Evaluation of an experimental filter designed for improving the quality of red blood cells (RBCs) during storage by simultaneously removing white blood cells and immunomodulators and improving RBC viscoelasticity and Band 3 proteins.

BACKGROUND: Storage age of red blood cells (RBCs) has been reported to be associated with increased mortality and morbidity. During storage, RBCs undergo changes in biochemical and functional properties. Stored RBCs may also contain white blood cells (WBCs), activated platelets (PLTs), cytokines, immunoglobulin, and other bioactive proteins. Transfusion of these bioactive proteins and cells with RBCs has the potential to cause serious adverse effects. We evaluated the performances of an experimental filter (EF) designed to remove immunoglobulins, cytokines, and other bioactive proteins in RBCs. STUDY DESIGN AND METHODS: Sixteen sets, each containing 3 units of ABO-identical RBCs in AS-3 were obtained from a blood bank. Three units of RBCs were combined together and then split into three equal aliquots, A, B, and C. Unit A was unfiltered while Units B and C were filtered with a leukoreduction filter and the EF, respectively. All the units were stored at 4°C in a blood bank refrigerator for 42 days. We measured RBC viscoelasticity, hemolysis, RBC adenosine triphosphate, Band 3 proteins, cytokines, PLTs, WBCs, and immunoglobulin before and after filtration and on Days 21 and 42 of storage. Data were analyzed by repeated-measures analysis of variance with Newman-Keuls multiple comparison test. RESULTS: The EF significantly (p<0.05) reduced the levels of immunoglobulin (control IgG, 2.184 ± 1.918 mg/mL; BPF4, 2.216 ± 1.956 mg/mL; and EF, 0.363 ± 0.391 mg/mL), PLTs, cytokines, and improved viscoelastic properties when compared to either control or leukoreduced RBCs. CONCLUSION: The EF achieved lower levels of WBCs, improved viscoelastic properties, and reduced levels of immunoglobulins and cytokines but significance will require clinical evaluation.

Antibodies to the HLA-A2 antigen prime neutrophils and serve as the second event in an in vitro model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is the most common cause of transfusion-related mortality and has been linked to the infusion of donor antibodies directed against recipient HLA class I antigens. We hypothesize that antibodies against HLA class I antigens bind to the antigens on the neutrophil (PMN) surface and induce priming and PMN cytotoxicity as the second event in a two-event in vitro model of PMN-mediated cytotoxicity. METHODS: Isolated PMNs from HLA-A2 homozygotes, heterozygotes and null donors were incubated with a monoclonal antibody to HLA-A2 and a human polyclonal IgG to HLA-A2 and priming of the oxidase was measured. The monoclonal antibodies and PMNs from these three groups were then used in a two-event model of PMN cytotoxicity. RESULTS: The antibodies to HLA-A2 both primed PMNs from HLA-A2 homozygotes but not from heterozygotes or nulls. Antibodies to HLA-A2 also served as the second event in a two-event model to induce PMN cytotoxicity of HLA-A2 homozygous PMNs. CONCLUSION: Antibodies to HLA class I antigens may directly prime/activate PMNs through the ligation of the antigen on the cell surface, and the antigen density appears to be important for these changes in PMN physiology.

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases.

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Crystallization and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael acceptor inhibitor.

Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. Whilst the native crystals were found to diffract only to medium resolution (2.9 Å), cocrystals of an inhibitor complex diffracted X-rays to 1.7 Šresolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

A simple filtration system for red blood cell depletion and volume reduction in routine processing of human umbilical cord blood.

BACKGROUND AND OBJECTIVES: Currently, stem cells and other progenitor cells are obtained from human umbilical cord blood (HUCB) using a variety of methods that are designed primarily for red blood cell depletion and volume reduction prior to freezing and storage. Some of these methods are very cumbersome and involve several steps that may result in significant cell loss. Therefore, processes that minimize the loss of haematopoietic stem and progenitor cells (HSPC) remains very critical. In the present study, we describe a simple filtration process for achieving both volume reduction and red blood cell depletion in a 'closed sterile system' with significant recovery of viable HSPC. MATERIALS AND METHODS: About 80-100 ml of HUCB were collected into citrate-phosphate-dextrose-adenine 1 anticoagulant. Each HUCB was divided into 25-70-ml aliquots and then either diluted with isotonic saline or filtered without any prior dilution with an experimental Red Cell Volume Reduction System (RCVRS). The HSPCs were recovered by retrograde rinsing of the filter with an isotonic stem cell recovery solution. The viability, colony forming properties, leucocytes and CD34+ cells recoveries were determined. RESULTS: The mean volume of the HUCB before processing was reduced from 43.9 +/- 7.9 ml to 11.8 +/- 0.7 ml (n = 55) with red blood cell depletion of 85.2 +/- 3.7%. Diluting the HUCB with isotonic saline prior to processing with RCVRS increased the red blood cell depletion to 91.9 +/- 3.0% (n = 7) without any significant loss in viability or cell recovery. The mean viability of the RCVRS-processed HUCB was not significantly different from the control unprocessed blood (96.60 +/- 1.90 vs. 96.63 +/- 2.12%; P > 0.05). The mean recoveries of the CD34+ and the haematopoietic clonogenic progenitor cells with the filter were 83.9 +/- 26.8 (n = 40) and 99.9 +/- 27.9% (n = 35), respectively. CONCLUSION: The present results show that the RCVRS provides a simple and easy-to-use process for obtaining red blood cell depletion and volume reduction of HUCB with good cell viability and recoveries.

Importance of vagally mediated bradycardia for the induction of torsade de pointes in an in vivo model.

BACKGROUND AND PURPOSE: Bradycardia is a risk factor for the development of torsade de pointes (TdP). The aim of this work was to compare the importance of changes in heart rate and arterial blood pressure in the development of drug-induced TdP and to investigate the role of vagal influences. EXPERIMENTAL APPROACH: Experiments were performed in open-chest, pentobarbital-anaesthetized, male rabbits which were given clofilium (20, 60 and 200 nmol kg(-1) min(-1)) with rising doses of either phenylephrine (75, 150, 225 and 300 nmol kg(-1) min(-1)), angiotensin II (0.25, 0.5, 0.75 and 1 nmol kg(-1) min(-1)) or saline. A fourth group received phenylephrine and cloflium after bilateral vagotomy. ECGs, haemodynamics and epicardial monophasic action potentials were recorded. KEY RESULTS: TdP occurred in 57% of rabbits given phenylephrine and clofilium. Replacement of phenylephrine with saline or angiotensin II reduced the incidence of TdP to 0 and 17%, respectively. Vagotomy prevented TdP in rabbits given phenylephrine and clofilium. Increases in blood pressure induced by phenylephrine and angiotensin II were similar. Bradycardia only occurred with phenylephrine and was reduced but not abolished by vagotomy. Neither short-term variability of repolarization nor action potential triangulation could predict TdP. CONCLUSIONS AND IMPLICATIONS: These results indicate that reflex activation of vagal nerve activity is essential for the induction of drug-induced TdP in alpha1-adrenoceptor-stimulated anaesthetized rabbits. This implies that alterations in vagal activity may also precipitate episodes of drug-induced TdP in man and that this should be considered in selecting models used in drug development.

Adrenaline reveals the torsadogenic effect of combined blockade of potassium channels in anaesthetized guinea pigs.

BACKGROUND AND PURPOSE: Torsade de pointes (TdP) can be induced in several species by a reduction in cardiac repolarizing capacity. The aim of this study was to assess whether combined I(Kr) and I(Ks) blockade could induce TdP in anaesthetized guinea pigs and whether short-term variability (STV) or triangulation of action potentials could predict TdP. EXPERIMENTAL APPROACH: Experiments were performed in open-chest, pentobarbital-anaesthetized, adrenaline-stimulated male Dunkin Hartley guinea pigs, which received three consecutive i.v. infusions of either vehicle, the I(Kr) blocker E-4031 (3, 10 and 30 nmol kg(-1) min(-1)), the I(Ks) blocker HMR1556 (75, 250, 750 nmol kg(-1) min(-1)) or E-4031 and HMR1556 combined. Phenylephrine-stimulated guinea pigs were also treated with the K(+) channel blockers in combination. Arterial blood pressure, ECGs and epicardial monophasic action potential (MAP) were recorded. KEY RESULTS: TdP was observed in 75% of adrenaline-stimulated guinea pigs given the K(+) channel blockers in combination, but was not observed in guinea pigs treated with either I(K) blocker alone, or in phenylephrine-stimulated guinea pigs. Salvos and ventricular tachycardia occurred with adrenaline but not with phenylephrine. No changes in STV or triangulation of the MAP signals were observed before TdP. CONCLUSIONS AND IMPLICATIONS: Combined blockade of both I(Kr) and I(Ks) plus the addition of adrenaline were required to induce TdP in anaesthetized guinea pigs. This suggests that there must be sufficient depletion of repolarization reserve and an appropriate trigger for TdP to occur.

Potentiation of E-4031-induced torsade de pointes by HMR1556 or ATX-II is not predicted by action potential short-term variability or triangulation.

BACKGROUND AND PURPOSE: Torsade de pointes (TdP) can be induced by a reduction in cardiac repolarizing capacity. The aim of this study was to assess whether IKs blockade or enhancement of INa could potentiate TdP induced by IKr blockade and to investigate whether short-term variability (STV) or triangulation of action potentials preceded TdP. EXPERIMENTAL APPROACH: Experiments were performed in open-chest, pentobarbital-anaesthetized, alpha 1-adrenoceptor-stimulated, male New Zealand White rabbits, which received three consecutive i.v. infusions of either the IKr blocker E-4031 (1, 3 and 10 nmol kg(-1) min(-1)), the IKs blocker HMR1556 (25, 75 and 250 nmol kg(-1) min(-1)) or E-4031 and HMR1556 combined. In a second study rabbits received either the same doses of E-4031, the INa enhancer, ATX-II (0.4, 1.2 and 4.0 nmol kg(-1)) or both of these drugs. ECGs and epicardial monophasic action potentials were recorded. KEY RESULTS: HMR1556 alone did not cause TdP but increased E-4031-induced TdP from 25 to 80%. ATX-II alone caused TdP in 38% of rabbits, as did E-4031; 75% of rabbits receiving both drugs had TdP. QT intervals were prolonged by all drugs but the extent of QT prolongation was not related to the occurrence of TdP. No changes in STV were detected and triangulation was only increased after TdP occurred. CONCLUSIONS AND IMPLICATIONS: Giving modulators of ion channels in combination substantially increased TdP but, in this model, neither STV nor triangulation of action potentials could predict TdP.

Greater antiarrhythmic activity of acute 17beta-estradiol in female than male anaesthetized rats: correlation with Ca2+ channel blockade.

BACKGROUND AND PURPOSE: Female sex hormones may protect pre-menopausal women from sudden cardiac death. We therefore investigated the effects of the main female sex hormone, 17beta-estradiol, on ischaemia-induced cardiac arrhythmias and on the L-type Ca2+ current (ICaL). EXPERIMENTAL APPROACH: In vivo experiments were performed in pentobarbital-anaesthetized rats subjected to acute coronary artery occlusion. ICaL was measured by the whole-cell patch-clamp technique, in rat isolated ventricular myocytes. KEY RESULTS: Acute intravenous administration of 17beta-estradiol as a bolus dose followed by a continuous infusion, commencing 10 min before coronary artery occlusion, had dose-dependent antiarrhythmic activity. In female rats 300 ng kg(-1) + 30 ng kg(-1) min(-1) 17beta-estradiol significantly reduced the number of ventricular premature beats (VPBs) and the incidence of ventricular fibrillation (VF). A ten fold higher dose of 17beta-estradiol was required to cause similar effects in male rats. In vitro 17beta-estradiol reduced peak ICaL in a concentration-dependent manner. The EC50 was ten-fold higher in male myocytes (0.66 microM) than in females (0.06 microM). CONCLUSIONS AND IMPLICATIONS: These results indicate that 17beta-estradiol has marked dose-dependent antiarrhythmic activity that is greater in female rats than in males. A similar differential potency in blocking ICaL in myocytes from female and male rats can account for this effect. This provides an explanation for the antiarrhythmic activity of 17beta-estradiol and gender-selective protection against sudden cardiac death.

The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion.

The Lambeth Conventions are guidelines intended to be of practical value in the investigation of arrhythmias induced by ischaemia, infarction, and reperfusion. They cover the design and execution of experiments and the definition, classification, quantification, and analysis of arrhythmias. Investigators are encouraged to adopt the conventions in the hope that this will improve uniformity and interlaboratory comparisons.

Effects of acute and chronic sunitinib treatment on cardiac function and calcium/calmodulin-dependent protein kinase II.

BACKGROUND AND PURPOSE: Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac contractile function and dysfunction and may be an unwanted secondary target for anti-cancer drugs such as sunitinib and imatinib that have been reported to alter cardiac performance. This study aimed to determine whether anti-cancer kinase inhibitors may affect CaMKII activity and expression when administered in vivo. EXPERIMENTAL APPROACH: Cardiovascular haemodynamics in response to acute and chronic sunitinib treatment, and chronic imatinib treatment, were assessed in guinea pigs and the effects compared with those of the known positive and negative inotropes, isoprenaline and verapamil. Parallel studies from the same animals assessed CaMKIIδ expression and CaMKII activity following drug treatments. KEY RESULTS: Acute administration of sunitinib decreased left ventricular (LV) dP/dtmax. Acute administration of isoprenaline increased LVdP/dtmax dose-dependently, while LVdP/dtmax was decreased by verapamil. CaMKII activity was decreased by acute administration of sunitinib and was increased by acute administration of isoprenaline, and decreased by acute administration of verapamil. CaMKIIδ expression following all acute treatments remained unchanged. Chronic imatinib and sunitinib treatments did not alter fractional shortening; however, both CaMKIIδ expression and CaMKII activity were significantly increased. Chronic administration of isoprenaline and verapamil decreased LV fractional shortening with parallel increases in CaMKIIδ expression and CaMKII activity. CONCLUSIONS AND IMPLICATIONS: Chronic sunitinib and imatinib treatment increased CaMKIIδ expression and CaMKII activity. As these compounds are associated with cardiac dysfunction, increased CaMKII expression could be an early indication of cellular cardiotoxicity marking potential progression of cardiac contractile dysfunction.

Paroxysmal, rhythmic lingual movements and chronic epilepsy.

Paroxysmal and rhythmic lingual movements were observed in three children during a study designed to investigate epileptiform movements of oropharyngeal muscles in patients with chronic epilepsy. The movements were confined to the tongue, occurred mainly during sleep, and were observed again in two children 7 and 18 months later. These movements corresponded to episodic desynchronization of the electroencephalogram and were attributed to an unusual form of subcortical seizures.

Phenacemide therapy of complex partial epilepsy in children: determination of plasma drug concentrations.

We used monotherapy with phenacemide to treat complex partial seizures in 13 children who were refractory to conventional antiepileptic drug therapy. Twelve patients responded with a reduction in seizure frequency, and 5 have been totally seizure free since the start of therapy. Phenacemide therapy was well tolerated with a minimum of untoward side effects and no evidence of irreversible drug toxicity. We developed a rapid and sensitive assay for the determination of plasma phenacemide concentrations by high performance liquid chromatography to monitor drug levels during therapy. Seizure control was achieved at plasma drug levels that ranged from 16 to 75 micrograms/ml. The median effective dose in our series was 52 micrograms/ml. The recurrence of seizures in three patients was, in each case, associated with trough plasma phenacemide levels below 50 micrograms/dl.

Early abnormalities of brainstem auditory evoked potentials in Friedreich's ataxia: evidence of primary brainstem dysfunction.

We studied five children with classic Friedreich's ataxia, using an audiologic test battery to determine the primary site of auditory dysfunction. None of the children had any hearing complaints, and all were tested soon after onset of symptoms. The audiologic test battery consisted of brainstem auditory evoked potential test, tympanometry, and acoustic reflex measurements. The results indicated that the brainstem was the primary site of auditory dysfunction.

Occluded fourth ventricle after multiple shunt revisions for hydrocephalus.

Trapped occluded fourth ventricle has been considered a rare occurrence. Intraventricular hemorrhage followed by repeated shunt revisions may increase the risk (8/47 cases). Because premature infants with intraventricular hemorrhage and shunted hydrocephalus often have preexisting neurologic abnormalities, dilation may produce clinically undetected further neurologic damage. Shunting improved function in both currently treated as well as 13 of 14 previously treated patients. In light of this observation, the importance of recognition is stressed.

Ventricular fibrillation is reduced in hypothyroid rats with enhanced myocardial alpha-adrenoceptor responsiveness.

1. The severity of ventricular arrhythmias induced by coronary artery occlusion and reperfusion has been examined in control rats and animals made hypothyroid by pretreatment with 6-propylthiouracil (PTU). The maximal driving frequency and sensitivity of isolated left atria and papillary muscles to isoprenaline and to phenylephrine in the presence of propranolol, were also examined in tissues from control and hypothyroid animals. 2. Pretreatment with PTU resulted in a potentiation of responses to the alpha-adrenoceptor agonist phenylephrine in both left atria and papillary muscles, while responses to isoprenaline were depressed in left atria but unaltered in papillary muscles from hypothyroid animals. 3. In rats subject to coronary artery occlusion, PTU pretreatment reduced the incidence of ventricular fibrillation during acute myocardial ischaemia and abolished reperfusion-induced ventricular fibrillation. Mortality during myocardial ischaemia and reperfusion was also abolished. Diastolic blood pressure was similar in hypothyroid and control animals, but there was a small reduction in systolic blood pressure and a marked decrease in heart rate in PTU pretreated animals. 4. These results demonstrate that PTU-induced hypothyroidism represents a condition where cardiac alpha-adrenoceptor-mediated responses are enhanced but the severity of ischaemia- and reperfusion-induced arrhythmias is reduced.

Comparison of the effects of isobutylmethylxanthine and milrinone on ischaemia-induced arrhythmias and platelet aggregation in anaesthetized rabbits.

1. The aim of this study was to compare the effects of the non-selective phosphodiesterase (PDE) inhibitor, isobutylmethylxanthine (IBMX) and the selective PDE III inhibitor, milrinone, in a rabbit model of acute myocardial ischaemia. 2. Coronary artery occlusion caused changes in the ST-segment of the ECG and ectopic activity in all control rabbits. Ventricular fibrillation occurred in 10 out of 14 (71%) of these animals. Pretreatment with IBMX 100 micrograms kg-1 plus 10 micrograms kg-1 min-1, starting 10 min before coronary artery occlusion, reduced ischaemia-induced ST-segment changes and ventricular fibrillation occurred in only 10% of this group (n = 10). A similar dose of milrinone had no antiarrhythmic activity, whereas with a lower dose of milrinone, 30 micrograms kg-1 plus 3 micrograms kg-1 min-1 (n = 10), only 30% of rabbits fibrillated and ST-segment changes were attenuated. 3. Acute administration of both IBMX and milrinone reduced arterial blood pressure. With the higher dose of milrinone a significant effect was still present after 10 min of drug infusion. A greater hypotensive response to the higher dose of milrinone was observed in the rabbits which subsequently fibrillated during ischaemia. A marked tachycardia was also observed after administration of the higher dose of milrinone. 4. At the end of the experiment platelet aggregation was studied ex vivo. ADP-induced aggregation was reduced by pretreatment of the rabbits with milrinone but not IBMX. Both PDE inhibitors enhanced the ability of isoprenaline to inhibit ADP-induced platelet aggregation but milrinone was more effective, particularly at the higher dose. The results demonstrate that IBMX was antiarrhythmic but that this activity was not directly related to inhibition of platelet aggregation. Adverse haemodynamic effects may explain the failure of milrinone to have similar activity during myocardial ischaemia.

The effects of timolol on arrhythmias and prostanoid release during canine myocardial ischaemia and reperfusion.

Timolol (50 micrograms kg-1), administered intravenously to chloralose-anaesthetized open-chest greyhounds 30 min prior to occlusion of the left anterior descending coronary artery, reduced heart rate and mean arterial blood pressure. This dose caused a 20 fold increase in the dose of isoprenaline required to increase heart rate by 25 beats min-1. During the first 30 min of myocardial ischaemia the number of extrasystoles in the timolol-treated dogs (327 +/- 179) was less than in the control group (888 +/- 168) and none of the dogs that received timolol fibrillated. The haemodynamic changes induced by coronary artery occlusion (decreased cardiac output and stroke volume, increased peripheral vascular resistance) were similar in both control and timolol-treated dogs as were the increases in PCO2 and decreases in PO2 and pH in blood draining from the ischaemic myocardium. Timolol did not alter the release during myocardial ischaemia, of either thromboxane B2 or prostacyclin (measured as 6-keto PGF1 alpha). Reperfusion-induced ventricular fibrillation occurred in 7 out of 8 control dogs and in 5 out of 10 timolol-treated dogs. The overall survival following occlusion and reperfusion was improved by 10% to 50% by timolol.

AH23848, a thromboxane antagonist, suppresses ischaemia and reperfusion-induced arrhythmias in anaesthetized greyhounds.

The effects of the thromboxane antagonist AH23848 (1 mg kg-1 i.v.) were examined in anaesthetized greyhounds prepared for occlusion of the left anterior descending coronary artery with subsequent reperfusion after 40 min of myocardial ischaemia. Pretreatment with AH23848 30 min before coronary artery occlusion reduced the number of ischaemia-induced extrasystoles to 339 +/- 111 compared with 736 +/- 153 in the control group. The incidence of ventricular fibrillation following reperfusion was also markedly reduced; 25% compared with 88% in the controls. Late intervention with AH23848, 25 min after the onset of myocardial ischaemia did not significantly alter the incidence of reperfusion-induced ventricular fibrillation; 70% of the control group died and 60% of those that received AH23848. The ischaemia-induced release of thromboxane A2 and prostacyclin was not altered by pretreatment with AH23848. The results suggest that blockade of thromboxane receptors before myocardial ischaemia is an effective antiarrhythmic strategy in this model.

The effects of prostaglandins E2, F2 alpha, prostacyclin, flurbiprofen and aspirin on arrhythmias resulting from coronary artery ligation in anaesthetized rats.

1 Various prostaglandins and inhibitors of prostaglandin synthesis were administered prior to acute coronary artery ligation in anaesthetized rats and their effects were assessed on the number and severity of the resulting early arrhythmias (ventricular ectopic activity; incidence and duration of ventricular tachycardia and of ventricular fibrillation). 2 Prostaglandin E2 (PGE2), PGF2 alpha and prostacyclin all showed antiarrhythmic activity; in contrast flurbiprofen increased the incidence of ventricular fibrillation and mortality. 3 Both the number of ventricular ectopic beats and the incidence of ventricular fibrillation were reduced by aspirin. 4 The results suggest that the release of endogenous PGE2, PGF2 alpha and prostacyclin could reduce early post-infarction ventricular arrhythmias whilst the protective effect of aspirin in this model adds further support for the hypothesis that thromboxane release is involved in the genesis of these arrhythmias.