Necessity of early and continuous monitoring for possible infectious complications in children undergoing therapeutic hypothermia.

AIM: Since therapeutic hypothermia (TH) is known for its inhibitory effects on leucocyte migration and cytokine synthesis, our aim was to underline the necessity of early monitoring for potential immunomodulatory risks. METHODS: Using a 13-year retrospective case-control study at the paediatric intensive care unit (PICU) of the Medical University in Vienna, all newborn infants and children receiving TH were screened and compared with a diagnosis-matched control group undergoing conventional normothermic treatment (NT). TH was accomplished by using a non-invasive cooling device. Target temperature was 32-34°C. Children with evident infections, a medical history of an immunodeficiency or undergoing immunosuppressive therapy, were excluded. RESULTS: During the observational period, 108 patients were screened, 27 of which underwent TH. Culture-proven infections occurred in 22% of the TH group compared with 4% of the normothermic controls (P = .1). From the second day following PICU admission, median C-reactive protein (CRP) values were higher in the TH group (day two P = .002, day three P = .0002, day six P = .008). CONCLUSION: Children undergoing TH showed earlier and higher increases in CRP levels when compared to normothermic controls. These data underline the necessity of early and continuous monitoring for possible infectious complications.

Peritoneal dialysis fluid induces p38-dependent inflammation in human mesothelial cells.

BACKGROUND: Noninfectious upregulation of proinflammatory pathways in mesothelial cells may represent an integral part of their stress response upon exposure to peritoneal dialysis fluids (PDF). OBJECTIVE: The aim of this study was to evaluate the role of the stress-inducible mitogen-activated protein kinase (MAPK) p38 in regulation of inflammatory and stress responses in mesothelial cells following in vitro exposure to PDF. MATERIALS AND METHODS: Human peritoneal mesothelial cells were exposed to Dianeal PD4 or Physioneal (Baxter AG, Vienna, Austria) containing 1.36% glucose and then allowed to recover. Phosphorylation of p38, induction of heat shock protein-70 (HSP70), release of lactate dehydrogenase (LDH), secretion of interleukin (IL)-8, gene transcription, and mRNA stability were assessed with and without the MAPK p38 inhibitor SB203580. RESULTS: Exposure to Dianeal resulted in phosphorylation of p38 within 30 minutes (309% of control, p < 0.05) and increased IL-8 release (370% of control, p < 0.05), HSP70 expression (151% of control, p < 0.05), and LDH release (180% of control, p < 0.05). Exposure to Physioneal resulted in attenuated changes in IL-8, HSP70, and LDH. Addition of the p38 inhibitor SB203580 to Dianeal resulted in dampened IL-8 release (-55%; p < 0.05) and basal HSP70 expression, and unchanged LDH release. Effects of p38 on IL-8 were at transcriptional, posttranscriptional, and translational levels. CONCLUSION: These data confirm concordant p38-dependent upregulation of IL-8 and HSP70 following exposure to bioincompatible PDF. The MAPK p38 pathway therefore links proinflammatory processes and the cellular stress response in human peritoneal mesothelial cells.

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.

Global health and social responsibility: a pilot project of the Medical University of Vienna in eastern Ethiopia.

The Medical University of Vienna and the eastern Ethiopian town of Gedamaytu have established a development collaboration with the aim of improving healthcare for the local population. A health survey of the study region was conducted by the university in February 2009 with the purpose of assessing the burden and spectrum of disease, and to evaluate the potential for improvement of healthcare and plan for future targeted health interventions. The most prevalent diseases in pediatric patients were found to be disorders of the respiratory system, febrile conditions and diarrhea. Adult patients suffered most frequently from disorders of the respiratory system, chronic pain, gastric discomfort and febrile conditions. De-worming was offered to all patients in the course of the survey. The anthropometric evaluation of patient data showed a high rate of growth retardation in children below the age of 5 years. Based on these data and on interviews with the local healthcare personnel, healthcare programs and interventions are discussed with the aim of further improving healthcare provision for the population of Gedamaytu.

Cellular stress-response modulators in the acute rat model of peritoneal dialysis.

Cytotoxicity of peritoneal dialysis fluids (PDF) not only results in cellular injury, but also induces heat-shock proteins (HSP), the main effectors of the cellular stress response. This study investigated effects of modulation of mesothelial HSP expression on peritoneal membrane integrity during acute PDF exposure. In the acute in vivo rat model of peritoneal dialysis (PD), either the HSP coinducer indomethacin or the HSP suppressor quercetin was added to standard PDF (CAPD 3, Fresenius, Germany). HSP-72 expression, number of detached mesothelial cells, and peritoneal protein loss were evaluated at the end of a 4-h dwell time. Compared with pure PDF exposure, addition of indomethacin resulted in increased expression of mesothelial HSP-72, reduced mesothelial cell exfoliation, and reduced peritoneal protein loss. Addition of quercetin resulted in decreased expression of HSP-72, increased mesothelial cell exfoliation, and higher peritoneal protein loss. Differences were statistically significant between indomethacin-treated and quercetin-treated rats. Mesothelial HSP expression was related to markers of peritoneal membrane integrity upon in vivo PDF exposure, consistent with HSP-mediated cytoprotection. These data clearly demonstrate the potential for clinically feasible pharmacologic interventions with the cellular stress response as a novel therapeutic approach to improve PD outcome.

Effects of epithelial-to-mesenchymal transition on acute stress response in human peritoneal mesothelial cells.

BACKGROUND: During peritoneal dialysis, mesothelial cells undergo epithelial-to-mesenchymal transition (EMT), resulting in markedly altered protein expression. This potentially includes heat-shock proteins (HSP), the main effectors of cellular repair. Thus, chronic cellular processes, such as EMT, may influence acute stress responses and thus survival of mesothelial cells following non-lethal injury upon exposure to peritoneal dialysis fluid (PDF). METHODS: In this study, we investigated the effects of EMT on acute stress responses and cytoresistance in human peritoneal mesothelial cells. In vivo EMT was defined as a fibroblast-like growth pattern in mesothelial cells grown from peritoneal effluents, and in vitro EMT was induced by TGF-beta1 in mesothelial cells grown from omental tissue. Morphologic EMT was validated by western blot analysis of EMT marker proteins (ezrin, alpha-SMA). Expression of HSP and cellular survival was evaluated in a simple in vitro PDF exposure model. RESULTS: In vivo and in vitro EMT resulted in marked effects on phenotypes of mesothelial cells, associated with differential HSP expression. In vivo 'chronic' EMT resulted in lower expression of HSP-27 and HSP-72, whereas in vitro 'acute' EMT was associated with increased HSP-27 and decreased HSP-72 expression. Following PDF exposure, there were no effects of in vivo EMT on the stress induction of HSP, and survival of epithelial versus fibroblast-like phenotypes was comparable. The non-stressful induction of HSP-27 following TGF-beta1 pretreatment resulted in the attenuated stress induction of HSP, and in improved survival in following PDF exposure. CONCLUSIONS: Taken together, this study confirms that mesothelial cells are not 'unchanged' or 'static targets' during the clinical course of PD treatment. The cellular processes during EMT play a complex role in acute cellular stress response and cytoresistance of mesothelial cells. Sequential analysis at different stages of EMT will be essential to provide more insights on cytoprotective cellular processes in in vitro and in vivo models of PD.

Quercetin protects human mesothelial cells against exposure to peritoneal dialysis fluid.

During peritoneal dialysis, mesothelial cells have been shown to undergo severe damage due to continuous exposure to peritoneal dialysis fluid (PDF) with cytotoxic physicochemical properties. In this study, we investigated the cytoprotective role of the bioflavonoid Quercetin in the in vitro model of peritoneal dialysis. Immortalized human mesothelial cells (Met5A) were exposed either to regular growth medium or to standard acidic lactate-buffered PDF (Dianeal PD4) or to a more biocompatible lactate-bicarbonate-buffered PDF (Physioneal 40). Parallel cell cultures were supplemented with 200 microM Quercetin. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by the release of cytoplasmic lactate dehydrogenase and fluorescence-activated cell sorting (FACS). PDF exposure with bioincompatible Dianeal PD4 resulted in severe disruption of cell cultures and in significantly increased lactate dehydrogenase (LDH) release (p=0.0007 vs. control). Addition of 200 microM Quercetin significantly decreased the LDH release (p=0.04 vs. "pure" Dianeal PD4 exposure), comparable to control exposure and to more biocompatible Physioneal 40 exposure (p=0.37) and resulted in marked preservation of cell culture monolayers and cellular viability as assessed by FACS. Introduction of cytoprotective agents such as Quercetin may represent an alternate approach to protect mesothelial cells from cytotoxicity of frequently used PDFs, comparably effective to the introduction of novel, more biocompatible, PDFs.

Ex vivo reversal of in vivo transdifferentiation in mesothelial cells grown from peritoneal dialysate effluents.

BACKGROUND: During peritoneal dialysis (PD), epithelial-mesenchymal transition (EMT) is likely involved in aberrant healing and progressive peritoneal fibrosis. Recently, EMT of the kidney was actively reversed into the opposite direction, into mesenchymal-epithelial transition (MET), by treatment with bone morphogenic protein-7 (BMP-7). In this study, the potential for ex vivo interconversion of in vivo transdifferentiation processes was investigated in mesothelial cells. METHODS: In vivo EMT was assessed in mesothelial cell cultures randomly grown from peritoneal effluents of seven patients on chronic PD. Then, ex vivo treatment with modulating factors was performed by incubating cobblestone-like cell cultures with transforming growth factor (TGF- beta1) and fibroblast-like cultures with BMP-7. Effects were assessed by morphological characterization, western analysis and reverse transcription-polymerase chain reaction of marker proteins ezrin and alpha-smooth muscle actin (alpha-SMA). RESULTS: PD caused progressive in vivo EMT with loss of the epithelial phenotype in the majority of mesothelial cell cultures over a 12-month period. EMT was reproducible by ex vivo treatment of cultured cells with TGF-beta1, converting the epithelial to the fibroblast-like phenotype. Ex vivo treatment with BMP-7 reversed in vivo and ex vivo EMT. During rhBMP-7 incubation the fibroblast-like growth pattern reversed into a more epithelial morphology, the expression of ezrin increased and alpha-SMA decreased. CONCLUSION: Our study shows that modulating factors of transdifferentiation, such as BMP-7, may be attractive tools in the balance between normal healing and aberrant profibrotic processes in mesothelial cells during peritoneal dialysis. Peritoneal-effluent-derived mesothelial cells are not mere biomarkers for in vivo EMT in the peritoneal cavity, but also represent an assay to test ex vivo interventions to reverse the profibrotic phenotype.

C4d in pediatric renal allograft biopsies: a marker for negative outcome in steroid-resistant rejection.

Recently, deposition of C4d, reflecting complement activation via the classical pathway, has been established as marker of antibody-mediated rejection. As C4d can be detected in paraffin sections, it allows for retrospective analysis in populations with low case loads, such as in pediatric transplantation. In this study we re-evaluated consecutive renal transplant biopsies obtained since 1990 in 36 children (18 boys, 18 girls) who had received their allograft (nine living, 27 cadaveric) at an age of 10.12+/-4.4 yr. Clinical indications for biopsy were 16 acute steroid resistant rejections (ASRs), 11 chronic rejections and nine other diagnoses. Overall, C4d deposition was found in nine cases (25%), eight of them with diagnosed ASR. Six out of these eight allografts were lost during 36 months of clinical follow-up, a significantly higher rate than in C4d-negative biopsies (p<0.05). C4d status therefore turned out to be an excellent predictor for inferior graft survival following ASR. None of the other histopathologic markers were sensitive for humoral rejections. In conclusion, the high prevalence of C4d-positive staining in ASR demonstrates the importance of the humoral part of the immune system in pediatric transplantation. The worse outcome of C4d-positive rejections despite massive immunosuppressive therapy clearly indicates the need for innovative therapies in this high-risk population.

Changes of blood pressure and left ventricular mass in pediatric renal transplantation.

Cardiovascular events are among the most frequent causes for long-term morbidity and mortality in children after renal transplantation. The aim of this study was to analyze the effects of post-transplant changes in arterial hypertension, as assessed by 24-h ambulatory blood pressure measurement (ABPM), on myocardial architecture, as assessed by echocardiography. In a retrospective chart review analysis, 39 children were identified in whom 24-h ABPM and echocardiography had been assessed within a 3-month interval after a mean of 4 years post transplantation; 20 repeated pairs of measurements after a mean of 2 years of follow-up were available to analyze the longitudinal effects of post-transplant changes of blood pressure control on left ventricular mass index (LVMI). Arterial hypertension (59%) and left ventricular hypertrophy (50%) were highly prevalent in children after renal transplantation. Renal allograft function and number of antihypertensive medications, but not ABPM variables, were correlated with LVMI at the initial observation. However, at repeat assessment, a significant correlation between ABPM and LVMI was found. In the longitudinal assessment, left ventricular remodeling was dependent on change of dosage of cyclosporine and interval changes of blood pressure levels. Hence, control of blood pressure correlates with changes of LVMI in children with renal allografts. These results clearly underline the importance of blood pressure control for the maintenance of the myocardial architecture.

ACE inhibition in the treatment of children after renal transplantation.

Currently, there are no data available on long-term effects of angiotensin-converting enzyme inhibitors (ACE-I) on graft function in children after renal transplantation. We therefore analyzed all children who were transplanted at our institution between 1989 and 1998 and followed for at least 2 years. Those treated with ACE-I, mainly because of failure of other antihypertensive medications, were compared to those without ACE-I. The ACE-I-treated children ( n=19) showed significantly better blood pressure control during the 1st year of follow-up ( p<0.05). In children with chronic allograft dysfunction ( n=8), treatment with ACE-I stabilized graft function, with improvement in creatinine clearance in 50% ( p<0.01). Serum potassium and hemoglobin levels remained stable. One patient discontinued ACE-I because of renal artery stenosis. Taken together, ACE-I were effective and safe in the treatment of hypertension in children following renal transplantation. Children with chronic allograft dysfunction experienced a stabilizing effect on graft function.