can affect outcomes. In the current study, influential factors identified were related to the physical environment, leadership, teachers, security personnel, and the students’ backgrounds.

     

The ΔF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTR(ΔF508/ΔF508) pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTR(ΔF508/ΔF508) airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3',5'-monophosphate (cAMP) agonists were less potent at stimulating current in CFTR(Δ)(F508/)(Δ)(F508) epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTR(ΔF508/ΔF508) pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.     

Advances in Cell and Gene-based Therapies for Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) is a disease characterized by airway infection, inflammation, remodeling, and obstruction that gradually destroy the lungs. Direct delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelia may offer advantages, as the tissue is accessible for topical delivery of vectors. Yet, physical and host immune barriers in the lung present challenges for successful gene transfer to the respiratory tract. Advances in gene transfer approaches, tissue engineering, and novel animal models are generating excitement within the CF research field. This review discusses current challenges and advancements in viral and nonviral vectors, cell-based therapies, and CF animal models.     

Tyrosine kinase receptor Axl enhances entry of Zaire ebolavirus without direct interactions with the viral glycoprotein

In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.     

Tuberculosis in indigenous peoples in the U.S., 2003-2008

Objectives

We examined trends and epidemiology of tuberculosis (TB) across racial/ethnic groups to better understand TB disparities in the United States, with particular focus on American Indians/Alaska Natives (AI/ANs) and Native Hawaiians/other Pacific Islanders (NH/PIs).

Methods

We analyzed cases in the U.S. National Tuberculosis Surveillance System and calculated TB case rates among all racial/ethnic groups from 2003 to 2008. Socioeconomic and health indicators for counties in which TB cases were reported came from the Health Resources and Services Administration Area Resource File.

Results

Among the 82,836 TB cases, 914 (1.1%) were in AI/ANs and 362 (0.4%) were in NH/PIs. In 2008, TB case rates for AI/ANs and NH/PIs were 5.9 and 14.7 per 100,000 population, respectively, rates that were more than five and 13 times greater than for non-Hispanic white people (1.1 per 100,000 population). From 2003 to 2008, AI/ANs had the largest percentage decline in TB case rates (−27.4%) for any racial/ethnic group, but NH/PIs had the smallest percentage decline (−3.5%). AI/ANs were more likely than other racial/ethnic groups to be homeless, excessively use alcohol, receive totally directly observed therapy, and come from counties with a greater proportion of people living in poverty and without health insurance. A greater proportion of NH/PIs had extrapulmonary disease and came from counties with a higher proportion of people with a high school diploma.

Conclusions

There is a need to develop flexible TB-control strategies that address the social determinants of health and that are tailored to the specific needs of AI/ANs and NH/PIs in the U.S.Among the 9.2 million new cases of tuberculosis (TB) and 1.7 million deaths due to TB globally each year,¹ the absolute magnitude of TB among the estimated 370 million indigenous people worldwide² is not known because few disaggregated surveillance data exist. However, evidence from nations where data are available, such as the U.S., Canada, Australia, and New Zealand, suggests that TB rates are higher among indigenous than nonindigenous people.^3–7Disparities in health, including differences in TB risk and burden, between indigenous and nonindigenous people are the result of the complex interplay among the individual, the community, and the social determinants of health.^8,9 Determinants of health extend beyond individual-level traits and behaviors and involve social and economic factors that operate at the community or population level.^10,11 It is critical to understand the factors that underlie poorer health status and outcomes among indigenous people to effectively address and narrow the health gap.In November 2008, indigenous leaders and TB experts from around the world met in Canada to develop a strategy for reducing the burden of TB among indigenous people globally.¹² Poverty and other social determinants of health were acknowledged as factors influencing indigenous people''s vulnerability to TB.¹³ Furthermore, participants highlighted the need for documenting and monitoring the burden of TB in indigenous people and better understanding the social realities of these groups to design more effective TB interventions.The term “indigenous” has many different usages. The United Nations approach uses various concepts, including self-identification and recognition by one''s community as indigenous and constituting non-dominant groups of society.¹⁴ Two broad categories of indigenous groups in the U.S. are the American Indians/Alaska Natives (AI/ANs) and the Native Hawaii-ans/other Pacific Islanders (NH/PIs). These groups encompass many people, each with distinct historical identities. In 2008, an estimated 2.3 million and 435,000 U.S. residents identified themselves as solely AI/AN and NH/PI, respectively.¹⁵TB is a public health concern for AI/ANs and NH/PIs in the U.S. For example, from 1993 to 2002, AI/ANs experienced the smallest decrease in TB case rates of any U.S.-born racial/ethnic group, and the rate was almost six times greater than for non-Hispanic white people in 2002.¹⁶ More recently, several large TB outbreaks among NH/PIs have occurred.¹⁷ However, to date, few studies have examined individual and community similarities and differences among TB cases in indigenous and nonindigenous groups at the national level.To update our understanding and further examine the magnitude of TB in indigenous people, and to inform public health approaches to address TB disparities in the U.S., we described the TB trends and epidemiology of AI/ANs and NH/PIs relative to other racial/ethnic groups during 2003–2008. We also examined county-level socioeconomic and health indicators from counties in which AI/AN and NH/PI TB cases were reported.