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Biomedical subjects

Gert Mayer

Publications and source records attributed to Gert Mayer.

At least 19 recordsLinked to original sources
PubMed2007

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells.

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.

Benzamides↗
PubMed2006

p21 and mTERT are novel markers for determining different ischemic time periods in renal ischemia-reperfusion injury.

In many clinical settings, the duration of renal ischemia and therefore the outcome of acute renal failure cannot be determined adequately. Renal ischemia reperfusion injury is known to shorten telomeres and upregulate stress-induced genes, such as the cyclin-dependent kinase (CDK) inhibitor p21. So far, the expression and role of CDK inhibitors, as well as mouse telomerase reverse transcriptase (mTERT), has not been investigated in a model with variable lasting ischemic periods. Male C57Bl/6 mice were subjected to renal ischemia reperfusion injury by clamping both renal pedicles for 10, 20, 30, and 45 min, and the kidneys were allowed to be reperfused for 3, 24, and 48 h. Expression of different CDK inhibitors and mTERT was evaluated. Mice developed signs of acute renal failure linear to the duration of the ischemic period. Real-time PCR revealed that mTERT was only significantly upregulated in kidneys after short ischemic periods (20 min). In contrast, p21 was constantly upregulated in kidneys after long ischemic intervals (30 and 45 min), but not in kidneys, which were clamped for shorter periods. Mainly, tubular cells contributed to the observed increase in p21 expression. Targeting p21 via the selective p53 inhibitor pifithrin-alpha was able to prevent acute renal failure when administered immediately before ischemia. The expression of another CDK inhibitor, namely p16, was differentially regulated, depending on the time of reperfusion. Taken together, we detected mTERT and p21 as "indicator" genes for short and long ischemic intervals, respectively. These two proteins might also be possible new therapeutic targets in the treatment and prevention of acute renal failure.

Animals↗
PubMed2006

Bloodstream infection following 217 consecutive systemic-enteric drained pancreas transplants.

BACKGROUND: Combined kidney pancreas transplantation (PTx) evolved as excellent treatment for diabetic nephropathy. Infections remain common and serious complications. METHODS: 217 consecutive enteric drained PTxs performed from 1997 to 2004 were retrospectively analyzed with regard to bloodstream infection. Immunosuppression consisted of antithymocyteglobuline induction, tacrolimus, mycophenolic acid and steroids for the majority of cases. Standard perioperative antimicrobial prophylaxis consisted of pipercillin/tazobactam in combination with ciprofloxacin and fluconazole. RESULTS: One year patient, pancreas and kidney graft survival were 96.4%, 88.5% and 94.8%, surgical complication rate was 35%, rejection rate 30% and rate of infection 59%. In total 46 sepsis episodes were diagnosed in 35 patients (16%) with a median onset on day 12 (range 1-45) post transplant. Sepsis source was intraabdominal infection (IAI) (n = 21), a contaminated central venous line (n = 10), wound infection (n = 5), urinary tract infection (n = 2) and graft transmitted (n = 2). Nine patients (4%) experienced multiple episodes of sepsis. Overall 65 pathogens (IAI sepsis 39, line sepsis 15, others 11) were isolated from blood. Gram positive cocci accounted for 50 isolates (77%): Coagulase negative staphylococci (n = 28, i.e. 43%) (nine multi-resistant), Staphylococcus aureus (n = 11, i.e. 17%) (four multi-resistant), enterococci (n = 9, i.e. 14%) (one E. faecium). Gram negative rods were cultured in twelve cases (18%). Patients with blood borne infection had a two year pancreas graft survival of 76.5% versus 89.4% for those without sepsis (p = 0.036), patient survival was not affected. CONCLUSION: Sepsis remains a serious complication after PTx with significantly reduced pancreas graft, but not patient survival. The most common source is IAI.

Adult↗
PubMed2006

Transforming omics data into context: bioinformatics on genomics and proteomics raw data.

Differential gene expression analysis and proteomics have exerted significant impact on the elucidation of concerted cellular processes, as simultaneous measurement of hundreds to thousands of individual objects on the level of RNA and protein ensembles became technically feasible. The availability of such data sets has promised a profound understanding of phenomena on an aggregate level, expressed as the phenotypic response (observables) of cells, e.g., in the presence of drugs, or characterization of cells and tissue displaying distinct patho-physiological states. However, the step of transforming these data into context, i.e., linking distinct expression or abundance patterns with phenotypic observables - and furthermore enabling a sound biological interpretation on the level of reaction networks and concerted pathways, is still a major shortcoming. This finding is certainly based on the enormous complexity embedded in cellular reaction networks, but a variety of computational approaches have been developed over the last few years to overcome these issues. This review provides an overview on computational procedures for analysis of genomic and proteomic data introducing a sequential analysis workflow: Explorative statistics for deriving a first, from the purely statistical viewpoint, relevant candidate gene/protein list, followed by co-regulation and network analysis to biologically expand this core list toward functional networks and pathways. The review on these procedures is complemented by example applications tailored at identification of disease-associated proteins. Optimization of computational procedures involved, in conjunction with the continuous increase in additional biological data, clearly has the potential of boosting our understanding of processes on a cell-wide level.

Animals↗
PubMed2005

Telomere length of in vivo expanded CD4(+)CD25 (+) regulatory T-cells is preserved in cancer patients.

PURPOSE: CD4(+)CD25(+) regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the upregulation of telomerase activity, whose regulation also remains unknown for Treg. EXPERIMENTAL DESIGN: Treg and CD4(+)CD25(-) T-cells were isolated from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17) and analyzed for their content of T-cell receptor excision circles (TREC) and for telomere length using flow-FISH, real-time PCR and Southern blotting. The in vitro regulation of telomerase of Treg was studied using PCR-ELISA in bulk cultures as well as in isolated proliferating and non-proliferating Treg. RESULTS: Treg isolated from peripheral blood of cancer patients exhibit significantly decreased levels of TREC when compared to Treg from healthy controls. Despite their in vivo proliferation, telomere length is not further shortened in Treg from cancer patients. Accordingly, telomerase activity of Treg was readily inducible in vitro. Notably, sorting of in vitro proliferating Treg revealed a significant telomere shortening in Treg with high-proliferative capacity. The latter are characterized by shortened telomeres despite high telomerase activity. CONCLUSIONS: Increased frequencies of Treg in peripheral blood of cancer patients are due to active proliferation rather than due to redistribution from other compartments (i.e., secondary lymphoid organs or bone marrow). In vivo expansion does not further shorten telomere length, probably due to induction of telomerase activity. In contrast, under conditions of strong in vitro stimulation telomerase induction seems to be insufficient to avoid progressive telomere shortening.

Aged↗
PubMed2005

Effect of tissue fixatives on telomere length determination by quantitative PCR.

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.

Cellular Senescence↗
PubMed2005

Detection of coregulation in differential gene expression profiles.

Genomics and proteomics approaches generate distinct gene expression and protein profiles, listing individual genes embedded in broad functional terms as gene ontologies. However, interpretation of gene profiles in a regulatory and functional context remains a major issue. Elucidation of regulatory mechanisms at the gene expression level via analysis of promoter regions is a prominent procedure to decipher such gene regulatory networks. We propose a novel genetic algorithm (GA) to extract joint promoter modules in a set of coexpressed genes as resulting from differential gene expression experiments. Algorithm design has focused on the following constraints: (I) identification of the major promoter modules, which are (II) characterized by a maximum number of joint motifs and (III) are found in a maximum number of coexpressed genes. The capability of the GA in detecting multiple modules was evaluated on various test data sets, analyzing the impact of the number of motifs per promoter module, the number of genes associated with a module, as well as the total number of distinct promoter modules encoded in a sequence set. In addition to the test data sets, the GA was evaluated on two biological examples, namely a muscle-specific data set and the upstream sequences of the beta-actin gene (ACTB) derived from different species, complemented by a comparison to alternative promoter module identification routines.

Actins↗
PubMed2005

CD4+CD25+ regulatory T cells inhibit experimental anti-glomerular basement membrane glomerulonephritis in mice.

CD4+CD25+ regulatory T cells (Treg) are of critical importance for the maintenance of tolerance. The kidney is frequently involved in autoimmune diseases, such as lupus erythematosus or glomerulonephritis (GN). Therefore, the therapeutic efficacy of Treg in a T cell-dependent murine model of experimental anti-glomerular basement membrane (anti-GBM) GN was tested. Transfer of 1 x 10(6) CD4+CD25+ T cells (day -1) into mice that were previously immunized with rabbit IgG (day -3) and subsequently received an injection of anti-GBM rabbit serum (day 0) significantly attenuated the development of proteinuria when compared with animals that received an injection of 1 x 10(6) CD4+CD25- T cells (control group). Treg injection induced a dramatic decrease of glomerular damage as well as a marked decrease of CD4+ T cell, CD8+ T cell, and macrophage infiltration. Of note, deposition of immune complexes was not prevented by Treg, showing that Treg rather inhibited cell-mediated organ damage than priming of the humoral immune response. Accordingly, a significant reduction of IFN-gamma, TNF-alpha, and TGF-beta1 mRNA in kidneys from animals that received Treg injection was observed. Tracking of enhanced green fluorescence protein-transgenic Treg revealed a predominant migration to secondary lymphoid organs with a significant increase of regulatory T cells (CD4+CD25+CD69-CD45RB(low)) in the lymph nodes. In contrast, enhanced green fluorescence protein-and FoxP3-positive cells by reverse transcription-PCR and CD4+CD25+CD69-CD45RB(low) T cells by flow cytometry in the kidney of nephritic animals were not detected. This report provides first evidence that Treg are potent suppressors of anti-GBM GN. Treg therefore might be of therapeutic value for the treatment of severe GN in humans.

Adoptive Transfer↗
PubMed2005

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells.

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1-phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte-derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720-P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th-priming capacity. We report that both FTY720 and FTY720-P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720-P showed an impaired immunostimmulatory capacity and reduced IL-12 but increased IL-10 production. T cells cultured in the presence of FTY720- or FTY720-P-treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen-presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid-derived drugs.

Actins↗
PubMed2005

Role of mast cells in experimental anti-glomerular basement membrane glomerulonephritis.

Recently, divergent reports on the role of mast cells (MC) in different glomerular diseases have brought our attention to their role in an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Genetically MC-deficient Kit(W)/Kit(W-v) mice, MC-reconstituted Kit(W)/Kit(W-v) mice and Kit+/+ control mice were subjected to anti-GBM GN. Kit(+/+) mice developed moderate proteinuria and glomerular damage following the induction of anti-GBM nephritis. In contrast, proteinuria and glomerular damage were dramatically increased in MC-deficient Kit(W)/Kit(W-v) mice. MC-reconstituted Kit(W)/Kit(W-v) mice showed proteinuria and glomerular damage comparable to Kit+/+ mice. A significant increase in infiltrating T cells and macrophages was detected in MC-deficient Kit(W)/Kit(W-v) mice as compared to Kit+/+ control mice and MC-reconstituted Kit(W)/Kit(W-v) mice. Accordingly, we observed an increase of TGF-beta1 mRNA in kidneys from Kit(W)/Kit(W-v) mice. Interestingly, we did not detect MC in the kidney using either Giemsa staining or RT-real-time PCR, but MC were found in the regional lymph nodes. Finally, mortality of Kit(W)/Kit(W-v) mice was significantly increased after the induction of anti-GBM GN due to uremia. Our report provides the first direct evidence that MC are protective in anti-GBM GN, possibly by modulating the influx of effector T cells and macrophages to inflammatory sites in the kidney.

Animals↗
PubMed2005

Proteinuria and hemoglobin levels in patients with primary glomerular disease.

BACKGROUND: Anemia in association with nephrotic syndrome has been described in small patient series and case reports. Whether nephrotic patients develop anemia has not been formally investigated. METHODS: We undertook a retrospective cross-sectional study of patients with biopsy-proven primary glomerular disease, various degrees of proteinuria, and creatinine levels less than 2 mg/dL (< 177 micromol/L). In addition to proteinuria, values for hemoglobin (Hb), age, body mass index (BMI), serum albumin and protein, and estimated glomerular filtration rate (eGFR) were derived from patient charts. RESULTS: We studied 297 patients, 187 men and 110 women, aged between 16 and 81 years. Univariate analysis showed no correlation between Hb level and proteinuria in either sex. Stratification of women and men into quartiles according to proteinuria showed no differences in Hb levels among the 4 groups. Three of 52 non-nephrotic women (6%) were anemic (Hb < 12 g/dL [< 120 g/L]) compared with 11 of 58 nephrotic women (19%; P = 0.047). Multiple regression analysis of all patients showed Hb level to have a correlation with sex (P < 0.001), BMI (P < 0.001), and eGFR (P = 0.005); negative correlation with age (P = 0.028); and borderline negative correlation with proteinuria (P = 0.054). In women, BMI showed a positive correlation with Hb level (P = 0.030). Proteinuria did not reach statistical significance (P = 0.093). In men, BMI (P = 0.001) and eGFR (P = 0.013) were associated positively and age (P = 0.031) was associated negatively with Hb level. CONCLUSION: These results indicate that nephrotic syndrome is not associated with anemia in men, but with a tendency to decrease Hb levels in women.

Adolescent↗
PubMed2004

Clearance of C4d deposition after successful treatment of acute humoral rejection in follow-up biopsies: a report of three cases.

Acute humoral rejection (AHR) is currently perceived as an immunological reaction against donor antigens mediated by complement-binding antibodies. C4d, a split product of complement activation and bound to endothelial cells of the peritubular capillaries, is used as a diagnostic marker for AHR. We report on three patients with biopsy-proven acute humoral rejection who were treated initially with plasmapheresis (PS). As two of the patients did not recover renal function, and biopsy showed persistent C4d staining after PS, immunoadsorption (IAS) was additionally performed on them. In all patients, renal function recovered, and follow-up biopsies in two patients showed complete disappearance of C4d, 29 days and 58 days after transplantation and only minimal residual C4d deposits in one patient 48 days after transplantation. We conclude that successful treatment of AHR is followed by complete resolution of serological and histological markers of AHR, displayed by the disappearance of C4d.

Adult↗
PubMed2004

Genome-wide gene-expression patterns of donor kidney biopsies distinguish primary allograft function.

Roughly 25% of cadaveric, but rarely living donor renal transplant recipients, develop postischemic acute renal failure, which is a main risk factor for reduced long-term allograft survival. An accurate prediction of recipients at risk for ARF is not possible on the basis of donor kidney morphology or donor/recipient demographics. We determined the genome-wide gene-expression pattern using cDNA microarrays in three groups of 36 donor kidney wedge biopsies: living donor kidneys with primary function, cadaveric donor kidneys with primary function and cadaveric donor kidneys with biopsy proven acute renal failure. The descriptive genes were characterized in gene ontology terms to determine their functional role. The validation of microarray experiments was performed by real-time PCR. We retrieved 132 genes after maxT adjustment for multiple testing that significantly separated living from cadaveric kidneys, and 48 genes that classified the donor kidneys according to their post-transplant course. The main functional roles of these genes are cell communication, apoptosis and inflammation. In particular, members of the complement cascade were activated in cadaveric, but not in living donor kidneys. Thus, suppression of inflammation in the cadaveric donor might be a cheap and promising intervention for postischemic acute renal failure.

Acute Kidney Injury↗
PubMed2004

In vitro treatment of dendritic cells with tacrolimus: impaired T-cell activation and IP-10 expression.

BACKGROUND: High doses (10(-6)-10(-8) M) of tacrolimus (FK506) were reported to induce a type-2 T-helper cell (Th2)-promoting function in developing dendritic cells (DC). We used a therapeutic dose (2.4 x 10(-9 )M) of tacrolimus to investigate its effect on human monocyte-derived DC. METHODS: Using untreated and treated immature and mature DC we compared T cell-activating capacity, surface marker expression, T cell and DC cytokine profile and transcription of genes coding for a panel of DC function-related molecules. RESULTS: Tacrolimus-treated mature DC had reduced T-cell stimulatory capacity. Although interleukin (IL)-12 production of DC was impaired, they did not promote Th2 development as T cells activated by tacrolimus-treated DC produced less interferon (IFN)-gamma, IL-4 and IL-10. The up-regulation of the T-cell activation marker CD69 and the production of IL-2 were impaired. In addition, tacrolimus-treated DC produced less IP-10 (CXCL10), which is known to be involved in allograft rejection. Other molecules related to DC function remained unchanged. CONCLUSIONS: Tacrolimus treatment reduces the ability of DC to stimulate T cells and the impaired production of DC-derived IP-10 (CXCL10) and IL-12 might play a role in the immunosuppressive action of tacrolimus.

Cell Culture Techniques↗