Klaus Kratochwill

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Xyr1 regulates xylanase but not cellulase formation in the head blight fungus Fusarium graminearum.

Fusarium graminearum is a plant pathogen that causes severe economical losses by infecting numerous agriculturally important plants and until now most culture plants have only low levels of Fusarium resistance. The plant cell wall can be assumed as the first target that has to be overcome by plant pathogens. Therefore pathogenic organisms are known to produce a complex cocktail of plant cell wall lytic enzymes. Xylanases are besides cellulases the most prominent enzymes secreted by Fusarium during growth on plant cell walls. We identified a putative regulator of xylanase production with high similarity to the Aspergillus niger XlnR and the Trichoderma reesei Xyr1 proteins. Disruptant strains of F. graminearum were heavily impaired in xylose utilization and xylanase production on wheat cell walls. In contrast to other filamentous fungi the lack of this transcriptional activator had no effect on the induction of cellulases.

IgG deposition and activation of the classical complement pathway involvement in the activation of human granulocytes by decellularized porcine heart valve tissue

Decellularization treatment of heart valves has been thought to eliminate tissue immunogenicity. Early failure of tissue-engineered xenogeneic heart valves was seen in children and has been a major drawback in this promising field of research. This study was designed to characterize the effects of acellular porcine heart valve tissue on immune activation in vitro. Incubation of decellularized porcine tissue with human plasma led to adsorption of IgG, activation of the classical complement pathway and adhesion of activated polymorphonuclear leukocytes (PMN). This inflammatory response was strongly inhibited by proteins extracted from native porcine tissue which might indicate that inhibitors of PMN activation present in the extracellular matrix (ECM) are lost during the decellularization process.

Alanyl–glutamine dipeptide restores the cytoprotective stress proteome of mesothelial cells exposed to peritoneal dialysis fluids

BACKGROUND Exposure of mesothelial cells to peritoneal dialysis fluids (PDF) results in cytoprotective cellular stress responses (CSR) that counteract PDF-induced damage. In this study, we tested the hypothesis that the CSR may be inadequate in relevant models of peritoneal dialysis (PD) due to insufficient levels of glutamine, resulting in increased vulnerability against PDF cytotoxicity. We particularly investigated the role of alanyl-glutamine (Ala-Gln) dipeptide on the cytoprotective PDF stress proteome. METHODS Adequacy of CSR was investigated in two human in vitro models (immortalized cell line MeT-5A and mesothelial cells derived from peritoneal effluent of uraemic patients) following exposure to heat-sterilized glucose-based PDF (PD4-Dianeal, Baxter) diluted with medium and, in a comparative proteomics approach, at different levels of glutamine ranging from depletion (0 mM) via physiological (0.7 mM) to pharmacological levels (8 mM administered as Ala-Gln). RESULTS Despite severe cellular injury, expression of cytoprotective proteins was dampened upon PDF exposure at physiological glutamine levels, indicating an inadequate CSR. Depletion of glutamine aggravated cell injury and further reduced the CSR, whereas addition of Ala-Gln at pharmacological level restored an adequate CSR, decreasing cellular damage in both PDF exposure systems. Ala-Gln specifically stimulated chaperoning activity, and cytoprotective processes were markedly enhanced in the PDF stress proteome. CONCLUSIONS Taken together, this study demonstrates an inadequate CSR of mesothelial cells following PDF exposure associated with low and physiological levels of glutamine, indicating a new and potentially relevant pathomechanism. Supplementation of PDF with pharmacological doses of Ala-Gln restored the cytoprotective stress proteome, resulting in improved resistance of mesothelial cells to exposure to PDF. Future work will study the clinical relevance of CSR-mediated cytoprotection.

Stress responses and conditioning effects in mesothelial cells exposed to peritoneal dialysis fluid.

Renal replacement therapy by peritoneal dialysis is frequently complicated by technical failure. Peritoneal dialysis fluids (PDF) cause injury to the peritoneal mesothelial cell layer due to their cytotoxicity. As only isolated elements of the involved cellular processes have been studied before, we aimed at a global assessment of the mesothelial stress response to PDF. Following single or repeated exposure to PDF or control medium, proteomics and bioinformatics techniques were combined to study effects in mesothelial cells (MeT-5A). Protein expression was assessed by two-dimensional gel electrophoresis, and significantly altered spots were identified by MALDI-TOF MS and MS2 techniques. The lists of experimentally derived candidate proteins were expanded by a next neighbor approach and analyzed for significantly enriched biological processes. To address the problem of an unknown portion of false positive spots in 2DGE, only proteins showing significant p-values on both levels were further interpreted. Single PDF exposure resulted in reduction of biological processes in favor of reparative responses, including protein metabolism, modification and folding, with chaperones as a major subgroup. The observed biological processes triggered by this acute PDF exposure mainly contained functionally interwoven multitasking proteins contributing as well to cytoskeletal reorganization and defense mechanisms. Repeated PDF exposure resulted in attenuated protein regulation, reflecting inhibition of stress responses by high levels of preinduced chaperones. The identified proteins were less attributable to acute cellular injury but rather to specialized functions with a reduced number of involved multitasking proteins. This finding agrees well with the concept of conditioning effects and cytoprotection. In conclusion, this study describes the reprogrammed proteome of mesothelial cells during recovery from PDF exposure and adaption to repetitive stress. A broad stress response with a number of highly overlapping processes and multitasking proteins shifts toward a more specific response of only few less overlapping processes.

HSP-MEDIATED CYTOPROTECTION OF MESOTHELIAL CELLS IN EXPERIMENTAL ACUTE PERITONEAL DIALYSIS

♦ Background: Low biocompatibility of peritoneal dialysis solution (PDS) injures mesothelial cells but also induces heat shock proteins (HSP), the main effectors of the cellular stress response. This study investigated whether overexpression of HSP upon pharmacologic induction results in cytoprotection of mesothelial cells in experimental PD. ♦ Methods: Stress response of mesothelial cells upon exposure to PDS was pharmacologically manipulated using glutamine as a co-inducer. In vitro, HSP-mediated cytoprotection was assessed by simultaneous measurements of HSP expression using Western blot analysis and viability testing using release of lactic dehydrogenase in cultured human mesothelial cells. In vivo, detachment of mesothelial cells from their peritoneal monolayer was assessed following exposure to PDS with and without the addition of glutamine in the acute rat model of PD. ♦ Results: In vitro, mesothelial cell viability following exposure to PDS was significantly improved upon pharmacologic co-induction of HSP expression by glutamine (226% ± 29% vs 190% ± 19%, p = 0.001). In vivo, mesothelial cell detachment during exposure to PDS was reduced upon pharmacologic induction of HSP expression by glutamine (93 ± 39 vs 38 ± 38 cells, p = 0.044), resulting in reduced peritoneal protein loss (75 ± 7 vs 65 ± 4 mg, p = 0.045). ♦ Conclusion: Our results represent the first study of pharmacologic manipulation of HSP expression for cytoprotection of mesothelial cells following acute in vitro and in vivo exposure to PDS. PDS with added glutamine might represent a promising therapeutic approach against low biocompatibility of PDS but needs validation in a chronic PD model.

Dynamic O-Linked N-Acetylglucosamine Modification of Proteins Affects Stress Responses and Survival of Mesothelial Cells Exposed to Peritoneal Dialysis Fluids

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD.

Peritoneal dialysis fluids can alter HSP expression in human peritoneal mesothelial cells

BACKGROUND Acute exposure of mesothelial cells to peritoneal dialysis fluid (PDF) has been shown not only to result in injury but also to induce cytoprotective heat shock proteins (HSP). The aim of the present study was to evaluate the expression of HSP in a more chronic in vitro PDF exposure system, searching for a role of glucose degradation products (GDP). METHODS Human peritoneal mesothelial cells (HPMC) were chronically incubated in filter- or heat-sterilized PDF (mixed 1:1 with cell culture medium), or in control cell culture medium. After incubation periods of 1, 3 and 10 days, cell extract was assessed for Ezrin, Hsp27 and Hsp72, and supernatant for IL-6 and IL-8. After 24-h exposure to the GDP 3.4-di-deoxyglucosone-3-ene (3.4-DGE), HPMC were assessed for expression of Hsp27 and Hsp72, and for release of LDH, IL-6 and IL-8. RESULTS In vitro PDF exposure for more than 1 day resulted in reduced cell mass, lower expression of the epithelial marker Ezrin and depressed cellular levels of both HSP, associated with increased IL-6 and IL-8 release. These effects occurred earlier and stronger with heat-sterilized than with filter-sterilized PDF. Exposure of HPMC to 3.4-DGE resulted in suppression of HSP, and increased release of LDH, IL-6 and IL-8. CONCLUSION Our data show that GDP (dys)regulate the mesothelial cell stress response. This was associated with reduced cell mass, loss of the epithelial phenotype and sterile cellular inflammation following extended exposure to heat-sterilized PDF. Toxic effects of PDF might thus be extended to reduced mesothelial cell stress responses.

Interleukin-1 Receptor-Mediated Inflammation Impairs the Heat Shock Response of Human Mesothelial Cells

Bioincompatibility of peritoneal dialysis fluids (PDF) limits their use in renal replacement therapy. PDF exposure harms mesothelial cells but induces heat shock proteins (HSP), which are essential for repair and cytoprotection. We searched for cellular pathways that impair the heat shock response in mesothelial cells after PDF-exposure. In a dose-response experiment, increasing PDF-exposure times resulted in rapidly increasing mesothelial cell damage but decreasing HSP expression, confirming impaired heat shock response. Using proteomics and bioinformatics, simultaneously activated apoptosis-related and inflammation-related pathways were identified as candidate mechanisms. Testing the role of sterile inflammation, addition of necrotic cell material to mesothelial cells increased, whereas addition of the interleukin-1 receptor (IL-1R) antagonist anakinra to PDF decreased release of inflammatory cytokines. Addition of anakinra during PDF exposure resulted in cytoprotection and increased chaperone expression. Thus, activation of the IL-1R plays a pivotal role in impairment of the heat shock response of mesothelial cells to PDF. Danger signals from injured cells lead to an elevated level of cytokine release associated with sterile inflammation, which reduces expression of HSP and other cytoprotective chaperones and exacerbates PDF damage. Blocking the IL-1R pathway might be useful in limiting damage during peritoneal dialysis.