Search

Three recently published sham-controlled studies proved the efficacy of renal denervation (RDN) in hypertensive patients. The study presented here analyzed a nationwide multicentre registry database to clarify which patient subgroups benefit most from radiofrequency RDN.This is a post hoc analysis from the multicentre Austrian Transcatheter Renal Denervation Registry hosted by the Austrian Society of Hypertension. We correlated change of SBP after RDN to sex and presence/absence of comorbidities. Univariable correlation and multiple linear regression analyses were performed.Two hundred and ninety-one patients (43% women, median age 64 years) undergoing RDN between April 2011 and September 2014 were included in this analysis. Mean baseline ambulatory 24 h BP (systolic/diastolic) was 150 ± 18/89 ± 14 mmHg and mean baseline office BP was 170 ± 16/94 ± 14 mmHg.After RDN, mean ambulatory 24 h BP reduction was 9 ± 19/6 ± 16 mmHg. The following features were associated with a good response to RDN: high baseline systolic ambulatory BP, high baseline diastolic office BP, female sex, absence of diabetes mellitus, and absence of peripheral artery disease. Multivariable analysis identified female sex and absence of diabetes mellitus as strongest predictors for ambulatory BP reduction, although those groups had the lowest baseline ambulatory BP.Ambulatory BP reductions after RDN were substantially more pronounced in female and in nondiabetic patients despite lower baseline BP. It is concluded that in terms of efficacy female patients and nondiabetic patients might benefit more from RDN.

Aging is a major driver for chronic kidney disease (CKD) and the counterbalancing of aging processes holds promise to positively impact disease development and progression. In this study we generated a signature of renal age-associated genes (RAAGs) based on six different data sources including transcriptomics data as well as data extracted from scientific literature and dedicated databases. Protein abundance in renal tissue of the 634 identified RAAGs was studied next to the analysis of affected molecular pathways. RAAG expression profiles were furthermore analysed in a cohort of 63 CKD patients with available follow-up data to determine association with CKD progression. 23 RAAGs were identified showing concordant regulation in renal aging and CKD progression. This set was used as input to computationally screen for compounds with the potential of reversing the RAAG/CKD signature on the transcriptional level. Among the top-ranked drugs we identified atorvastatin, captopril, valsartan, and rosiglitazone, which are widely used in clinical practice for the treatment of patients with renal and cardiovascular diseases. Their positive impact on the RAAG/CKD signature could be validated in an in-vitro model of renal aging. In summary, we have (i) consolidated a set of RAAGs, (ii) determined a subset of RAAGs with concordant regulation in CKD progression, and (iii) identified a set of compounds capable of reversing the proposed RAAG/CKD signature., Graphical abstract Unlabelled Image

Background/Introduction Three recently published sham-controlled studies proved the efficacy of renal denervation (RDN) in hypertensive patients. However, there it is still unclear which patients should be selected for RDN. Purpose This study seeks to clarify which patient subgroups benefit most from radiofrequency RDN by analysing a nationwide multicentre registry database. Methods This is a post-hoc analysis from the multicentre Austrian Transcatheter Renal Denervation Registry hosted by the Austrian Society of Hypertension. We correlated change of systolic blood pressure (BP) after RDN to gender and presence/absence of comorbidities. Bivariate correlation and multiple linear regression analyses were performed. Results 291 patients (43% female, median age 64 years) undergoing RDN between April 2011 and September 2014 were included in this analysis. Mean baseline ambulatory 24-hour BP (systolic/diastolic) was 150±18/89±14 mmHg and mean baseline office BP was 170±16/94±14 mmHg. After RDN, mean ambulatory 24-hour BP reduction was 9±19/6±16 mmHg. The following features were associated with a good response to RDN: high baseline systolic ambulatory BP (ρ=0.53, p Figure 1 Discussion Ambulatory BP reductions after RDN were substantially more pronounced in female and in non-diabetic patients despite lower baseline BP. It is concluded that in terms of efficacy female patients and non-diabetics might be more suitable for RDN. Acknowledgement/Funding The Austrian Renal Denervation Registry was funded by the Austrian Society of Hypertension.

Background Several studies reported that the mode of delivery may induce changes to the immune system. Our hypothesis was that the delivery mode may influence mainly the naive T cell subpopulation. Aims Particular focus was set on the proportions and peripheral replicative history of naive T cells and cord blood serum concentrations of IL-7, a cytokine involved in peripheral naive T cell homeostasis. Study design, subjects and outcome measures In a prospective cohort study, proportions of lymphocyte populations were measured in mothers and newborns delivered by spontaneous vaginal delivery (SD), vacuum extraction (VE), primary (PCS) and secondary Cesarean sections (SCS) by flow cytometry. T-cell-receptor-excision-circles (TRECs) and relative telomere lengths (RTLs) were used to estimate the replicative history of peripheral naive T cells. The cytokine profile was assessed by ELISA. Results The study demonstrated that leukocytes, neutrophils and NK cells were increased in spontaneously delivered newborns compared to PCS, whereas circulating T cells were relatively lower. TRECs and RTLs were not significantly influenced by the delivery mode. IL-2, IL-8 and IFN-γ were increased in VD. IL-7 production tends to be increased in more stress-associated delivery modes, such as VE and SCS. Conclusions Our results demonstrate proportional changes in newborns delivered by PCS and diminished cytokine production. It has to be proven whether these alterations may be of disadvantage regarding early defense of infectious diseases. Understanding the physiological role of these changes may help to find preventive strategies for neonatal infectious risks and the development of atopy or other immune diseases.

Introduction In transplantation medicine calcineurin inhibitors (CNI) still represent the backbone of immunosuppressive therapy. The nephrotoxic potential of the CNI Cyclosporine A (CsA) and Tacrolimus (FK506) is well recognized and CNI not only have been linked with toxicity, but also with cellular senescence which hinders parenchymal tissue regeneration and thus may prime kidneys for subsequent insults. To minimize pathological effects on kidney grafts, alternative immunosuppressive agents like mTOR inhibitors or the T-cell co-stimulation blocker Belatacept have been introduced. Methods We compared the effects of CsA, FK506 and Sirolimus on the process of cellular senescence in different human renal tubule cell types (HK2, RPTEC). Telomere length (by real time PCR), DNA synthesis (by BrdU incorporation), cell viability (by Resazurin conversion), gene expression (by RT-PCR), protein (by western blotting), Immuncytochemistry and H2O2 production (by Amplex Red® conversion) were evaluated. Results DNA synthesis was significantly reduced when cells were treated with cyclosporine but not with tacrolimus and sirolimus. Resazurin conversion was not altered by all three immunosuppressive agents. The gene expression as well as protein production of the cell cycle inhibitor p21 (CDKN1A) but not p16 (CDKN2A) was significantly induced by cyclosporine compared to the other two immunosuppressive agents when determined by western blotting an immuncytochemistry. Relative telomere length was reduced and hydrogen peroxide production increased after treatment with CsA but not with FK506 or sirolimus. Conclusion In summary, renal tubule cells exposed to CsA show clear signs of cellular senescence where on the contrary the second calcineurin inhibitor FK506 and the mTOR inhibitor sirolimus are not involved in such mechanisms. Chronic renal allograft dysfunction could be in part triggered by cellular senescence induced by immunosuppressive medication and the choice of drug could therefore influence long term outcome. Tacrolimus and Sirolimus are equally effective in avoiding cellular senescence compared to cyclosporine at least in parts due to a lack of induction of reactive oxygen species.

Pathfinder cells (PCs) are a novel class of adult-derived cells that facilitate functional repair of host tissue. We used rat PCs to demonstrate that they enable the functional mitigation of ischemia reperfusion (I/R) injury in a mouse model of renal damage. Female C57BL/6 mice were subjected to 30 min of renal ischemia and treated with intravenous (i.v.) injection of saline (control) or male rat pancreas-derived PCs in blinded experimentation. Kidney function was assessed 14 days after treatment by measuring serum creatinine (SC) levels. Kidney tissue was assessed by immunohistochemistry (IHC) for markers of cellular damage, proliferation, and senescence (TUNEL, Ki67, p16ink4a, p21). Fluorescence in situ hybridization (FISH) was performed to determine the presence of any rat (i.e., pathfinder) cells in the mouse tissue. PC-treated animals demonstrated superior renal function at day 14 post-I/R, in comparison to saline-treated controls, as measured by SC levels (0.13 mg/dL vs. 0.23 mg/dL, p

In this review we discuss the current knowledge on Biomarkers of Aging (BoAs) in the context of human diseases and their value as predictive or prognostic markers. The vast majority of studies using BoAs in a clinical context have been undertaken by determining telomere length in peripheral blood mononuclear cells (PBMCs), whereas the expression of cell cycle inhibitors and an increase in advanced glycation end products (AGEs) have rarely been used. Here we summarize the impact of BoAs on non-oncological, hematological, cardiovascular, metabolic, renal and neurological diseases, as well as on overall survival. The specific methodologies utilized are described and evaluated for their high-throughput potential.

Background. It is unclear whether the choice of maintenance immunosuppression modulates the negative effect of advanced donor age on outcome after renal transplantation. Methods. All 1829 patients who received their first transplant between 1990 and 2003 at the Vienna Medical Centre and had a functioning graft after 90 days were studied. At this time point, 1587 received calcineurin inhibitors (CNI+), 242 did not (CNI−). Actual and functional graft survival was analyzed in subgroups based on donor age ( 64 years) and immunosuppressive therapy. Results. The median follow-up time was 7 years. In total, we observed 312 deaths and 275 graft losses. After adjusting for several variables considered as potential confounders, actual graft survival was better in CNI+ patients compared with CNI− patients only if donor age was less than 36 years (adjusted hazard ratio 0.25, 95% confidence interval 0.17-0.38) or 36 to 49 years (0.43, 95% confidence interval 0.29-0.62). Similar results were obtained for functional graft survival. Patient survival was significantly better in CNI+ subjects irrespective of donor age (0.41, 95% confidence interval 0.30-0.57). Discussion. Use of CNI 90 days after transplantation is associated with improved patient survival even after adjustment for confounders, but its beneficial association with actual and functional graft survival is lost or at least reduced if kidneys from donors older than 50 years are used.

OBJECTIVE To analyse the morphological appearance of horseshoe kidneys (HKs) and crossed fused ectopia (CFE) and to assess the frequency and clinical significance of associated anomalies and diseases. PATIENTS AND METHODS The findings and images of 209 patients with fused kidneys (FKs) were reviewed; in all, 244 scans from computed tomography (CT), 233 ultrasonograms and 89 micturition cysto-urethrograms, urograms, magnetic resonance images and angiograms were taken. RESULTS HKs (found in one of 474 abdominal CT scans) and CFEs (found in one of 3078 CT scans) showed a high variability of vasculature that could not be classified. However, some generalized conclusions were possible about the renal vasculature (430 arteries in 103 kidneys). Variants of the most cephalad artery of both sides were rare. The second artery on the right had a pre-caval course. The origins of vessels located further caudal were more ventral. CFEs were anatomically different from HKs with respect to lower position, greater axial rotation, smaller pelvic width, more caudal origin, and fewer vessels, but not in accompanying anomalies. Severe anomalies or malformations were found in 23% of patients, with half of them in the urogenital system. Malformations were found considerably more often in children than in adults. There was no increased incidence of diseases such as stones or inflammation of the renal pelvis. CONCLUSION Concomitant anomalies and diseases were equally frequent for HK and CFE, but less frequent than generally assumed. Individual cases of complex anatomical situations require special examination strategies, and CT appears to be the most reliable imaging method.

Summary Although chronological donor age is the most potentpredictor of long-term outcome after renal transplantation,it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimateof biological organ age as derived from markers of cellularsenescence in zero hour biopsies would be of higherpredictive value. Telomere length and mRNA expressionlevels of the cell cycle inhibitors CDKN2A (p16INK4a) andCDKN1A (p21WAF1) were assessed in pre-implantationbiopsies of 54 patients and the association of these andvarious other clinical parameters with serum creatinineafter 1 year was determined. In a linear regression analysis,CDKN2A turned out to be the best single predictorfollowed by donor age and telomere length. A multiplelinear regression analysis revealed that the combinationof CDKN2A values and donor age yielded even higherpredictive values for serum creatinine 1 year after trans-plantation. We conclude that the molecular aging markerpredict renal allograft function after 1 year significantlybetter than chronological donor age alone.Key words: biological organ age, CDKN1A, CDKN2A, kidneytransplantation, long-term allograft function, telomerelength, zero hour biopsy.

The nephrotoxic potential of the widely used immunosuppressive agent cyclosporine A (CsA) is well recognized. However, the mechanism of renal tubular toxicity is not yet fully elucidated. Chronic CsA nephropathy and renal organ aging share some clinical features, such as renal fibrosis and tubular atrophy, raising the possibility that CsA may exert some of its deleterious effects via induction of a stress-induced senescent phenotype. We investigated this hypothesis in HK-2 cells and primary proximal tubular cells in vitro. CsA induced the production of H2O2, caused cell cycle arrest in the G0/G1 phase, and inhibited DNA synthesis. Furthermore, CsA exposure lead to a reduction of telomere length, increased p53 serine 15 phosphorylation, and caused an upregulation of the cell cycle inhibitor p21Kip1(CDKN1A) mRNA levels. CsA caused an increase in p16INK4a(CDKN2A) expression after a 13-day exposure in primary proximal tubular cells but not in HK-2 cells. Coincubation of cells with CsA and catalase was able to prevent telomere shortening and partially restored DNA synthesis. In summary, CsA induces cellular senescence in human renal tubular epithelial cells, which can be attenuated by scavenging reactive oxygen species.

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-α 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.

Background Human lifespan is increasing continuously and about one-third of the population >70 years of age suffers from chronic kidney disease. The pathophysiology of the loss of renal function with ageing is unclear. Methods We determined age-associated gene expression changes in zero-hour biopsies of deceased donor kidneys without laboratory signs of impaired renal function, defined as a last serum creatinine >0.96 mg/dL in females and >1.18 mg/dL in males, using microarray technology and the Significance Analysis of Microarrays routine. Expression changes of selected genes were confirmed by quantitative polymerase chain reaction and in situ hybridization and immunohistochemistry for localization of respective mRNA and protein. Functional aspects were examined in vitro. Results Donors were classified into three age groups ( 59 years; Groups 1, 2 and 3, respectively). In Group 3 especially, genes encoding for metallothionein (MT) isoforms were more significantly expressed when compared with Group 1; localization studies revealed predominant staining in renal proximal tubular cells. RPTEC/TERT1 cells overexpressing MT2A were less susceptible towards cadmium chloride-induced cytotoxicity and hypoxia-induced apoptosis, both models for increased generation of reactive oxygen species. Conclusions Increased expression of MTs in the kidney with ageing might be a protective mechanism against increased oxidative stress, which is closely related to the ageing process. Our findings indicate that MTs are functionally involved in the pathophysiology of ageing-related processes.

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-β-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes. peerReviewed

A 16-year-old man presented with severe nephrotic syndrome complicated by massive perirenal fluid. Percutaneous drainage of fluid was performed 3 times, followed by improvement in renal function and hypertension, but perirenal fluid recurred within days. Nephrotic syndrome was unresponsive to steroid therapy. A laparoscopic bilateral fenestration of Gerota's fascia and peritoneum allowed permanent drainage of fluid into the peritoneal cavity. During the same procedure, a renal wedge biopsy was performed. Histological examination showed advanced focal glomerular sclerosis of the tip lesion variant. The glomerular disease was refractory to further treatment with cyclophosphamide, mycophenolate, and rituximab. However, perirenal fluid did not recur despite persistent nephrotic syndrome, showing that fenestration of Gerota's fascia is a successful treatment of floating kidneys in such patients.

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.

Background: Life expectancy, as well as the average age of patients undergoing solid organ transplantation, increases constantly. Consequently, immunosuppressive therapy is no longer limited to young organ recipients. Objective: Here, we investigate how different types of immunosuppressive therapy, namely the calcineurin inhibitors cyclosporin A and tacrolimus, as well as the mTOR inhibitor rapamycin, affect the function of immune cells in young and elderly persons. Methods: Proliferation, cell viability, cytokine production (IL-2, IFN-γ), H2O2 production and telomere length of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMCs) of young (n = 13; median age 27 years) and old (n = 19; median age 71 years) healthy donors were analyzed. Results: The inhibition of proliferation was dampened in PBMCs from elderly donors, especially after incubation with rapamycin. All three immunosuppressive drugs inhibited the production of IL-2 equally well, whereas the production of IFN-γ was less well inhibited by rapamycin. Both calcineurin inhibitors increased H2O2 concentrations after stimulation with PHA and led to a shortening of telomeres in PBMCs from young and old individuals. Rapamycin had only minor effects on H2O2 production and telomere length. Conclusion: Our results demonstrate that the effects of immunosuppressive drugs on PBMCs differ between young and elderly persons. Calcineurin inhibitors compared to rapamycin have a more pronounced prosenescence effect. These data indicate that specific treatment regimens for the elderly might therefore be considered.

Background Significant immunomodulatory effects have been described as result of cigarette smoking in adults and pregnant women. However, the effect of cigarette smoking during pregnancy on the lymphocyte subpopulations in newborns has been discussed, controversially. Methods In a prospective birth cohort, we analyzed the peripheral lymphocyte subpopulations of smoking (SM) and non-smoking mothers (NSM) and their newborns and the replicative history of neonatal, mostly naive CD4 + CD45RA + T cells by measurements of T-cell-receptor-excision-circles (TRECs), relative telomere lengths (RTL) and the serum cytokine concentrations. Results SM had higher lymphocyte counts than NSM. Comparing SM and NSM and SM newborns with NSM newborns, no significant differences in proportions of lymphocyte subpopulations were seen. Regardless of their smoking habits, mothers had significantly lower naive T cells and higher memory and effector T cells than newborns. NSM had significantly lower percentages of CD4 + CD25++ T cells compared to their newborns, which was not significant in SM. There were no differences regarding cytokine concentrations in newborns of SM and NSM. However, NSM had significantly higher Interleukin-7 concentrations than their newborns. Regardless of smoking habits of mothers, newborns had significantly longer telomeres and higher TRECs than their mothers. Newborns of SM had significantly longer telomeres than newborns of NSM. Conclusions Apart from higher lymphocyte counts in SM, our results did not reveal differences between lymphocyte subpopulations of SM and NSM and their newborns, respectively. Our finding of significantly longer RTL in newborns of SM may reflect potential harm on lymphocytes, such as cytogenetic damage induced by smoking.

Introduction Juvenile idiopathic arthritis is a heterogeneous T cell-mediated autoimmune disease with symptoms of premature aging of the immune system (immunosenescence). The present work is an investigation of immunosenescence parameters, such as quantity of naive and CD28- T cells, T cell receptor excision circles, relative telomere length and alterations of peripheral T cell replication, and was performed via comparison of a case of acute exacerbation of juvenile idiopathic arthritis against six patients with juvenile idiopathic arthritis with disease remission and six age-matched healthy donors over a follow-up course of 12 months. Case presentation Phenotypical T cell characterization and intracellular interferon γ, tumor necrosis factor α, and interleukin 2 production were studied in peripheral blood mononuclear cells from seven patients with juvenile idiopathic arthritis and six healthy control donors, with findings determined by flow cytometry. T cell receptor excision circles and relative telomere length quantification were performed on deoxyribonucleic acid isolated from naive (CD4+CD28+CD45RA+) T cells and investigated via reverse transcription polymerase chain reaction. Ki67 expression was studied by immunohistochemistry on naive T cells. The non-parametric Mann-Whitney U test and Wilcoxon test for two independent groups of variables were used to compare healthy donors with patients with juvenile idiopathic arthritis. During follow-up, patients with juvenile idiopathic arthritis showed lower total counts of naive and CD28-expressing T cells compared to healthy donors. Acute exacerbation led to low naive and CD28+ T cell populations and elevated proportions of Ki67-expressing CD4+ naive T cells. In conditions of exacerbation, T cell receptor excision circle numbers were in the lower range in patients with juvenile idiopathic arthritis and increased after follow-up. Healthy donors showed significantly higher relative telomere lengths compared to patients with juvenile idiopathic arthritis. Conclusions This investigation illustrates that the changes in T cell homeostasis in patients with juvenile idiopathic arthritis may be the result of several mechanisms, such as diminished thymus function and peripheral exertions to maintain the peripheral T cell pool. The results also demonstrate that hallmarks of immunosenescence such as decreased naive T cell levels and lower T cell receptor excision circle numbers can only be interpreted together with replication markers such as relative telomere length or Ki67 expression.

It is unclear, whether pediatric patients with type 1 diabetes (T1DM) show immunological alterations typically found in autoimmune conditions resembling immune dysfunction of the thymus, such as decrease of naïve T cells, lower T cell receptor excision circle (TREC) numbers, telomeric erosion, and diminished interleukin-7 (IL-7) levels. Furthermore, it is unknown, whether long-term therapy with insulin, a thymic growth factor, interferes with these changes. Therefore, the aim of this study was to analyze the quantity of the naïve T cell subset and its TREC content, relative telomere length (RTL) of naïve T cells, and peripheral IL-7 levels in patients with recent-onset T1DM (n = 5), long-standing T1DM (n = 33), and age-matched healthy donors (HD) (n = 37). In long-standing T1DM, TREC numbers/CD8+CD45RA+ T cells were enhanced (p0.01) compared to HD and correlated with disease duration (p0.02), an independent factor for increased thymic output (p0.01), and insulin dosage at blood withdrawal (p0.05). IL-7 serum levels were elevated in long-standing T1DM (p0.001) and positively correlated with TREC numbers (p0.01) and disease duration (p0.0001). RTLs in CD8+CD45RA+ T cells were significantly increased compared to HD (p0.02). Our data suggest that longterm insulin therapy may serve as a driving factor for thymic function and rejuvenation of the naïve T cell compartment. The ability of the immune system to reconstitute the naïve T cell compartment under well-adjusted insulin therapy may be of major importance for recognition of new antigens, response to vaccinations, and defense of infectious complications.

Background: Recent studies indicate an increased mortality of anemic patients with renal failure when near-normal hemoglobin levels are aimed for by treatment with erythropoiesis stimulating agents. Aortic pulse wave velocity (aPWV) is a strong predictor of all-cause and cardiovascular mortality in patients with end-stage renal disease. The relationships between aPWV, hemoglobin levels and erythropoiesis stimulating agent dosage have not been evaluated to date. Methods: In 75 patients, aPWV was measured by applanation tonometry. Associations of aPWV and a broad range of clinical, laboratory and therapeutic parameters were determined by stepwise linear regression analysis. Results: aPWV was positively correlated to age (r = 0.55, p < 0.001), whereas the association with hemoglobin was significant, but negative (r = –0.31, p = 0.01). Multivariate analysis determined age (β = 0.513, p < 0.001), mean blood pressure (β = 0.255, p = 0.01), the presence of heart failure (β = 0.188, p = 0.03), hemoglobin (β = –0.226, p = 0.01), daily calcium load (β = –0.230, p = 0.01) and the presence of diabetes mellitus (β = 0.179, p = 0.04) to have a significant and independent influence on aPWV. Conclusions: This study demonstrates that in hemodialysis patients, aPWV is significantly but negatively associated with the serum hemoglobin concentration, even after multiple adjustments for other covariates.

Juvenile idiopathic arthritis (JIA) is an autoimmune disease of the young. The pathogenesis is not completely understood. Premature aging, associated thymic involution, and compensatory autoproliferation could play important roles in the pathogenesis of autoimmunity. We undertook this study to determine whether patients with JIA demonstrate premature immunosenescence.To test this hypothesis, we measured 3 indicators of aging: the percentages and total counts of peripheral blood naive T cells, the frequency of T cell receptor excision circles (TRECs) in naive T cells, and telomeric erosion and Ki-67 expression as estimates of the replicative history of homeostatic proliferation.JIA patients showed an accelerated loss of CD4+,CD45RA+,CD62L+ naive T cells with advancing age and a compensatory increase in the number of CD4+,CD45RO+ memory T cells. JIA patients demonstrated a significantly decreased frequency of TRECs in CD4+,CD45RA+ naive T cells compared with age-matched healthy donors (P = 0.002). TREC numbers correlated with age only in healthy donors (P = 0.0001). Telomeric erosion in CD4+,CD45RA+ naive T cells was increased in JIA patients (P = 0.01). The percentages of Ki-67-positive CD4+,CD45RA+ naive T cells were increased in JIA patients (P = 0.001) and correlated with disease duration (P = 0.003), which was also an independent factor contributing to telomeric erosion (P = 0.04).Our findings suggest that age-inappropriate T cell senescence and disturbed T cell homeostasis may contribute to the development of JIA. In patients with JIA, dysfunction in the ability to reconstitute the T cell compartment should be considered when exploring new therapeutic strategies.

To investigate the influence of latent cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections on CD28-expressing T-cell subpopulations and replicative senescence of naive T-cells as a marker for aging of the immune system in children with juvenile ideopathic arthritis (JIA).T-cell subpopulations were analyzed from 24 patients with JIA and 61 healthy age-matched controls by fluorescence activated cell sorting. Relative telomere length (RTL) in CD4+CD28+CD45RA+ (naive) T-cells was measured by quantitative polymerase chain reaction.Although confirming known data of expansions of CD28- T-cells and tendency of decreasing naive T-cells in CMV-seropositive healthy individuals, our findings did not show a marked influence of latent EBV or CMV infection on CD28-expressing T-cells in patients with JIA. In contrast, CMV was an independent factor for loss of CD28, regardless of age, in healthy controls. Irrespective of serology results for CMV or EBV, patients with JIA showed signficantly decreased RTL compared to age-matched controls. Regression lines for RTL and age revealed decreased RTL with advancing age in CMV-positive and EBV-positive subjects. The evidence that findings for CMV-positive JIA patients did not resemble the findings of healthy CMV-positive controls, namely expansion of CD28- T-cells and decrease of naive T-cells, may support the theory of a disturbed peripheral T-cell homeostasis in JIA.Diminished mechanisms of T-cell homeostasis and premature aging of the immune system may play a role in the pathogenesis of JIA.

Circulating endothelial precursor cells (CEP) are interesting candidates for the treatment of ischemic diseases and for tumor targeting/imaging. We isolated a homogeneous population of CEP from CD34+/CD133− cells of peripheral blood that can be expanded easily on collagen-type-I coated plastic. CEP displayed a phenotype of mature endothelial cells (vWF, CD31, CD34, VEGF-R2, CD105, CD146) similar to that of cord-blood CEP and umbilical vein endothelial cells. They bound UEA-1 lectin, incorporated acetylated LDL and formed tube-like structures with capillary lumens in vitro. Weibel-Palade bodies were observed by electron microscopy. After 40–60 cell population doublings, CEP cultures underwent a terminal growth arrest, had shorter telomeres, up-regulated cell cycle inhibitory proteins, such as p21CIP1 and stained positive for senescence-associated-beta galactosidase. During the whole expansion period CEP retained their endothelial phenotype and a normal karyotype. CEP had the capacity to home to ischemic tissue in vivo after systemic injection in nude rats. The convenient expandability, the homogenous phenotype, the functional cellular senescence program, the regular karyotype and the homing capacity to ischemic myocardium suggest autologous CEP cultures as a safe and promising tool for cell-based therapeutic approaches in targeting ischemic tissue and tumors.

In vitro models for chronic toxicity, defined as a recurring exposure to compounds over a prolonged period of time, are still underrepresented in drug evaluation processes. The classical approach to cell culture is not readily suitable to long term repetitive applications. Therefore, we assessed the use of a commercially available perfusion cell culture apparatus in its applicability to chronic renal toxicity testing and describe the technical aspects of adopting the perfusion cell culture system to our purposes. It was apparent that there is a subtle dynamic difference between human renal proximal tubular cells cultured under perfusion and static conditions as illustrated by the accumulation of lactate dehydrogenase (LDH) and the secondary metabolism of resazurin to hydroresorufin, which occurred only under static conditions. The major achievement was the standardisation of the handling of this system with regard to cell cultivation, pH regulation, temperature regulation, and reproducibility of common toxicity endpoints.

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 up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.

The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.