Porcine endogenous retrovirus is not transmitted in a discordant porcine-to-cynomolgus xenokidney transplantation model with long-term survival of organ recipients.
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Biomedical subjects
Publications and source records attributed to M Winkler.
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The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.
Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8 - 10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase-3-like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non-resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase-3-like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross-resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.
AIMS: The aim of the study was to investigate the pharmacokinetics and metabolism of the new immunosuppressant SDZ RAD during concomitant therapy with cyclosporin in stable renal transplant patients. Furthermore, we studied the influence of SDZ RAD on the pharmacokinetics of cyclosporin at steady state levels. METHODS: SDZ RAD was administered orally in different doses (0.25-15 mg day-1) to seven patients, who were on standard cyclosporin-based immunosuppression. The blood concentrations of both drugs including their main groups of metabolites were measured simultaneously by LC/electrospray-mass spectrometry. RESULTS: The mean area under the blood concentration-time curve to 12 h (AUC(0,12 h)) was 4244 +/- 1311 microg l-1 h for cyclosporin before SDZ RAD treatment and 4683 +/- 1174 microg l-1 h (P = 0.106) on the day of SDZ RAD treatment (95% CI for difference -126, 1003). On both study days Cmax, and tmax of cyclosporin were not significantly different. The metabolite pattern of cyclosporin did not change. The pharmacokinetic data of SDZ RAD dose-normalized to 1 mg SDZ RAD were as follows: AUC(0,24 h): 35.4 +/- 13.1 microg l-1 h, Cmax: 7.9 +/- 2.7 microg l-1 and tmax: 1.5 +/- 0.9 h. The metabolites of SDZ RAD found in blood were hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and a ring-opened form of SDZ RAD. CONCLUSIONS: A single dose of SDZ RAD did not influence significantly the pharmacokinetics of cyclosporin. The most important metabolite of SDZ RAD was the hydroxy-SDZ RAD, its AUC(0,24 h) being nearly half that of the parent compound SDZ RAD.
A study of the characterization of the phenotypic patterns of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Israeli dairy herds and their correlation with the severity of the disease was undertaken. A total of 400 chronically S. aureus-infected Israeli-Holstein cows, from 15 dairy herds were included in this study. Based on the results of the biochemical reactions, of the anti-biogram and phage typing, one major type of S. aureus was determined in each herd, its prevalence being between 54 and 100% of the total isolates from that same herd. The majority of the isolates were found to be non-haemolytic (62.7%). The most common phage type was 3/A,3/C,55,71, which was predominant in five herds. In two herds none of the isolates (24) were typable by this set of phages. All isolates were susceptible to methicillin, erythromycin, cephalotin, norfloxacin, trimethoprin-sulphamethoxazole and novobiocin. Most isolates were resistant to penicillin (96.6%) and 52% to oxytetracyclin. Differences in protein patterns between 50 and 36 kDa were found by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. No correlation between any combination of the phenotypic characteristics was found when correlation was done with milk yield and somatic cell count, corresponding to the 6 months before sampling. Otherwise, a positive correlation was found between type of haemolysis and the N-acetyl-beta-glucosaminidase (NAGase) values. In milk from quarters infected with the-non-haemolytic strains, the level of NAGase was significantly lower (P < 0.05) than that from quarters infected with the haemolytic strains (69.7 and 105.9, respectively). However, the level of NAGase activity in the milk of the quarters infected with the non-haemolytic strains was significantly higher (P < 0.05) when compared to the milk of quarters infected with coagulase-negative staphylococci (43.5).
UNLABELLED: We evaluated the effects of aggressive warming and maintenance of normothermia on surgical blood loss and allogeneic transfusion requirement. We randomly assigned 150 patients undergoing total hip arthroplasty with spinal anesthesia to aggressive warming (to maintain a tympanic membrane temperature of 36.5 degrees C) or conventional warming (36 degrees C). Autologous and allogeneic blood were given to maintain a priori designated hematocrits. Blood loss was determined by a blinded investigator based on sponge weight and scavenged cells; postoperative loss was determined from drain output. Results were analyzed on an intention-to-treat basis. Average intraoperative core temperatures were warmer in the patients assigned to aggressive warming (36.5 degrees +/- 0.3 degrees vs 36.1 degrees +/- 0.3 degrees C, P< 0.001). Mean arterial pressure was similar in each group preoperatively, but was greater intraoperatively in the conventionally warmed patients: 86+/-12 vs 80+/-9 mm Hg, P<0.001. Intraoperative blood loss was significantly greater in the conventional warming (618 mL; interquartile range, 480-864 mL) than the aggressive warming group (488 mL; interquartile range, 368-721 mL; P: = 0.002), whereas postoperative blood loss did not differ in the two groups. Total blood loss during surgery and over the first two postoperative days was also significantly greater in the conventional warming group (1678 mL; interquartile range, 1366-1965 mL) than in the aggressively warmed group (1,531 mL; interquartile range, 1055-1746 mL, P = 0.031). A total of 40 conventionally warmed patients required 86 units of allogeneic red blood cells, whereas 29 aggressively warmed patients required 62 units (P = 0.051 and 0.061, respectively). We conclude that aggressive intraoperative warming reduces blood loss during hip arthroplasty. IMPLICATIONS: Aggressive warming better maintained core temperature (36.5 degrees vs 36.1 degrees C) and slightly decreased intraoperative blood pressure. Aggressive warming also decreased blood loss by approximately 200 mL. Aggressive warming may thus, be beneficial in patients undergoing hip arthroplasty.
OBJECTIVE: To compare plasma concentrations and cardiovascular effects of epinephrine after application via a conventional endotracheal airway and via the esophageal lumen of a new emergency airway, the esophageal tracheal Combitube. DESIGN: Prospective, randomized study. SETTING: Center for Biomedical Research, University of Vienna. SUBJECTS: Fourteen juvenile swine received either an endotracheal tube (Group A) or a Combitube in esophageal position (Group B). INTERVENTIONS: In Part I of the study, epinephrine was administered during spontaneous beating of the heart; in Part II, epinephrine was administered during cardiopulmonary resuscitation, using a ten-fold higher dosage in Group B, respectively. MEASUREMENTS: Plasma epinephrine levels were measured 1, 2, 3, 5, 7, 10, 15, and 30 mins after application. Systolic arterial blood pressure and cardiac output in Part I, and end-tidal CO2 and coronary perfusion pressure in Part II were recorded. MAIN RESULTS: In Part I, increased levels of plasma epinephrine and systolic arterial pressure were maintained significantly longer in Group B when compared with Group A. In Part II, no significant differences between the groups were found with regard to plasma epinephrine levels and hemodynamic variables. CONCLUSION: Epinephrine applied via the esophageal lumen of the Combitube in a ten-fold higher dosage has similar effects on plasma epinephrine levels and hemodynamic variables compared to endotracheal administration.
The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing beta-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-beta-D-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.
Women with pseudomyxoma peritonei may have both, appendiceal and ovarian mucinous tumors, and there is a considerable debate regarding the site of origin of the tumor in such cases. Recent studies which have investigated the histological and immunohistochemical profile of these tumors showed, that ovarian tumors in most cases are secondary to the appendiceal tumors. In our case report we demonstrate that, despite of extensive immunohistochemical examinations (analysis of CK 7, 18, 20, CEA, HAM 56), it may be difficult to determine the histogenetic origin of pseudomyxoma peritonei.
We report for the first time on a puerperal ovarian vein thrombophlebitis associated with heparin-induced thrombocytopenia type II. Two weeks after cesarean section with subsequent prophylactic application of heparin a thrombosis in the left deep femoral and pelvic veins and in the right ovarian vein occurred. Despite only moderately reduced platelet count a heparin-induced thrombocytopenia type II was diagnosed by heparin-induced platelet activation test. By treatment with plasmin activator lysis the thrombus was diminished. An operative procedure had been prevented. We conclude that also in cases with only moderately reduced platelet count during and after administration of heparin, especially in patients with wide spread and accumulating thrombosis, the presence of a heparin-induced thrombocytopenia should be taken into consideration. In contrast to difficulties in the management of puerperal ovarian vein thrombophlebitis--occurring independently from heparin-induced thrombocytopenia (surgical treatment necessary in most cases)--occurrence of puerperal ovarian vein thrombophlebitis in relation to heparin-induced thrombocytopenia seems to response better to fibrinolysis and anticoagulation.
HISTORY AND ADMISSION FINDINGS: A 17-year-old girl had swallowed 80-100 tablets of Nebivolol, 5 mg each, with suicidal intent. She was referred to hospital 8 hours later by an emergency duty physician. On admission she was sweaty and pale, but there were no other obvious abnormalities. Neurological examination revealed decreased responsiveness and slowed movements. She was known to have type 1 diabetes mellitus. INVESTIGATIONS: Blood pressure was 105/55 mmHg, the ECG showed sinus bradycardia of 55 beats/min. Biochemical tests revealed hypoglycaemia (2.1 mmol/l), hypokalaemia (3.4 mmol/l) and respiratory failure (pO2 6.16 kPa, O2 saturation 82%, pCO2 6.55 kPA). Heart and lung were unremarkable on physical examination as were chest radiogram and echocardiogram. Plasma level of nebivolol was 480 ng/ml on admission (therapeutic range 88-195 ng/ml). TREATMENT AND COURSE: After gastric lavage and administration of charcoal and sodium sulphate a temporary pacemaker was connected and glucagon infused intravenously as an antidote. The cardiovascular state stabilized with falling plasma level of nebivolol. Glucose was administered initially, but transient intravenous insulin infusion became necessary to counteract hyperglycaemia. The patient was transferred from the intensive care unit in a stable cardiovascular state after 2 days. CONCLUSION: This case demonstrates that swallowing 400-500 mg nebivolol, resulting in a plasma level of 480 ng/ml, need not be fatal. But the outcome in this patient should not be taken as necessarily applying to similar cases. It depends on the individual patient's metabolic state whether higher plasma levels might be reached with the same amount of ingested nebivolol.
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An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.
OBJECTIVE: Superoxide production by polymorphonuclear neutrophils (PMNs) under cyclosporin A (CsA) therapy following kidney transplantation is impaired. We investigated if the respiratory burst of PMNs is similarly depressed in patients undergoing CsA treatment following orthotopic liver transplantation (OLTx). Additionally, the in vitro influence of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the superoxide anion production was examined during the respiratory burst. PATIENTS: 10 patients after OLTx and 10 healthy blood donors (control group). MEASUREMENTS AND RESULTS: PMNs were stimulated with bacteria (Escherichia coli) or a combination of tumour necrosis factor alpha (TNFalpha) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). The respiratory burst was measured by oxidation of non-fluorescent dihydrorhodamine to the fluorescent rhodamine by means of flow cytometry. No differences in respiratory bursts from OLTx patients compared to those from healthy blood donors could be seen. Under TNFalpha/FMLP stimulation, the respiratory burst was significantly increased after in vitro incubation with GM-CSF (500 U ml(-1)) in patients following OLTx (from 58.2 to 74.5 %) as well as in the control group (from 47.4 to 61.9%). CONCLUSIONS: Our results demonstrate that superoxide production is not impaired under CsA treatment following OLTx. The respiratory burst of these patients' PMNs can even be augmented by GM-CSF in vitro.
OBJECTIVE: The objective of this study was to quantify the extent of neutrophil infiltration and the concentrations of enzymes involved in collagenolysis in the lower uterine segment in relation to the degree of cervical dilatation and the duration of labor. STUDY DESIGN: Biopsy specimens of the lower uterine segment were obtained from 62 women undergoing cesarean section at term. The number of extravascular neutrophils was assessed with enzyme histochemical evaluation, and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 by were assessed by enzyme-linked immunosorbent assay. RESULTS: The neutrophil count and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 increased with increasing cervical dilatation. At >6 cm the neutrophil count and the concentrations of matrix metalloproteinase-8, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinases-1 were significantly higher than at <2 cm. An association with the duration of labor was found for the neutrophil count and the concentrations of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1. Multiple linear regression analysis showed that the degree of cervical dilatation is more closely related to the 4 laboratory parameters investigated than to the duration of labor. CONCLUSION: The findings support the hypothesis that local changes (ie, collagenolysis) in the lower uterine segment unrelated to uterine activity play a crucial role in cervical dilatation at term.
Early postoperative infections and septic complications are predominant causes of morbidity and mortality in patients following orthotopic liver transplantation (OLTx). Prophylactic granulocyte colony-stimulating factor (G-CSF) administration after OLTx was found to decrease the number of sepsis episodes and sepsis-related mortality. Since polymorphonuclear neutrophils (PMNs) are one of the major determinants of antimicrobial defense, alteration of their functions may influence the development of sepsis in these patients. Therefore, we investigated in vitro whether or not priming with G-CSF affects the neutrophils' respiratory burst (RB) in immunosuppressed liver-transplanted patients. Venous blood was drawn from liver allograft recipients (n=12) between the 5th and 15th day postoperatively. Patients without clinical signs of infection or rejection were included in this study. Leukocytes were obtained as supernatant following sedimentation and incubated with 1000 IE ml-1 G-CSF. The RB was measured by the intracellular oxidation of non-fluorescent dihydrorhodamine to the fluorescent rhodamine by flow cytometry. The results were expressed as a percentage of increasing stimulation compared to the control responses, which are made up of the percentage of cells with RB reaction after stimulation with phorbol ester (PMA), bacteria (E. coli), or the combination of a cytokine (TNF-alpha) and a bacterial peptide (FMLP) in the absence of G-CSF. In vitro priming with G-CSF resulted in significantly increased activity of the RB after PMA (from 71.7% to 85.6%) and TNF-alpha/FMLP (from 58.4% to 72.7%) stimulation. These data demonstrate that G-CSF in vitro augments the RB of PMNs, thereby suggesting a possible therapeutic role for G-CSF as immunomodulating agent during bacterial and fungal infections following OLTx.
AIMS: To evaluate the tolerability of single oral SDZ RAD doses in stable renal transplant recipients and the pharmacokinetics of ascending SDZ RAD doses when coadministered with steady-state cyclosporin A microemulsion (Neoral). METHODS: This randomized, double-blind, placebo-controlled, sequential study involved 54 patients in six treatment groups; a different SDZ RAD dose (0.25, 0. 75, 2.5, 7.5, 15, 25 mg) was assessed in each group. Patients received a single oral dose of SDZ RAD (n=6) or placebo (n=3) with their usual Neoral dose. SDZ RAD and cyclosporin A pharmacokinetic parameters were determined. RESULTS: All SDZ RAD doses were well tolerated, with no discontinuations due to adverse events, serious adverse events, or deaths. Similar proportions of patients receiving SDZ RAD and placebo had at least one adverse event (44% and 50%, respectively). Mean changes in laboratory variables (baseline to endpoint) showed no clinically meaningful differences between SDZ RAD and placebo groups. SDZ RAD was absorbed rapidly and showed dose-proportional pharmacokinetics (dose: 2.5-25 mg), based on systemic exposure. Multiple postabsorptive phases in the pharmacokinetic profile indicate tissue distribution. The elimination half-life ranged from 24 to 35 h across the five highest dose groups. Pharmacokinetics were similar in men and women. Co-administration of escalating single oral SDZ RAD doses did not affect steady-state cyclosporin A pharmacokinetics. CONCLUSIONS: SDZ RAD was well tolerated; safety profiles of SDZ RAD and placebo were similar. SDZ RAD pharmacokinetics were dose-proportional across the range 2.5-25 mg in conjunction with cyclosporin A-based therapy, according to systemic exposure. Cyclosporin A pharmacokinetics were not affected by coadministration of single oral doses of 0.25-25 mg SDZ RAD.
Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria from bovine mammary gland milk samples. The objective of this study was to determine the type of inflammation evoked by CNS in the mammary gland of cows during their first lactation. Twenty-four Israeli-Holstein heifers in their first lactation were tested for bacteriological status, somatic cell count (SCC) and differential leucocyte count in milk 60-120 days postparturition and every 50-60 days after until drying off. Following the first testing, the 96 quarters of the 24 heifers were classified as follows: 69.8% as no bacterial growth (NBG), 27.1% infected with CNS and 3.1% infected with Staphylococcus aureus. During lactation, 84.5% quarters had no change in their classification, 6.2% were newly infected with other pathogens, 3.1% were classified as self-cured and in 6.2% sporadic bacteria were isolated. Among the CNS, S. intermedius, S. chromogenes and S. haemolyticus were the most frequently isolated. Milk from CNS-infected quarters had significantly higher SCC than milk from NBG quarters. An analysis of the leucocyte pattern in milk from CNS vs. NBG quarters revealed a significant increase in polymorphonuclears and a significant decrease in the percentage of total lymphocytes and lymphocytes bearing CD4+ or CD8+. The high percentage of CNS-infected quarters that remained unchanged in their bacterial status during the first lactation, indicates that those CNS have the ability to elude the immune system and persist in the mammary gland for a long time. The persisting infection, resulting to some extent from an increase of SCC by some CNS strains, suggests that in the near future control steps will have to be taken into consideration, in order to enhance the improvement of milk quality.