Impact of spontaneous donor hypothermia on graft outcomes after kidney transplantation.

A previous donor intervention trial found that therapeutic hypothermia reduced delayed graft function (DGF) after kidney transplantation. This retrospective cohort study nested in the randomized dopamine trial (ClinicalTrials.gov identifier: NCT000115115) investigates the effects of spontaneous donor hypothermia (core body temperature <36°C) on initial kidney graft function, and evaluates 5-year graft survival. Hypothermia assessed by a singular measurement in the intensive care unit 4-20 hours before procurement was associated with less DGF after kidney transplantation (odds ratio [OR] 0.56, 95% confidence interval [CI] 0.34-0.91). The benefit was greater when need for more than a single posttransplant dialysis session was analyzed (OR 0.48, 95%CI 0.28-0.82). Donor dopamine ameliorated dialysis requirement independently from hypothermia in a temporal relationship with exposure (OR 0.93, 95%CI 0.87-0.98, per hour). A lower core body temperature in the donor was associated with lower serum creatinine levels before procurement, which may reflect lower systemic inflammation and attenuated renal injury from brain death. Despite a considerable effect on DGF, our study failed to demonstrate a graft survival advantage (hazard ratio [HR] 0.83, 95%CI 0.54-1.27), whereas dopamine treatment was associated with improved long-term outcome (HR 0.95, 95%CI 0.91-0.99 per hour).

Impact of Steroids on the Inflammatory Response after Ischemic Acute Kidney Injury in Rats.

Inflammation plays a crucial role in acute kidney injury (AKI). The current study was designed to analyze the influence of prednisolone treatment on the inflammatory reaction during the first 96 h after AKI induction in a rat model. AKI was induced by unilateral clipping of the renal vessels. The treatment group received prednisolone 5 mg/kg s.c. daily. Infiltration rates of macrophages, leukocytes, and T-cells (24, 96 h) as well as plasma concentrations of the inflammatory markers intercellular adhesion molecule, interleukin-1 beta (IL-1ß), IL-18, IL-6, and tumor necrosis factor-alpha (0, 6, 24, 96 h) were determined by fluorescence-activated cell sorting (FACS) analysis only. Ninety-six hours after AKI induction, the prednisolone group demonstrated significantly lower creatinine concentrations compared to the control group (P < 0.05). Twenty-four hours after induction of AKI, a significantly higher rate of infiltrating leukocytes was detectable with FACS analysis in the control group (P < 0.01) with a corresponding significantly higher rate of macrophages after 96 h (P < 0.01). IL-6 and IL-1ß demonstrated a peak after 6 h with a significantly higher release in the control group (IL-6: P < 0.01; IL-1ß: P < 0.05). In contrast to the control group, the prednisolone group demonstrated no further incline of IL-18 after 24 h. The results demonstrate the importance of stretching the observation period in an ischemia-reperfusion-induced AKI setting beyond the first 24 h. Despite the demonstrated protective effects of a continuous prednisolone application, it seems that this single anti-inflammatory agent will not be able to completely suppress the inflammatory response after an ischemia-reperfusion-induced AKI.

Donor Preconditioning After the Onset of Brain Death With Dopamine Derivate n-Octanoyl Dopamine Improves Early Posttransplant Graft Function in the Rat.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Dopamine treatment of brain-dead Fisher rats improves renal histology but not early renal function in Lewis recipients after prolonged static cold storage.

BACKGROUND: Brain death (BD) and cold preservation are major risk factors for an unfavorable transplantation outcome. Although donor dopamine treatment in brain-dead rats improves renal function and histology in allogeneic recipients, it remains to be assessed if this also holds true for the combinations of BD and prolonged static cold preservation. METHODS: BD was induced in F344 donor rats, which were subsequently treated with NaCl 1 mL/h (BD, n = 11), NaCl/hydroxy ethyl starch (BD-norm, n = 10), or 10 µg/min/kg dopamine (BD-dopa, n = 10). Renal grafts were harvested 4 h after BD and transplanted into bilateral nephrectomized Lewis recipients 6 h after cold preservation in University of Wisconsin solution. Renal function was evaluated by use of serum creatinine and urea concentrations at days 0, 1, 3, 5, and 10. Ten days after transplantation, recipients were killed and the renal allografts were processed for light microscopy and immune histology. RESULTS: Serum urea concentrations at days 5 and 10 were significantly lower in recipients that received a renal graft from dopamine-treated rats; for serum creatinine, only a trend was observed at day 10. Immune histology revealed a lower degree of ED1-positive cells in the donor dopamine-treated group. Under light microscopy, Banff classification revealed significantly less intimal arteritis in these grafts (P < .05). CONCLUSIONS: Although donor dopamine treatment clearly improves renal histology in this model, the beneficial effect on early renal function was marginal. It remains to be assessed if donor dopamine treatment has a beneficial effect on renal function in long-term follow-up.

Carnosine treatment in combination with ACE inhibition in diabetic rats.

In humans, we reported an association of a certain allele of carnosinase gene with reduced carnosinase activity and absence of nephropathy in diabetic patients. CN1 degrades histidine dipeptides such as carnosine and anserine. Further, we and others showed that treatment with carnosine improves renal function and wound healing in diabetic mice and rats. We now investigated the effects of carnosine treatment alone and in combination with ACE inhibition, a clinically established nephroprotective drug in diabetic nephropathy. Male Sprague-Dawley rats were injected i.v. with streptozotocin (STZ) to induce diabetes. After 4 weeks, rats were unilaterally nephrectomized and randomized for 24 weeks of treatment with carnosine, lisinopril or both. Renal CN1 protein concentrations were increased under diabetic conditions which correlated with decreased anserine levels. Carnosine treatment normalized CN1 abundance and reduced glucosuria, blood concentrations of glycosylated hemoglobin (HbA1c), carboxyl-methyl lysine (CML), N-acetylglucosamine (GlcNac; all p<0.05 vs. non-treated STZ rats), reduced cataract formation (p<0.05) and urinary albumin excretion (p<0.05), preserved podocyte number (p<0.05) and normalized the increased renal tissue CN1 protein concentration. Treatment with lisinopril had no effect on HbA1C, glucosuria, cataract formation and CN1 concentration, but reduced albumin excretion rate more effectively than carnosine treatment (p<0.05). Treatment with both carnosine and lisinopril combined the effects of single treatment, albeit without additive effect on podocyte number or albuminuria. Increased CN1 amount resulted in decreased anserine levels in the kidney. Both carnosine and lisinopril exert distinct beneficial effects in a standard model of diabetic nephropathy. Both drugs administered together combine the respective effects of single treatment, albeit without exerting additive nephroprotection.

Different design of enzyme-triggered CO-releasing molecules (ET-CORMs) reveals quantitative differences in biological activities in terms of toxicity and inflammation.

Acyloxydiene-Fe(CO)3 complexes can act as enzyme-triggered CO-releasing molecules (ET-CORMs). Their biological activity strongly depends on the mother compound from which they are derived, i.e. cyclohexenone or cyclohexanedione, and on the position of the ester functionality they harbour. The present study addresses if the latter characteristic affects CO release, if cytotoxicity of ET-CORMs is mediated through iron release or inhibition of cell respiration and to what extent cyclohexenone and cyclohexanedione derived ET-CORMs differ in their ability to counteract TNF-α mediated inflammation. Irrespective of the formulation (DMSO or cyclodextrin), toxicity in HUVEC was significantly higher for ET-CORMs bearing the ester functionality at the outer (rac-4), as compared to the inner (rac-1) position of the cyclohexenone moiety. This was paralleled by an increased CO release from the former ET-CORM. Toxicity was not mediated via iron as EC50 values for rac-4 were significantly lower than for FeCl2 or FeCl3 and were not influenced by iron chelation. ATP depletion preceded toxicity suggesting impaired cell respiration as putative cause for cell death. In long-term HUVEC cultures inhibition of VCAM-1 expression by rac-1 waned in time, while for the cyclohexanedione derived rac-8 inhibition seems to increase. NFκB was inhibited by both rac-1 and rac-8 independent of IκBα degradation. Both ET-CORMs activated Nrf-2 and consequently induced the expression of HO-1. This study further provides a rational framework for designing acyloxydiene-Fe(CO)3 complexes as ET-CORMs with differential CO release and biological activities. We also provide a better understanding of how these complexes affect cell-biology in mechanistic terms.

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

Enzyme-triggered CO-releasing molecules (ET-CORMs): evaluation of biological activity in relation to their structure.

Acyloxydiene-Fe(CO)3 complexes act as enzyme-triggered CO-releasing molecules (ET-CORMs) and can deliver CO intracellularly via esterase-mediated hydrolysis. The protective properties of structurally different ET-CORMs on hypothermic preservation damage and their ability to inhibit VCAM-1 expression were tested on cultured human umbilical vein endothelial cells (HUVEC) and renal proximal tubular epithelial cells (PTEC) using a structure-activity approach. Cytotoxicity of ET-CORMs, protection against hypothermic preservation damage, and inhibition of VCAM-1 expression were assessed. Cytotoxicity of 2-cyclohexenone and 1,3-cyclohexanedione-derived ET-CORMs was more pronounced in HUVEC compared to PTEC and was dependent on the position and type of the ester (acyloxy) substituent(s) (acetate>pivalate>palmitate). Protection against hypothermic preservation injury was only observed for 2-cyclohexenone-derived ET-CORMs and was not mediated by the ET-CORM decomposition product 2-cyclohexenone itself. Structural requirements for protection by these ET-CORMs were different for HUVEC and PTEC. Protection was affected by the nature of the ester functionality in both cell lines. VCAM-1 expression was inhibited by both 2-cyclohexenone- and 1,3-cyclohexanedione-derived ET-CORMs. 2-Cyclohexenone, but not 1,3-cyclohexanedione, also inhibited VCAM-1 expression. We demonstrate that structural alterations of ET-CORMs significantly affect their biological activity. Our data also indicate that different ET-CORMs behave differently in various cell types (epithelial vs endothelial). These findings warrant further studies not only to elucidate the structure-activity relation of ET-CORMs in mechanistic terms but also to assess if structural optimization will yield ET-CORMs with restricted cell specificity.

Aerosolized semifluorinated alkanes as excipients are suitable for inhalative drug delivery--a pilot study.

Semifluorinated alkanes (SFAs) have been described as potential excipients for pulmonary drug delivery, but proof of their efficacy is still lacking. We tested whether SFA formulations with the test drug ibuprofen can be nebulised and evaluated their pharmacokinetics. Physico-chemical properties of five different ibuprofen formulations were evaluated: an aqueous solution (H2O), two different SFAs (perfluorohexyloctane (F6H8), perfluorobutylpentane (F4H5)) with and without ethanol (SFA/EtOH). Nebulisation was performed with a jet catheter system. Inhalative characteristics were evaluated by laser diffraction. A confirmative animal study with an inhalative single-dose (6 mg/kg) of ibuprofen with each formulation was performed in anaesthetised healthy rabbits. Plasma samples at defined time points and lung tissue harvested after the 6-h study period were analyzed by HPLC-MS/MS. Pharmacokinetics were calculated using a non-compartment model. All formulations were nebulisable. No differences in aerodynamic diameters (MMAD) were detected between SFA and SFA/EtOH. The ibuprofen plasma concentration-time curve (AUC) was highest with F4H5/EtOH. In contrast, F6H8/EtOH had the highest deposition of ibuprofen into lung tissue but the lowest AUC. All tested SFA and SFA/EtOH formulations are suitable for inhalation. F4H5/EtOH formulations might be used for rapid systemic availability of drugs. F6H8/EtOH showed intrapulmonary deposition of the test drug.

Semifluorinated alkanes--a new class of excipients suitable for pulmonary drug delivery.

INTRODUCTION: Semifluorinated alkanes (SFAs) are considered as diblock molecules with fluorocarbon and hydrocarbon segments. Unlike Perfluorocarbons (PFCs), SFAs have the potential to dissolve several lipophilic or water-insoluble substances. This makes them possibly suitable as new excipients for inhalative liquid drug carrier systems. PURPOSE: The aim of the study was to compare physico-chemical properties of different SFAs and then to test their respective effects in healthy rabbit lungs after nebulisation. METHODS: Physico-chemical properties of four different SFAs, i.e. Perfluorobutylpentane (F4H5), Perfluorohexylhexane (F6H6), Perfluorohexyloctane (F6H8) and Perfluorohexyldodecane (F6H12) were measured. Based on these results, aerosol characteristics of two potential candidates suitable as excipients for pulmonary drug delivery, i.e. F6H8 and F4H5, were determined by laser light diffraction. Tracheotomised and ventilated New Zealand White rabbits were nebulised with either a high- or a low dose of SFAs (F6H8(low/high) and F4H5(low/high)) or saline (NaCl). Ventilated healthy animals served as controls (Sham). Arterial blood gases, lung mechanics, heart rate and blood pressure were recorded prior to nebulisation and in 30 min intervals during the 6-h study period. RESULTS: Out of the four SFAs studied initially, no satisfactory behaviour as a solvent has to be expected because of low lipophilicity for F6H6. Output rate during aerosolisation was very low for F6H12. F6H8 and F4H5 presented comparable aerosolisation characteristics and lipophilicity and were therefore tested in the in vivo model. Aerosol therapy, either SFAs or saline, impaired paO2/FiO2 ratio, dynamic lung compliance and respiratory mechanics in all groups, except for F4H5(low) group which behaved like the control group (Sham). F4H5(low) had no adverse effects on gas exchange or pulmonary mechanics. CONCLUSIONS: Perfluorobutylpentane (F4H5) in a low-dose application may be suitable as a new inhalable excipient in SFA-based pulmonary drug delivery systems for lipophilic or water-insoluble substances.

Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients.

Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) patients' dysregulation of this process might underlie vascular inflammation. Peripheral blood mononuclear cells (PBMC) and neutrophils from AAV patients (n = 12) and healthy controls (HC, n = 12) were isolated. The influence of human umbilical vein endothelial cells (HUVEC) on neutrophil/monocytes function was tested by N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-mediated ROS production, degranulation and interleukin (IL)-8 production. In addition, the ability of lipopolysaccharide (LPS)-stimulated PBMC to produce tumour necrosis factor (TNF)-alpha in the presence or absence of HUVEC was tested. HUVEC inhibited ROS production dose-dependently by fMLP-stimulated neutrophils but did not influence degranulation. No differences between neutrophils from HC and AAV were found. However, in only one active patient was degranulation inhibited significantly by HUVEC only before cyclophosphamide treatment, but not 6 weeks later. Co-cultures of HUVEC with LPS-stimulated neutrophils/monocytes increased IL-8 production while TNF-alpha production was inhibited significantly. There was no apparent difference between AAV patients and HC in this respect. Our findings demonstrate that HUVEC are able to inhibit ROS and modulate cytokine production upon stimulation of neutrophils or monocytes. Our data do not support the hypothesis that endothelial cells inhibit ROS production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation may exist in active patients, but this finding needs to be confirmed.

Modulation of brain dead induced inflammation by vagus nerve stimulation.

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

Donor treatment with a PHD-inhibitor activating HIFs prevents graft injury and prolongs survival in an allogenic kidney transplant model.

Long-term survival of renal allografts depends on the chronic immune response and is probably influenced by the initial injury caused by ischemia and reperfusion. Hypoxia-inducible transcription factors (HIFs) are essential for adaptation to low oxygen. Normoxic inactivation of HIFs is regulated by oxygen-dependent hydroxylation of specific prolyl-residues by prolyl-hydroxylases (PHDs). Pharmacological inhibition of PHDs results in HIF accumulation with subsequent activation of tissue-protective genes. We examined the effect of donor treatment with a specific PHD inhibitor (FG-4497) on graft function in the Fisher-Lewis rat model of allogenic kidney transplantation (KTx). Orthotopic transplantation of the left donor kidney was performed after 24 h of cold storage. The right kidney was removed at the time of KTx (acute model) or at day 10 (chronic model). Donor animals received a single dose of FG-4497 (40 mg/kg i.v.) or vehicle 6 h before donor nephrectomy. Recipients were followed up for 10 days (acute model) or 24 weeks (chronic model). Donor preconditioning with FG-4497 resulted in HIF accumulation and induction of HIF target genes, which persisted beyond cold storage. It reduced acute renal injury (serum creatinine at day 10: 0.66 +/- 0.20 vs. 1.49 +/- 1.36 mg/dL; P < 0.05) and early mortality in the acute model and improved long-term survival of recipient animals in the chronic model (mortality at 24 weeks: 3 of 16 vs. 7 of 13 vehicle-treated animals; P < 0.05). In conclusion, pretreatment of organ donors with FG-4497 improves short- and long-term outcomes after allogenic KTx. Inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves clinical evaluation.

The carbon monoxide releasing molecule (CORM-3) inhibits expression of vascular cell adhesion molecule-1 and E-selectin independently of haem oxygenase-1 expression.

BACKGROUND AND PURPOSE: Although carbon monoxide (CO) can modulate inflammatory processes, the influence of CO on adhesion molecules is less clear. This might be due to the limited amount of CO generated by haem degradation. We therefore tested the ability of a CO releasing molecule (CORM-3), used in supra-physiological concentrations, to modulate the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on endothelial cells and the mechanism(s) involved. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor (TNF)-alpha in the presence or absence of CORM-3. The influence of CORM-3 on VCAM-1 and E-selectin expression and the nuclear factor (NF)-kappaB pathway was assessed by flow cytometry, Western blotting and electrophoretic mobility shift assay. KEY RESULTS: CORM-3 inhibited the expression of VCAM-1 and E-selectin on TNF-alpha-stimulated HUVEC. VCAM-1 expression was also inhibited when CORM-3 was added 24 h after TNF-alpha stimulation or when TNF-alpha was removed. This was paralleled by deactivation of NF-kappaB and a reduction in VCAM-1 mRNA. Although TNF-alpha removal was more effective in this regard, VCAM-1 protein was down-regulated more rapidly when CORM-3 was added. CORM-3 induced haem oxygenase-1 (HO-1) in a dose- and time-dependent manner, mediated by the transcription factor, Nrf2. CORM-3 was still able to down-regulate VCAM-1 expression in HUVEC transfected with siRNA for HO-1 or Nrf2. CONCLUSIONS AND IMPLICATIONS: Down-regulation of VCAM and E-selectin expression induced by CORM-3 was independent of HO-1 up-regulation and was predominantly due to inhibition of sustained NF-kappaB activation.

Rosiglitazone reduces angiotensin II and advanced glycation end product-dependent sustained nuclear factor-kappaB activation in cultured human proximal tubular epithelial cells.

Tubular damage is a major feature in the development of diabetic nephropathy. This study investigates the effects of the thiazolidindione rosiglitazone on angiotensin II and advanced glycation end product-induced tubular activation in human proximal tubular epithelial cells IN VITRO. Angiotensin II and advanced glycation end products, both induced a dose-dependent sustained activation of the redox-sensitive transcription factor, Nuclear Factor KAPPA B (NF-kappaB). Nuclear translocation of NF-kappaB was evident already after one hour and persistent for more than four days. Co-incubation of proximal tubular epithelial cells with rosiglitazone significantly reduced angiotensin II and advanced glycation end product-mediated generation of reactive oxygen species, angiotensin II-dependent advanced glycation end product formation, NF-kappaB activation, and NF-kappaB-dependent pro inflammatory gene expression. Most importantly, rosiglitazone effects on NFkappaB activation were maximal at later time points, indicating that rosiglitazone treatment confers long lasting renoprotective effects.

High mobility group box 1 and adenosine are both released by endothelial cells during hypothermic preservation.

Hypothermic preservation of solid allografts causes profound damage of vascular endothelial cells. This, in turn, might activate innate immunity. In the present study we employed an in vitro model to study to what extent supernatants of damaged endothelial cells are able to activate innate immunity and to study the nature of these signals. The expression of high mobility group box 1 (HMGB1) and adhesion molecules on human umbilical vein endothelial cell was studied by immunofluorescence, fluorescence activated cell sorter and Western blotting. Cytokine production was performed by enzyme-linked immunosorbent assay. HMGB1 expression was lost completely in endothelial cells after hypothermic preservation. This was associated with cell damage as it occurred only in untreated endothelial cell but not in cells rendered resistant to hypothermia-mediated damage by dopamine treatment. Only supernatants from hypothermia susceptible cells up-regulated the expression of interleukin (IL)-8 and adhesion molecules in cultured endothelial cells in an HMGB1-dependent manner. In whole blood assays, both supernatants of hypothermia susceptible and resistant cells inhibited tumour necrosis factor (TNF)-alpha production concomitantly with an increased IL-10 secretion. The activity of the supernatants was already found after 6 h of hypothermic preservation, and paralleled the decrease in intracellular adenosine triphosphate (ATP) levels. Modulation of TNF-alpha and IL-10 production by these supernatants was abrogated completely by prior treatment with adenosine deaminase and was similar to the response of an A2R agonist. Our study demonstrates that both HMGB1 and adenosine are released during hypothermic preservation. While release of HMGB1 is caused by cell damage, release of adenosine seems to be related to ATP hydrolysis, occurring in both susceptible and resistant cells.

Imatinib mesylate, a new kid on the block for the treatment of anti-neutrophil cytoplasmic autoantibodies-associated vasculitis?

Persistent T cell activation is a common finding in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AAV) patients. Because imatinib, a selective inhibitor of the ABL, ARG, PDGFR and c-KIT tyrosine kinases, inhibits T cell activation, this study was conducted to evaluate the potential use of imatinib for the treatment AAV patients refractory to conventional therapy. In particular, we investigated the inhibition of T cell activation by this drug and its efficacy on activated T cells from anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitides (AASV) patients. T cell stimulation has been induced by anti-CD3/anti-CD28 antibodies or by phorbol myristate acetate (PMA)/ionomycin. T cell proliferation was analysed by tritiumthymidine incorporation. Cell cycle progression was determined by propidium iodide staining using fluorescence activated cell sorter (FACS) analysis and by RNAse protection assay (RPA). Cytokine levels were assessed by enzyme-linked immunosorbent assay. T cell proliferation was inhibited significantly by imatinib, due most probably to cell cycle arrest in the G1-phase. This was paralleled by inhibition in the expression of cyclin-dependent kinases 1 and 2 mRNA. The expression of CD25 in naive and memory T cells was decreased significantly by imatinib in activated T cells. Similarly, conversion from naive to memory T cells after T cell activation was impaired by imatinib. Imatinib did not influence interleukin-2 and tumour necrosis factor-alpha production but increased interferon-gamma production. These observed effects of imatinib were similar in T cells from AASV patients and from healthy individuals. Imatinib might be an alternative therapeutical option for AASV patients refractory to conventional therapy.

Hypothermic preservation of lung allograft inhibits cytokine-induced chemoattractant-1, endothelial leucocyte adhesion molecule, vascular cell adhesion molecule-1 and intracellular adhesion molecule-1 expression.

Organ dysfunction is a major clinical problem after lung transplantation. Prolonged cold ischaemia and reperfusion injury are believed to play a central role in this complication. The influence of cold preservation on subsequent warm reperfusion was studied in an isolated, ventilated and perfused rat lung. Rat lungs were flushed with cold Perfadex-solution and stored at 4 degrees C for different time periods. Thereafter lungs were perfused and ventilated for up to 3 h. Physiological parameters, production of inflammatory mediators and leucocyte infiltration were measured before and after perfusion. Lungs subjected to a cold ischaemia time of up to 6 h showed stable physiological conditions when perfused for 3 h. However, cold-ischaemia time beyond 6 h resulted in profound tissue oedema, thereby impairing ventilation and perfusion. Warm reperfusion and ventilation per se induced a strong inflammatory response, as demonstrated by a significant up-regulation of chemokines and adhesion molecules (cytokine-induced chemoattractant-1, intracellular adhesion molecule and endothelial leucocyte adhesion molecule), accompanied by enhanced leucocyte infiltration. Although the up-regulation of inflammatory mediators was blunted in lungs that were subjected to cold ischaemia, this did not influence leucocyte infiltration. In fact, cold ischaemia time correlated with leucocyte sequestration. Although cold preservation inhibits the expression of inflammatory mediators it does not affect leucocyte sequestration during warm reperfusion. Cold preservation might cause impairment of the endothelial barrier function, as evidenced by tissue oedema and profound leucocyte infiltration.

[The role of endothelial progenitor cells in sepsis]. / Die Rolle endothelialer Vorläuferzellen in der Sepsis.

In sepsis and septic shock a series of immunological events are initiated that alter endothelial function in the macrocirculation and microcirculation. Endothelial swelling, deformation and apoptosis with detachment from the vasculature occur and endothelial cells (EC) appear in the circulation. Simultaneous to these pathological processes, reconstitution of the endothelial layer is initiated which can occur via migration and proliferation of surrounding mature ECs. However, terminally differentiated ECs have a low proliferative potential, hence their capacity to substitute damaged endothelium is limited. Therefore, adequate vascular repair requires additional support. Many studies have now convincingly demonstrated that vascular maintenance, repair, angiogenesis and neovascularization are partly mediated by recruitment of endothelial progenitor cells (EPCs) from the basal membrane. However, it seems that EPCs play a pivotal role not only in re-endothelialization after vascular damage, but also after severe inflammation. Recently, evidence was found that EPCs are increasingly mobilized during sepsis and that this mobilization is associated with clinical outcome. In septic patients the number of EPCs was significantly higher than in controls and was correlated with survival and the concentration of cytokines. In summary EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial layer by replacing denuded parts of the microcirculation or by stimulation of EC proliferation. Therefore, EPC enumeration seems to be a valuable prognostic and diagnostic marker for the outcome in these patients and the induction of enhanced EPC mobilization a therapeutic option.

In vivo effects of cyclic administration of 15-deoxyspergualin on leucocyte function in patients with Wegener's granulomatosis.

15-Deoxyspergualin (DSG) is an alternative treatment modality for Wegener's granulomatosis (WG) patients refractory to conventional treatment. Nevertheless, it is unclear how DSG modulates disease activity in these patients. This study was conducted to investigate which parameters of adaptive and acquired immunity were influenced during two subsequent cycles of DSG treatment. Emphasis was put upon T cell and monocyte activation, neutrophil function and surface expression of proteinase-3 (PR-3). Anti-CD3/anti-CD28 and interleukin (IL)-15/IL-7-mediated T cell proliferation were assessed by fluorescence activated cell sorter (FACS) analysis using carboxyfluorescein succinimidyl ester (CSFE) labelling. Interferon (IFN)-gamma and IL-10 production were determined in the supernatants of these cultures by enzyme-linked immunosorbent assay. Monocyte activation was assessed in lipopolysaccharide (LPS)-stimulated whole blood, using tumour necrosis factor (TNF)-alpha as read-out. Neutrophil function was determined by measuring oxidative burst, chemotaxis and phagocytosis. T cell activation markers and PR3 expression were measured by FACS. All parameters were determined directly before and after each DSG cycle. Anti-CD3/anti-CD28-mediated T cell proliferation was reduced directly after DSG treatment. Directly before a subsequent cycle of DSG was started, T cell proliferation was increased. Similar findings were observed for IFN-gamma and IL-10 production by T cells. DSG did not influence IL-15/IL-7-mediated T cell proliferation. LPS-mediated TNF-alpha production was also impaired directly after DSG treatment. No influence on T cell activation markers, neutrophil function and surface PR-3 expression was observed in peripheral blood of these patients. Our data demonstrate that DSG influences T cell and monocyte activation in a reversible fashion. Although DSG causes neutropenia in these patients, it does not influence neutrophil function.