Experimental Model of Isolated Lung Perfusion in Rats: First Brazilian Experience Using the IL-2 Isolated Perfused Rat or Guinea Pig Lung System

Introduction. Lung transplantation has become the mainstay therapy for patients with end-stage lung disease refractory to medical management. However, the number of patients listed for lung transplantation largely exceeds available donors. The study of lung preservation requires accurate, cost-effective small animal models. We have described a model of ex vivo rat lung perfusion using a commercially available system. Methods. Male Wistar rats weighing 250 g-300 g were anesthetized with intraperitoneal sodium thiopental (50 mg/kg body weight). The surgical technique included heart-lung block extraction, assembly, and preparation for perfusion and data collection. We used an IL-2 Isolated Perfused Rat or Guinea Pig Lung System (Harvard Apparatus, Holliston, Mass, United States; Hugo Sachs Elektronik, Alemanha). Results. Preliminary results included hemodynamic and pulmonary mechanics data gathered in the experiments. Conclusion. The isolated rat lung perfusion system is a reliable method to assess lung preservation.

Mechanisms of hydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney.

The vasoconstrictor effect of hydrogen peroxide (H(2)O(2)) on isolated perfused rat kidney was investigated. H(2)O(2) induced vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effects of H(2)O(2) were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response to H(2)O(2). The H(2)O(2) dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10(-4) mol/l), whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium (3.10(-3) mol/l). Separately, removal of extracellular Ca(2+), administration of a mixture of calcium desensitizing agents (nitroprusside, papaverine, and diazoxide), and administration of a protein kinase C (PKC) inhibitor (chelerythrine, 10(-5) mol/l) each significantly attenuated the vasoconstrictor response to H(2)O(2), which was virtually suppressed when they were performed together. The pressor response to H(2)O(2) was not affected by: dimethyl sulfoxide (7.10(-5) mol/l) plus mannitol (3.10(-5) mol/l); intracellular Ca(2+) chelation using BAPTA (10(-5) mol/l); calcium store depletion after repeated doses of phenylephrine (10(-5) g/g kidney); or the presence of indomethacin (10(-5) mol/l), ODYA (2.10(-6) mol/l) or genistein (10(-5) mol/l). We conclude that the vasoconstrictor response to H(2)O(2) in the rat renal vasculature comprises the following components: 1) extracellular calcium influx, 2) activation of PKC, and 3) stimulation of pathways leading to sensitization of contractile elements to calcium. Moreover, a reduced pressor responsiveness to H(2)O(2) in female kidneys was observed.

High sodium intake enhances insulin-stimulated glucose uptake in rat epididymal adipose tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity. Methods and Procedures: Male Wistar rats were fed on normal- (0.5% Na+; NS), high- (3.12% Na+; HS), or low-sodium (0.06% Na+; LS) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail-cuff system. An intravenous insulin tolerance test (ivITT) was performed in fasted animals. At the end of each period, rats were killed and blood samples were collected for glucose and insulin determinations. The white adipose tissue (WAT) from abdominal and inguinal subcutaneous (SC) and periepididymal (PE) depots were weighed and processed for adipocyte isolation and measurement of in vitro rates of insulin-stimulated 2-deoxy-d-[H-3]-glucose uptake (2DGU) and conversion of -[U-C-14]-glucose into (CO2)-C-14. Results: After 6 weeks, HS diet significantly increased the BP, SC and PE WAT masses, PE adipocyte size, and plasma insulin concentration. The sodium dietary content did not influence the whole-body insulin sensitivity. A higher half-maximal effective insulin concentration (EC50) from the dose - response curve of 2DGU and an increase in the insulin-stimulated glucose oxidation rate were observed in the isolated PE adipocytes from HS rats. Discussion: The chronic salt overload enhanced the adipocyte insulin sensitivity for glucose uptake and the insulin-induced glucose metabolization, contributing to promote adipocyte hypertrophy and increase the mass of several adipose depots, particularly the PE fat pad.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model†

OBJECTIVES The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability; however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC); brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury; however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. METHODS Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF); 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. RESULTS Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively; P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%; P < 0.01 for all) in LoR subgroups. CONCLUSIONS Effects of MPC depend on energy substrate availability; MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model

Gebiet: Chirurgie Abstract: OBJECTIVES: – The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability, however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC), brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury, however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. – – METHODS: – Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF), 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. – – RESULTS: – Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively, P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%, P < 0.01 for all) in LoR subgroups. – – CONCLUSIONS: – Effects of MPC depend on energy substrate availability, MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Antioxidant activity of flavonoids in isolated mitochondria

Mitochondria are important intracellular sources and targets of reactive oxygen species (ROS), while flavonoids, a large group of secondary plant metabolites, are important antioxidants. Following our previous study on the energetics of mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, the present work addressed the antioxidant activity of these compounds (1-50 mu mol/L) on Fe2+/citrate-mediated membrane lipid peroxidation (LPO) in isolated rat liver mitochondria, running in parallel studies of their antioxidant activity in non-organelle systems. Only quercetin inhibited the respiratory chain of mitochondria and only galangin caused uncoupling. Quercetin and galangin were far more potent than taxifolin and catechin in affording protection against LPO (IC50 = 1.23 +/- 0.27 and 2.39 +/- 0.79 mu mol/L, respectively), although only quercetin was an effective scavenger of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals. These results, together with the previous study, suggest that the 2,3-double bond in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids in mitochondria, the presence of an o-di-OH structure on the B-ring, as occurs in quercetin, favours this activity via superoxide scavenging, while the absence of this structural feature in galangin, favours it via a decrease in membrane fluidity and/or mitochondrial uncoupling. Copyright (c) 2008 John Wiley & Sons, Ltd.

Acidosis induces relaxation mediated by nitric oxide and potassium channels in rat thoracic aorta

We investigated the mechanism by which extracellular acidification promotes relaxation in rat thoracic aorta. The relaxation response to HCl-induced extracellular acidification (7.4 to 6.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10(-6) M) or KCl (45 mM). The vascular reactivity experiments were performed in endothelium-intact and denuded rings, in the presence or absence of indomethacin (10(-5) M), L-NAME (10(-4) M), apamin (10(-6) M), and glibenclamide (10(-5) M). The effect of extracellular acidosis (pH 7.0 and 6.5) on nitric oxide (NO) production was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 mu M). The extracellular acidosis failed to induce any changes in the vascular tone of aortic rings pre-contracted with KCl, however, it caused endothelium-dependent and independent relaxation in rings pre-contracted with Phe. This acidosis induced-relaxation was inhibited by L-NAME, apamin, and glibenclamide, but not by indomethacin. The acidosis (pH 7.0 and 6.5) also promoted a time-dependent increase in the NO production by the isolated endothelial cells. These results suggest that extracellular acidosis promotes vasodilation mediated by NO, K(ATP) and SK(Ca), and maybe other K(+) channels in isolated rat thoracic aorta. (C) 2011 Elsevier B.V. All rights reserved.

Isolated limb perfusion with melphalan for human melanoma xenografts in the hindlimb of nude rats: A surviving animal model

We have established a surviving model of isolated limb perfusion using xenografts of the human melanoma cell line MM 96L injected subcutaneously into the hindlimb of a nude rat, The femoral artery and vein were cannulated via the left renal artery and vein and the hind limb was isolated using tourniquets. The limb was perfused with Krebs Heinseleit buffer at 37 degrees C containing 4.7% bovine serum albumin at a constant flow rate of 4 mi per min for 30-60 min with 100% survival of the animals, Tumour vascularization and blood flow were demonstrated using vascular casts and [Cr-51]-microspheres. Following the addition of melphalan (15 or 100 mu g/ml), drug concentrations in the perfusate, tissues and systemic circulation were determined using high pressure liquid chromatography (HPLC), Systemic leakage, assessed using [I-125]albumin and melphalan and detected by a gamma-counter and HPLC respectively, was <0.5%. The melphalan concentration and tissue flow rate in the tumour deposits were 40 and 30% respectively, when compared with the surrounding subcutaneous tissue, At a dose of 15 mu g/ml, melphalan caused a reduction in tumour growth after 60 min perfusion, and a significant reduction in tumour size was seen when the melphalan dose was 100 mu g/ml. The surviving nude rat model of isolated limb perfusion for recurrent melanoma will allow examination of optimal perfusion conditions, along with the pharmacokinetics, pharmacodynamics and efficacy of melphalan and other drugs.

PHOSPHODIESTERASE-4 INHIBITION REDUCES PROTEOLYSIS AND ATROGENES EXPRESSION IN RAT SKELETAL MUSCLES

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371-381, 2011

Toxicity and uptake mechanism of cylindrospermopsin and lophyrotomin in primary rat hepatocytes

The toxicities and uptake mechanisms of two hepatotoxins, namely cylindrospermopsin and lophyrotomin, were investigated on primary rat hepatocytes by using microcystin-LIZ (a well-known hepatotoxin produced by cyanobacteria) as a comparison. Isolated rat hepatocytes were incubated with different concentrations of hepatotoxins for 0, 24, 48 and 72 h. The cell viability was assayed by the tetrazolium-based (MTT) assay. Microcystin-LR, cylindrospermopsin and lophyrotomin all exhibited toxic effects on the primary rat hepatocytes with 72-h LC50 of 8, 40 and 560 ng/ml, respectively. The involvement of the bile acid transport system in the hepatotoxin-induced toxicities was tested in the presence of two bile acids, cholate and taurocholate. Results showed that the bile acid transport system was responsible for the uptake, and facilitated the subsequent toxicities of lophyrotomin on hepatocytes. This occurred to a much lesser extent with cylindrospermopsin. With its smaller molecular weight, passive diffusion might be one of the possible mechanisms for cylindrospermopsin uptake into hepatocytes. This was supported by incubating a permanent cell line, KB (devoid of bile acid transport system), with cylindrospermopsin which showed cytotoxic effects. No inhibition of protein phosphatase 2A by cylindrospermopsin or lophyrotomin was found. This indicated that other toxic mechanisms besides protein phosphatase inhibition were producing the toxicities of cylindrospermopsin and lophyrotomin, and that they were unlikely to be potential tumor promoters. (C) 2001 Elsevier Science Ltd. All rights reserved.

The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.