Pulmonary instillation of multi-walled carbon nanotubes promotes coronary vasoconstriction and exacerbates injury in isolated hearts

The growing use of multi-walled carbon nanotubes (MWCNTs) across industry has increased human exposures. We tested the hypothesis that pulmonary instillation of MWCNTs would exacerbate cardiac ischaemia/reperfusion (I/R) injury. One day following intratracheal instillation of 1, 10 or 100 μg MWCNT in Sprague-Dawley rats, we used a Langendorff isolated heart model to examine cardiac I/R injury. In the 100 μg MWCNT group we report increased premature ventricular contractions at baseline and increased myocardial infarction. This was associated with increased endothelin-1 (ET-1) release and depression of coronary flow during early reperfusion. We also tested if isolated coronary vascular responses were affected by MWCNT instillation and found trends for enhanced coronary tone, which were dependent on ET-1, cyclooxygenase, thromboxane and Rho-kinase. We concluded that instillation of MWCNTs promoted cardiac injury and depressed coronary flow by invoking vasoconstrictive mechanisms involving ET-1, cyclooxygenase, thromboxane and Rho-kinase.     

Establishing biennial paramedic experience benchmarks

Objective. Since stroke symptoms are often vague, and acute therapies for stroke are more recently available, it has been hypothesized that stroke patients may not be treated with the same urgency as myocardial infarction (MI) patients by emergency medical services (EMS). To examine this hypothesis, EMS transport times were examined for both stroke and MI patients who used a paramedic-level, county-based EMS system for transportation to a single hospital during 1999. Methods. Patients were first identified by their hospital discharge diagnosis as stroke (ICD-9 430–436, n = 50) or MI (ICD-9 410, n = 55). Trip sheets with corresponding transport times were retrospectively obtained from the 911 center. A separate analysis was performed on patients identified by dispatchers with a chief complaint of stroke (n = 85) or MI (n = 372). Results. Comparing stroke and MI patients identified by ICD-9 codes, mean EMS transport times in minutes did not meaningfully differ with respect to dispatch to scene arrival time (8.3 vs 8.9, p = 0.61), scene time (19.5 vs 21.4, p = 0.23), and transport time (13.7 vs 16.2, p = 0.10). Mean total call times in minutes from dispatch to hospital arrival were similar between stroke and MI patients (41.5 vs 46.4, p = 0.22). Results were similar when comparing patients identified by dispatchers with a chief complaint indicative of stroke or MI. Conclusion. In this single county, EMS response times were not different between stroke and MI patients. Replication in other EMS settings is needed to confirm these findings.     

Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.     

Combinatorial detection of autoreactive CD8<Superscript>+</Superscript> T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop

Aims/hypothesis

Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8⁺ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting.

Methods

The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers.

Results

The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8⁺ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8⁺ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors.

Conclusions/interpretation

Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

     

Control of mammalian glycogen synthase by PAS kinase

The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy.     

Cumulative Marijuana Use and Carotid Intima-Media Thickness at Middle Age: The CARDIA Study

BackgroundLong-term cardiovascular health effects of marijuana are understudied. Future cardiovascular disease is often indicated by subclinical atherosclerosis for which carotid intima-media thickness is an established parameter.MethodsUsing the data from the Coronary Artery Risk Development in Young Adults (CARDIA) study, a cohort of 5115 Black and white women and men at Year 20 visit, we studied the association between carotid intima-media thickness in midlife and lifetime exposure to marijuana (1 marijuana year = 365 days of use) and tobacco smoking (1 pack-year = 20 cigarettes/day for 365 days). We measured carotid intima-media thickness by ultrasound and defined high carotid intima-media thickness at the threshold of the 75th percentile of all examined participants. We fit logistic regression models stratified by tobacco smoking exposure, adjusting for demographics, cardiovascular risk factors, and other drug exposures.ResultsData was complete for 3257 participants; 2722 (84%) reported ever marijuana use; 374 (11%) were current users; 1539 (47%) reported ever tobacco smoking; 610 (19%) were current smokers. Multivariable adjusted models showed no association between cumulative marijuana exposure and high carotid intima-media thickness in never or ever tobacco smokers, odds ratio (OR) 0.87 (95% confidence interval [CI]: 0.63-1.21) at 1 marijuana-year among never smokers and OR 1.11 (95% CI: 0.85-1.45) among ever tobacco smokers. Cumulative exposure to tobacco was strongly associated with high carotid intima-media thickness, OR 1.88 (95%CI: 1.20-2.94) for 20 pack-years of exposure.ConclusionsThis study adds to the growing body of evidence that there might be no association between the average population level of marijuana use and subclinical atherosclerosis.