Development and Qualification of Analytical Methods to Support Low Concentration Drug Product in-use Studies.

The emergence of highly potent therapeutics with low expected clinical doses creates a challenge for analytical characterization of simulated drug product in-use samples. The low expected protein concentration (often µg/mL) and highly charged and sub-optimal sample matrices like 0.9% saline or 5% dextrose make ensuring dose solution stability and characterizing product quality changes difficult. Health authority expectations require analysis of low concentration in-use samples to be completed with suitable assays to ensure little to no changes are occurring during drug product dose preparation and administration, thus ensuring patient safety. However, characterization of these samples for protein concentration, size variants, charge variants and potency often necessitates additional analytical method development to improve sensitivity and compatibility with in-use samples. Here we report the development and qualification of reliable in-use methods to characterize simulated in-use samples to assist during drug product development.

Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization.

CRISPR (clustered regularly interspaced short palindromic repeats)-associated proteins (Cas) are powerful gene-editing tools used in therapeutic applications. Efforts to minimize off-target cleavage by CRISPR-Cas9 have motivated the development of engineered Cas9 variants. The wild-type (WT) Streptococcus pyogenes (SpCas9) has been engineered into a high-fidelity Cas9 (SpyFi Cas9) that shows promising results in providing high on-target activity (targeting efficiency) while reducing off-target editing (unwanted mutations). This work describes for the first time the development of ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE)-based methods for a full characterization of different engineered Cas9 variants, including determination of purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities. The purity and size variant characterization were first determined by CE-sodium dodecyl sulfate (SDS). An in vitro DNA cleavage assay using an automated electrophoresis tool was employed to investigate the functional activity of ribonucleoprotein (RNP) complexes derived from Cas9 variants. The pIs of the engineered Cas9 proteins were determined by imaged capillary isoelectric focusing (icIEF), while intact mass measurements were performed by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS). A peptide mapping assay based on LC-UV-MS/MS using endoproteinase Lys-C under non-reducing conditions was developed to confirm amino acid sequences, allowing differentiation of SpyFi Cas9 from WT SpCas9. The potential of using a low-resolution MS detector, especially for a GMP environment, as a low-cost and simple method to identify SpyFi Cas9 is discussed.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Using the delayed spatial alternation task to assess environmentally associated changes in working memory in very young children.

BACKGROUND: Working memory (WM) is critical for problem solving and reasoning. Beginning in infancy, children show WM capacity increasing with age but there are few validated tests of WM in very young children. Because rapid brain development may increase susceptibility to adverse impacts of prenatal neurotoxicant exposure, such as lead, tests of WM in very young children would help to delineate onset of developmental problems and windows of susceptibility. PURPOSE: Our objective was to assess the feasibility of administering a Delayed Spatial Alternation Task (DSAT) to measure WM among 18- and 24-month old children enrolled in an ongoing longitudinal birth cohort study and compare DSAT performance with age and general cognitive development. We further explored whether prenatal lead exposure impacted DSAT performance. METHODS: We assessed 457 mother-child pairs participating in the Programming Research in Obesity, GRowth, Environment and Social Stressors (PROGRESS) Study in Mexico City. The DSAT and Bayley Scales of Infant Development (BSID-III) were administered at 18- and 24-months. Lead was measured in maternal blood collected during pregnancy (MBPb) and in a subsample of children at 24-months (CBPb). We regressed DSAT measures on MBPb and CBPb, child sex, and maternal age, education, socioeconomic status, and household smoking. We compared DSAT performance to BSID-III performance with adjusted residuals. RESULTS: 24-month children perform better on the DSAT than 18-month children; 24-month subjects reached a higher level on the DSAT (3.3 (0.86) vs. 2.4 (0.97), p < 0.01), and had a higher number of correct responses (20.3 vs. 17.2, p < 0.01). In all DSAT parameters, females performed better than males. Maternal education predicted better DSAT performance; household smoking predicted worse DSAT performance. A higher number of correct responses was associated with higher BSID-III Cognitive scales at 18 months (r = 0.20, p < 0.01) and 24 months (r = 0.27, p < 0.01). MBPb and CPBb did not significantly predict DSAT performance. CONCLUSION: Improved performance on the DSAT with increasing age, the positive correlation with the BSID-III cognitive and language scales and the correlation with common sociodemographic predictors of neurodevelopment demonstrate the validity of the DSAT as a test of infant development.

E-cigarettes in airports and on flights: Europe and the US. / Cigarrillos electrónicos en aeropuertos y en vuelos: Europa y EEUU.

BACKGROUND: While progress has been made to create smoke-free airports, sales of e-cigarettes at airports and airplanes and the presence of advertisements might detract from these smoke-free policies. The objective of this study is to describe the presence of policies, advertising, sales and use of e-cigarettes in airports and on flights in Europe and the US. METHODS: A cross-sectional study was conducted between March-May, 2014. The study included 21 large and mid-sized airports of Europe and the US as well as 19 flights. A standardised protocol was used to observe points of sales and advertisements and to collect information on the implementation of policies on e-cigarette use. In addition, a series of questions were developed to obtain policy details from airport personnel and flight attendants. RESULTS: Retail outlets selling e-cigarettes in airports were present in approximately 20% and 40% of the observed pre and post-security areas, respectively. In post-security, 27.8% of the airport staff reported that the use of e-cigarettes indoors was not allowed, 22.2% reported that they did not know, 27.8% reported that it was only allowed in the smoking room, and 22.2% reported that it was allowed anywhere. Smoking ban announcements were made on all flights. However, only 15.8% of the flights made a specific announcement regarding the ban of using e-cigarettes. Conclusions. In light of our results, it seems necessary to reinforce in-flight e-cigarette smoking ban announcements and to instruct airport employees about the existence of e-cigarette smoking policies. Furthermore, airports themselves should also be encouraged to adopt smoke-free policies.

Antecedentes. Pese a los avances en las políticas libres de humo en los aeropuertos, las ventas de cigarrillos electrónicos en aeropuertos y aviones y la presencia de publicidad pueden suponer un paso atrás en la implementación de dichas políticas. El objetivo de este estudio es describir la presencia de políticas, publicidad, ventas y el uso de cigarrillos electrónicos en aeropuertos y en vuelos de Europa y los EE.UU.Métodos. Estudio transversal realizado entre marzo y mayo del año 2014. El estudio incluyó 21 aeropuertos grandes y medianos de Europa y los EE.UU., así como 19 vuelos. Se utilizó un protocolo estandarizado para observar puntos de venta y publicidad y se recogió información sobre la implementación de políticas sobre el uso de cigarrillos electrónicos. Además, obtuvo información más detallada del personal del aeropuerto y de los asistentes de vuelo sobre las políticas de uso de cigarrillo electrónicos.Resultados. Los puntos de venta de cigarrillos electrónicos en los aeropuertos estaban presentes en aproximadamente el 20% y el 40% de las áreas observadas antes y después del control de seguridad, respectivamente. Después del control, el 27,8% del personal del aeropuerto declaró que no estaba permitido el uso los cigarrillos electrónicos en el interior, el 22,2% declaró que no sabía si se podían usar, el 27,8% declaró que sólo estaba permitido en el área de fumadores y el 22,2% declaró que se podía fumar en cualquier parte. Todos los vuelos anunciaron la prohibición de fumar. Sin embargo, sólo el 15,8% de los vuelos específicamente anunció la prohibición de usar cigarrillos electrónicos.Conclusiones. Nuestros resultados muestran que sería necesario reforzar los avisos de prohibición del uso de cigarrillos electrónicos durante los vuelos y de instruir a los empleados del aeropuerto sobre la existencia de políticas sobre el uso de cigarrillos electrónicos. Además, también se debería promover políticas libres de humo sin excepciones en todos los aeropuertos.

Characterization of Ring-Opening Reaction of Succinimide Linkers in ADCs.

A new class of highly potent biopharmaceutical drugs, antibody-drug conjugates (ADCs), has been proven to be clinically effective to treat oncologic diseases. ADCs contain 3 major components: the monoclonal antibody, cytotoxic drug, and chemical linker. THIOMAB™ drug conjugates and interchain-cysteine ADCs are common ADC platforms that apply thiol-maleimide chemistry via Michael addition to conjugate linker-drugs to cysteine residues. However, the resulting succinimide ring in the linker is susceptible to ring-opening reactions via hydrolysis, especially at high pH and elevated temperatures. Once the succinimide ring is opened, in vivo stability of the ADCs can be changed and the therapeutic activity will be altered. In this study, we investigated the impact of conjugation sites on succinimide ring opening for ADCs. A new methodology based on imaged capillary isoelectric focusing was developed to monitor the formation of succinimide ring-opened products. In addition, a reverse-phase high-performance liquid chromatography method was used to monitor site-specific ring-opening reactions. Our data confirmed that succinimide ring-opening rates in ADCs are conjugation-site dependent. With a good understanding of the conjugation site impact on final product's stability, it is potentially feasible to modify ring-opening rates in vitro to achieve desirable in vivo stability and biological activity.

Cigarrillos electrónicos en aeropuertos y en vuelos: Europa y EEUU / E-cigarettes in airports and on flights: Europe and the US

Antecedentes. Pese a los avances en las políticas libres de humo en los aeropuertos, las ventas de cigarrillos electrónicos en aeropuertos y aviones y la presencia de publicidad pueden suponer un paso atrás en la implementación de dichas políticas. El objetivo de este estudio es describir la presencia de políticas, publicidad, ventas y el uso de cigarrillos electrónicos en aeropuertos y en vuelos de Europa y los EE.UU. Métodos. Estudio transversal realizado entre marzo y mayo del año 2014. El estudio incluyó 21 aeropuertos grandes y medianos de Europa y los EE.UU., así como 19 vuelos. Se utilizó un protocolo estandarizado para observar puntos de venta y publicidad y se recogió información sobre la implementación de políticas sobre el uso de cigarrillos electrónicos. Además, obtuvo información más detallada del personal del aeropuerto y de los asistentes de vuelo sobre las políticas de uso de cigarrillo electrónicos. Resultados. Los puntos de venta de cigarrillos electrónicos en los aeropuertos estaban presentes en aproximadamente el 20% y el 40% de las áreas observadas antes y después del control de seguridad, respectivamente. Después del control, el 27,8% del personal del aeropuerto declaró que no estaba permitido el uso los cigarrillos electrónicos en el interior, el 22,2% declaró que no sabía si se podían usar, el 27,8% declaró que sólo estaba permitido en el área de fumadores y el 22,2% declaró que se podía fumar en cualquier parte. Todos los vuelos anunciaron la prohibición de fumar. Sin embargo, sólo el 15,8% de los vuelos específicamente anunció la prohibición de usar cigarrillos electrónicos. Conclusiones. Nuestros resultados muestran que sería necesario reforzar los avisos de prohibición del uso de cigarrillos electrónicos durante los vuelos y de instruir a los empleados del aeropuerto sobre la existencia de políticas sobre el uso de cigarrillos electrónicos. Además, también se debería promover políticas libres de humo sin excepciones en todos los aeropuertos

Background. While progress has been made to create smoke-free airports, sales of e-cigarettes at airports and airplanes and the presence of advertisements might detract from these smoke-free policies. The objective of this study is to describe the presence of policies, advertising, sales and use of e-cigarettes in airports and on flights in Europe and the US. Methods. A cross-sectional study was conducted between March-May, 2014. The study included 21 large and mid-sized airports of Europe and the US as well as 19 flights. A standardised protocol was used to observe points of sales and advertisements and to collect information on the implementation of policies on e-cigarette use. In addition, a series of questions were developed to obtain policy details from airport personnel and flight attendants. Results. Retail outlets selling e-cigarettes in airports were present in approximately 20% and 40% of the observed pre and post-security areas, respectively. In post-security, 27.8% of the airport staff reported that the use of e-cigarettes indoors was not allowed, 22.2% reported that they did not know, 27.8% reported that it was only allowed in the smoking room, and 22.2% reported that it was allowed anywhere. Smoking ban announcements were made on all flights. However, only 15.8% of the flights made a specific announcement regarding the ban of using e-cigarettes. Conclusions. In light of our results, it seems necessary to reinforce in-flight e-cigarette smoking ban announcements and to instruct airport employees about the existence of e-cigarette smoking policies. Furthermore, airports themselves should also be encouraged to adopt smoke-free policies

Impact of Blood Collection Tubes and Sample Handling Time on Serum and Plasma Metabolome and Lipidome.

Background: Metabolomics is emerging as a valuable tool in clinical science. However, one major challenge in clinical metabolomics is the limited use of standardized guidelines for sample collection and handling. In this study, we conducted a pilot analysis of serum and plasma to determine the effects of processing time and collection tube on the metabolome. Methods: Blood was collected in 3 tubes: Vacutainer serum separator tube (SST, serum), EDTA (plasma) and P100 (plasma) and stored at 4 degrees for 0, 0.5, 1, 2, 4 and 24 h prior to centrifugation. Compounds were extracted using liquid-liquid extraction to obtain a hydrophilic and a hydrophobic fraction and analyzed using liquid chromatography mass spectrometry. Differences among the blood collection tubes and sample processing time were evaluated (ANOVA, Bonferroni FWER ≤ 0.05 and ANOVA, Benjamini Hochberg FDR ≤ 0.1, respectively). Results: Among the serum and plasma tubes 93.5% of compounds overlapped, 382 compounds were unique to serum and one compound was unique to plasma. There were 46, 50 and 86 compounds affected by processing time in SST, EDTA and P100 tubes, respectively, including many lipids. In contrast, 496 hydrophilic and 242 hydrophobic compounds differed by collection tube. Forty-five different chemical classes including alcohols, sugars, amino acids and prenol lipids were affected by the choice of blood collection tube. Conclusion: Our results suggest that the choice of blood collection tube has a significant effect on detected metabolites and their overall abundances. Perhaps surprisingly, variation in sample processing time has less of an effect compared to collection tube; however, a larger sample size is needed to confirm this.

Environmental exposures and pediatric kidney function and disease: A systematic review.

BACKGROUND: Environmental chemical exposures have been implicated in pediatric kidney disease. No appraisal of the available evidence has been conducted on this topic. METHODS: We performed a systematic review of the epidemiologic studies that assessed association of environmental exposures with measures of kidney function and disease in pediatric populations. The search period went through July 2016. RESULTS: We found 50 studies that met the search criteria and were included in this systematic review. Environmental exposures reviewed herein included lead, cadmium, mercury, arsenic, fluoride, aflatoxin, melamine, environmental tobacco, bisphenol A, dental procedures, phthalates, ferfluorooctanoic acid, triclosan, and thallium/uranium. Most studies assessed environmental chemical exposure via biomarkers but four studies assessed exposure via proximity to emission source. There was mixed evidence of association between metal exposures, and other non-metal environmental exposures and pediatric kidney disease and other kidney disease biomarkers. The evaluation of causality is hampered by the small numbers of studies for each type of environmental exposure, as well as lack of study quality and limited prospective evidence. CONCLUSION: There is a need for well-designed epidemiologic studies of environmental chemical exposures and kidney disease outcomes.

Tobacco Smoke Exposure in Indoor and Outdoor Locations in Airports Across Europe and the United States: A Cross-Sectional Study.

OBJECTIVE: Conduct a systematic evaluation of indoor and outdoor areas of selected airports, assess compliance and identify areas of improvement with smoke-free policies in airports. METHODS: Cross-sectional observational study conducted at 21 airports in Europe (11) and the United States (10). Using a standardized protocol, we assessed compliance (smoking, cigarette butts, smoke smell), and the physical environment (signage, ashtrays, designated smoking rooms [DSRs], tobacco sales). RESULTS: Cigarette butts (45% vs. 0%), smoke smell (67% vs. 0%), ashtrays (18% vs. 10%), and DSRs (63% vs. 30%) were observed more commonly indoors in Europe than in the United States. Poor compliance indoors was related to the presence of DSRs (OR 4.8, 95% CI 0.69, 33.8) and to cigarettes sales in pre-security areas (OR 6.0, 95% CI 0.57, 64.7), although not significantly different. Smoking was common in outdoor areas of airports in Europe and the United States (mean (SD) number of smokers 27.7 (23.6) and 6.3 (7.7), respectively, p value < .001). Around half (55%) of airports in Europe and all airports in the United States had some/partial outdoor smoking restrictions. CONCLUSIONS: Exposure to secondhand smoke (SHS) remains a public health problem in major airports across Europe and in some airports in the United States, specifically related to the presence of DSRs and SHS exposure in outdoor areas. Airports must remove DSRs. Research is needed in low- and middle-income countries and on the effectiveness of outdoor smoking-restricted areas around entryways. Eliminating smoking at airports will protect millions of people from SHS exposure and promote social norms that discourage smoking. IMPLICATIONS: Airports are known to allow exceptions to smoke-free policy by providing DSRs. We found that smoking still occurs in indoor areas in airports, particularly in the context of DSRs. Smoking, moreover, is widespread in outdoor areas and compliance with smoking restrictions is limited. Advancing smoke-free policy requires improvements to the physical environment of airports, including removal of DSRs and implementation of stricter outdoor smoking restrictions.

Factors that influence attitude and enforcement of the smoke-free law in Turkey: a survey of hospitality venue owners and employees.

INTRODUCTION: In 2009, Turkey extended the smoke-free legislation to hospitality venues. Compliance, however, remains low in some hospitality venues. We identified characteristics associated with knowledge of health effects that can be prevented by the smoke-free law, the attitude towards and enforcement of the law. METHODS: In 2014, we conducted 400 interviews with hospitality venue owners and employees in 7 cities in Turkey. The venues were identified based on a random sampling strategy in a previous phase of the study. RESULTS: Over one-third (37.3%) of hospitality owners and employees had adequate knowledge of the health effects from secondhand smoke (SHS), 71.3% had a positive attitude towards the law and 19.5% had personally enforced the law. Participants who worked 70 hours or more per week were more likely to have a positive attitude towards the law. Older individuals, women, participants working in bars/nightclubs, venue owners receiving fines for non-compliance and current smokers were less likely to have a positive attitude towards the law. Participants working in traditional coffee houses, former smokers, and participants with a high school education or greater were more likely to enforce the law. Smokers who quit or reduced smoking because of the law were more likely to enforce the law compared with those who were not influenced by the law. CONCLUSIONS: Although the attitude towards the law was positive, interventions are needed to increase knowledge on the health effects of SHS and facilitate enforcement of the law, particularly among subgroups less likely to have a positive attitude and enforce the law.

Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla).

The antibody-drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the N-hydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da. This series is consistent with the presence of a population containing a bound linker without DM1 ("unconjugated linker"). Extended characterization of trastuzumab emtansine was performed using capillary isoelectic focusing, CE-SDS, peptide mapping, and LC/MS following (18)O labeling of peptide digests to identify this family of product variants. These studies demonstrate that the presence of these +222 Da species is due to an unexpected reaction of the maleimide moiety in the MCC linker with antibody lysine residues to produce cross-linked species that cannot conjugate to DM1.

Investigation of low recovery in the free drug assay for antibody drug conjugates by size exclusion-reversed phase two dimensional-liquid chromatography.

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the selectivity of monoclonal antibodies and highly efficacious small molecule drugs to successfully eliminate tumor cells while limiting the general toxicity and side effects of the therapeutic to protect patient safety. One unique attribute critical to the safety of ADCs is the residual content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. Typically for quality control assays, quantifying the amount of the free drug is performed through precipitation of the protein species using an organic solvent and then assaying the amount of free drug left in the supernatant. During the validation of an assay of this type for a maleimide based linker drug, issues were experienced with low and variable recovery in the spiked samples of the drug substance and drug product. A two-dimensional heart-cutting method coupling Size Exclusion Chromatography (SEC) with Reverse Phase (RP) chromatography was utilized to explore possible mechanisms leading to the low recovery of the free linker drug. The results of the investigation indicated that the spiked linker drug reacts with residual reactive groups on the ADC; a conclusion which was confirmed by the observed increase of average Drug to Antibody Ratio (DAR) determined by Hydrophobic Interaction Chromatography (HIC). Finally, several approaches were evaluated to minimize the recovery loss. Capping the residual reactive groups on the ADC with maleimide containing reagents effectively mitigated the low recovery issue.

The association between airline flight and sinonasal symptoms.

BACKGROUND: Airplane cabin supply air has been shown to contain multiple possible respiratory irritants. In addition, changes in barometric pressure in flight may contribute to specific respiratory conditions. Therefore, there may be an association between commercial airline flight and sinus disease. METHODS: Participants of the Secondhand-Smoke, Air Quality and Respiratory Health Among Flight Attendants Study were administered an online questionnaire pertaining to their flight experience and respiratory health. Working years, working days per month, and number of trips per month were quantified, as well as smoking exposure and self-reported physician diagnoses of sinusitis, asthma, and rhinitis. The sinonasal outcomes were quantified using a Respiratory Questionnaire Survey (RQS) score. Multivariable analyses were performed to evaluate the associations between flight time and sinus disease. RESULTS: A total of 579 participants met the inclusion criteria for this study, with cohort prevalence of sinusitis, asthma, and rhinitis of 25.3%, 14.4%, and 20.5%, respectively. Tertiles 2 and 3 of working days per month were associated with higher RQS scores compared to tertile 1 (p for trend <0.01). Individual symptoms significantly associated with increasing number of working days per month included "need to blow nose," "sneezing," and "thick nasal discharge," and the number of international trips per month was significantly associated with "coughing" and "facial pain and pressure," among other symptoms. CONCLUSION: This is the largest study to analyze the relations between airline flight time and sinonasal disease. The results suggest a possible association between sinusitis diagnosis, symptom scores, and specific sinonasal symptoms, and airline flight time.

Clear Skies and Grey Areas: Flight Attendants' Secondhand Smoke Exposure and Attitudes toward Smoke-Free Policy 25 Years since Smoking was Banned on Airplanes.

Our objective was to provide descriptive data on flight attendant secondhand smoke (SHS) exposure in the work environment, and to examine attitudes toward SHS exposure, personal health, and smoke-free policy in the workplace and public places. Flight attendants completed a web-based survey of self-reported SHS exposure and air quality in the work environment. We assessed the frequency and duration of SHS exposure in distinct areas of the workplace, attitudes toward SHS exposure and its health effects, and attitudes toward smoke-free policy in the workplace as well as general public places. A total of 723 flight attendants participated in the survey, and 591 responded to all survey questions. The mean level of exposure per flight attendant over the past month was 249 min. The majority of participants reported being exposed to SHS always/often in outdoor areas of an airport (57.7%). Participants who worked before the in-flight smoking ban (n=240) were more likely to support further smoking policies in airports compared to participants who were employed after the ban (n=346) (76.7% versus 60.4%, p-value<0.01). Flight attendants are still being exposed to SHS in the workplace, sometimes at concerning levels during the non-flight portions of their travel. Flight attendants favor smoke-free policies and want to see further restrictions in airports and public places.

Validation of PR interval length as a criterion for development of atrial fibrillation in non-Hispanic whites, African Americans and Hispanics.

BACKGROUND: PR interval prolongation on electrocardiogram (ECG) increases the risk of atrial fibrillation (AF). Non-Hispanic Whites are at higher risk of AF compared to African Americans and Hispanics. However, it remains unknown if prolongation of the PR interval for the development of AF varies by race/ethnicity. Therefore, we determined whether race affects the PR interval length's ability to predict AF and if the commonly used criterion of 200 ms in AF prediction models can continue to be used for non-White cohorts. METHODS: This is a retrospective epidemiological study of consecutive inpatient and outpatients. An ECG database was initially interrogated. Patients were included if their initial ECG demonstrated sinus rhythm and had two or more electrocardiograms and declared a race and/or ethnicity as non-Hispanic White, African American or Hispanic. Development of AF was stratified by race/ethnicity along varying PR intervals. Cox models controlled for age, gender, race/ethnicity, systolic blood pressure, BMI, QRS, QTc, heart rate, murmur, treatment for hypertension, heart failure and use of AV nodal blocking agents to assess PR interval's predictive ability for development of AF. RESULTS: 50,870 patients met inclusion criteria of which 5,199 developed AF over 3.72 mean years of follow-up. When the PR interval was separated by quantile, prolongation of the PR interval to predict AF first became significant in Hispanic and African Americans at the 92.5th quantile of 196-201 ms (HR: 1.42, 95% CI: 1.09-1.86, p=0.01; HR: 1.32, 95% CI: 1.07-1.64, p=0.01, respectively) then in non-Hispanic Whites at the 95th quantile at 203-212 ms (HR: 1.24, 95% CI: 1.24-1.53, p=0.04). For those with a PR interval above 200 ms, African Americans had a lower risk than non-Hispanic Whites to develop AF (HR: 0.80, 95% CI: 0.64-0.95, p=0.012), however, no significant difference was demonstrated in Hispanics. CONCLUSIONS: This is the first study to validate a PR interval value of 200 ms as a criterion in African Americans and Hispanics for the development of AF. However, a value of 200 ms may be less sensitive as a predictive measure for the development of AF in African Americans compared to non-Hispanic Whites.

The association of urine arsenic with prevalent and incident chronic kidney disease: evidence from the Strong Heart Study.

BACKGROUND: Few studies have evaluated associations between low to moderate arsenic levels and chronic kidney disease (CKD). The objective was to evaluate the associations of inorganic arsenic exposure with prevalent and incident CKD in American Indian adults. METHODS: We evaluated the associations of inorganic arsenic exposure with CKD in American Indians who participated in the Strong Heart Study in 3,851 adults ages 45-74 years in a cross-sectional analysis, and 3,119 adults with follow-up data in a prospective analysis. Inorganic arsenic, monomethylarsonate, and dimethylarsinate were measured in urine at baseline. CKD was defined as estimated glomerular filtration rate ≤ 60 ml/min/1.73 m, kidney transplant or dialysis. RESULTS: CKD prevalence was 10.3%. The median (IQR) concentration of inorganic plus methylated arsenic species (total arsenic) in urine was 9.7 (5.8, 15.7) µg/L. The adjusted odds ratio (OR; 95% confidence interval) of prevalent CKD for an interquartile range in total arsenic was 0.7 (0.6, 0.8), mostly due to an inverse association with inorganic arsenic (OR: 0.4 [0.3, 0.4]). Monomethylarsonate and dimethylarsinate were positively associated with prevalent CKD after adjustment for inorganic arsenic (OR: 3.8 and 1.8). The adjusted hazard ratio of incident CKD for an IQR in sum of inorganic and methylated arsenic was 1.2 (1.03, 1.41). The corresponding HRs for inorganic arsenic, monomethylarsonate, and dimethylarsinate were 1.0 (0.9, 1.2), 1.2 (1.00, 1.3), and 1.2 (1.0, 1.4). CONCLUSIONS: The inverse association of urine inorganic arsenic with prevalent CKD suggests that kidney disease affects excretion of inorganic arsenic. Arsenic species were positively associated with incident CKD. Studies with repeated measures are needed to further characterize the relation between arsenic and kidney disease development.

Arsenic and Chronic Kidney Disease: A Systematic Review.

In epidemiologic studies, high arsenic exposure has been associated with adverse kidney disease outcomes. We performed a systematic review of the epidemiologic evidence of the association between arsenic and various kidney disease outcomes. The search period was January 1966 through January 2014. Twenty-five papers (comprising 24 studies) meeting the search criteria were identified and included in this review. In most studies, arsenic exposure was assessed by measurement of urine concentrations or with an ecological indicator. There was a generally positive association between arsenic and albuminuria and proteinuria outcomes. There was mixed evidence of an association between arsenic exposure and chronic kidney disease (CKD), ß-2 microglobulin (ß2MG), and N-acetyl-ß-D-glucosaminidase (NAG) outcomes. There was evidence of a positive association between arsenic exposure and kidney disease mortality. Assessment of a small number of studies with three or more categories showed a clear dose-response association between arsenic and prevalent albuminuria and proteinuria, but not with CKD outcomes. Eight studies lacked adjustment for possible confounders, and two had small study populations. The evaluation of the causality of the association between arsenic exposure and kidney disease outcomes is limited by the small number of studies, lack of study quality, and limited prospective evidence. Because of the high prevalence of arsenic exposure worldwide, there is a need for additional well-designed epidemiologic and mechanistic studies of arsenic and kidney disease outcomes.

The effect of ex vivo CDDO-Me activation on nuclear factor erythroid 2-related factor 2 pathway in white blood cells from patients with septic shock.

Nuclear factor erythroid 2-related factor 2 (NRF2) has been shown to protect against experimental sepsis in mice and lipopolysaccharide (LPS)-induced inflammation in ex vivo white blood cells from healthy subjects by upregulating cellular antioxidant genes. The objective of this study was to test the hypothesis that ex vivo methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me) activates NRF2-regulated antioxidant genes in white blood cells from patients with septic shock and protects against LPS-induced inflammation and reactive oxidative species production. Peripheral blood was collected from 18 patients with septic shock who were being treated in medical and surgical intensive care units. Real-time polymerase chain reaction was used to quantify the expression of NRF2 target genes (NQO1, HO-1, GCLM, and FTL) and IL-6 in peripheral blood mononuclear cells (PBMCs), monocytes, and neutrophils after CDDO-Me treatment alone or after subsequent LPS exposure. Superoxide anion (O2) was measured to assess the effect of CDDO-Me pretreatment on subsequent LPS exposure. Treatment with CDDO-Me increased the gene expression of NQO1 (P = 0.04) and decreased the expression of HO-1 (P = 0.03) in PBMCs from patients with septic shock. Purified monocytes exhibited significant increases in the expression of NQO1 (P = 0.01) and GCLM (P = 0.003) after CDDO-Me treatment. Levels of other NRF2 target genes (HO-1 and FTL) remained similar to those of vehicle-treated cells. Peripheral blood mononuclear cells showed a trend toward increased IL-6 gene expression after CDDO-Me treatment, whereas purified monocytes showed a trend toward decreased IL-6. There was no discernible trend in the IL-6 expression subsequent to LPS treatment in either vehicle-treated or CDDO-Me-treated PBMCs and monocytes. Treatment with CDDO-Me significantly increased O2 production in PBMCs (P = 0.04). Although CDDO-Me pretreatment significantly attenuated O2 production to subsequent LPS exposure (P = 0.03), the change was comparable to that observed in vehicle-treated PBMCs. Pretreatment with CDDO-Me followed by LPS exposure had no significant effect on O2 levels in purified monocytes. These data suggest that the NRF2 pathway is differentially responsive to CDDO-Me activation in peripheral blood cells from patients with septic shock and results in increased O2 production. The data may also suggest a suppressed NRF2 pathway in white blood cells from critically ill patients.