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).

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.

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.

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.

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.

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.

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.

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.

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.

The role of T lymphocytes in ANCA associated vasculitis: neglected or revisited?

"Antineutrophil cytoplasmic antibodies (ANCA) are pathogenic--Oh yes they are!" is the title of a recent review [Falk et al. 2002], discussing the current evidence on the pathogenic role of ANCA in vasculitis. But what about T lymphocytes? Do these cells also contribute to disease manifestation and if so to what extend? T-cells most likely play a role in delivering proper signals to autoreactive B cells for the production of ANCA, but, in the efferent arm of the immune response the involvement of T cells is less obvious and controversially discussed. Numerous studies provide evidence that peripheral T-cell phenotypes are dramatically changed in ANCA associated vasculitis (AAV) patients. How these changes relate to disease manifestation is still a matter of discussion. In an attempt to provide a better understanding of how T cells might play a role in AAV, the present paper will review recent data presented at the 12th international vasculitis and ANCA workshop.

Nongenomic effects of aldosterone on human renal cells.

The development of chronic renal insufficiency may be partially mediated by the nongenomic action of aldosterone. Here we investigate whether aldosterone could evoke a nongenomic action in primary cultures of human renal cells. Intracellular Ca(2+) ([Ca(2+)](i)) and cAMP were measured in human mesangial cells (MC), glomerular visceral epithelial cells (GVEC), and proximal and distal tubular epithelial cells (Ptec and Dtec) in the presence of aldosterone (10-100 nmol/liter) by fura-2 fluorescence and RIA, respectively. In MC, Ptec, and Dtec, aldosterone increased [Ca(2+)](i) within 1 min, whereas in GVEC, only a minor effect was found. Preincubation of cells with spironolactone did not blunt this effect. Hydrocortisone, used at a concentration 100-fold higher than that of aldosterone, did not affect [Ca(2+)](i.) In MC, Ptec, and Dtec, a dose-dependent increase ( approximately 1.3- to 1.5-fold) in intracellular cAMP levels was found. These data demonstrate a nongenomic action of aldosterone in human MC, Ptec, and Dtec. As these effects occur at concentrations close to free plasma aldosterone levels in man, they may be of physiological relevance and may contribute to renal injury.

Sulfation-dependent down-regulation of interferon-gamma-induced major histocompatibility complex class I and II and intercellular adhesion molecule-1 expression on tubular and endothelial cells by glycosaminoglycans.

BACKGROUND: Previously, it has been demonstrated that heparin inhibits major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1) expression on interferon-gamma (IFN-gamma)-stimulated human umbilical vein endothelial cells (HUVECs). Inasmuch as proximal tubular epithelial cells (PTECs) are prime targets in acute renal allograft rejection, we investigated whether there is a difference in the ability of heparin to influence MHC and ICAM-1 expression on PTECs as compared to HUVECs. We also studied whether the degree of sulfation of heparin is of relevance for the binding to IFN-gamma and inhibition of MHC and ICAM-1 expression after IFN-gamma stimulation. METHODS: Cultured HUVECs and PTECs were stimulated with IFN-gamma for 72 hr in the presence or absence of various heparinoids. MHC and ICAM-1 expression were thereafter determined by fluorescence-activated cell sorting. RESULTS: Heparin was able to inhibit the up-regulation of MHC and ICAM-1 in a dose-dependent fashion on both IFN-gamma-stimulated HUVECs and PTECs. In PTEC cultures, higher concentrations of heparin were required for the inhibition of MHC class I. Heparin and supersulfated glycosaminoglycans (GAGs) were able to bind to IFN-gamma, whereas N-desulfated N-acetylated GAGs with a low amount of sulfate were not. Inhibition of cell-bound heparan sulfate proteoglycan sulfation with NaClO3 resulted in an impaired MHC and ICAM-1 expression after IFN-gamma stimulation. CONCLUSION: We postulate that IFN-gamma binds to cell-bound heparan sulfate proteoglycan in a sulfation-dependent fashion. This binding may facilitate the interaction of IFN-gamma with its receptor. Supersulfated GAGs with low anti-coagulant activity could be used therapeutically to decrease MHC and ICAM-1 expression on organ grafts.

The clinical significance of allospecific antibodies against endothelial cells detected with an antibody-dependent cellular cytotoxicity assay for vascular rejection and graft loss after renal transplantation.

Serum samples of 64 consecutive patients who underwent renal transplantation in our institution were examined for the presence of antibody-dependent cellular cytotoxicity (ADCC) activity against endothelial cells (EC). From each patient serum samples were obtained immediately before transplantation and 1 week, 1 month and 1 year thereafter. The results were evaluated in the context of tests to measure donor-specific humoral immunity against lymphocytes and monocytes, and related to parameters of presensitization, graft survival, and histology. Sera from 10 patients were positive for ADCC on a panel of HLA-typed endothelial cells. In 8 patients sera were already positive before transplantation and remained positive thereafter. In 4 patients a positive crossmatch with donor T and B cells and monocytes could be observed after transplantation. In only one patient were these crossmatches positive before transplantation. A significant correlation was found between ADCC positivity and vascular rejection (P = 0.015); in addition graft survival was significantly better in the ADCC negative group vs. the positive group (P = 0.0004). These data demonstrate the significance of allospecific anti EC antibodies for the occurrence of vascular rejection and graft loss after renal transplantation.

Prevention of chronic renal allograft rejection in rats with an oral endothelin A receptor antagonist.

BACKGROUND: Chronic rejection is the most common cause of graft loss in renal transplantation. The pathomechanisms underlying chronic rejection are poorly understood, and no treatment has yet successfully been established. We hypothesized that, in analogy to models of reduced renal mass, the administration of a selective endothelin (ET) A receptor antagonist could improve the course of chronic rejection in renal allografts. METHODS: Experiments were performed in the Fisher-to-Lewis rat model of chronic rejection. Lewis-->Lewis isografts served as controls. Animals were treated with either the oral selective ET-A receptor antagonist LU135252 (50 mg/kg/day) or vehicle. Animal survival, blood pressure, creatinine clearance, proteinuria, and urinary ET excretion were investigated for 24 weeks. Kidneys were removed for light microscopical evaluation, determination of ET mRNA expression and tissue protein concentration, and immunohistochemical assessment of cell surface markers. RESULTS: Rats with chronic rejection showed an increase in renal ET mRNA synthesis and ET protein content. Treatment with LU135252 resulted in a significant improvement in survival after 24 weeks (0.92 vs. 0.38, P<0.01 by log-rank test). Creatinine clearance was higher in animals treated with the selective ET-A receptor antagonist (P<0.05). LU135252 had no influence on blood pressure and proteinuria. Selective ET-A blockade was associated with significantly less morphological changes and a significant reduction of expression of cell surface markers for macrophages (ED1), T cells (R73), and MHC II (F17-23-2). CONCLUSION: The renal ET-A system plays an important role in the pathomechanisms underlying chronic renal allograft rejection, because the treatment with a selective ET-A receptor antagonist dramatically improves the course of chronic renal failure after allograft transplantation. These results offer a novel therapeutical option for treatment of chronic renal allograft rejection, for which so far no therapy is known.

Inhibition of LPS-induced chemokine production in human lung endothelial cells by lipid conjugates anchored to the membrane.

1. In acute respiratory distress syndrome (ARDS) induced by endotoxins, a high production of inflammatory mediators by microvascular lung endothelial cells (LMVEC) can be observed. Activation of cells by endotoxins may result in elevated secretion of phospholipase A(2) (sPLA(2)) which is thought to contribute to tissue damage. The present study was undertaken to investigate the role of sPLA(2) in chemokine production in human lung microvascular endothelial cells (LMVEC) stimulated with the endotoxins lipopolysaccharide (LPS) and lipoteichoic acid (LTA). In particular, we investigated the effects of sPLA(2) inhibitors, specifically, the extracellular PLA(2) inhibitors (ExPLIs), composed of N-derivatized phosphatidyl-ethanolamine linked to polymeric carriers, and LY311727, a specific inhibitor of non-pancreatic sPLA(2). 2. ExPLIs markedly inhibited LPS and LTA induced production and mRNA expression of the neutrophile attracting chemokines IL-8, Gro-alpha and ENA-78, as well as of the adhesion molecules ICAM-1 and E-selectin. Concomitantly, ExPLIs inhibited the LPS-induced activation of NF-kappaB by LPS but not its activation by TNF-alpha or IL-1. 3. Endotoxin mediated chemokine production in LMVEC seems not to involve PLA(2) activity, since LPS stimulation was not associated with activation of intracellular or secreted PLA(2). It therefore seems that the inhibitory effect of the ExPLIs was not due to their PLA(2) inhibiting capacity. This was supported by the finding that the LPS-induced chemokine production was not affected by the selective sPLA(2) inhibitor LY311727. 4. It is proposed that the ExPLIs may be considered a prototype of potent suppressors of specific endotoxin-induced inflammatory responses, with potential implications for the therapy of subsequent severe inflammation.

Tissue antigens in tubulointerstitial and vascular rejection.

We propose that tissue-specific alloantigens are of importance in interstitial and vascular rejection. To study this hypothesis we took the following approaches: multivariate analysis on our database (N = 482) was performed, the specificity of T cells cultured from kidneys with rejection was analyzed, and non-anti-HLA antibodies reactive with endothelium were studied. First we observed that in a cohort study of 482 patients receiving a cadaveric renal allograft 76 (15.8%) patients developed vascular rejection and 115 (23.9%) developed interstitial rejection. The incidence of vascular rejection was increased in patients with delayed graft function, HLA-DR mismatches, a prolonged cold ischemia period, and previous transplantations. Next we examined 40 graft infiltrating cell (GIC) lines cultured from renal biopsies taken during rejection episodes. Thirteen GIC lines reacted in a donor-specific fashion to proximal tubular cells (PTEC) but not to donor splenocytes. These GIC recognize polymorphic tissue-specific peptides in the context of allo-MHC Class I. Finally, we studied if non-conventional allo-antigen systems on endothelial cells could be the target of the humoral immune response during vascular rejection. We found the endothelial monocyte (EM) system, and another system that is present on endothelial cells and platelets, which can be tested in an antibody-dependent cellular cytotoxicity assay (ADCC).

Analysis of T cell lines from rejecting renal allografts.

Acute rejection after renal allotransplantation is characterized by the presence of a mononuclear cell infiltrate in the interstitium with involvement of tubuli. In a previous study on renal histology we showed that tubular damage by graft infiltrating cells (GIC) is a sign of clinically significant rejection. We cultured proximal tubular epithelial cells (PTEC) and T cells bearing the interleukin 2 (IL-2) receptor from biopsies after transplantation. In vitro outgrowth of T cells from the biopsy was significantly (P = 0.0014) related to histological signs of graft rejection. Of the T cell lines generated from 25 biopsies, only five lines showed no or low cytotoxicity against donor PTEC. Three cell lines were cytotoxic towards donor PTEC, but not against PHA stimulated donor splenocytes, suggesting tissue specificity of GIC. Treatment of PTEC with interferon (IFN) gamma for 72 hours to upregulate MHC class I and to induce MHC class II expression did not necessarily result in an increased susceptibility to lysis. However, three PTEC lines displayed an increment of susceptibility to lysis after IFN gamma treatment. Analysis of one T cell line from the same graft revealed a high percentage of CD4 positive cells, compatible with a class II restricted cytotoxicity. This was confirmed by blocking experiments using anti-CD4, anti-CD8, anti-class I, and anti-class II antibodies. Blocking experiments were done with 12 of these 25 lines. Anti-CD3 and anti-CD18 antibodies inhibited cytotoxicity in every case, showing that cytotoxicity was T cell mediated.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of peptides eluted from the groove of HLA-B27 from Salmonella infected and non-infected cells.

Reactive arthritis (ReA) is associated with the MHC class-I molecule HLA-B27 and caused by certain Gram-negative bacteria. The mechanism by which HLA-B27 confers a higher susceptibility for this disease compared to other MHC Class-I alleles is still not known. We investigated whether infection of human HLA-B27+ cells is able to change the peptide repertoire presented by these HLA-B27 molecules. To this end large quantities of a B-cell line (C1R-B27) transfected with HLA-B2705 were infected with S. typhimurium. Peptides were eluted from the B27 molecules and separated by Reversed Phase Chromatography (RPC). We then compared the peptide profiles obtained from S. typhimurium infected CIR B-cells with that obtained from non infected cells. Apart from a few additional peaks present in the profile derived from the infected batch the peptide profiles were almost identical. A few fractions were subjected to sequencing by Edman degradation. All peptides found were nonameres with arginine (Arg) at position 2 which is in agreement with the previously described HLA-B27 peptide binding motif. The majority of peaks expressed a mixture of at least four different peptides. The analysis of differences between HLA-B27 bound peptides from Salmonella infected and non infected cells might lead to the identification of T-cell epitopes shared by Salmonella and autoantigens.

Immunization of HLA-B27 transgenic and non transgenic mice with Salmonella typhimurium results predominantly in the generation of proliferative T cell responses.

Reactive arthritis (ReA) due to Gram-negative intestinal bacteria or Chlamydia, is associated by an unknown mechanism with HLA-B27. Like other MHC class I molecules, HLA-B27 presents antigenic peptides derived from intracellular proteins to CD8+ cytotoxic T cells (CTL). In humans however, CTL specific for ReA associated bacteria have been reported in a limited number of studies. This may be caused by an inefficient in vivo induction of CTL against such micro-organisms. In the present study we addressed the question whether and to what extend mice transgenic for HLA-B27 are able to generate CTL against Salmonella typhimurium after immunization. To this end both HLA-B27 transgenic and non transgenic mice were immunized i.p., i.v. or orally, receiving a secondary challenge four weeks later. One day after infection with Salmonella, bacteria could be cultured from spleen and liver. There was no significant difference in the number of bacteria cultured from these organs between both groups of mice. Spleen cells from all immunized mice proliferated specifically in the presence of heat killed Salmonella but not in the presence of heat killed Yersinia. No proliferation of spleen cells from naive mice was observed in the presence of heat killed Salmonella, excluding the possibility that Salmonella antigens were mitogenic. Only in one out of 6 mice immunized i.v. with Salmonella Salmonella specific CTL could be generated. In order to rule out the possibility that in HLA-B27 transgenic mice the HLA-B27 molecule is not used as a restriction element by murine T cells, CTL were raised against the male minor histocompatibility (mH) antigen H-Y. Both murine class I as well as HLA-B27 restricted CTL could be generated. In conclusion this study demonstrates that MHC class I restricted CTL specific for the Gram-negative bacterium Salmonella typhimurium are difficult to generate in contrast to proliferative responses which can be easily demonstrated. This may comparable in humans where in the majority of studies bacteria specific T cells isolated from ReA patients appear to be CD4+ and class II restricted.

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.