Formulation development of a stable influenza recombinant neuraminidase vaccine candidate.

Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Apolipoprotein A1 and high-density lipoprotein limit low-density lipoprotein transcytosis by binding SR-B1.

Atherosclerosis results from the deposition and oxidation of LDL and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Studies have shown that transcytosis transports circulating LDL across endothelial cells lining blood vessels. LDL transcytosis is initiated by binding to either scavenger receptor B1 (SR-B1) or activin A receptor-like kinase 1 on the apical side of endothelial cells leading to its transit and release on the basolateral side. HDL is thought to partly protect individuals from atherosclerosis due to its ability to remove excess cholesterol and act as an antioxidant. Apolipoprotein A1 (APOA1), an HDL constituent, can bind to SR-B1, raising the possibility that APOA1/HDL can compete with LDL for SR-B1 binding, thereby limiting LDL deposition in the sub-arterial space. To examine this possibility, we used in vitro approaches to quantify the internalization and transcytosis of fluorescent LDL in coronary endothelial cells. Using microscale thermophoresis and affinity capture, we find that SR-B1 and APOA1 interact and that binding is enhanced when using the cardioprotective variant of APOA1 termed Milano (APOA1-Milano). In male mice, transiently increasing the levels of HDL reduced the acute deposition of fluorescently labeled LDL in the atheroprone inner curvature of the aorta. Reduced LDL deposition was also observed when increasing circulating wild-type APOA1 or the APOA1-Milano variant, with a more robust inhibition from the APOA1-Milano. The results suggest that HDL may limit SR-B1-mediated LDL transcytosis and deposition, adding to the mechanisms by which it can act as an atheroprotective particle.

Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities.

Vesicle-mediated transcellular transport or simply "transcytosis" is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between 2 neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells, the process often involves receptor-mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.

SR-BI Mediated Transcytosis of HDL in Brain Microvascular Endothelial Cells Is Independent of Caveolin, Clathrin, and PDZK1.

The vascular endothelium supplying the brain exhibits very low paracellular and transcellular permeability and is a major constituent of the blood-brain barrier. High-density lipoprotein (HDL) crosses the blood-brain barrier by transcytosis, but technical limitations have made it difficult to elucidate its regulation. Using a combination of spinning-disc confocal and total internal reflection fluorescence microscopy, we examined the uptake and transcytosis of HDL by human primary brain microvascular endothelial cell monolayers. Using these approaches, we report that HDL internalization requires dynamin but not clathrin heavy chain and that its internalization and transcytosis are saturable. Internalized HDL partially co-localized with the scavenger receptor BI (SR-BI) and knockdown of SR-BI significantly attenuated HDL internalization. However, we observed that the adaptor protein PDZK1-which is critical to HDL-SR-BI signaling in other tissues-is not required for HDL uptake in these cells. Additionally, while these cells express caveolin, the abundance of caveolae in this tissue is negligible and we find that SR-BI and caveolin do not co-fractionate. Furthermore, direct silencing of caveolin-1 had no impact on the uptake of HDL. Finally, inhibition of endothelial nitric oxide synthase increased HDL internalization while increasing nitric oxide levels had no impact. Together, these data indicate that SR-BI-mediated transcytosis in brain microvascular endothelial cells is distinct from uptake and signaling pathways described for this receptor in other cell types.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Cell and molecular biology of septins.

Septins are a family of GTP-binding proteins that assemble into cytoskeletal filaments. Unlike other cytoskeletal components, septins form ordered arrays of defined stoichiometry that can polymerize into long filaments and bundle laterally. Septins associate directly with membranes and have been implicated in providing membrane stability and serving as diffusion barriers for membrane proteins. In addition, septins bind other proteins and have been shown to function as multimolecular scaffolds by recruiting components of signaling pathways. Remarkably, septins participate in a spectrum of cellular processes including cytokinesis, ciliogenesis, cell migration, polarity, and cell-pathogen interactions. Given their breadth of functions, it is not surprising that septin abnormalities have also been linked to human diseases. In this review, we discuss the current knowledge of septin structure, assembly and function, and discuss these in the context of human disease.

Mitotic regulation of SEPT9 protein by cyclin-dependent kinase 1 (Cdk1) and Pin1 protein is important for the completion of cytokinesis.

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.

Effects of socioeconomic status on colon cancer treatment accessibility and survival in Toronto, Ontario, and San Francisco, California, 1996-2006.

OBJECTIVES: We examined the differential effects of socioeconomic status on colon cancer care and survival in Toronto, Ontario, Canada, and San Francisco, California. METHODS: We analyzed registry data for colon cancer patients from Ontario (n = 930) and California (n = 1014), diagnosed between 1996 and 2000 and followed until 2006, on stage, surgery, adjuvant chemotherapy, and survival. We obtained socioeconomic data for individuals' residences from population censuses. RESULTS: Income was directly associated with lymph node evaluation, chemotherapy, and survival in San Francisco but not in Toronto. High-income persons had better survival rates in San Francisco than in Toronto. After adjustment for stage, survival was better for low-income residents of Toronto than for those of San Francisco. Middle- to low-income patients were more likely to receive indicated chemotherapy in Toronto than in San Francisco. CONCLUSIONS: Socioeconomic factors appear to mediate colon cancer care in urban areas of the United States but not in Canada. Improvements are needed in screening, diagnostic investigations, and treatment access among low-income Americans.

Associations of physician supplies with colon cancer care in Ontario and California, 1996 to 2006.

BACKGROUND: This study examined the differential effects of physician supplies on colon cancer care in Ontario and California. The associations of physician supplies with colon cancer stage at diagnosis, receipt of surgery and adjuvant chemotherapy, and 5-year survival were observed within each country and compared between-country. METHODS: Random samples of Ontario and California cancer registries provided 2,461 and 2,200 colon cancer cases that were diagnosed between 1996 and 2000, and followed until 2006. Both registries included data on the stage of disease at the time of diagnosis, receipt of cancer-directed surgery, receipt of adjuvant chemotherapy, and survival. Census tract-level data on low-income prevalence were, respectively, taken from 2001 and 2000 Canadian and United States population censuses. County-level primary care physician and gastroenterologist densities were computed for the same years. RESULTS: Significant income-adjusted, gastroenterologist density threshold effects (2.0 or more vs. less than 2.0 per 100,000 inhabitants) were observed for early diagnosis (OR = 1.57) and 5-year survival (OR = 1.63) in Ontario, but not in California. Significant incremental threshold effects of primary care physician densities on chemotherapy receipt (8.0 and 9.0 or more per 10,000 inhabitants, respective ORs of 1.79 and 2.37) were also only observed in Ontario. CONCLUSIONS: These colon cancer care findings support the theory that while personal economic resources are more predictive in America, community-level resources such as physician supplies are more predictive of health care access and effectiveness in Canada.

Mining pharmacovigilance data using Bayesian logistic regression with James-Stein type shrinkage estimation.

Spontaneous adverse event reporting systems are widely used to identify adverse reactions to drugs following their introduction into the marketplace. In this article, a James-Stein type shrinkage estimation strategy was developed in a Bayesian logistic regression model to analyze pharmacovigilance data. This method is effective in detecting signals as it combines information and borrows strength across medically related adverse events. Computer simulation demonstrated that the shrinkage estimator is uniformly better than the maximum likelihood estimator in terms of mean squared error. This method was used to investigate the possible association of a series of diabetic drugs and the risk of cardiovascular events using data from the Canada Vigilance Online Database.

Income and long-term breast cancer survival: comparisons of vulnerable urban places in Ontario and California.

Effects of socioeconomic status on the long-term survival of 808 women with node-negative breast cancer in Canada and the United States were observed. Ontario and California samples diagnosed between 1988 and 1990 were followed until 2006. Socioeconomic data were taken from population censuses. Compared with their California counterparts, residents of low-income urban areas in Ontario experienced a significant 15-year survival advantage (RR = 1.66 [95% CI: 1.00, 2.76]). In these and other vulnerable, lower-middle- to working-class neighborhoods, significantly more Ontario residents gained access to adjuvant radiation therapy (RR = 1.75 [1.21, 2.53]) which seemed associated with better long-term survival (RR = 1.36 [0.99, 1.86]). This stage-adjusted, historical cohort analysis suggests much greater cancer care equity in Canada than in the United States.

Physician supply and breast cancer survival.

BACKGROUND: This study tested the hypothesis that physician supply thresholds are associated with breast cancer survival in Ontario. METHODS: The 5-year survival of 17,820 female breast cancer patients diagnosed between 1995 and 1997 was surveilled until 2003 for all-cause mortality. Physician supply densities in 1991 and 2001 were computed for 49 Ontario regions. RESULTS: There were independent threshold effects for general practitioners (GP; 7.25 per 10,000) and obstetrician/gynecologists (OB/GYN; 6 per 100,000) at or above which women with breast cancer were more likely to survive for 5 years. The respective risk of living in areas undersupplied with OB/GYN and GP increased 30% to nearly 5-fold during the 1990s. Five-year survival tended to be lower in provincial areas outside of Toronto, which experienced GP (odds ratio, 0.83; 90% CI, 0.70-0.99) and OB/GYN (odds ratio, 0.76; 95% CI, 0.61-0.96) supply decreases. CONCLUSION: As they do in America, primary care physician supplies in Canada seem to matter in the effective provision of cancer care. Community resources such as health care service endowments, including physician supplies, may be particularly critical to the performance of health care systems such as Canada's, which aim to provide medically necessary care for all.

Breast cancer care in the Canada and the United States: ecological comparisons of extremely impoverished and affluent urban neighborhoods.

This study examined the differential effect of extreme impoverishment on breast cancer care in urban Canada and the United States. Ontario and California registry-based samples diagnosed between 1998 and 2000 were followed until 2006. Extremely poor and affluent neighborhoods were compared. Poverty was associated with non-localized disease, surgical and radiation therapy (RT) waits, non-receipt of breast conserving surgery, RT and hormonal therapy, and shorter survival in California, but not in Ontario. Extremely poor Ontario women were consistently advantaged on care indices over their California counterparts. More inclusive health insurance coverage in Canada seems the most plausible explanation for such Canadian breast cancer care advantages.

Associations of physician supplies with breast cancer stage at diagnosis and survival in Ontario, 1988 to 2006.

BACKGROUND: The authors examined whether the supply of primary care physicians had protective effects on breast cancer stage and survival in Ontario and whether supply losses during the 1990s were associated with diminished protection. METHODS: Random samples of the Ontario Cancer Registry, respectively, provided 879 women and 951 women who were diagnosed with breast cancer between 1988 and 1990 (followed until 1996) and 1998 and 2000 (followed until 2006), respectively. Active physician supply data (1991 and 2001) joined to each woman's census division of residence was taken from the Scott's Medical Database. RESULTS: Protective thresholds were observed among the earlier cohort for supplies of general practitioners (7 per 10,000 population) and supplies of obstetricians/gynecologists (6 per 100,000 population) at or above which women with breast cancer were significantly more likely to have been diagnosed with localized disease and to have survived for >or=5 years. These protective effects seemed generally attenuated among the more recent cohort. The risk of living in primary care physician-undersupplied areas increased significantly between 1991 and 2001 (10%-30%), and such physician supply losses were associated with reduced cancer care protection, including less prevalent early diagnoses (odds ratio [OR], 1.60; 95% confidence interval [95% CI], 1.00-2.58) and lower 5-year survival rates (OR, 1.62; 95% CI, 1.03-2.55). CONCLUSIONS: Primary care physician supplies appeared to matter very much in the effective provision of cancer care in Canada. Community healthcare service endowments that include adequate physician supplies may be particularly critical to the performance of a healthcare system such as that in Canada, which provides universal accessibility to medically necessary care.

Wait times for surgical and adjuvant radiation treatment of breast cancer in Canada and the United States: greater socioeconomic inequity in America.

PURPOSE: The demand for cancer care has increased among aging North American populations as cancer treatment innovations have proliferated. Gaps between supply and demand may be growing. This study examined whether socioeconomic status has a differential effect on waits for surgical and adjuvant radiation treatment (RT) of breast cancer in Canada and the US. METHODS: Ontario and California cancer registries provided 929 and 984 breast cancer cases diagnosed between 1998 and 2000 in diverse urban and rural places. Residence-based socioeconomic data were taken from censuses. Cancer care variables were reliably abstracted from health records: stage, receipt of surgery and RT, and waits from diagnosis to initial and initial to adjuvant treatment. Median waits were compared within- and between-country with the non-parametric Mann-Whitney U-test. Categorically long, age-adjusted wait comparisons used the Mantel-Haenszel chi-square test. RESULTS: There were significant associations between lower socioeconomic status and longer surgical waits, lower access to adjuvant RT and to longer RT waits across diverse places in California. None were observed in Ontario. The two cohorts did not practically differ on access to surgery or on surgical waits. Compared with their counterparts in California, low-income Ontarians, particularly those in small urban places, gained greater access to RT, while high-income Americans had shorter waits for RT. CONCLUSIONS: This historical study contextualized Canada's "waiting-list problems" with evidence on breast cancer care, where lower income Americans seemed to have waited as long as similar Canadians. Many more low-income Americans seemed to experience the longest wait of all for adjuvant care. They simply did not receive it. In contrast to stark American socioeconomic inequity, this study evidenced remarkable equity in Canadian breast cancer care.

Breast cancer survival in ontario and california, 1998-2006: socioeconomic inequity remains much greater in the United States.

This study re-examined the differential effect of socioeconomic status on the survival of women with breast cancer in Canada and the United States. Ontario and California cancer registries provided 1,913 cases from urban and rural places. Stage-adjusted cohorts (1998-2000) were followed until 2006. Socioeconomic data were taken from population censuses. SES-survival associations were observed in California, but not in Ontario, and Canadian survival advantages in low-income areas were replicated. A better controlled and updated comparison reaffirmed the equity advantage of Canadian health care.

Increased racial differences on breast cancer care and survival in America: historical evidence consistent with a health insurance hypothesis, 1975-2001.

PURPOSE: This study examined whether race/ethnicity had differential effects on breast cancer care and survival across age strata and cohorts within stages of disease. METHODS: The Detroit Cancer Registry provided 25,997 breast cancer cases. African American and non-Hispanic white, older Medicare-eligible and younger non-eligible women were compared. Successive historical cohorts (1975-1980 and 1990-1995) were, respectively, followed until 1986 and 2001. RESULTS: African American disadvantages on survival and treatments increased significantly, particularly among younger women who were much more likely to be uninsured. Within node positive disease all treatment disadvantages among younger African American women disappeared with socioeconomic adjustment. CONCLUSIONS: Growth of this racial divide implicates social, rather than biological, forces. Its elimination will require high quality health care for all.

Cancer survival in Ontario, 1986-2003: evidence of equitable advances across most diverse urban and rural places.

OBJECTIVES: This study examined whether place and socio-economic status had differential effects on the survival of women diagnosed with breast cancer in Ontario during the 1980s and the 1990s. METHODS: The Ontario Cancer Registry provided 29,934 primary malignant breast cancer cases. Successive historical cohorts (1986-1988 and 1995-1997) were, respectively, followed until 1994 and 2003. Diverse places were compared: the greater metropolitan Toronto area, other cities, ranging in size from 50,000 to a million people, smaller towns and villages, and rural and remote areas. Socio-economic data for each woman's residence at the time of diagnosis were taken from population censuses. RESULTS: Very small cities (6%) with populations between 50,000 and 100,000 were the only places where breast cancer survival had advanced less compared to the province as a whole. Income gradients began to appear, however, in larger cities. Urban residents in the lowest income areas were significantly disadvantaged compared to the highest income areas during the 1990s, but not during the 1980s. CONCLUSION: This historical analysis of breast cancer survival evidenced remarkably equitable advances across nearly all of Ontario's diverse places. The most likely explanation for such substantial equity seems to be Canada's universally accessible, single-payer, health care system.

Impact of air pollution on hospital admissions in Southwestern Ontario, Canada: generating hypotheses in sentinel high-exposure places.

BACKGROUND: Southwestern Ontario (SWO) in Canada has been known as a 'hot spot' in terms of environmental exposure and potential effects. We chose to study 3 major cities in SWO in this paper. We compared age-standardized hospital admission ratios of Sarnia and Windsor to London, and to generate hypotheses about potential pollutant-induced health effects in the 'Chemical Valley', Sarnia. METHODS: The number of daily hospital admissions was obtained from all hospitals in London, Windsor and Sarnia from January 1, 1996 to December 31, 2000. We used indirect age adjustment method to obtain standardized admissions ratios for males and females and we chose London as the reference population. This process of adjustment was to apply the age-specific admission rates of London to the population of Sarnia and Windsor in order to yield expected admissions. The observed number of admissions was then compared to the expected admissions in terms of a ratio. These standardized admissions ratios and their corresponding confidence intervals were calculated for Sarnia and Windsor. RESULTS: Our findings showed that Sarnia and Windsor had significantly higher age-adjusted hospital admissions rates compared to London. This finding was true for all admissions, and especially pronounced for cardiovascular and respiratory admissions. For example, in 1996, the observed number of admissions in Sarnia was 3.11 (CI: 2.80, 3.44) times for females and 2.83 (CI: 2.54, 3.14) times for males as would be expected by using London's admission rates. CONCLUSION: Since hospital admissions rates were significantly higher in 'Chemical Valley' as compared to both London and Windsor, we hypothesize that these higher rates are pollution related. A critical look at the way ambient air quality and other pollutants are monitored in this area is warranted. Further epidemiological research is needed to verify our preliminary indications of harmful effects in people living in 'Chemical Valley'.

A comparison of methods for the analysis of recurrent health outcome data with environmental covariates.

Recurrent events such as repeated hospital admissions for the same health outcome occur frequently in environmental health studies. Dewanji and Moolgavkar proposed a flexible parametric model and a conditional likelihood analysis for recurrent events based on a Poisson process formulation. In this paper, we examine the statistical properties of the Dewanji-Moolgavkar (DM) estimator of the risk of an adverse health outcome associated with environmental exposures based on recurrent event data using computer simulation. We also compare the DM approach with both case-crossover analysis for multiple observations and time series analysis when there are no subject-specific covariates. When using a correctly specified model, the DM method produced better estimates with respect to relative mean square error when each subject had constant or curved baseline intensity functions than it did when baseline intensities were increasing or decreasing in a linear fashion. For under-specified models, the DM method outperformed case-crossover analysis for decreasing straight line intensity functions, was outperformed by case-crossover analysis for increasing straight line intensity functions, and was roughly equivalent to case-crossover analysis for constant and curved intensity functions. Case-crossover analysis produced superior risk estimates more frequently than the other two methods in the cases considered here, especially for linear representations of the baseline intensities.