Rapid fall in circulating non-classical monocytes in ST elevation myocardial infarction patients correlates with cardiac injury.

Myocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes. We retrospectively analyzed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical, and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand, circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (P < .05) and correlated inversely with left ventricular function (r = 0.41, P = .04). The early fall in NC monocytes post-reperfusion was confirmed in a second prospective study of 13 STEMI patients. Furthermore, in a mouse cardiac ischemia model, there was significant monocyte adhesion to coronary vessel endothelium at 2 hours post-reperfusion pointing to a specific and rapid vessel margination response to cardiac injury. In conclusion, rapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature.

A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.

A collaborative Canadian-United Kingdom evaluation of an immunohistochemistry protocol to diagnose bovine spongiform encephalopathy.

Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.