Unlocking the Potential of Purinergic Signaling in Transplantation.

Purinergic signaling has been recognized as playing an important role in inflammation, angiogenesis, malignancy, diabetes and neural transmission. Activation of signaling pathways downstream from purinergic receptors may also be implicated in transplantation and related vascular injury. Following transplantation, the proinflammatory "danger signal" adenosine triphosphate (ATP) is released from damaged cells and promotes proliferation and activation of a variety of immune cells. Targeting purinergic signaling pathways may promote immunosuppression and ameliorate inflammation. Under pathophysiological conditions, nucleotide-scavenging ectonucleotidases CD39 and CD73 hydrolyze ATP, ultimately, to the anti-inflammatory mediator adenosine. Adenosine suppresses proinflammatory cytokine production and is associated with improved graft survival and decreased severity of graft-versus-host disease. Furthermore, purinergic signaling is involved both directly and indirectly in the mechanism of action of several existing immunosuppressive drugs, such as calcineurin inhibitors and mammalian target of rapamycin inhibitors. Targeting of purinergic receptor pathways, particularly in the setting of combination therapies, could become a valuable immunosuppressive strategy in transplantation. This review focuses on the role of the purinergic signaling pathway in transplantation and immunosuppression and explores possible future applications in clinical practice.

Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).

Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

Effect of vildagliptin compared to glimepiride on postprandial proinsulin processing in the ß cell of patients with type 2 diabetes mellitus.

This study compared the effect of Glimepiride versus Vildagliptin on ß-cell function and the release of intact proinsulin (PI) in patients with type 2 diabetes mellitus. Patients on metformin monotherapy were randomized to add on treatment with Vildagliptin or Glimepiride. A standardized test meal was given at baseline, after 12 and 24 weeks of treatment. Insulin, PI and blood glucose values were measured in the fasting state and postprandial for 300 min. Fasting PI levels significantly decreased in the Vildagliptin group. The area under the curve for the postprandial release of PI decreased during Vildagliptin and increased during Glimepiride treatment. The proinsulin to insulin ratio declined in the Vildagliptin group, whereas it did not change significantly in the Glimepiride group. Addition of Vildagliptin to ongoing Metformin treatment reconstitutes the disproportionality of the proinsulin to insulin secretion from the ß cell.

Treatment persistence, hypoglycaemia and clinical outcomes in type 2 diabetes patients with dipeptidyl peptidase-4 inhibitors and sulphonylureas: a primary care database analysis.

AIMS: To investigate therapy persistence, frequency of hypoglycaemia and macrovascular outcomes among type 2 diabetes patients with dipeptidyl peptidase-4 (DPP-4) inhibitors (DPP-4) and sulphonylureas (SU). METHODS: Data from 19,184 DPP-4 (mean age: 64 years; 56% males) and 31,110 SU users (69 years; 51%) with new prescriptions (index date), without additional antidiabetics except metformin, in 1201 general practises in Germany were analysed. Therapy discontinuation (prescription gap >90 days), hypoglycaemia [International Classification of Diseases (ICD-10)] and macrovascular outcomes (ICD-10) (2-year follow-up) were compared adjusting for age, sex, diabetes duration, metformin, previous hypoglycaemia, health insurance, hypertension, hyperlipidaemia, antihypertensives, lipid-lowering and antithrombotic drugs, microvascular complications and Charlson co-morbidity score using logistic or Cox regression models. RESULTS: Two years after index date, DDP-4 (non-persistence: 39%) were associated with a lower risk of discontinuation compared to SU (49%) [adjusted hazard ratio (HR): 0.74; 95% confidence interval (CI): 0.71-0.76]. Hypoglycaemias (≥1) were documented in 0.18% patients with DPP-4 and in 1.00% with SU [odds ratio (OR): 0.21; 95%CI: 0.08-0.57]. Hypoglycaemias were significantly associated with incident macrovascular complications (HR: 1.6; 95% CI: 1.1-2.2). Risk of macrovascular events was 26% lower in DPP-4 than in SU users. CONCLUSIONS: Lack of persistence with antidiabetic therapy is frequently found in primary care patients. DPP-4 was associated with lower therapy discontinuation and a fivefold reduced frequency of patients with hypoglycaemia compared to SU. The low absolute numbers of hypoglycaemias are most likely due to the fact that only severe events were documented. DPP-4 treatment was associated with reduced incidence of macrovascular events relative to SU in type 2 diabetes patients in primary care practises.

Prevention of embolic stroke by catheter ablation of atrial fibrillation.

BACKGROUND AND PURPOSE: Radiofrequency-catheter-ablation of atrial fibrillation is now commonly performed. Aim of this short review is to summarize questions and uncertainties concerning radiofrequency ablation of atrial fibrillation with respect to therapeutic mechanisms, long-term efficacy and stroke-prevention. RESULTS: The majority of atrial fibrillation patients is too old for radiofrequency ablation. Candidates for radiofrequency ablation belong to a subgroup with a low embolic risk. The radiofrequency ablation procedure itself may increase the embolic risk, and at present it is uncertain how long this embolic risk persists after the procedure. CONCLUSION: We doubt if radiofrequency ablation prevents embolism in atrial fibrillation.

Intense exercise increases adenosine concentrations in rat brain: implications for a homeostatic sleep drive.

Intense exercise and sleep deprivation affect the amount of homeostatically regulated slow wave sleep in the subsequent sleep period. Since brain energy metabolism plays a decisive role in the regulation of behavioral states, we determined the concentrations of nucleotides and nucleosides: phosphocreatine, creatine, ATP, ADP, AMP, adenosine, and inosine after moderate and exhaustive treadmill exercise as well as 3 and 5 h of sleep deprivation and sleep in the rat brain using the freeze-clamp technique. High intensity exercise resulted in a significant increase of the sleep-promoting substance adenosine. In contrast, following sleep, inosine and adenosine levels declined considerably, with an accompanied increase of ADP after 3 h and ATP after 5 h. Following 3 h and 5 h sleep deprivation, ADP and ATP did not differ significantly, whereas inosine increased during the 3 and 5-h period. The concentrations of AMP, creatine and phosphocreatine remained unchanged between experimental conditions. The present results are in agreement with findings from other authors and suggest that depletion of cerebral energy stores and accumulation of the sleep promoting substance adenosine after high intensity exercise may play a key role in homeostatic sleep regulation, and that sleep may play an essential role in replenishment of high-energy compounds.

Persistence of non-typeable Haemophilus Influenzae in the pharynx of children with adenotonsillar hypertrophy after treatment with azithromycin.

This study was performed in children with adenotonsillar hypertrophy to evaluate the effect of azithromycin (AZT) on the presence of NTHi in monocyte/macrophages (CD14(+) cells) of adenoids/tonsils and the persistence of NTHi after adenotonsillectomy. A total of 36 pediatric patients participated in the study: 20 children were treated with AZT before adenotonsillectomy, and 16 children did not receive the antibiotic prior to surgery. NTHi were identified by culture and PCR in swabs and tissue samples. NTHi was detected in the lysates of CD14(+) cells by fluorescence in situ hybridization (FISH) and by culture. The molecular typing was used to cluster NTHi isolates from each child. The intracellular NTHi was found in 10 (62.5%) untreated patients and was identified in three (15%) azithromycin-treated patients (P = 0.003). The proportion of the persistent NTHi strains was similar in both groups. AZT treatment followed by adenotonsillectomy did not completely eliminate NTHi from pharynges; however, it significantly reduced the risk of carriage of Haemophilus influenzae inside the CD14(+) cells.

Unsaturated fatty acids isolated from human lipoproteins activate protein phosphatase type 2Cbeta and induce apoptosis in endothelial cells.

Activity of serine/threonine protein phosphatase type 2C is known to be stimulated by certain unsaturated fatty acids and this enzyme dephosphorylates Bad, thus acting on apoptosis. This prompted us to investigate endothelial cell death. Here, we present evidence for the presence of protein phosphatase type 2Cbeta (PP2Cbeta) in human umbilical vein endothelial cells (HUVECs) and report on colocalization of PP2Cbeta and Bad in the cytosol of endothelial cells. Lipophilic compounds that stimulated PP2Cbeta activity in vitro were found to induce cell death of HUVECs. Lipoproteins did neither influence PP2Cbeta activity nor affect cell behaviour. Lipoproteins treated with the lipoprotein lipase, however, stimulated the activity of PP2Cbeta at least 10-fold concomitantly triggering cell death. Analytical methods revealed that both effects - stimulation of PP2Cbeta and apoptosis - were caused by free fatty acids liberated from VLDL, LDL and HDL with oleic acid and linoleic acid as major constituents. The results provide novel insights in endothelial apoptosis and suggest that PP2Cbeta participates in the development and progress of atherosclerosis.

Disrupted sleep-wake regulation in type 1 equilibrative nucleoside transporter knockout mice.

The type 1 equilibrative nucleoside transporter (ENT1) is implicated in regulating levels of extracellular adenosine ([AD]ex). In the basal forebrain (BF) levels of [AD]ex increase during wakefulness and closely correspond to the increases in the electroencephalogram (EEG) delta (0.75-4.5Hz) activity (NRδ) during subsequent non-rapid eye movement sleep (NREMS). Thus in the BF, [AD]ex serves as a biochemical marker of sleep homeostasis. Waking EEG activity in theta range (5-9Hz, Wθ) is also described as a marker of sleep homeostasis. An hour-by-hour temporal relationship between the Wθ and NRδ is unclear. In this study we examined the relationship between these EEG markers of sleep homeostasis during spontaneous sleep-wakefulness and during sleep deprivation (SD) and recovery sleep in the ENT1 gene knockout (ENT1KO) mouse. We observed that baseline NREMS amount was decreased during the light period in ENT1KO mice, accompanied by a weak correlation between Wθ of each hour and NRδ of its subsequent hour when compared to their wild-type (WT) littermates. Perfusion of low dose of adenosine into BF not only strengthened the Wθ-NRδ relationship, but also increased NREMS to match with the WT littermates suggesting decreased [AD]ex in ENT1KO mice. However, the SD-induced [AD]ex increase in the BF and the linear correlation between the EEG markers of sleep homeostasis were unaffected in ENT1KO mice suggesting that during SD, sources other than ENT1 contribute to increase in [AD]ex. Our data provide evidence for a differential regulation of wakefulness-associated [AD]ex during spontaneous vs prolonged waking.

Real-life efficacy and safety of vildagliptin compared with sulfonylureas as add-on to metformin in patients with type 2 diabetes mellitus in Germany.

OBJECTIVE: Metformin is an established first-line treatment for type 2 diabetes mellitus (T2DM) patients, but intensification of oral anti-diabetes therapy is usually required over time. A large observational study of 45,868 T2DM patients in 27 countries (EDGE) was conducted to compare the effectiveness and safety of vildagliptin as add-on therapy to another oral anti-diabetes drug (OAD) vs other dual OAD combinations. This report presents results from a post-hoc analysis of patients in Germany who received vildagliptin or a sulfonylurea (SU) in combination with metformin. RESEARCH DESIGN AND METHODS: Patients inadequately controlled with monotherapy became eligible only after the add-on treatment was finalized. Patients included were assigned to receive either vildagliptin or another OAD (SUs, thiazolidinediones, glinides, α-glucosidase inhibitors, or metformin; DPP-4 inhibitors or glucagon-like peptide-1 [GLP-1] mimetics/analogs were excluded). The primary end-point was the proportion of patients achieving a reduction in HbA1c >0.3% without peripheral edema, hypoglycemia, discontinuation due to gastrointestinal event, or weight gain ≥5%. RESULTS: Of 8887 patients enrolled in Germany, 6439 received vildagliptin and 971 received SUs as add-on to metformin. The primary end-point was reached in 34.9% and 29.6% of patients in the vildagliptin and SU groups, respectively, with an unadjusted odds ratio of 1.27 (95% CI = 1.09, 1.47; p = 0.001). HbA1c decreased in both cohorts from baseline (-0.7% with vildagliptin vs -0.5% with SUs), with a mean between-group difference of -0.2% (95% CI = -0.22, -0.09). The number of hypoglycemic events was 4-fold higher in the SU group than in the vildagliptin group (vildagliptin = 0.11%; SU = 0.41%). CONCLUSIONS: In a real-life setting, vildagliptin was associated with a numerically greater reduction in HbA1c, less hypoglycemia, and more patients reaching target HbA1c without hypoglycemia or weight gain compared with SUs. Open-label design and under reporting of adverse events are limitations of this post hoc analysis.

Sleep, brain energy levels, and food intake: Relationship between hypothalamic ATP concentrations, food intake, and body weight during sleep-wake and sleep deprivation in rats.

BACKGROUND: The feeling of hunger and feeding, a wake-state-dependent behavior, is regulated by specific centers within the hypothalamus. While paraventricular nucleus (PVN), arcuate nucleus (ARC), and dorso- and ventromedial hypothalamus (DMH/VMH) regulate feeding, the lateral hypothalamus (LH) is associated both with feeding and wake/REM sleep regulation. In order to examine the effects of sleep and wakefulness on food intake and body weight, we also measured hypothalamic ATP concentrations, which are known to be involved in feeding behavior and sleep-wake regulation. METHODS: In rats, food intake and body weight was measured during a 24-h light-dark cycle and during 6 h of sleep deprivation (SD) performed by gentle handling. Tissue samples from the PVN, ARC/DMH/VMH, and LH were collected after 6 h of SD and from time-matched diurnal controls. ATP was measured by luciferin-luciferase bioluminescence assay. RESULTS: Across the 24-h light-dark period, rats consumed approximately 28.13±4.48 g of food and gained 5.22±1.65 g with a positive correlation between food intake and body weight. During SD, while food intake increased significantly +147.31±6.13%, they lost weight significantly (-93.29±13.64%) when compared to undisturbed controls. SD resulted in a significant decrease in ATP levels only in LH (-44.60±21.13%) with no change in PVN, ARC/DMH/VMH region when compared with undisturbed controls. CONCLUSION: The results indicate a strong overall correlation between ATP concentrations in the LH and individual food intake and suggest a sleep-wake dependent neuronal control of food intake and body weight.

Delta oscillations induced by ketamine increase energy levels in sleep-wake related brain regions.

Neuronal signaling consumes much of the brain energy, mainly through the restoration of the membrane potential (MP) by ATP-consuming ionic pumps. We have reported that, compared with waking, ATP levels increase during the initial hours of natural slow-wave sleep, a time with prominent electroencephalogram (EEG) delta oscillations (0.5-4.5 Hz). We have hypothesized that there is a delta oscillation-ATP increase coupling, since, during delta waves, neurons exhibit a prolonged hyperpolarizing phase followed by a very brief phase of action potentials. However, direct proof of this hypothesis is lacking, and rapid changes in EEG/neuronal activity preclude measurement in the naturally sleeping brain. Thus, to induce a uniform state with pure delta oscillations and one previously shown to be accompanied by a similar pattern of neuronal activity during delta waves as natural sleep, we used ketamine-xylazine treatment in rats. We here report that, with this treatment, the high-energy molecules ATP and ADP increased in frontal and cingulate cortices, basal forebrain, and hippocampus compared with spontaneous waking. Moreover, the degree of ATP increase positively and significantly correlated with the degree of EEG delta activity. Supporting the hypothesis of decreased ATP consumption during delta activity, the ATP-consuming Na+-K+-ATPase mRNA levels were significantly decreased, whereas the mRNAs for the ATP-producing cytochrome c oxidase (COX) subunits COX III and COX IVa were unchanged. Taken together, these data support the hypothesis of a cortical delta oscillation-dependent reduction in ATP consumption, thus providing the brain with increased ATP availability, and likely occurring because of reduced Na+-K+-ATPase-related energy consumption.

Coronary angiography in noncompaction with and without neuromuscular disorders.

BACKGROUND AND AIMS: The prevalence of coronary pathologic findings in patients with left ventricular hypertrabeculation/noncompaction (LVHT) is unknown. The study in a cohort of consecutive LVHT patients aimed to assess how often coronary angiography (CA) had been performed, if clinical findings and prognosis differed between patients with and without CA, how often pathologic findings of the coronary arteries were found, and if there were differences between LVHT patients with and without coronary pathologic findings. METHODS AND RESULTS: Between 1995-2007 LVHT was diagnosed in 113 patients (mean age 53 years, 29% females, 67% neuromuscular disorders). CA had been performed in 52. Patients with CA had more exertional dyspnoea (79 vs. 61%, p < 0.05), angina pectoris (42 vs. 10%, p < 0.001), hypertension (44 vs. 25%, p < 0.05) and larger left ventricles (66 vs. 60 mm, p < 0.001) than patients without. No anomalously originating coronary arteries were found. Patients with coronary arteriosclerosis (n = 8) had more right-bundle-branch block than patients without (25 vs. 0%, p < 0.05). Mortality was 5.8% per year and did neither differ between patients with and without CA nor with and without coronary arteriosclerosis. CONCLUSIONS: Coronary arteriosclerosis is rarely associated with LVHT and does not seem to affect prognosis.

Cardiac and neuromuscular implications of left bundle branch block in left ventricular hypertrabeculation/noncompaction.

BACKGROUND: Left bundle branch block (LBB) is frequently found in left ventricular hypertrabeculation/noncompaction (LVHT). OBJECTIVES: To compare LVHT patients with and without LBB regarding LVHT location and extension, left ventricular function, symptoms, electrocardiographic findings, prevalence of neuromuscular disorders (NMDs) and mortality during follow-up. METHODS: The charts of patients who underwent transthoracic echocardiographic examination at the Krankenanstalt Rudolfstiftung (Wien, Austria) between June 1995 and November 2006 were examined. RESULTS: LVHT was diagnosed in 102 patients (30 women) with a mean (+/- SD) age of 53+/-16 years (range 14 to 94 years). A specific NMD was diagnosed in 21 patients and an NMD of unknown etiology was diagnosed in 47. The neurological investigation was normal in 14 patients and 20 patients refused the investigation. The 24 patients with LBB were older (61 versus 51 years of age; P<0.01), and suffered from exertional dyspnea (96% versus 59%; P<0.01) and heart failure (79% versus 46%; P<0.01) more often than patients without LBB. LBB patients had less frequent tall QRS complexes (8% versus 47%; P<0.01) and ST-T wave abnormalities (4% versus 50%; P<0.01) than patients without LBB. Patients with LBB had a larger left ventricular end-diastolic diameter (73 mm versus 61 mm; P<0.01), worse left ventricular fractional shortening (15% versus 26%; P<0.01) and more extensive LVHT (1.8 versus 1.5 ventricular segments; P<0.05). The prevalence of NMDs did not differ between patients with and without LBB. Survival did not differ between patients with and without LBB during follow-up. CONCLUSIONS: LBB is associated with increased age, decreased systolic function and increased extension of LVHT. Whether LBB is a prognostic factor in LVHT remains speculative.

AB0 system and D(Rh o) antigen frequencies in left ventricular hypertrabeculation/noncompaction in relation to cardiac and neuromuscular findings.

BACKGROUND: Left ventricular hypertrabeculation/noncompaction (LVHT) is a cardiac abnormality with suspected genetic background, characterized by trabeculations and intertrabecular recesses. Aim of the study in LVHT patients was to assess the AB0 system and D(Rh(o)) antigen frequencies, to look for an association between the prevalence of cardiac and neuromuscular abnormalities and to compare distribution of the AB0 system and D(Rh(o)) antigen frequencies with the Austrian general population. METHODS AND RESULTS: In 77/102 LVHT patients (75%) information about blood group could be obtained. There were no differences in the prevalence of clinical, electrocardiographic and echocardiographic findings and neuromuscular disorders between the AB0 blood groups. When comparing 67 LVHT patients who were D(Rh(o)) antigen positive with 10 patients who were D(Rh(o)) antigen negative, no differences could be found. Among LVHT patients, 0 D(Rh(o)) antigen positive was the most frequent (39%) followed by A D(Rh(o)) antigen positive (34%). In the Austrian general population A D(Rh(o)) antigen positive was the most frequent (33%) followed by 0 D(Rh(o)) antigen positive (30%). CONCLUSION: This study shows that LVHT patients do not differ according the blood groups, and that the distribution of blood groups is only minimally different between LVHT patients and the general Austrian population.