Ensuring hemodialysis adequacy by dialysis dose monitoring with UV spectroscopy analysis of spent dialyzate.

INTRODUCTION: Patients' session-to-session variation has been shown to influence outcomes, making critical the monitoring of dialysis dose in each session. The aim of this study was to detect the intra-patient variability of blood single pool Kt/V as measured from pre-post dialysis blood urea and from the online tool Adimea®, which measures the ultraviolet absorbance of spent dialyzate. METHODS: This open, one-armed, prospective non-interventional study, evaluates patients on bicarbonate hemodialysis or/and on hemodiafiltration. Dialysis was performed with B. Braun Dialog+ machines equipped with Adimea®. In the course of the prospective observation, online monitoring with Adimea® in each session was established without the target warning function being activated. A sample size of 97 patients was estimated. RESULTS: A total of 120 patients were enrolled in six centers in China (mean age 51.5 ± 12.2 years, 86.7% males, 24.2% diabetics). All had an AV-fistula. The proportion of patients with blood Kt/V < 1.20 at baseline was 48.3%. During follow-up with Adimea®, the subgroup with Kt/V > 1.20 at baseline remains at the same adequacy level for more than 90% of the patients. Those with a Kt/V < 1.20 at baseline, showed a significant increase of Kt/V to 60% of the patients reaching the adequacy level >1.20. The coefficient of variation for spKt/V as evaluated by Adimea® was 9.6 ± 3.4%, not significantly different from the 9.6 ± 8.6% as blood Kt/V taken at the same time. CONCLUSION: Online monitoring of dialysis dose by Adimea® improves and maintains dialysis adequacy. Implementing online monitoring by Adimea into daily practice moves the quality of dialysis patient care a significant step forward.

Effectiveness of a New Single-Needle Single-Pump Dialysis System with Simultaneous Monitoring of Dialysis Dose.

To date, single-needle (SN) hemodialysis (HD) requires a dialysis machine equipped with two blood pumps-one controlling arterial blood flow (Qb) and one controlling venous Qb. B. Braun has developed an innovative single-pump SN HD system. Therefore, usability is improved by reducing complexity. The aim of this study was to compare dialysis parameters of the new single-pump SN HD system with a double-pump SN HD system available on the market (Fresenius Medical Care [FMC] 5008). In this two-armed crossover study, patients were randomized into two groups (B. Braun - FMC/FMC - B. Braun). Study period was 2 weeks (6 HD sessions) for each SN HD system. Both B. Braun and FMC dialysis machines were operated in the single-needle auto mode. With the FMC dialysis machines, Qb was optimized manually, whereas for B. Braun machines it was optimized automatically using the auto-mode functionality. A phase volume of 25 mL, treatment time, needle type and size, and dialyzer type and size were kept constant per patient throughout the study. Due to technical prerequisites in the SN mode, online dialysis adequacy (Kt/V: K - dialyzer clearance of urea; t - dialysis time; V - volume of distribution of urea) monitoring could only be performed in the B. Braun group. Twelve HD patients (5 male/7 female, mean age 75.5 ± 8.8 years, mean time on dialysis 4.97 ± 3.86 years, 3× weekly HD) were enrolled. Total number of treatments performed: n = 132 (65 B. Braun, 67 FMC) and the mean online Kt/V value in the B. Braun group was 1.26 ± 0.29 (n = 63). Mean dialysis time per session: B. Braun 253.4 ± 19.9 min, FMC 251.6 ± 18.8 min. Mean phase volume: B. Braun 25.1 ± 0.2 mL, FMC 25.4 ± 3.1 mL. Mean cumulated blood volume (CBV): B. Braun 55.0 ± 5.5 L, FMC 40.5 ± 5.9 L (P < 0.0001). Mean Qb: B. Braun 217.8 ± 12.9 mL/min, FMC 178.6 ± 14.9 mL/min (effective Qb) (P < 0.0001), which corresponds to a difference of 39.3 mL/min (22.0%). Higher Qb has an influence on the CBV. To evaluate this effect, CBV was corrected for the difference in Qb by calculating the CBV/Qb rate. The mean CBV/Qb rate was 252.2 ± 19.4 min (B. Braun) and 226.8 ± 27.6 min (FMC) (P < 0.0001) per session. This represents a highly significant difference of 11.4%. To support the in vivo data the dead time for opening/closure of the clamps of the FMC 5008 was measured, resulting in 364 milliseconds. Over a 240 min dialysis session, with a blood flow rate of 250 mL/min and a phase volume of 25 mL, it was estimated at about 14.56 min (6.1% of the session). Similarly, it was estimated that the dead time of the pumps of the FMC 5008 during 240 min dialysis session was 4.7 min (1.9% of the session). In case single needle therapy is the only practical option for a patient, the advantages of the new single-pump single needle system-namely the proven higher cumulative blood volume, the alarm-free auto-regulation of the blood flow and the easier handling for the nursing staff-ensure higher treatment efficiency than conventional double-pump single needle systems.

Caspase-8 and caspase-7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF-R1 receptosomes.

We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.

Real-time Kt/V determination by ultraviolet absorbance in spent dialysate: technique validation.

Real-time determination of Kt/V can be provided by monitoring ultraviolet absorbance of solutes in spent dialysate. This not only overcomes dependency on error-prone pre- and post-dialysis blood sampling; it circumvents inaccuracies associated with estimating the urea distribution volume and its high measurement frequency tightly reflects the course of the dialysis. Our study compared the ultraviolet-based spKt/V and eKt/V with the commonly used blood-based counterparts. A first study of 16 patients compared ultraviolet Kt/Vs against blood Kt/Vs obtained by using the 'gold standard' of serial blood samples. A second study included 18 patients and compared the ultraviolet and blood values under routine conditions. Both studies showed mean blood-based spKt/V and eKt/V values statistically indistinguishable from their ultraviolet-based counterparts. Hence, on-line monitoring of ultraviolet absorption of spent dialysate is applicable in routine hemodialysis allowing continuous measure of removed solutes from spent dialysate without disturbing the patient or treatment session.

Interaction with XIAP prevents full caspase-3/-7 activation in proliferating human T lymphocytes.

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.

Recent patents on cardiovascular stem cells.

Chronic heart failure has emerged as a leading cause of morbidity and mortality worldwide. Conceptually, replacement of akinetic scar tissue by viable myocardium and improvement of neovascularization should improve cardiac function and impede progressive left ventricular remodelling. Experimental and clinical studies suggest that transfer or mobilization of stem and progenitor cells can have a favourable impact on tissue perfusion and contractile cardiac performance. The aim of the present review was to screen various available patent data bases to give an overview on different patent applications of cell-based and non-cell based therapies in regenerative cardiovascular medicine. The first part describes cell based methods and use of growth factors to improve cardiovascular function. Secondly, patents on methods to improve angiogenesis, re-endothelialization and vascular function are presented. Finally, we describe patents describing methods for improved differentiation of stem cells to cardiovascular cells, including the generation of cardiomyocytes from embryonic or adult stem cells. A systematic overview on the current patent situation about use of stem cells in cardiovascular medicine should facilitate future decision making in the development of novel therapeutic strategies in regenerative medicine.

Growth hormone treatment improves markers of systemic nitric oxide bioavailability via insulin-like growth factor-I.

CONTEXT AND OBJECTIVE: Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I. DESIGN, PATIENTS, AND RESULTS: Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment, we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P < 0.05). Likewise, plasma nitrate and nitrite levels were increased, whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly, IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P < 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly, blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability. CONCLUSIONS: GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus, GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases.

Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone-mediated increase of insulin-like growth-factor-1.

Aging is associated with an increased risk for atherosclerosis. A possible cause is low numbers and dysfunction of endothelial progenitor cells (EPC) which insufficiently repair damaged vascular walls. We hypothesized that decreased levels of insulin-like growth factor-1 (IGF-1) during age contribute to dysfunctional EPC. We measured the effect of growth hormone (GH), which increases endogenous IGF-1 levels, on EPC in mice and human subjects. We compared EPC number and function in healthy middle-aged male volunteers (57.4+/-1.4 years) before and after a 10 day treatment with recombinant GH (0.4 mg/d) with that of younger and elderly male subjects (27.5+/-0.9 and 74.1+/-0.9 years). Middle-aged and elderly subjects had lower circulating CD133(+)/VEGFR-2(+) EPC with impaired function and increased senescence. GH treatment in middle-aged subjects elevated IGF-1 levels (126.0+/-7.2 ng/mL versus 241.1+/-13.8 ng/mL; P<0.0001), increased circulating EPC with improved colony forming and migratory capacity, enhanced incorporation into tube-like structures, and augmented endothelial nitric oxide synthase expression in EPC comparable to that of the younger group. EPC senescence was attenuated, whereas telomerase activity was increased after GH treatment. Treatment of aged mice with GH (7 days) or IGF-1 increased IGF-1 and EPC levels and improved EPC function, whereas a two day GH treatment did not alter IGF-1 or EPC levels. Ex vivo treatment of EPC from elderly individuals with IGF-1 improved function and attenuated cellular senescence. IGF-1 stimulated EPC differentiation, migratory capacity and the ability to incorporate into forming vascular networks in vitro via the IGF-1 receptor. IGF-1 increased telomerase activity, endothelial nitric oxide synthase expression, phosphorylation and activity in EPC in a phosphoinositide-3-kinase/Akt dependent manner. Small interference RNA-mediated knockdown of endothelial nitric oxide synthase in EPC abolished the IGF-1 effects. Growth hormone-mediated increase in IGF-1 reverses age-related EPC dysfunction and may be a novel therapeutic strategy against vascular disorders with impairment of EPC.

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism.

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-kappaB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

TNF-receptor I defective in internalization allows for cell death through activation of neutral sphingomyelinase.

The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI DeltaTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI DeltaTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI DeltaTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis.

Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles.

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.