Browsing by Author "Ringe, B."

Background. Corticosteroids have been used traditionally for immunosuppression after solid organ transplantation. The variety of modern immunosuppressive agents offers the chance to replace drugs with an unfavorable risk-benefit ratio. The objective of this prospective pilot study was to investigate a novel steroid-free immunosuppressive regimen after clinical liver transplantation. Methods. 30 adult liver graft recipients were included in an intent-to-treat analysis. Dual induction immunosuppression consisted of tacrolimus and mycophenolate mofetil. Prophylactic steroids were not given. Efficacy and safety parameters analyzed were patient and graft survival, incidence and severity of rejection, and adverse events in correlation to immunosuppressive drug levels. Results. Patient and graft survival at 2 years was 86.7 and 83.9%, respectively. Acute rejection occurred in 26.2%, and was associated with subtherapeutic tacrolimus blood levels and diarrhea, All rejections were completely reversible by temporary addition of steroids. Acute renal failure was seen in 10/30 patients, and was related to high tacrolimus blood levels together with primary liver graft dysfunction, 43% of all patients never received any steroids, and 73% were on a steroid-free maintenance regimen. Conclusions. These results confirm that corticosteroids can be completely avoided from the beginning after liver transplantation. Double drug immunosuppression with tacrolimus and mycophenolate mofetil is effective and safe in terms of patient and graft survival as well as incidence and severity of rejection. In order to avoid under- or over-immunosuppression, which may be caused by impaired absorption or metabolism, close drug monitoring is advised.

The technique of segmental liver transplantation (s-LTx) provides a method to overcome the shortage of suitable livers for small recipients. Patient survival rates are parallel to those obtained with whole liver transplantation (w-LTx), For long-term rehabilitation, adaptive liver growth and adequate perfusion is crucial; however, morphometric and hemodynamic parameters in growing children with s-LTx are not available. Seventeen children who received a s-LTx and 25 with a w-LTx who had follow-up evaluation 1 and 2 yr after LTx were studied. Mean age at time of transplantation was 4.3 +/-3.5 yr for s-LTx and 10.3 +/-6.0 yr for w-LTx, mean height 98 +/- 21 cm and 122 +/- 30 cm respectively. At follow-up evaluation mean values for liver enzymes, bilirubin and prothrombin time were in the normal ranges for both groups. Liver dimensions were measured by gray scale ultrasound, and hemodynamic parameters by Doppler sonography in the portal vein and hepatic artery using an Acuson 128 machine. Maximal (V-max), minimal (V-min) and time-average velocity (TAV) were measured and the resistive index (RI) calculated. We found that 1 and 2 yr after LTx liver dimensions were at a mean in the upper normal range of healthy controls. Spleen size was above the normal range and did not show any tendency towards regression. Mean V-max in the hepatic artery in s-LTx and w-LTx was 48 cm/sec vs. 28 cm/sec after 1 yr and 30 cm/sec vs. 35 cm/sec after 2 yr, the RI 0.66 vs. 0.55 and 0.59 vs. 0.73, respectively (p for all parameters >0.05), Maximal portal vein flow was 25 cm/sec in s-LTx vs. 29 cm/sec in w-LTx. Blood flow calculated by vessel diameter and TAV showed no statistical difference between both groups. In conclusion, liver size after s-LTx and w-LTx was increased to the upper normal range, and portal vein blood flow velocities were within the normal range. V-max in the hepatic artery was reduced in s-LTx; however, the reduction was to the same extent as in w-LTx. In the view of long-term functional adaptation, s-LTx is not inferior to w-LTx.

Background. Ischemia-reperfusion injury (IRI) can result in severe organ dys- or nonfunction. Interaction of leukocytes and endothelial cells mediated by E-selectin appears to be a key step for disturbed microcirculation. Therefore we studied gene and protein expression as well as localization of E-selectin during intestinal IRI. Methods. Intestinal tissue samples were obtained from extracorporeal perfused intestines (cold ischemia time [CIT] 2 or 20 hours, each n = 5) and additionally in intestinal transplanted pigs (CIT 2 or 20 hours, each n = 1). Mucosal damage was graded according to the Chin classification. E-selectin mRNA was determined by PCR and quantitative RT-PCR. Localization of E-selectin mRNA was performed by in situ hybridization and of the protein by immunohistochemistry. Results. Histologically, mucosal damage occurred during reperfusion and was earlier and more severe after 20 hours of CIT. E-selectin mRNA expression was detected by PCR already after laparotomy and was elevated after reperfusion. Interestingly, mRNA expression was already increased after 20 hours of CIT. E-selectin mRNA was localized to the luminal surface of muscular, submucosal, and mucosal endothelial cells and the protein was detected on submucosal arterial endothelium as early as 2 hours after reperfusion. Conclusion. Prolongation of CIT results in more severe mucosal damage during reperfusion, which is associated with protein expression of E-selection that might be used as a marker for activated endothelial cells. Increased E-selectin mRNA at end of 20 hours of CIT might indicate a preactivated state of endothelial cells potentially triggered by bacterial translocation or products.

Based on experimental work and clinical small studies, histidine-tryptophan-ketoglutarate (HTK) solution was found to be suitable not only for heart and kidney preservation but also for liver preservation. We decided, therefore, to use this preservation solution for clinical liver preservation in a prospective multi-centre trial. Enrolment to the study was from 1996 to 1999 in four European centres, and the results of 214 patients with HTK-preserved organs were analysed. Analysis showed a primary dysfunction (PDF) rate of 8.8%, with a primary non-function (PNF) rate of 2.3% and initial poor function (IPF) in 6.5%. Patient survival rate at 1 year was 83% and 1-year graft survival rate was 80%. In a univariate and a multivariate analysis PDF, early surgical complications and tendentiously severe infections (septicaemia, pneumonia, cholangitis) were identified as independent risk factors for graft and patient survival. Preservation with HTK can be regarded as an established alternative to preservation with University of Wisconsin (UW) solution when preservation times are short. Definitive assessment of the efficacy of preservation solutions requires further prospective randomised clinical trials that compare HTK and UW.

Background. Intestinal ischemia-reperfusion injury (IRI) represents an exaggerated inflammatory cascade with a complex pathophysiology. IL-2, IL-6, HSP70, and INF-gamma are mediators of the inflammatory process. Therefore, we investigated their kinetics and localization during intestinal IRI. Methods. Pig intestinal specimens were obtained during cold preservation (cold ischemia time 2 hours) and extracorporeal perfusion. Mucosal damage was graded according to the Chiu classification. MRNA expression was determined by Northern blot (IL-2, IL-6, IFN-gamma) or by quantitative RT-PCR (IL-6, HSP70) and localized by in situ hybridization. Results. Histologically, mucosal damge occurred during reperfusion. Expression of IL-2 mRNA was up-regulated after HTK perfusion and was highest at the start and 7 hours after reperfusion. Expression of IL-6 mRNA increased at 2 hours after reperfusion and HSP70 at 3 hours after reperfusion. IFN-gamma mRNA was expressed after HTK perfusion, with expression of this cytokine increasing to 1 hour after the start of reperfusion, and decreasing thereafter. IL-2 mRNA was localized to endothelial cells (EC) and leukocytes and in close relation to ganglion cells (GC): IL-6 mRNA in EC, smooth muscle cells (SMC), and GC: HSP70 mRNA in EC and SMC and IFN-gamma mRNA in leukocytes. Conclusion. IL-2, IL-6, HSP70, and INF-gamma are parameters of early mRNA expression during intestinal IRI. EC, SMC, leukocytes, and GC have been identified as sources of transcripts that might afford potential targets for intervention strategies to attenuate IRI.

Background. Urine cytology, although considered a valuable diagnostic tool in the monitoring of kidney graft function, is hampered by difficulty in differentiating the nucleated non-squamous cells in urine using conventional techniques. We have now developed a method for the simple identification of urinary cell types by lectin staining. Methods. Acetone-fixed cytopreparations of urinary sediments were incubated with the lectin combination Sophora Japonica agglutinin (SJA; rhodamine-labelled) and Erythrina cristagalli agglutinin (ECA; fluorescein isothiocyanate (FITC)-labelled) for 15 min, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole (DAPI). The courses of 38 patients were serially monitored after kidney transplantation during the period in hospital. Results. Nucleated urinary cell types could be easily identified from one specimen by their characteristic lectin-binding pattern using triple-immunofluorescence microscopy (FITC/rhodamine/ultra violet), permitting a differentiation between proximal (SJA+/ECA+) and distal tubules (SJA-/ECA+), collecting ducts (SJA+/ECA-) and lymphocytes (SJA-/ECA-). Stable graft function was characterized by low numbers of lymphocytes, tubular cells and urothelia. During rejection episodes, but not graft dysfunction unrelated to rejection, urinary excretion of lymphocytes as well as of distal tubular cells (from 1.0 to 6.0 and from 1.4 to 4.0 per 10 high-power fields, respectively) increased significantly up to 3 days prior to clinical diagnosis. Conclusions. Lectin staining facilitates unambiguous differentiation of the urinary cell types, in particular the various tubular epithelial cells, which are otherwise difficult to identify. This technique provides a rapid and easily applicable tool to evaluate the significance of the respective cell types in the monitoring of kidney graft function.

Background: A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration. A polymorphism in the CYP3A4 promoter region, termed "variant" allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity. A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp. Methods: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping. Results: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection. Conclusions: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients. (C) 2001 American Association for Clinical Chemistry.