A method for assaying poly (ADP-ribose) polymerase (PADPRP) activity in lymphocytes of systemic lupus erythematosus (SLE) patients has been developed. Using this method, PADPRP activity has been studied in lymphocytes from 15 patients and 13 controls. The mean activity in SLE lymphocytes was significantly lower than that in controls and 60% of the SLE patients demonstrated activities below the minimum of the control population. Possible mechanisms for this altered metabolism were investigated. The Km app of PADPRP for NAD; size distribution, branch frequency, and rates of turnover of polymers; competition for substrate; and number of PADPRP molecules were studied. The data demonstrated that SLE lymphocytes have a decreased synthetic capacity rather than alterations in the substrate or in turnover of the product.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Objectives: Diagnosis of pleural tuberculosis (PT) is still a challenge, particularly in resource-constrained settings. Alternative diagnostic tools are needed. We aimed at evaluating the utility of Clinical Prediction Rules (CPRs) for diagnosis of pleural tuberculosis in Peru. Methods: We identified CPRs for diagnosis of PT through a structured literature search. CPRs using high-complexity tests, as defined by the FDA, were excluded. We applied the identified CPRs to patients with pleural exudates attending two third-level hospitals in Lima, Peru, a setting with high incidence of tuberculosis. Besides pleural fluid analysis, patients underwent closed pleural biopsy for reaching a final diagnosis through combining microbiological and histopathological criteria. We evaluated the performance of the CPRs against this composite reference standard using classic indicators of diagnostic test validity. Results: We found 15 eligible CPRs, of which 12 could be validated. Most included ADA, age, lymphocyte proportion and protein in pleural fluid as predictive findings. A total of 259 patients were included for their validation, of which 176 (67%) had PT and 50 (19%) malignant pleural effusion. The overall accuracy of the CPRs varied from 41% to 86%. Two had a positive likelihood ratio (LR) above 10, but none a negative LR below 0.1. ADA alone at a cut-off of ≥40 IU attained 87% diagnostic accuracy and had a positive LR of 6.6 and a negative LR of 0.2. Conclusion: Many CPRs for PT are available. In addition to ADA alone, none of them contributes significantly to diagnosis of PT.

Abstract Interest in off-pump coronary surgery and ischaemic preconditioning has been increasing. The aim of this study was to evaluate surrogate indicators of haemodynamic, myocardial, and cerebral outcome during off-pump surgery and preconditioning. Haemodynamics and myocardial preservation were monitored in a pilot study of twelve patients undergoing off-pump coronary surgery. Indicators of myocardial metabolism and tissue injury as well as cerebral damage were evaluated in a randomized study of thirty-three patients undergoing on-pump (11) or off-pump surgery with (11) or without (11) preceding myocardial ischaemic preconditioning for five minutes followed by reperfusion for five minutes. The pilot study showed minimal haemodynamic changes and myocardial derangements during off-pump surgery as evaluated intraoperatively based on transcardiac differences of ATP degradation products and lactate and postoperatively based on MB mass of creatine kinase and troponin T. In the following studies, myocardial ischaemic metabolism was evaluated intraoperatively by measuring transcardiac differences of ATP degradation products, lactate, and pH, which increased significantly from the baseline values in all study groups. However, the maximum values of lactate and pH were significantly higher in the cardiopulmonary bypass group (p = 0.02 and p = 0.007, respectively). There were no statistical differences between the preconditioning and non-preconditioning groups. Myocardial tissue injury was evaluated by postoperative leakage of MB mass of creatine kinase and troponin I. Their peak values were significantly higher (p < 0.001 and p = 0.008) after cardiopulmonary bypass (15.1 μg/l and 13.8 μg/l) than after off-pump surgery without preconditioning (6.3 μg/l and 5.2 μg/l). The respective values were 14.8 μg/l and 7.4 μg/l after preconditioning, and there were no statistically significant differences between the off-pump groups with and without preconditioning. Cerebral damage was evaluated based on the intra- and postoperative serum concentrations of neuron-specific enolase, which were corrected with respect to haemolysis. The corrected values were significantly higher after on-pump than off-pump surgery (p = 0.003 and p = 0.005). In conclusion, multi-vessel off-pump coronary artery surgery is a haemodynamically feasible procedure offering better myocardial preservation compared to on-pump surgery. Ischaemic preconditioning of the myocardium does not seem to improve myocardial preservation in off-pump surgery. The slightly lower levels of neuron-specific enolase also suggest less cerebral damage.

Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery

Please read the abstract in the section 00front of this document / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2006. / Immunology / unrestricted

No description available.

Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

PART I Background: For over forty years, depolarizing, hyperkalemic cardioplegia solutions have served as the standard of care for cardiac surgery. While effective in inducing cardiac arrest, potassium-based solutions are associated with an array of negative consequences, such as coagulopathies, conduction dysfunction, inflammation, coronary vasoconstriction, myocardial edema, and ischemic injury. Adenosine-lidocaine-magnesium, a non-depolarizing, non-potassium-containing solution, has recently entered the clinical arena. Animal research suggests that this agent may provide a method of diastolic arrest that is as effective as potassium-based cardioplegia but with improved protective benefits.Purpose: The aim is to assess the safety and efficacy of adenosine-lidocaine-magnesium as a cardioplegia solution in terms of overall patient outcomes.Methodology: In June 2014, Banner University Medical Center Tucson became the first American institution to adopt the use of PolarShot (ALM)--adenosine-lidocaine-magnesium - as a cardioplegia solution. This one-year, retrospective study compares patients receiving adenosine-lidocaine-magnesium to those receiving high-potassium/low-potassium cardioplegia during adult cardiac surgery. Cases compared in this study include isolated coronary artery bypass, isolated aortic/mitral valve repair/replacement, and combination coronary artery bypass/valve replacement surgery only. A propensity-weighted regression model was used for analysis to determine whether or not cardioplegia treatment affected clinical outcome. To assess overall clinical outcome, major morbidity and mortality and post-procedural length of stay were chosen as primary endpoints. Results: In terms of treatment (adenosine-magnesium-lidocaine vs. high-potassium/low-potassium), no statistically significant difference was found between groups in regard to major morbidity and mortality event occurrences nor was a significant difference found between post-procedural length of stay. Discussion: After comparing postoperative outcomes between cardioplegia treatment groups, PolarShot (ALM) cardioplegia produced postoperative outcomes that were statistically similar to those of high-potassium/low-potassium cardioplegia. The confidence in these results is limited by low case volume, surgical case variability, and retrospective nature of this study. Conclusion: According to this propensity-weighted regression model, PolarShot (ALM) cardioplegia appears to be a safe and effective alternative to traditional potassium-based cardioplegia for the purpose of adult cardiac surgery. More research, including prospective randomized trials, is necessary to confirm or deny the findings of this study. PART II Background: Historically, surgical cardioplegia compounding was accomplished by filling patient-tailored prescriptions on-demand. Modern day compounding has become a manufacturing process to improve quality and accommodate physician demand. Additionally, sterile compounding standards have become more stringent, further necessitating a standardized compounding approach. In 2013, scrutiny of sterile drug compounding increased with passage of the Drug Quality and Security Act (DQSA) and subsequent Federal Drug Administration oversight. This federal mandate requires all compounded sterile preparations distributed by 503B Outsourcing Facilities be tested for potency, stability, and sterility. To accomplish this, compounders must significantly reduce batched formula variability. Purpose: A review of 2014 sales data from a large 503B outsourcing facility and cardioplegia compounder will be conducted. The study will identify solution differences and detail its findings. The aim of this study is to assess cardioplegia variability on a national level. Methodology: Results will be summarized by cardioplegia strategy (Buckberg, high-potassium/low-potassium, crystalloid, del Nido, Adenocaine, and microplegia), dilution strategy (4:1 blood-crystalloid, 8:1 blood-crystalloid, 1:4 crystalloid-blood, all-blood, and all-crystalloid), formula constituents (base solutions, additives, buffers), potassium concentrations. Any observed patterns in formula usage will also be reported, geographical or otherwise. Results / Discussion: Based on institutional use, high-potassium/low-potassium (two-solution) multidose strategy was the most common. Based on solutions ordered, the most common cardioplegia ingredient was potassium chloride, present in almost ninety percent (89.64%) of all units sold. After looking at potassium content, extensive variability was noted in terms of potassium added to the bag (undiluted) and potassium to-be delivered (post-dilutional). Additionally, unique solution formulations identified in multiple institutions were often found in neighboring states or within a single state. Conclusion: The results of this analysis illustrate the extent to cardioplegia formula variability nationwide. Variability exists in both methodology and formulation on a state-to-state, institution-to-institution, even across-single-institution basis. This formula customization appears to be institution- and surgeon-specific, suggesting empirical influence in formula adaptation. Formula standardization may be necessary to combat the compounded issue of formula customization moving forward.

From the standpoint of physiologists, emphasis has been placed on viewing the chemical nature of the membrane as an operational barrier to the free diffusion of ions. This tends to explain the fact that the ionic composition of the cytoplasm of the animal cell differs from its external fluid environment. It is well know that where sodium is the principal cation of the extracellular fluid, potassium has such a role inside the cell. Since there appears to be differential distribution of these ions across the cell membrane, this infers a concentration gradient of these ions. This differential distribution is important for certain life processes, for example, the propagation of nervous impulses mainly is dependent on the changes in concentration of sodium and potassium ions on both sides of the axonal membrane. Much effort has been put into elucidating the mechanism which cells maintain and change such concentration gradients. The enzyme system investigated in this study, namely, the sodium-activated adenosine triphosphatase, might be involved in maintaining this ionic gradient.<\p>