Epoxy Resins for Flooring Applications, an Optimal Host for Recycling Deactivated Cement Asbestos.

Cement asbestos slates, commonly known as Eternit® and still abundant in private and public buildings, were deactivated through a thermal process. The resulting deactivated cement asbestos powder (DCAP), a mixture of Ca-Mg-Al silicates and glass, was compounded with Pavatekno Gold 200 (PT) and Pavafloor H200/E (PF), two different epoxy resins (bisphenol A epichlorohydrin) for flooring applications. The addition of the DCAP filler to the PF samples causes a slight but acceptable decrease in the relevant mechanical properties (compressive, tensile, and flexural strengths) upon increasing DCAP content. The addition of the DCAP filler to pure epoxy (PT resin) causes a slight decrease in the tensile and flexural strengths with increasing DCAP content, while the compressive strength is almost unaffected, and the Shore hardness increases. The main mechanical properties of the PT samples are significantly better than those of the filler-bearing sample of normal production. Overall, these results suggest that DCAP can be advantageously used as filler in addition to, or in substitution for, commercial barite. In particular, the sample with 20 wt% of DCAP is the best performing in terms of compressive, tensile, and flexural strengths, whereas the sample with 30 wt% of DCAP shows the highest Shore hardness, which is an important property to be considered in flooring applications.

Implementation of a Commercial Deep Learning-Based Auto Segmentation Software in Radiotherapy: Evaluation of Effectiveness and Impact on Workflow.

Proper delineation of both target volumes and organs at risk is a crucial step in the radiation therapy workflow. This process is normally carried out manually by medical doctors, hence demanding timewise. To improve efficiency, auto-contouring methods have been proposed. We assessed a specific commercial software to investigate its impact on the radiotherapy workflow on four specific disease sites: head and neck, prostate, breast, and rectum. For the present study, we used a commercial deep learning-based auto-segmentation software, namely Limbus Contour (LC), Version 1.5.0 (Limbus AI Inc., Regina, SK, Canada). The software uses deep convolutional neural network models based on a U-net architecture, specific for each structure. Manual and automatic segmentation were compared on disease-specific organs at risk. Contouring time, geometrical performance (volume variation, Dice Similarity Coefficient-DSC, and center of mass shift), and dosimetric impact (DVH differences) were evaluated. With respect to time savings, the maximum advantage was seen in the setting of head and neck cancer with a 65%-time reduction. The average DSC was 0.72. The best agreement was found for lungs. Good results were highlighted for bladder, heart, and femoral heads. The most relevant dosimetric difference was in the rectal cancer case, where the mean volume covered by the 45 Gy isodose was 10.4 cm3 for manual contouring and 289.4 cm3 for automatic segmentation. Automatic contouring was able to significantly reduce the time required in the procedure, simplifying the workflow, and reducing interobserver variability. Its implementation was able to improve the radiation therapy workflow in our department.

Hypofractionation and Concomitant Boost in Ductal Carcinoma In Situ (DCIS): Analysis of a Prospective Case Series with Long-Term Follow-Up.

We previously reported on a cohort of breast cancer patients affected with ductal carcinoma in situ (DCIS) that were treated with breast conservative surgery and hypofractionated whole-breast radiotherapy with a concomitant boost to the lumpectomy cavity. We now report on the long-term results of the oncological and toxicity outcomes, at a median follow-up of 11.2 years. We also include an analysis of the predictive factors for local recurrence (LR). Eighty-two patients with long-term observation were considered for this report. All received hypofractionated post-operative radiotherapy with a concomitant boost (45 Gy/20 fractions to the whole breast and 50 Gy/20 fractions to the lumpectomy cavity). We report on LC rates at 5 and 10 years, overall survival (OS), and breast-cancer-specific survival (BCSS), employing the Kaplan-Meier method. Cox proportional regression analysis was used to determine the role of selected clinical parameters on the risk of local recurrence, by the univariate and multivariate models. After a median follow-up of 11.2 years (range 5-15 years), 9 pts (11%) developed LR. The LR rates at 5 years and 10 years were 2.4% and 8.2%, respectively. The 5- and 10-year overall survival rates were 98.8% and 91.6%, respectively. The 5- and 10-year breast-cancer-specific survival rates were 100.0% and 99.0%. Late skin and subcutaneous toxicities were generally mild, and cosmetic results were good-excellent for most patients. For the univariate regression analysis, ER positive status (HR; 95% CI, p = 0.021), PgR positive status (HR; 95% CI, p = 0.012), and the aggregate data of positive hormonal status (HR; 95% CI, p = 0.021) were inversely correlated to LR risk. Conversely, a high tumor grade (G3) was directly correlated with the risk of LR (HR; 95% CI, p = 0.048). For the multivariate regression analysis, a high tumor grade (G3) confirmed its negative impact on LR (HR 0.40; 95% CI 0.19-0.75, p = 0.047). Our long-term data demonstrate hypofractionated whole-breast radiotherapy with a concomitant boost to be feasable, effective, and tolerable. Our experience suggests positive hormonal status to be protective with respect to LR risk. A high tumor grade is a risk factor for LR.

Effect of delaying surgery by more than 10 weeks after neoadjuvant therapy in rectal cancer: a single institution experience.

The optimal timing of surgery after neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer is still controversial. Aim of this study was to evaluate the effect of increasing time interval between the end of CRT and surgery on pathological outcomes. This is a retrospective analysis on 114 patients treated with long-course neoadjuvant RT with or without chemotherapy between January 2005 and September 2020. 43 patients underwent surgery within 10 weeks from the end of CRT (1st group), whereas 71 patients underwent total mesorectal excision with a time interval equal or greater than 10 weeks (2nd group). Primary endpoint was pCR (pathological complete response). Secondary endpoints were near pCR (ypT0-1 N0), tumor downstaging (ypT less than cT), nodal downstaging (ypN less than cN), and overall response comparing clinical with pathological TN stage. Overall, the pCR rate was 8.8%, whereas we observed no significantly difference in primary endpoint between the two groups. Considering near pCR, a trend toward significant difference in favor of 2nd group was seen (p = 0.072). Tumor and nodal downstaging rates were 39.5%, 41.9%, 59.2%, and 56.3% in the 1st and 2nd group, respectively, with a statistically significant difference for T category (p = 0.042). Overall response rates (TN stage) showed a trend toward significant difference in favor of patients of the ≥ 10 week group (p = 0.059). Our study suggests that a prolonged time interval between the end of CRT and surgery (≥ 10 weeks) increases pathological response rates.

Delta4 Discover transmission detector: A comprehensive characterization for in-vivo VMAT monitoring.

OBJECTIVE: To investigate the dosimetric behaviour, influence on photon beam fluence and error detection capability of Delta4 Discover transmission detector. METHODS: The transmission detector (TRD) was characterized on a TrueBeam linear accelerator with 6 MV beams. Linearity, reproducibility and dose rate dependence were investigated. The effect on photon beam fluence was evaluated in terms of beam profiles, percentage depth dose, transmission factor and surface dose for different open field sizes. The transmission factor of the 10x10 cm2 field was entered in the TPS's configuration and its correct use in the dose calculation was verified recalculating 17 clinical IMRT/VMAT plans. Surface dose was measured for 20 IMRT fields. The capability to detect different delivery errors was investigated evaluating dose gamma index, MLC gamma index and leaf position of 15 manually modified VMAT plans. RESULTS: TRD showed a linear dependence on MU. No dose rate dependence was observed. Short-term and long-term reproducibility were within 0.1% and 0.5%. The presence of the TRD did not significantly affect PDDs and profiles. The transmission factor of the 10x10 cm2 field size was 0.985 and 0.983, for FF and FFF beams respectively. The 17 recalculated plans met our clinical gamma-index passing rate, confirming the correct use of the transmission factor by the TPS. The surface dose differences for the open fields increase for shorter SSDs and greater field size. Differences in surface dose for the IMRT beams were less than 2%. Output variation ≥2%, collimator angle variations within 0.3°, gantry angle errors of 1°, jaw tracking and leaf position errors were detected. CONCLUSIONS: Delta4 Discover shows good linearity and reproducibility, is not dependent on dose rate and does not affect beam quality and dose profiles. It is also capable to detect dosimetric and geometric errors and therefore it is suitable for monitoring VMAT delivery.

Moderately Hypofractionated Radiotherapy with Simultaneous Integrated Boost in Prostate Cancer: A Comparative Study with Conventionally Fractionated Radiation.

BACKGROUND: To report 5-year clinical outcomes and toxicity in organ-confined prostate cancer (PCa) for low- and intermediate-risk patients treated with a moderately hypofractionated schedule of radiotherapy (RT) delivered with simultaneous integrated boost (SIB) compared to a conventionally fractionated RT regimen. METHODS: Data of 384 patients with PCa treated between August 2006 and June 2017 were retrospectively reviewed. The treatment schedule consisted of hypofractionated RT (HYPO FR) with SIB up to 70 Gy to the prostate gland and 63 Gy to seminal vesicles delivered in 28 fractions or in conventionally fractionated RT (CONV FR) up to a total dose of 80 Gy in 40 fractions. Patient allocation to treatment was based on the time period considered. For intermediate-risk patients, androgen deprivation was given for a median duration of 6 months. The 5-year biochemical relapse-free survival (bRFS), cancer-specific survival (CSS), and overall survival (OS) were assessed. Furthermore, we evaluated gastrointestinal (GI) and genitourinary (GU) toxicities. Uni- and multivariate Cox regression analyses were used to test the impact of clinical variables on both outcome and toxicity. RESULTS: A total of 198 patients was treated with hypofractionated RT and 186 with the conventional schedule. At a median follow-up of 5 years, no significant differences were observed in terms of GI toxicity and outcome between the two groups. Early GU toxicity was significantly increased in HYPO FR, while late GU toxicity was significantly higher in CONV FR. In HYPO FR, a biochemical relapse occurred in 12 patients (6.1%), and 9 patients (4.5%) reported a clinical relapse (4 local, 2 locoregional, and 3 systemic recurrence). In CONV FR, 15 patients (8.1%) experienced a biochemical relapse and 11 patients (5.9%) showed a clinical relapse (5 local, 4 locoregional, and 3 systemic recurrences). Early grades 1-2 GU and GI toxicities were observed in 60 (30.3%) and 37 (18.7%) patients, respectively, in the hypofractionated group and in 33 (17.7%) and 27 (14.5%) patients, respectively, in the conventionally fractionated RT group. Late GU and GI toxicities occurred in 1 (0.51%) and 8 (4.1%) patients, respectively, in HYPO FR. In CONV FR, 5 (2.7%) and 6 (3.2%) patients experienced late GU and GI toxicities, respectively. The 5-year OS, bRFS, and CSS were 98.9%, 94.1%, and 99.5%, respectively, in HYPO FR, and 94.5%, 92.1%, and 99.0%, respectively, in CONV FR. CONCLUSIONS: Results obtained in this study showed that moderately hypofractionated RT employing SIB can be an effective approach providing valuable clinical outcomes with an acceptable toxicity profile.

[Giant parathyroid adenoma: a rare cause of severe hypercalcemia].

We report the case of a 37-year-old woman that developed severe hypercalcemia due to a parathyroid gland mass. After the initial medical treatment, only a minimal reduction of calcemia was observed and her clinical condition worsened; thus, she required continuous renal replacement therapy (CRRT) that resulted in the normalization of calcium serum level. She then underwent a left thyroid lobectomy with exeresis of the associated parathyroid glands; the histological diagnosis revealed a giant parathyroid adenoma (GPA). CRRT, initially recommended only in case of severe refractory hypercalcemia poorly responsive to pharmacological approaches, is now being evaluated in the first line treatment of life-threatening cases, with or without associated acute kidney injury (AKI).

Pancreatitis Is a Silent Killer in Peritoneal Dialysis With Difficult Diagnostic Approach.

Until 2018, 236 cases of acute pancreatitis have been reported in patients who underwent peritoneal dialysis. Here, we presented a patient with double renal transplantation with chronic renal failure, under renal replacement therapy by peritoneal dialysis, who developed acute pancreatitis with abdominal pain, nausea, vomiting, leukocytosis with neutrophil left shift which is complicated by pancreatic pseudocyst, candida peritonitis, fungal sepsis, overlapping of Acinetobacter baumannii sepsis, and pneumonitis. After the percutaneous cystogastrostomy drainage of pancreatic pseudocyst, changes from peritoneal dialysis to hemodialysis, various thoracentesis, and polyantibiotics therapy, the resolution of the sepsis state was seen. The particular aspect of our case is the various comorbidity risks, severe pancreatitis associated with candida and A baumannii sepsis, and treatment strategy that lead to heal this kind of the high mortality rate condition.

Correction to: Ten-year results of accelerated hypofractionated adjuvant whole-breast radiation with concomitant boost to the lumpectomy cavity after conserving surgery for early breast cancer.

An error inadvertently occurred in the discussion of the original publication when citing the local relapse rates of the EORTC 22881-10882 trial ('boost vs no boost trial').

Ten-year results of accelerated hypofractionated adjuvant whole-breast radiation with concomitant boost to the lumpectomy cavity after conserving surgery for early breast cancer.

Accelerated hypofractionated whole-breast radiotherapy (WBRT) is considered a standard therapeutic option for early breast cancer (EBC) in the postoperative setting after breast conservation (BCS). A boost to the lumpectomy cavity may further increase local control. We herein report on the 10-year results of a series of EBC patients treated after BCS with hypofractionated WBRT with a concomitant photon boost to the surgical bed over 4 weeks. Between 2005 and 2007, 178 EBC patients were treated with a basic course of radiotherapy consisting of 45 Gy to the whole breast in 20 fractions (2.25 Gy daily) with an additional boost dose of 0.25 Gy delivered concomitantly to the lumpectomy cavity, for an additional dose of 5 Gy. Median follow-up period was 117 months. At 10-year, overall, cancer-specific, disease-free survival and local control were 92.2% (95% CI 88.7-93.4%), 99.2% (95% CI 96.7-99.7%), 95.5% (95% CI 91.2-97.2%) and 97.3% (95% CI 94.5-98.9%), respectively. Only eight patients recurred. Four in-breast recurrences, two axillary node relapses and two metastatic localizations were observed. Fourteen patients died during the observation period due to other causes while breast cancer-related deaths were eight. At last follow-up, ≥G2 fibrosis and telangiectasia were seen in 7% and 5% of patients. No major lung and heart toxicities were observed. Cosmetic results were excellent/good in 87.8% of patients and fair/poor in 12.2%. Hypofractionated WBRT with concomitant boost to the lumpectomy cavity after BCS in EBC led to consistent clinical results at 10 years. Hence, it can be considered a valid treatment option in this setting.

No increase in toxicity of pelvic irradiation when intensity modulation is employed: clinical and dosimetric data of 208 patients treated with post-prostatectomy radiotherapy.

OBJECTIVE: To compare the toxicity of image-guided intensity-modulated radiotherapy (IG-IMRT) to the pelvis or prostate bed (PB) only. To test the hypothesis that the potentially injurious effect of pelvic irradiation can be counterbalanced by reduced irradiated normal tissue volume using IG-IMRT. METHODS: Between February 2010 and February 2012, 208 patients with prostate cancer were treated with adjuvant or salvage IG-IMRT to the PB (102 patients, Group PB) or the pelvis and prostate bed (P) (106 patients, Group P). The Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer criteria were used to evaluate toxicity. RESULTS: Median follow-up was 27 months. Toxicity G ≥ 2 in Group PB: in the bowel acute and late toxicities were 11.8% and 10%, respectively; urinary acute and late toxicities were 10.8% and 15%, respectively. Toxicity G ≥ 2 in Group P: in the bowel acute and late toxicities were both 13.2%; urinary acute and late toxicities were 13.2% and 15.1%, respectively. No statistical difference in acute or late toxicity between the groups was found (bowel: p = 0.23 and p = 0.89 for acute and late toxicity, respectively; urinary: p = 0.39 and p = 0.66 for acute and late toxicity, respectively). Of the clinical variables, only previous abdominal surgery was correlated with acute bowel toxicity. Dosimetric parameters that correlated with bowel toxicity were identified. CONCLUSION: The toxicity rates were low and similar in both groups, suggesting that IG-IMRT allows for a safe post-operative irradiation of larger volumes. Further investigation is warranted to exclude bias owing to non-randomized character of the study. ADVANCES IN KNOWLEDGE: Our report shows that modern radiotherapy technology and careful planning allow maintaining the toxicity of pelvic lymph node treatment at the acceptable level, as it is in the case of PB radiotherapy.

Neutrophil Gelatinase Associated Lipocalin Is an Early and Accurate Biomarker of Graft Function and Tissue Regeneration in Kidney Transplantation from Extended Criteria Donors.

BACKGROUND: Delayed graft function (DGF) is an early complication of kidney transplantation (KT) associated with increased risk of early loss of graft function. DGF increases using kidneys from extended criteria donors (ECD). NGAL is a 25KDa protein proposed as biomarker of acute kidney injury. The aim of this study was to investigate the role of NGAL as an early and accurate indicator of DGF and Tacrolimus (Tac) toxicity and as a mediator of tissue regeneration in KT from ECD. METHODS: We evaluated plasma levels of NGAL in 50 KT patients from ECD in the first 4 days after surgery or after Tac introduction. RESULTS: Plasma levels of NGAL at day 1 were significantly higher in DGF group. In the non DGF group, NGAL discriminated between slow or immediate graft function and decreased more rapidly than serum creatinine. NGAL increased after Tac introduction, suggesting a role as marker of drug toxicity. In vitro, hypoxia and Tac induced NGAL release from tubular epithelial cells (TEC) favoring an autocrine loop that sustains proliferation and inhibits apoptosis (decrease of caspases and Bax/Bcl-2 ratio). CONCLUSIONS: NGAL is an early and accurate biomarker of graft function in KT from ECD favoring TEC regeneration after ischemic and nephrotoxic injury.

[Candida peritonitis and sepsis due to Acinetobacter baumannii in peritoneal dialysis: an association with prognosis not always unfavourable]. / Peritonite da Candida e sepsi da Acinetobacter baumannii in dialisi peritoneale: un'associazione con prognosi non sempre infausta.

Fungal infections have a high incidence in patients receiving peritoneal dialysis. (1)Peritoneal dialysis is often complicated by peritonitis which has only minimally mycotic etiology, but nonetheless it is associated with 15-45% mortality (8). The opportunistic pathogens such as Candida can cause infection in immunocompromised conditions. Even the Acinetobacter tends to infect immunocompromised individuals and it has the same risk factors for infection as Candida: immunosuppression, malignancy, HIV positivity and all the other conditions of immunosuppression, central venous catheterization, mechanical ventilation and prolonged antibiotic therapy. The sepsis by Acinetobacter predicts a negative prognosis with the mortality rate between 20 to 60% (12), especially in cases of isolation of multi-resistant germs. We present a case report of a CKD patient undergoing peritoneal dialysis therapy who was hospitalized for acute pancreatitis, later complicated by the development of pancreatic pseudocysts, C. albicans peritonitis with hematologic spread of the fungus, superimposed Acinetobacter baumannii sepsis and pneumonia. She has been subjected to percutaneous drainage of pseudocysts, to switch from peritoneal dialysis to hemodialysis, to various evacuative thoracentesis, and to polymicrobial therapy (meropenem, teicoplanina, tigeciclina, linezolid, colimicina, fluconazolo, etc.) that allowed the resolution of sepsis. The peculiarity of this case is represented by the numerous morbidity that the patient developed simultaneously, with the genesis of a complex clinical picture, by the combination of infections due to Candida albicans and Acinetobacter baumannii. Successful treatment strategies allowed to fight and cure a medical condition associated with a high mortality rate.

Interaction between systemic inflammation and renal tubular epithelial cells.

Systemic inflammation is known to target tubular epithelial cells (TECs), leading to acute kidney injury. Tubular cells have been implicated in the response to inflammatory mediators in ischaemic and septic renal damage. Moreover, loss of tubular cells by apoptosis or epithelial-to-mesenchymal transition may ingenerate conditions that lead to progression towards chronic kidney disease. On the other hand, TECs may actively contribute to the production of inflammatory mediators that may propagate the injury locally or in distant organs. In the present review, we discuss the tubular cell response and its contribution to systemic inflammation.

What is the price of functional surgical organ preservation in local-regionally advanced supraglottic cancer? Long-term outcome for partial laryngectomy followed by radiotherapy in 32 patients.

AIMS AND BACKGROUND: To achieve the goal of organ preservation, both a chemoradiotherapy and a conservative surgical approach can be proposed. The aim of the study was to review all patients treated in our Institute with conservative surgery and postoperative radiotherapy for locally advanced supraglottic tumor. METHODS AND STUDY DESIGN: A retrospective analysis of 32 patients treated between 2000 and 2010 was performed. Overall survival, disease-free survival and late laryngeal toxicity were evaluated. The impact of surgical procedures, radiotherapy characteristics and addition of chemotherapy on late laryngeal toxicity was studied. RESULTS: The median follow-up was 38 months. Overall survival and disease-free survival at 5 years were 73% and 66%, respectively. Three (9%) patients experienced local recurrence (after 22, 25 and 40 months, respectively) and were treated with total laryngectomy. The larynx preservation rate was 93%. Severe treatment-related late laryngeal toxicity (grade 3 and 4 laryngeal edema, laryngeal stenosis, presence of tracheotomy at last follow-up because of treatment-related toxicity, and the need for enteral nutrition) was experienced by 34% of patients. The functional larynx preservation rate was 81%. The statistically significant risk factors for severe late toxicity were: female gender, extension of the surgical procedure, removal of one arytenoid and association with concomitant chemotherapy. CONCLUSIONS: We confirmed literature data on the feasibility and efficacy of a surgical organ preservation strategy. However, the high incidence of severe late toxicity requires further studies to improve patient selection and to reduce side effects.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.

Functional characterization of two isoforms of the P2Y-like receptor GPR17: [35S]GTPgammaS binding and electrophysiological studies in 1321N1 cells.

The previously "orphan" G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a "short" and a "long" receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer NH(2) terminus. The short isoform has been recently "deorphanized," revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [35S]GTPgammaS binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to K+ channels. [35S]GTPgammaS binding in long-GPR17-expressing 1321N1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LTC4 > LTD4), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATPgammaS also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, Gq proteins. UDP-glucose and LTD(4) also induced increases in overall outward K+ currents, which were antagonized by the purinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward K+ currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury.

Opposite effects of uracil and adenine nucleotides on the survival of murine cardiomyocytes.

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor.

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors.

Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y(1,2,4,6,11,12,13,14) receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 microM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 microM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y(1,2,4,6) receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 microM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 microM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.