Mutation-independent rescue of a novel mouse model of Retinitis Pigmentosa.

Retinitis Pigmentosa (RP) is the leading cause of inherited blindness in the developed world, affecting approximately 1 in 3000 individuals. Although there is currently no cure for RP, the genetic pathology has been well established. In this study, we developed a novel mouse model of RP (huRhoP347S) expressing a pathogenic human rhodopsin gene with a Pro347Ser (P347S) mutation on a rhodopsin knockout background. These mice undergo severe retinal degeneration at 1 month of age. In contrast to prior studies, this model was administered a gene therapy treatment at 19 days postnata. We evaluated several self-complementary adeno-associated virus (AAV) serotypes for photoreceptor tropism, including scAAV2/2, scAAV2/5, scAAV2/6.2 and scAAV2/9, and found that scAAV2/9 transduced photoreceptors with greater efficiency and expression than other vectors. We engineered an scAAV2/9 vector to contain a microRNA sequence specifically targeting the human rhodopsin gene and demonstrated its ability to silence rhodopsin by 60.2±8.2% in vitro. In addition, we constructed an scAAV2/9 vector to contain a replacement 'codon-modified' rhodopsin transgene (RhoR2) that was resistant to degradation by the microRNA. We found that delivery of the RhoR2 by scAAV2/9 is capable of restoring vision to rhodopsin knockout mice, and rescuing our novel transgenic huRhoP347S mouse model of dominant RP. Average a-wave responses of RhoR2-injected eyes were 1.8-fold higher than those of control-injected eyes. We found that delivery of the microRNA and replacement rhodopsin in a 1:2 ratio produced an average electroretinography (ERG) a-wave response of 17.4±2.9 compared to 6.5±2.8 µV for eyes injected with negative control virus.

A mutation in CFTR produces different phenotypes depending on chromosomal background.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene but the association between mutation (genotype) and disease presentation (phenotype) is not straightforward. We have been investigating whether variants in the CFTR gene that alter splicing efficiency of exon 9 can affect the phenotype produced by a mutation. A missense mutation, R117H, which has been observed in three phenotypes, was found to occur on two chromosome backgrounds with intron 8 variants that have profoundly different effects upon splicing efficiency. A close association is shown between chromosome background of the R117H mutation and phenotype. These findings demonstrate that the genetic context in which a mutation occurs can play a significant role in determining the type of illness produced.

Adenovirus-mediated delivery of CD46 attenuates the alternative complement pathway on RPE: implications for age-related macular degeneration.

Activation of the alternative pathway of the complement system has been implicated in the pathogenesis of age-related macular degeneration. Membrane attack complex (MAC) has been identified mainly on the Bruch's membrane and drusen underlying the retinal pigment epithelium (RPE). Membrane cofactor protein (CD46) preferentially regulates the alternative pathway of complement. The aim of this study was to evaluate the potential of increasing CD46 expression on RPE cells using an adenovirus as a gene therapy approach to reduce alternative pathway-mediated damage to RPE cells. We generated a recombinant adenovirus vector expressing human CD46 (hCD46) and delivered the vector to murine hepatocytes and RPE cells in vitro. After incubation in human serum in conditions in which the classical pathway of complement was blocked, we measured alternative pathway-mediated damage of these cells by quantifying lysis and MAC formation. Adenovirus expressing hCD46 was delivered to the subretinal space of adult mice, and 1 week later, ocular flat mounts were challenged with human serum and the levels of complement-mediated damage was quantified. Adenovirus-mediated delivery of hCD46 localizes to the basal and lateral surfaces of RPE cells where it offers protection from alternative pathway-mediated damage, but not classical, allowing the classical pathway to function unhindered.

A multi-centre cohort study evaluating the role of inflammatory markers in patient's presenting with acute ureteric colic (MIMIC).

BACKGROUND: Spontaneous Stone Passage (SSP) rates in acute ureteric colic range from 47 to 75%. There is conflicting evidence on the role of raised inflammatory markers in acute ureteric colic. The use of an easily applicable biomarker that could predict SSP or need for intervention would improve the management of obstructing ureteric stones. Thus, there is a need to determine in an appropriately powered study, in patients who are initially managed conservatively, which factors at the time of acute admission can predict subsequent patient outcome such as SSP and the need for intervention. Particularly, establishing whether levels of white cell count (WBC) at presentation are associated with likelihood of SSP or intervention may guide clinicians on the management of these patients' stones. DESIGN: Multi-center cohort study disseminated via the UK British Urology Researchers in Surgical Training (BURST) and Australian Young Urology Researchers Organisation (YURO). PRIMARY RESEARCH QUESTION: What is the association between WBC and SSP in patients discharged from emergency department after initial conservative management? PATIENT POPULATION: Patients who have presented with acute renal colic with CT KUB evidence of a solitary ureteric stone. A minimum sample size of 720 patients across 15 centres will be needed. HYPOTHESIS: A raised WBC is associated with decreased odds of spontaneous stone passage. PRIMARY OUTCOME: The occurrence of SSP within six months of presentation with acute ureteric colic (YES/NO). SSP was defined as absence of need for intervention to assist stone passage. STATISTICAL ANALYSIS PLAN: A multivariable logistic regression model will be constructed, where the outcome of interest is SSP using data from patients who do not undergo intervention at presentation. A random effect will be used to account for clustering of patients within hospitals/institutions. The model will include adjustments for gender, age as control variables.

Excretion of metabolites of prostacyclin and thromboxane by rats with nephrotoxic nephritis: effects of interleukin-1.

1. To obtain direct evidence of abnormal eicosanoid biosynthesis in rats injected with anti-glomerular-basement-membrane antibodies (a-GBM), products derived from thromboxane A2 (TXA2) and prostacyclin (PGI2) were measured in 24 h urine collections before and after a-GBM. 2. Administration of a-GBM (9.5 mg) caused albuminuria, decreased creatinine clearance, increased numbers of intra-glomerular neutrophils and increased excretion of TXB2, 2,3-dinor-TXB2 (products of TXA2) and 6-oxo-PGF 1 alpha and 2,3-dinor-6-oxo-PGF 1 alpha (products of PGI2) at 24 h. 3. Interleukin-1 (IL-1 beta; 5 micrograms) alone caused an increase in PGI2 metabolite excretion but had no effect on TXA2 metabolites. It had no effect on creatinine clearance but increased numbers of glomerular neutrophils by approximately 4-5 fold compared to a-GBM. 4. Pretreatment of rats with IL-1 beta before a-GBM synergistically increased albumin excretion but only additively increased eicosanoid excretion. Numbers of intra-glomerular neutrophils and creatinine clearance were unchanged compared to IL-1 beta alone. 5. The cyclo-oxygenase inhibitor, ibuprofen (10 mgkg-1 i.p., twice daily for 4 days) inhibited both serum TXB2 production and urinary prostaglandin excretion. It also caused an almost complete attenuation of albumin excretion. Creatinine clearance and glomerular neutrophils remained unchanged after a-GBM/IL-1 beta. 6. We conclude that the 50% inhibition of thromboxane production induced by ibuprofen does not modify the fall in creatinine clearance of accumulation of neutrophils in the glomerulus caused by the a-GBM. This degree of inhibition of eicosanoid production was associated with a striking decrease in proteinuria, but this may reflect a haemodynamic rather than a disease modifying action.

The cluster committee: setting the stage for community-responsive care.

Recent reviews of medical education at the undergraduate and graduate level call for a greater emphasis on community-relevant teaching and development of partnerships between providers, academic health centers, and the community. The cluster committee, developed by the Center for Community Responsive Care, Inc., sets the stage. The cluster committee is an activity in which preventive medicine residents and fellows are trained in a community setting to participate in community-responsive health care. It is designed to meet the following goals: initiate the fellow in the steps of community-oriented primary care (COPC); develop a fellow's leadership skills; encourage relationships and coalitions within the community and among providers; and bring together the perspectives of community members, public health practitioners, academicians, and local clinicians regarding community health, as well as teach each about COPC. This article describes the cluster committee process, provides examples of a series of meetings, and presents lessons learned from the first six years of implementation.

Special contribution: transforming a neighborhood health center into a community-oriented primary care practice.

We describe a community-oriented primary care (COPC) preventive medicine residency. Through the residency, medical, nursing, dental, and other health professionals work as fellows to combine clinical and public health skills in primary care practices. We describe the steps that fellows used to begin transforming one community health center into a COPC practice, and we highlight the activities of one specific environmental cleanliness project as an example. COPC activities have laid the foundation for further advances in developing a professional partnership between the center and the community it serves.

Carrying out the Medicine/Public Health Initiative: the roles of preventive medicine and community-responsive care.

Leaders in medicine and public health, recognizing the inherent interdependency of these fields, established the Medicine/Public Health Initiative in the mid-1990s as "an evolving forum in which representatives of both sectors can explore their mutual interests in improving health and [can] define collaborative mechanisms to achieve that goal." The Initiative's participants developed six goals that they and others in medicine and public health across the nation should implement: engage the community; change the education process; create joint research efforts by clinical, public health, and preventive medicine investigators; develop a shared view of illness between medicine and public health; work together to provide health care; and work jointly to develop health care assessment measures. The authors describe the six goals in depth and explain the important combined roles of clinically-oriented preventive medicine and community-oriented preventive medicine--as practiced in a model of health care delivery called community-oriented primary care (COPC)--in implementing the Initiative's goals. They then report recent efforts, including two in Boston and Dallas, to merge medicine and public health, and state that academic health centers, which are in the process of reshaping themselves, can help themselves as well as the public by embracing their key role in the effort to integrate medicine and public health. In particular, they can expand and strengthen existing training programs in preventive medicine and COPC or add these programs to their curricula.

An integrated residency in internal and preventive medicine.

The importance of preventive and population-based principles in clinical practice is widely acknowledged. The challenge of imparting these principles in either undergraduate or postgraduate medical education has, however, not been fully met. The necessary skills are provided comprehensively by preventive medicine residency programs, but at the expense of clinical training. Sequential residencies in primary care and preventive medicine, the currently available means of obtaining thorough preparation in both clinical and population-based principles, represent an inefficient, generally unappealing, and non-integrated approach. In response to these concerns, and in an effort to make preventive medicine training appeal to a wider audience, the authors developed and implemented a residency program fully integrating internal and preventive medicine. The program meets, and generally exceeds, the requirements of both specialty boards over a four-year period. The program provides extensive training in clinical, preventive, and public health skills, along with case management and cost-effective care, conferring the MPH degree and leading to dual board eligibility. The model is ideally wed to the demands of the modern health care environment in the United States, is extremely attractive to applicants, and may warrant replication both to train academic and administrative leaders and to raise the standards of preventive and public health practice in primary care.

Integrating Healthy Communities concepts into health professions training.

To meet the demands of the evolving health care system, health professionals need skills that will allow them to anticipate and respond to the broader social determinants of health. To ensure that these skills are learned during their professional education and training, health professions institutions must look beyond the medical model of caring for communities. Models in Seattle and Roanoke demonstrate the curricular changes necessary to ensure that students in the health professions are adequately prepared to contribute to building Healthy Communities in the 21st century. In addition to these models, a number of resources are available to help promote the needed institutional changes.

The patient population of a major chain of investor-owned ambulatory care walk-in centers.

In sum, people go to a walk-in office for quick, convenient service, and overall they are happy with their experience. Although we cannot generalize from this case study of one chain of walk-in centers to walk-ins nationally, results from other studies of walk-in patient populations are approximately similar to ours.

Baseline metal concentrations in coastal Labrador sediments.

Levels of Cr, Cu, Fe, Pb, Ni, and Zn in surface sediment from the Voisey's Bay area of coastal Labrador showed no evidence of recent anthropogenic input of metals. Metal concentrations in surface sediments, normalized to Li, fell within the 95% confidence limits of the background levels. Further analysis showed that the Li-metal regression lines from the surface sediments and sediments from 30-cm depth had the same slope and intercepts, suggesting that there was no difference in the metal content of the sediments at the two depths. Li-metal relationships can be used as a measure of the natural variability of the metal concentrations for the region and will serve as a baseline against which future anthropogenic affects may be assessed.

Towards mutation-independent silencing of genes involved in retinal degeneration by RNA interference.

More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases. We describe use of RNA interference (RNAi) and specifically short hairpin RNAs (shRNAs) expressed from DNA templates to silence both normal and mutant (P23H) human rhodopsin alleles by 94.34+/-2.17 and 94.9+/-1.9%, respectively, in human embryonic retinoblasts. Degeneracy of the genetic code was used to engineer a codon-exchanged mRNA (cmRNA) that demonstrated complete resistance to silencing by the shRNA. Simulation of autosomal dominant retinitis pigmentosa in cell culture through triple transfection of DNAs expressing a cmRNA, a P23H mRNA and an shRNA revealed shRNA-mediated silencing, specifically of P23H rhodopsin by 90.64+/-5.19% and no loss of rhodopsin translation from the cmRNA in those cells. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin. Our results have implications for the treatment of a very large variety of retinal degenerations in a mutation-independent manner.

Nomenclatures of anatomical distortions of the spine: a comparison.

A review of the major chiropractic, osteopathic and medical nomenclatures for describing anatomical distortions of the spine is presented. A survey of the institutions demonstrates 10 separate nomenclatures currently being employed by the three professions to describe similar anatomical distortions of the spine. This wide variety of nomenclature can only be seen as creating barriers to communication both inter- and intraprofessionally. Because there are two differing conceptual principles for the definition of an anatomical distortion of the spine, an argument is presented for the adoption of two universal nomenclatures.

Investor-owned ambulatory care walk-in centers: how have primary care physicians responded?

Investor-owned walk-in centers are a recent innovation in ambulatory care. The authors surveyed private practice primary care physicians about their marketing strategies before and after the advent of a local walk-in center. Respondents were more likely to report that they accepted walk-ins and that they advertised in the latter than in the former time period. Findings are discussed in the framework of previous predictions about the impact of walk-in centers on the delivery of care in traditional primary care practice settings.