Cardiac myocytes rendered ischemia resistant by expressing the human adenosine A1 or A3 receptor

Adenosine is an important mediator of the endogenous defense against ischemia-induced injury in the heart. Adenosine can achieve cardioprotection by mediating the effect of ischemic preconditioning and by protecting against myocyte injury when it is present during the infarct-producing ischemia. A novel adenosine A3 receptor can mediate this protective function. One approach to achieve cardioprotection is to enhance myocardial sensitivity to the endogenous adenosine by increasing the number of adenosine receptors instead of administering an adenosine receptor agonist. The objective of the present study was to investigate whether genetic manipulation of the cardiac myocyte, achieved by gene transfer and overexpression of the human A3 receptor cDNA, renders the myocytes resistant to the deleterious effect of ischemia. Prolonged hypoxia with glucose deprivation, causing myocyte injury and adenosine release, was used to simulate ischemia in cultured chick embryo ventricular myocytes. During simulated ischemia, cultured myocytes with enhanced expression of the human A3 receptor and showed significantly higher ATP content, fewer cells killed, and less creatine kinase released into the medium than either control or mock-transfected myocytes. Also, increased expression of the A3 receptor caused an enhanced cardioprotective effect by the preconditioning ischemia. Overexpressing the adenosine A1 receptor also led to increased protection against ischemia-induced myocyte injury as well as an enhanced preconditioning effect. Thus, increasing the receptor level improves the myocyte sensitivity to the endogenous adenosine, which in turn causes all of the cardioprotective effects found for exogenously administered adenosine agonists. The study provides the first proof for the new concept that an increased expression of the human A3 receptor in the cardiac myocyte can be an important cardioprotective therapeutic approach.     

Development and population study of an eight-locus short tandem repeat (STR) multiplex system

Amplification of short tandem repeat (STR) loci has become a useful tool for human identification applications. To improve throughput and efficiency for such uses, the polymorphic STR loci CSF1PO, TPOX, TH01, vWA, D16S539, D7S820, D13S317, D5S818, F13A01, FESFPS, F13B, and LPL have been evaluated, developed, and configured into fluorescently labeled multiplex systems. Eight of these STR loci were combined to generate the PowerPlex System, a two-color multiplex system that supports rapid, accurate, reliable analysis and designation of alleles. The remaining four loci comprise the FFFL System, a one-color multiplex system. The PowerPlex System may be evaluated alternatively as two one-color, four-locus multiplex systems, CTTv Multiplex and GammaSTR Multiplex. The products of multiplex amplification may be analyzed with a variety of fluorescence detection instruments. Determination of genotypes of over 200 individuals from each of three different population/ethnic groups revealed independence of inheritance of the loci and allowed calculation of matching probability, typical paternity index, and power of exclusion for each multiplex.     

Blocking of MHC class I antigens on leukemic B-cells enhances their conjugate formation with cytotoxic lymphocytes and their susceptibility to lysis

The role of major histocompatibility complex (MHC) class I antigens and adhesion molecules (AM) in the resistance of leukemic B-cells to cell-mediated cytotoxicity was investigated using cells from eight patients with B-chronic lymphocytic leukemia (B-CLL) and six patients with immunocytoma (IC). Both CLL and IC cells were completely resistant to natural killer (NK) and lymphokine activated killer (LAK) cytotoxicity and no binding to effector cells was observed, irrespectively of AM expression. Blocking of MHC class I antigens with monoclonal antibodies or their temporary elimination from leukemic B-cell surface by acid treatment resulted in a significant (p < 0.005) increase in both conjugate formation and susceptibility to lysis, thus suggesting the relevance of MHC class I expression on leukemic B-cells for the NK/LAK resistance phenomenon.     

Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum

We have explored the role of excitatory amino acids in the increased dopamine (DA) release that occurs in the neostriatum during stress-induced behavioral activation. Studies were performed in awake, freely moving rats, using in vivo microdialysis. Extracellular DA was used as a measure of DA release; extracellular 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase provided a measure of apparent DA synthesis. Mild stress increased the synthesis and release of DA in striatum. DA synthesis and release also were enhanced by the intra-striatal infusion of N-methyl-D-aspartate (NMDA), an agonist at NMDA receptors, and kainic acid, an agonist at the DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)/kainate site. Stress-induced increase in DA synthesis was attenuated by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and AMPA/kainate receptors, respectively. In contrast, intrastriatal APV, CNQX, or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) did not block the stress-induced increase in DA release. Stress-induced increase in DA release was, however, blocked by administration of tetrodotoxin along the nigrostriatal DA projection. It also was attenuated when APV was infused into substantia nigra. Thus, glutamate may act via ionotropic receptors within striatum to regulate DA synthesis, whereas glutamate may influence DA release via an action on receptors in substantia nigra. However, our method for monitoring DA synthesis lowers extracellular DA and this may permit the appearance of an intra-striatal glutamatergic influence by reducing a local inhibitory influence of DA. If so, under conditions of low extracellular DA glutamate may influence DA release, as well as DA synthesis, by an intrastriatal action. Such conditions might occur during prolonged severe stress and/or DA neuron degeneration. These results may have implications for the impact of glutamate antagonists on the ability of patients with Parkinson's disease to tolerate stress.     

Fibrinogen coating density affects the conformation of immobilized fibrinogen: implications for platelet adhesion and spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.     

Prospective use of platelet glycoprotein IIb/IIIa receptor blockers in the emergency department setting

Platelets play a pivotal role in the pathophysiology of acute coronary syndromes (ACS) and thus are logical therapeutic targets for treatment of this disease process. Platelet glycoprotein (GP IIb/IIIa receptor antagonists, which interrupt the final common pathway of platelet aggregation, have been proven to reduce the 30-day incidence of death, acute myocardial infarction (MI), and urgent revascularization in both high-risk and low-risk patients undergoing percutaneous intervention procedures. Three-year follow-up has indicated that these benefits appear durable. Recent large-scale randomized trials have demonstrated the value of GP IIb/IIIa receptor inhibitors in reducing the risk of death and MI in unstable angina/non-Q-wave MI patients receiving pharmacologic management. And, emerging evidence suggests a future role for GP IIb/IIIa receptor inhibitors as an adjunct to low-dose fibrinolytic therapy in patients with acute MI. As the list of indications for GP IIb/IIIa receptor antagonists expands to encompass the full spectrum of ACS, there is increasing interest in the potential use of these agents in the emergency department setting. The integration of GP IIb/IIIa receptor inhibitors into emergency department protocols will ultimately depend largely on whether these drugs prove to be safe and effective regardless of the direction of ST-segment deviation, and irrespective of whether definitive therapy will be invasive or conservative.     

Individualized assessment and intervention in bilateral siderail use

The use of bilateral siderails, similar to physical restraints, can be safely reduced by a comprehensive assessment process. This article presents an individualized assessment for evaluating siderail use to guide nurses in managing resident characteristics for falling out of bed and intervening for high-risk residents. The individualized assessment is consistent with federal resident assessment instrument requirements and includes risk factors specific to falls from bed.     

Effect of visual cues, vital signs, and protocols on triage: a prospective randomized crossover trial

The purpose of this study is to review published data regarding gender differences in cardiac electrophysiology and in the occurrence of clinical arrhythmias. ECG differences between men and women include a faster resting heart rate in women, a longer corrected QT interval, and a lower QT dispersion than in men. The faster resting heart rate in women appears to be primarily related to differences in physical conditioning. The mechanism for the longer corrected QT interval in women is not completely known, but does not appear to be related to acute effects of estrogen or progesterone or differences in autonomic innervation. Women also appear to have a lower incidence of atrial fibrillation, a difference in the age distribution of supraventricular tachycardia, and a lower incidence of sudden death than men. Much of the lower incidence of sudden death in women may relate to a difference in the prevalence of coronary artery disease, but other factors such as inherent differences in repolarization, which may be reflected by a gender difference in the corrected QT interval, also may be operative. The paradox of a longer corrected QT interval and higher incidence of torsades de pointes, but lower population-based incidence of sudden death in women, has not been completely resolved. Further studies will be required to help better understand the basic mechanisms involved in gender differences in electrophysiology and arrhythmias and determine the extent to which these differences have implications for clinical management of cardiac arrhythmias.     

Conditioning heat stress reduces excitotoxic and apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro

Both accidental and experimental lesions of the spinal cord suggest that neuronal processes occurring in the spinal cord modify the relay of information through the dorsal column-lemniscal pathway. How such interactions might occur has not been adequately explained. To address this issue, the receptive fields of mechanosensory neurons of the dorsal column nuclei were studied before and after manipulation of the spinal dorsal horn. After either a cervical or lumbar laminectomy and exposure of the dorsal column nuclei in anesthetized cats, the representation of the hindlimb or of the forelimb was defined by multiunit recordings in both the dorsal column nuclei and in the ipsilateral spinal cord. Next, a single cell was isolated in the dorsal column nuclei, and its receptive field carefully defined. Each cell could be activated by light mechanical stimuli from a well-defined cutaneous receptive field. Generally the adequate stimulus was movement of a few hairs or rapid skin indentation. Subsequently a pipette containing either lidocaine or cobalt chloride was lowered into the ipsilateral dorsal horn at the site in the somatosensory representation in the spinal cord corresponding to the receptive field of the neuron isolated in the dorsal column nuclei. Injection of several hundred nanoliters of either lidocaine or cobalt chloride into the dorsal horn produced an enlargement of the receptive field of the neuron being studied in the dorsal column nuclei. The experiment was repeated 16 times, and receptive field enlargements of 147-563% were observed in 15 cases. These data suggest that the dorsal horn exerts a tonic inhibitory control on the mechanosensory signals relayed through the dorsal column-lemniscal pathway. Because published data from other laboratories have shown that receptive field size is controlled by signals arising from the skin, we infer that the control of neuronal excitability, receptive field size and location for lemniscal neurons is determined by tonic afferent activity that is relayed through a synapse in the dorsal horn. This influence of dorsal horn neurons on the relay of mechanosensory information through the lemniscal pathways must modify our traditional views concerning the relative independence of these two systems.