Manfred Wiesel

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: A gene expression analysis on total and microdissected prostate tissue

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.

Serial peripheral blood perforin and granzyme B gene expression measurements for prediction of acute rejection in kidney graft recipients.

In the present study we investigated whether peripheral blood gene expression measurements may serve as an early and non‐invasive tool to predict renal allograft rejection. Peripheral blood was collected twice weekly after transplantation and gene expression was measured using real‐time polymerase chain reaction (PCR). Recipients with acute rejection (n = 17) had higher levels of perforin and granzyme B transcript on days 5–7, 8–10, 11–13, 17–19, 20–22, and 26–29, as compared to patients without rejection (n = 50, p < 0.05 in all cases). Rejection diagnosis using gene expression criteria, determined with receiver operating characteristic (ROC) curves, was possible 2–30 days before traditional diagnosis (median 11 days). The best diagnostic result was obtained from samples taken on days 8–10, with a specificity of 90% and a sensitivity of 82% for perforin, and a specificity of 87% and sensitivity of 72% for granzyme B. Decreases in perforin (p < 0.01) and granzyme B expression (p < 0.05) were observed after initiation of anti‐rejection therapy. Our data indicate that gene expression measurement is a useful tool for the recognition of graft rejection in its earliest stages. Serial measurements could be implemented as a monitoring system to highlight patients at higher risk of rejection, making them candidates for biopsy or pre‐emptive anti‐rejection therapy.

Area under the plasma concentration-time curve for total, but not for free, mycophenolic acid increases in the stable phase after renal transplantation: a longitudinal study in pediatric patients. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Mycophenolate mofetil, an ester prodrug of the immunosuppressant mycophenolic acid (MPA), is widely used for maintenance immunosuppressive therapy in pediatric renal transplant recipients. However, little is known about the pharmacokinetics of MPA in this patient population in the stable transplant phase, and dosage guidelines are preliminary. The authors therefore compared the pharmacokinetics of MPA, free MPA, and the renal metabolite MPA glucuronide (MPAG) in the initial (sampling at 1 and 3 weeks) and stable phases (sampling at 3 and 6 months) posttransplant in 17 children (age, 12.0 +/- 0.77 years; range, 5.9 to 15.8 years), receiving the currently recommended dose of 600 mg MMF/m2 body surface area (BSA) twice a day. Plasma concentrations of MPA and MPAG were measured by reverse phase HPLC. Because MPA is extensively bound to serum albumin and only the free drug is presumed to be pharmacologically active, the authors also analyzed the MPA free fraction by HPLC after separation by ultrafiltration. The intraindividual variability of the area under the concentration-time curves (AUC0-12) of MPA throughout the 12-hour dosing interval was high in the immediate posttransplant period, but declined in the stable phase, whereas the interindividual variability remained unchanged. The median MPA-AUC0-12 values increased 2-fold from 32.4 (range, 13.9 to 57.0) mg x h/L at 3 weeks to 65.1 (range, 32.6 to 114) mg x h/L at 3 months after transplantation, whereas the median AUC0-12 values of free MPA did not significantly change over time. This discrepancy can be attributed to a 35% decline of the MPA free fraction from 1.4% in the initial phase posttransplant to 0.9% (p < 0.01) in the stable phase. In conclusion, pediatric renal transplant recipients given a fixed MMF dose exhibit a 2-fold increase of the AUC0-12 of total MPA in the stable phase posttransplant and a 35% decrease of the MPA free fraction, whereas the AUC0-12 of free MPA remains unchanged over time. Because the latter pharmacokinetic variable is theoretically best predictive of the clinical immunosuppressive efficacy of MMF, these findings may have consequences for the dosing recommendations of MMF in renal transplant recipients.

Soluble CD30 as a predictor of kidney graft outcome.

Background. In the present study, we investigated whether the soluble form of CD30 (sCD30), a marker for T helper 2‐type cytokine‐producing T cells, is increased in sera of potential kidney graft recipients. We also investigated whether the pretransplantation serum sCD30 content is related to kidney graft survival. Methods. Pretransplantation sera of 844 cadaver kidney recipients from three transplant centers in Germany were tested for serum sCD30 content using a commercially available ELISA kit. Results. Kidney graft recipients showed a significantly higher serum sCD30 content than healthy controls (P<0.0001). High sCD30 serum content was associated with graft rejection. The 2‐year graft survival rate in recipients with a high pretransplantation serum sCD30 was 68±6%, significantly lower than the 86±1% rate in recipients with a low sCD30 (P<0.0001). Importantly, high sCD30 was indicative of an increased risk of graft loss even in recipients without lymphocytotoxic alloantibodies. Conclusion. These data show that an elevated pretransplantation serum sCD30 reflects an immune state that is detrimental for kidney graft survival.

Evaluation of posttransplantation soluble CD30 for diagnosis of acute renal allograft rejection1

Background. Posttransplantation measurement of soluble CD30 (sCD30) may be useful for identifying kidney graft recipients at risk of impending graft rejection in the early posttransplantation period. Methods. We measured plasma sCD30 levels and evaluated the levels in relation to the diagnosis of rejection. Results. Receiver operating characteristic curves demonstrated that on posttransplantation days 3 to 5, sCD30 allowed a differentiation of recipients who subsequently developed acute allograft rejection (n=25) from recipients with an uncomplicated course (n=20, P <0.0001) (area under the receiver operating characteristic curve 0.96, specificity 100%, sensitivity 88%) and recipients with acute tubular necrosis in the absence of rejection (n=11, P =0.001) (area under the receiver operating characteristic curve 0.85, specificity 91%, sensitivity 72%). Conclusions. sCD30 measured on posttransplantation days 3 to 5 offers a noninvasive means for differentiating patients with impending acute allograft rejection from patients with an uncomplicated course or with acute tubular necrosis.

Psychological consultation before living kidney donation: finding out and handling problem cases

Background. Since 1996, a team of medical psychologists, nephrologists, and urologists at Heidelberg University Hospital has developed a family-oriented consultation procedure for donors, recipients, and family members before living kidney transplantation. Qualitative content analyses of these consultations and their follow-up histories are presented, with particular focus on “problem cases.” Methods. Sixty-seven consultation interviews were explored by rating family interaction, consultee-consultant interaction, decision-making process, and intervention strategies in problem cases. Subsequently, 33 catamnestic interviews 1 year or more after living donation were explored by qualitative content analysis for donor and recipient quality of life, quality of relationships, and health status. Results. Generally, donors show themselves to be eager; recipients appear more reluctant. Expectations focus on spontaneity and a “normal life.” Fears are usually expressed not about oneself but about the partner involved. Types of confrontation with possible complications are anxious avoidance, active consideration, and optimistic fatalism. Past family experiences of medical traumata may influence content and level of anxiety. Problem cases are characterized by unilaterally dependent close relationships, unrealistic expectations, anxious avoidance of problem confrontation, and negative experiences with the medical system. At follow-up, the majority are in good medical and psychological health. Few donors and recipients are suffering from disappointed expectations or unexpected treatment side effects. Conclusions. The Heidelberg consultation setting has proven useful for allowing open discussion about critical issues. In problem cases, prescribing a moratorium instead of rejecting donation helps to relax consultation anxiety. Psychological support after transplantation seems to be indicated for a minority with typical first-year problems.

Pediatric renal transplantation with mycophenolate mofetil-based immunosuppression without induction: results after three years1,2

Background. Mycophenolate mofetil (MMF)-based immunosuppression has reduced the acute rejection rate in adults and in children in the early posttransplantation period. Three-year posttransplantation results have been reported for adults but not for children thus far. In the present open-labeled study, patients 18 years old and younger were evaluated prospectively for up to 3 years after renal transplantation (RTX). Methods. Eighty-six patients receiving MMF in combination with cyclosporine and prednisone without induction were evaluated for patient survival, transplant survival, renal function, arterial blood pressure, adverse events, and opportunistic infections. These patients were compared with a historic control group (n=54) receiving azathioprine (AZA) instead of MMF. Results. Patient survival after 3 years was 98.8% in the MMF group and 94.4% in the AZA group (NS). Intent-to-treat analysis of graft survival demonstrated superiority for MMF (98% vs. 80%; P<0.001). Cumulative acute rejection episodes occurred in 47% of patients in the MMF group versus 61% in the AZA group (P<0.05). Renal function was not significantly different, neither after 3 years nor in the long-term calculation. Antihypertensive medication was administered to 73% to 84% of patients, similar in both groups. Opportunistic infections were recorded only for MMF. Infection rates were comparable to those reported in adults. Conclusions. These results suggest that MMF is safe and beneficial as a longer term maintenance immunosuppressive drug in children and adolescents.

DETECTION OF HEMATOGENOUS MICROMETASTASIS IN PATIENTS WITH TRANSITIONAL CELL CARCINOMA

PURPOSE Cytokeratin 20 (CK 20) is selectively expressed in urothelium, gastric intestinal epithelium, in Merkel cells and in a variety of malignant neoplasms. CK 20 RT-PCR assay has been extensively used to detect isolated cancer cells in peripheral blood, lymph nodes and bone marrow samples of patients with colorectal carcinoma. Since CK-20 is also actively expressed in transitional cell carcinoma (TCC), we analyzed, whether CK 20 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is suitable to detect residual tumor cells in patients with transitional cell carcinoma of the bladder and the upper urinary tract. MATERIALS AND METHODS Nested Reverse Transcriptase Polymerase Chain Reaction assay was used to analyze CK 20 transcripts in peripheral venous blood samples and tumor biopsies of 49 patients with transitional cell carcinoma. Blood samples of 22 healthy volunteers served as negative controls. RESULTS CK 20 mRNA was detectable in blood samples of 12 of 49 patients with TCC. All blood samples of the control group tested negative. The detection rate for CK 20 mRNA significantly correlated (p = 0.0019, Cochran-Armitage Trend Test) to the stage of disease and increased from 0% in stage pTa to 63% in stage pT4. CONCLUSIONS These results suggest that CK 20 is a suitable marker for the detection of disseminated TCC cells in peripheral venous blood samples and may be helpful in the molecular staging of TCC patients. The prognostic relevance has to be evaluated in further followup.

Pre-transplant Th1 and post-transplant Th2 cytokine patterns are associated with early acute rejection in renal transplant recipients

Abstract:  In this retrospective study, we tried to define pre‐ and post‐transplant immunological parameters that identify patients at risk for early acute rejection. Lymphocyte subpopulations and plasma levels of cytokines and neopterin were determined pre‐ and post‐transplant in 32 renal transplant recipients with biopsy‐proven early acute graft rejection. Recipients without early acute rejection served as controls. High pre‐transplant interferon‐γ (IFN‐γ) plasma levels (p = 0.006), consistently high levels of neopterin early post‐transplant (p = 0.008), a post‐transplant switch from a Th1 to a Th2 cytokine pattern with decreasing IFN‐γ (p = 0.02), low CD8+ lymphocyte counts (p = 0.006) and consistently high CD19+ B lymphocyte counts were associated with acute rejection. Our data suggest that patients with a pre‐transplant Th1 and an early post‐transplant Th2 cytokine pattern are pre‐disposed for early acute rejection.

Strong inflammatory cytokine response in male and strong anti-inflammatory response in female kidney transplant recipients with urinary tract infection

Urinary tract infection (UTI) is the most common post‐transplant infection in renal transplant recipients. The relationship of plasma and urine cytokines with UTI after kidney transplantation has not yet been delineated and literature reports on cytokine and UTI are rare. In a retrospective study, we compared post‐transplant plasma and urine cytokine levels of 132 outpatient renal transplant recipients with or without UTI. Soluble interleukin‐1 receptor antagonist (sIL‐1RA), IL‐2, sIL‐2R, IL‐3, IL‐4, IL‐6, sIL‐6R, IL‐8, IL‐10, transforming growth factor‐β2 (TGF‐β2), interferon‐γ (IFN‐γ), and tumor necrosis factor‐α (TNF‐α) levels were determined using commercially available enzyme‐linked immunosorbent assay (ELISA) kits. We found gender‐related urine cytokine patterns. Anti‐inflammatory sIL‐1RA was significantly higher in females than in males and this gender‐related difference was more pronounced in bacteriuric (P < 0.0001) than in nonbacteriuric (P = 0.001) patients. Urine proinflammatory cytokines IL‐6 (P = 0.001) and IL‐8 (P = 0.007) were significantly higher in male patients with bacteriuria than in males without bacteriuria and sIL‐2R (P = 0.001) and sIL‐6R (P = 0.03) were significantly higher in males with leukocyturia than in males without leukocyturia. Bacteriuria in males was associated with higher doses of immunosuppressive drugs (P = 0.02). Male renal transplant recipients with UTI have a strong inflammatory cytokine response with activation of IL‐6, IL‐8, sIL‐2R and sIL‐6R producing cells, whereas female patients with UTI block the inflammatory response to UTI by production of sIL‐1RA.