Hemostasis Laboratory: Assays for Platelet Function and Von Willebrand Disease

Journal of Thrombosis and Thrombolysis -     

Evidence for the contribution of non-covalent steroid interactions between DNA and topoisomerase in the genotoxicity of steroids

Fifty two steroids and 9 Vitamin D analogs were docked into ten crystallographically-defined DNA dinucleotide sites and two human topoisomerase II ATP binding sites using two computational programs, Autodock and Surflex. It is shown that both steroids and Vitamin D analogs exhibit a propensity for non-covalent intercalative binding to DNA. A higher predicted binding affinity was found, however, for steroids and the ATP binding site of topoisomerase; in fact these drugs exhibited among the highest topo II binding observed in over 1370 docked drugs. These findings along with genotoxicity data from 26 additional steroids not subjected to docking analysis, support a mechanism wherein the long known, but poorly understood, clastogenicity of steroids may be attributable to inhibition of topoisomerase. A “proof of principle” experiment with dexamethasone demonstrated this to be the likely mechanism of clastogenicity of, at least, this steroid. The generality of this proposed mechanism of genotoxicity across the steroids and vitamin-D analogs is discussed.     

The effects of biphasic waveform design on post-resuscitation myocardial function

OBJECTIVES: This study examined the effects of biphasic truncated exponential waveform design on survival and post-resuscitation myocardial function after prolonged ventricular fibrillation (VF). BACKGROUND: Biphasic waveforms are more effective than monophasic waveforms for successful defibrillation, but optimization of energy and current levels to minimize post-resuscitation myocardial dysfunction has been largely unexplored. We examined a low-capacitance waveform typical of low-energy application (low-energy biphasic truncated exponential [BTEL]; 100 microF, < or =200 J) and a high-capacitance waveform typical of high-energy application (high-energy biphasic truncated exponential [BTEH]; 200 microF, > or =200 J). METHODS: Four groups of anesthetized 40- to 45-kg pigs were investigated. After 7 min of electrically induced VF, a 15-min resuscitation attempt was made using sequences of up to three defibrillation shocks followed by 1 min of cardiopulmonary resuscitation. Animals were randomized to BTEL at 150 J or 200 J or to BTEH at 200 J or 360 J. RESULTS: Resuscitation was unsuccessful in three of the five animals treated with BTEH at 200 J. All other attempts were successful. Significant therapy effects were observed for survival (p = 0.035), left ventricular ejection fraction (p < 0.001), stroke volume (p < 0.001), fractional area change (p < 0.001), cardiac output (p = 0.044), and mean aortic pressure (p < 0.001). Hemodynamic outcomes were negatively associated with energy and average current but positively associated with peak current. Peak current was the only significant predictor of survival (p < 0.001). CONCLUSIONS: Maximum survival and minimum myocardial dysfunction were observed with the low-capacitance 150-J waveform, which delivered higher peak current while minimizing energy and average current.     

Phorbol ester effects on neurotransmission: interaction with neurotransmitters and calcium in smooth muscle.

Stimulation of the phosphatidylinositol cycle by neurotransmitters generates diacylglycerol, an activator of protein kinase C, which may regulate some forms of neurotransmission. Phorbol esters, potent inflammatory and tumor-promoting compounds, also activate protein kinase C. We demonstrate potent and selective effects of phorbol esters on smooth muscle, indicating a role for protein kinase C in neurotransmission. In rat vas deferens and dog basilar artery, phorbol esters synergize with calcium to mimic the contractile effects of neurotransmitters that act through the phosphatidylinositol cycle. In guinea pig ileum and rat uterus, phorbol esters block contractions produced by these neurotransmitters.     

Competition between T cells maintains clonal dominance during memory inflation induced by MCMV

Both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) establish persistent infections that induce the accumulation of virus‐specific T cells over time in a process called memory inflation. It has been proposed that T cells expressing T‐cell receptors (TCRs) with high affinity for HCMV‐derived peptides are preferentially selected after acute HCMV infection. To test this in the murine model, small numbers of OT‐I transgenic T cells, which express a TCR with high affinity for the SIINFEKL peptide, were transferred into congenic mice and recipients were challenged with recombinant MCMV expressing SIINFEKL. OT‐I T cells were selectively enriched during the first 3 weeks of infection. Similarly, in the absence of OT‐I T cells, the functional avidity of SIINFEKL‐specific T cells increased from early to late times postinfection. However, even when exceedingly small numbers of OT‐I T cells were transferred, their inflation limited the inflation of host‐derived T cells specific for SIINFEKL. Importantly, subtle minor histocompatibility differences led to late rejection of the transferred OT‐I T cells in some mice, which allowed host‐derived T cells to inflate substantially. Thus, T cells with a high functional avidity are selected shortly after MCMV infection and continuously sustain their clonal dominance in a competitive manner.     

Automated preparation for identification and antimicrobial susceptibility testing: evaluation of a research use only prototype,the BD Kiestra IdentifA/SusceptA system

ObjectiveThe current BD Kiestra? total laboratory automation (TLA) system automates specimen inoculation, incubation, and digital visualization of cultures prior to initiation of manual or semi-automated identification (ID) and antimicrobial susceptibility testing (AST). The current study aimed to compare the performance, in a clinical setting, of a fully automated research-use-only prototype, BD Kiestra? IdentifA/SusceptA (automated system), to our current BD Kiestra? TLA which utilizes manual or semi-automated IDs and ASTs (current system).MethodsClinical samples yielding significant growth after processing by the BD Kiestra? TLA were tested in parallel for ID and AST by both systems. IDs and ASTs were determined by Bruker matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and BD Phoenix, respectively, with data stored and managed in the BD EpiCenter?. The automated system used a common inoculum preparation for both tests, whereas the current system used separate inocula. Results were compared to assess agreement between the systems.ResultsOn initial testing, 89% of IDs (466/523) and 92.4% of IDs (484/523) for the automated and current ID systems, respectively, yielded acceptable MALDI-TOF log scores of ≥1.7. On repeat testing, the respective acceptable scores were 97.1% (508/523) and 98.1% (513/523). For initial ASTs, the automated and current systems yielded 97.5% categorical agreement for 7325 drug–organism tests. After omitting discrepant MICs that differed by only one dilution and categorical discrepancies that were not reproducible, 0.2% unresolved discrepancies remained thus (99.8% categorical agreement).ConclusionsThe automated prototype is suitable for development into technology that will provide clinical microbiology laboratories with significant advantages such as improved efficiency, standardization, reproducibility, reduced technical error and greater safety.     

Clickable Poly(ethylene glycol)‐Microsphere‐Based Cell Scaffolds

Clickable poly(ethylene glycol) (PEG) derivatives are used with two sequential aqueous two‐phase systems to produce microsphere‐based scaffolds for cell encapsulation. In the first step, sodium sulfate causes phase separation of the clickable PEG precursors and is followed by rapid geleation to form microspheres in the absence of organic solvent or surfactant. The microspheres are washed and then deswollen in dextran solutions in the presence of cells, producing tightly packed scaffolds that can be easily handled while also maintaining porosity. Endothelial cells included during microsphere scaffold formation show high viability. The clickable PEG‐microsphere‐based cell scaffolds open up new avenues for manipulating scaffold architecture as compared with simple bulk hydrogels.