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.

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.

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.

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.

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.

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.

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'.

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.

Association between air pollution and multiple respiratory hospitalizations among the elderly in Vancouver, Canada.

Recurrent events, such as repeated hospital admissions for the same health outcome, occur frequently in environmental health studies. In this study, we conducted an analysis of data on repeated respiratory hospitalizations among the elderly in Vancouver, Canada, for the period of June 1, 1995, to March 31, 1999, using a new method proposed by (Dewanji and Moolgavkar 2000, 2002) for recurrent events, and compared it with some traditional methods. In particular, we assessed the impact of ambient gaseous (SO2, NO2, CO, and O3) and particulate pollutants (PM10, PM2.5, and PM10-2.5) as well as the coefficient of haze (CoH) on recurrent respiratory hospital admissions. Using the new procedure, significant associations were found between admissions and 3-day, 5-day, and 7-day moving averages of the ambient SO2 concentrations, with the strongest association observed at the 7-day lag (RR = 1.044, 95% CI: 1.018-1.070). We also found PM10-2.5 for 3-day and 5-day lag to be significant, with the strongest association at 5-day lag (RR = 1.020, 95% CI: 1.001-1.039). No significant associations with admission were found with current day exposure.

Association of ambient air pollution with respiratory hospitalization in a government-designated "area of concern": the case of Windsor, Ontario.

This study is part of a larger research program to examine the relationship between ambient air quality and health in Windsor, Ontario, Canada. We assessed the association between air pollution and daily respiratory hospitalization for different age and sex groups from 1995 to 2000. The pollutants included were nitrogen dioxide, sulfur dioxide, carbon monoxide, ozone, particulate matter 10 microm in diameter (PM10), coefficient of haze (COH), and total reduced sulfur (TRS). We calculated relative risk (RR) estimates using both time-series and case-crossover methods after controlling for appropriate confounders (temperature, humidity, and change in barometric pressure). The results of both analyses were consistent. We found associations between NO2, SO2, CO, COH, or PM10 and daily hospital admission of respiratory diseases especially among females. For females 0-14 years of age, there was 1-day delayed effect of NO2 (RR = 1.19, case-crossover method), a current-day SO2 (RR = 1.11, time series), and current-day and 1- and 2-day delayed effects for CO by case crossover (RR = 1.15, 1.19, 1.22, respectively). Time-series analysis showed that 1-day delayed effect of PM10 on respiratory admissions of adult males (15-64 years of age), with an RR of 1.18. COH had significant effects on female respiratory hospitalization, especially for 2-day delayed effects on adult females, with RRs of 1.15 and 1.29 using time-series and case-crossover analysis, respectively. There were no significant associations between O3 and TRS with respiratory admissions. These findings provide policy makers with current risks estimates of respiratory hospitalization as a result of poor ambient air quality in a government designated "area of concern."

Air pollution and daily hospital admissions for cardiovascular diseases in Windsor, Ontario.

OBJECTIVE: To examine the role that ambient air pollution plays in exacerbating cardiovascular disease hospitalization in Windsor, Ontario. METHODS: The number of daily cardiac hospital admissions was obtained from all Windsor hospitals from April 1, 1995 to December 31, 2000 and linked to concentrations of ambient air pollutants and weather variables. The logarithm of daily counts of hospitalization was regressed on the levels of pollutants, after adjusting for seasonal, weekly cycles, and weather variables using time series analysis with natural splines as smoothing functions. RESULTS: Of all the pollutants considered, sulphur dioxide (SO2) had the strongest effect on cardiac hospitalization among the > or = 65 age group. The percentage increase in daily admission was 2.6% for current day sulphur dioxide level (95% CI: 0.5-6.4), 4.0% for 2-day mean level (95% CI: 0.1-6.9), and 5.6% (95% CI: 1.5-9.9) for 3-day mean level for an increase in interquartile range of 19.3 ppb. When particulate PM10 was included in the model, the contributing effect of sulphur dioxide remained significant for the > or = 65 age group for all three levels. CONCLUSIONS: Short-term effects of sulphur dioxide are associated significantly to daily cardiac hospital admissions for people > or = 65 years of age living in Windsor. Since Windsor is a border city, additional monitoring and assessment is recommended to determine if air quality and resultant health effects have deteriorated since traffic congestion at the border has increased following the events of September 11, 2001.

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.

Comparison of time series and case-crossover analyses of air pollution and hospital admission data.

BACKGROUND: Time series analysis is the most commonly used technique for assessing the association between counts of health events over time and exposure to ambient air pollution. Recently, case-crossover analysis has been proposed as an alternative analytical approach. While each technique has its own advantages and disadvantages, there remains considerable uncertainty as to which statistical methodology is preferable for evaluating data of this type. METHODS: The objective of this paper is to evaluate the performance of different variations of these two procedures using computer simulation. Hospital admission data were generated under realistic models with known parameters permitting estimates based on time series and case-crossover analyses to be compared with these known values. RESULTS: While accurate estimates can be achieved with both methods, both methods require some decisions to be made by the researcher during the course of the analysis. With time series analysis, it is necessary to choose the time span in the LOESS smoothing process, or degrees of freedom when using natural cubic splines. For case-crossover studies using either uni- or bi-directional control selection strategies, the choice of time intervals needs to be made. CONCLUSIONS: We prefer the times series approach because the best estimates of risk that can be obtained with time series analysis are more precise than the best estimates based on case-crossover analysis.

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.

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.