[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

[3H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [3H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [3H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [3H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [3H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [3H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided

Hyperoxic Treatment Induces Mesenchymal-to-Epithelial Transition in a Rat Adenocarcinoma Model

Tumor hypoxia is relevant for tumor growth, metabolism and epithelial-to-mesenchymal transition (EMT). We report that hyperbaric oxygen (HBO) treatment induced mesenchymal-to-epithelial transition (MET) in a dimetyl-α-benzantracene induced mammary rat adenocarcinoma model, and the MET was associated with extensive coordinated gene expression changes and less aggressive tumors. One group of tumor bearing rats was exposed to HBO (2 bar, pO2 = 2 bar, 4 exposures à 90 minutes), whereas the control group was housed under normal atmosphere (1 bar, pO2 = 0.2 bar). Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation, cell death, collagen fibrils and gene expression profile. Tumor growth was significantly reduced (∼16%) after HBO treatment compared to day 1 levels, whereas control tumors increased almost 100% in volume. Significant decreases in tumor cell proliferation, tumor blood vessels and collagen fibrils, together with an increase in cell death, are consistent with tumor growth reduction and tumor stroma influence after hyperoxic treatment. Gene expression profiling showed that HBO induced MET. In conclusion, hyperoxia induced MET with coordinated expression of gene modules involved in cell junctions and attachments together with a shift towards non-tumorigenic metabolism. This leads to more differentiated and less aggressive tumors, and indicates that oxygen per se might be an important factor in the “switches” of EMT and MET in vivo. HBO treatment also attenuated tumor growth and changed tumor stroma, by targeting the vascular system, having anti-proliferative and pro-apoptotic effects

Hyperbaric treatment for children with autism: a multicenter, randomized, double-blind, controlled trial

<p>Abstract</p> <p>Background</p> <p>Several uncontrolled studies of hyperbaric treatment in children with autism have reported clinical improvements; however, this treatment has not been evaluated to date with a controlled study. We performed a multicenter, randomized, double-blind, controlled trial to assess the efficacy of hyperbaric treatment in children with autism.</p> <p>Methods</p> <p>62 children with autism recruited from 6 centers, ages 2–7 years (mean 4.92 ± 1.21), were randomly assigned to 40 hourly treatments of either hyperbaric treatment at 1.3 atmosphere (atm) and 24% oxygen ("treatment group", n = 33) or slightly pressurized room air at 1.03 atm and 21% oxygen ("control group", n = 29). Outcome measures included Clinical Global Impression (CGI) scale, Aberrant Behavior Checklist (ABC), and Autism Treatment Evaluation Checklist (ATEC).</p> <p>Results</p> <p>After 40 sessions, mean physician CGI scores significantly improved in the treatment group compared to controls in overall functioning (p = 0.0008), receptive language (p < 0.0001), social interaction (p = 0.0473), and eye contact (p = 0.0102); 9/30 children (30%) in the treatment group were rated as "very much improved" or "much improved" compared to 2/26 (8%) of controls (p = 0.0471); 24/30 (80%) in the treatment group improved compared to 10/26 (38%) of controls (p = 0.0024). Mean parental CGI scores significantly improved in the treatment group compared to controls in overall functioning (p = 0.0336), receptive language (p = 0.0168), and eye contact (p = 0.0322). On the ABC, significant improvements were observed in the treatment group in total score, irritability, stereotypy, hyperactivity, and speech (p < 0.03 for each), but not in the control group. In the treatment group compared to the control group, mean changes on the ABC total score and subscales were similar except a greater number of children improved in irritability (p = 0.0311). On the ATEC, sensory/cognitive awareness significantly improved (p = 0.0367) in the treatment group compared to the control group. Post-hoc analysis indicated that children over age 5 and children with lower initial autism severity had the most robust improvements. Hyperbaric treatment was safe and well-tolerated.</p> <p>Conclusion</p> <p>Children with autism who received hyperbaric treatment at 1.3 atm and 24% oxygen for 40 hourly sessions had significant improvements in overall functioning, receptive language, social interaction, eye contact, and sensory/cognitive awareness compared to children who received slightly pressurized room air.</p> <p>Trial Registration</p> <p>clinicaltrials.gov NCT00335790</p

Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA system

Abstract Background A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody. Methods A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. Results A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches. Conclusions Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches

Cardiovascular Risk Reduction with Icosapent Ethyl for Hypertriglyceridemia

BACKGROUND Patients with elevated triglyceride levels are at increased risk for ischemic events. Icosapent ethyl, a highly purified eicosapentaenoic acid ethyl ester, lowers triglyceride levels, but data are needed to determine its effects on ischemic events. METHODS We performed a multicenter, randomized, double-blind, placebo-controlled trial involving patients with established cardiovascular disease or with diabetes and other risk factors, who had been receiving statin therapy and who had a fasting triglyceride level of 135 to 499 mg per deciliter (1.52 to 5.63 mmol per liter) and a low-density lipoprotein cholesterol level of 41 to 100 mg per deciliter (1.06 to 2.59 mmol per liter). The patients were randomly assigned to receive 2 g of icosapent ethyl twice daily (total daily dose, 4 g) or placebo. The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina. The key secondary end point was a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. RESULTS A total of 8179 patients were enrolled (70.7% for secondary prevention of cardiovascular events) and were followed for a median of 4.9 years. A primary end-point event occurred in 17.2% of the patients in the icosapent ethyl group, as compared with 22.0% of the patients in the placebo group (hazard ratio, 0.75; 95% confidence interval [CI], 0.68 to 0.83; P<0.001); the corresponding rates of the key secondary end point were 11.2% and 14.8% (hazard ratio, 0.74; 95% CI, 0.65 to 0.83; P<0.001). The rates of additional ischemic end points, as assessed according to a prespecified hierarchical schema, were significantly lower in the icosapent ethyl group than in the placebo group, including the rate of cardiovascular death (4.3% vs. 5.2%; hazard ratio, 0.80; 95% CI, 0.66 to 0.98; P=0.03). A larger percentage of patients in the icosapent ethyl group than in the placebo group were hospitalized for atrial fibrillation or flutter (3.1% vs. 2.1%, P=0.004). Serious bleeding events occurred in 2.7% of the patients in the icosapent ethyl group and in 2.1% in the placebo group (P=0.06). CONCLUSIONS Among patients with elevated triglyceride levels despite the use of statins, the risk of ischemic events, including cardiovascular death, was significantly lower among those who received 2 g of icosapent ethyl twice daily than among those who received placebo. (Funded by Amarin Pharma; REDUCE-IT ClinicalTrials.gov number, NCT01492361

Supplementary Material for: Elevated Triglycerides (≥150 mg/dL) and High Triglycerides (200–499 mg/dL) Are Significant Predictors of Hospitalization for New-Onset Kidney Disease: A Real-World Analysis of High-Risk Statin-Treated Patients

Background: Dyslipidemia in kidney disease (KD) involves increased levels of triglycerides (TG) and TG-rich lipoproteins, with only minor changes in low-density lipoprotein cholesterol. The increasing prevalence of diabetic KD and the shared atherogenic lipid profile between KD and diabetes underscore the importance of understanding dyslipidemia in these patients. Previous studies suggest an association between elevated TG and new-onset KD. Additional data are needed to better define the relationship between hypertriglyceridemia and new-onset KD. Objective: To evaluate the real-world impact of elevated and high TG on risk of KD in high-risk statin-treated patients. Methods: This retrospective administrative claims analysis of the Optum Research Database included statin-treated patients (age ≥45 years) with diabetes and/or atherosclerotic cardiovascular disease who were followed for ≥6 months. Cohorts included patients with elevated TG (≥150 mg/dL; n = 27,471) or high TG (200–499 mg/dL; subgroup of elevated TG cohort; n = 13,411), and a comparator cohort (TG 40 mg/dL; n = 32,506). The probability of hospitalization for new-onset KD was calculated post hoc from multivariate analyses controlled for patient characteristics and comorbidities using a Cox proportional hazards model. Results: The rate of hospitalization for new-onset KD was 31% higher in the elevated-TG cohort (hazard ratio [HR], 1.311; 95% confidence interval [CI], 1.228–1.401; p p Conclusions: In a real-world analysis of statin-treated patients with high cardiovascular risk, both elevated TG (≥150 mg/dL) and high TG (200–499 mg/dL) were significant predictors of hospitalization for new-onset KD, identifying hypertriglyceridemia as a potential KD risk factor