Neoadjuvant hormone therapy for radical prostate radiotherapy:bicalutamide monotherapy vs. luteinizing hormone-releasing hormone agonist monotherapy: a single-institution matched-pair analysis

The purpose of this study was to compare the prostate-specific antigen (PSA) response to either neoadjuvant bicalutamide (BC) monotherapy or neoadjuvant luteinizing hormone-releasing hormone agonist (LHRHa) monotherapy and the subsequent effect on biochemical failure-free survival (BFFS) in men receiving radical radiotherapy (RT) for localized prostate cancer.

Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

The Beta Agonist Lung Injury Trial Prevention:A Randomized Controlled Trial

Rationale: Experimental studies suggest that pretreatment with b-agonists might prevent acute lung injury (ALI).

Objectives: To determine if in adult patients undergoing elective esophagectomy, perioperative treatment with inhaled b-agonists effects the development of early ALI.

Methods:We conducted a randomized placebo-controlled trial in 12 UK centers (2008-2011). Adult patients undergoing elective esophagectomy were allocated to prerandomized, sequentially numbered treatment packs containing inhaled salmeterol (100 mg twice daily) or a matching placebo. Patients, clinicians, and researchers were masked to treatment allocation. The primary outcome was development of ALI within 72 hours of surgery. Secondary outcomes were ALI within 28 days, organ failure, adverse events, survival, and health-related quality of life. An exploratory substudy measured biomarkers of alveolar-capillary inflammation and injury.

Measurements and Main Results: A total of 179 patients were randomized to salmeterol and 183 to placebo. Baseline characteristics were similar. Treatment with salmeterol did not prevent early lung injury (32 [19.2%] of 168 vs. 27 [16.0%] of 170; odds ratio [OR], 1.25; 95% confidence interval [CI], 0.71-2.22). There was no difference in organ failure, survival, or health-related quality of life.Adverse events were less frequent in the salmeterol group (55 vs. 70; OR, 0.63; 95% CI, 0.39-0.99), predominantly because of a lower number of pneumonia (7 vs. 17; OR, 0.39; 95% CI, 0.16-0.96). Salmeterol reduced some biomarkers of alveolar inflammation and epithelial injury.

Conclusion: Perioperative treatment with inhaled salmeterol was well tolerated but did not prevent ALI.

Clinical trial registered with International Standard Randomized Controlled Trial Register (ISRCTN47481946) and European Union database of randomized Controlled Trials (EudraCT 2007-004096-19).Copyright © 2014 by the American Thoracic Society.

Internalization and desensitization of the human glucose-dependent-insulinotropic receptor is affected by N-terminal acetylation of the agonist

How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modelled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide.

Identification and validation of the dopamine agonist bromocriptine as a novel therapy for high-risk myelodysplastic syndromes and secondary acute myeloid leukemia

Myelodysplastic syndromes (MDS) represent a broad spectrum of diseases characterized by their clinical manifestation as one or more cytopenias, or a reduction in circulating blood cells. MDS is predominantly a disease of the elderly, with a median age in the UK of around 75. Approximately one third of MDS patients will develop secondary acute myeloid leukemia (sAML) that has a very poor prognosis. Unfortunately, most standard cytotoxic agents are often too toxic for older patients. This means there is a pressing unmet need for novel therapies that have fewer side effects to assist this vulnerable group. This challenge was tackled using bioinformatic analysis of available transcriptomic data to establish a gene-based signature of the development and progression of MDS. This signature was then used to identify novel therapeutic compounds via statistically-significant connectivity mapping. This approach suggested re-purposing an existing and widely-prescribed drug, bromocriptine as a novel potential therapy in these disease settings. This drug has shown selectivity for leukemic cells as well as synergy with current therapies.

Gingival fibroblasts respond to the TRPV1 agonist capsaicin

Background: The oral cavity is a frontline barrier which is often exposed to physical trauma and noxious substances, leading to pro-inflammatory responses designed to be protective in nature. The transient receptor potential (TRP) super family of ion channels is believed to play a critical role in sensory physiology, acting as transducers for thermal, mechanical and chemical stimuli. Our understanding of the role of TRP channel activation in gingival and periodontal inflammation is currently limited. Gingival fibroblasts are the most abundant structural cell in periodontal tissues and we hypothesised that they may have a role in the inflammatory response associated with TRP channel activation. Objectives: The present study was designed to determine whether the TRPV1 agonist capsaicin could elicit a pro-inflammatory response in gingival fibroblasts in vitro by up-regulation of interleukin-8 (IL-8) production. Methods: Gingival fibroblasts were derived by explant culture from surgical tissues following ethical approval. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) in 5% CO2. Following treatment of gingival fibroblasts with capsaicin, IL-8 levels were measured by ELISA. The potential cytotoxicity of capsaicin was determined by the MTT assay. Results: In gingival fibroblasts treated with the TRPV1 agonist capsaicin (10µM), IL-8 production was significantly increased compared with untreated control cells. Capsaicin was shown not to be toxic to gingival fibroblasts at the concentrations studied. Conclusion: The identification of factors that modulate pro-inflammatory cytokine production is important for our understanding of gingival and periodontal inflammation. This study reports for the first time that gingival fibroblasts respond to the TRPV1 agonist capsaicin by increased production of IL-8. Activation of TRPV1 on gingival fibroblasts could therefore have an important role in initiating and sustaining the inflammatory response associated with periodontal diseases

Ca2+- and volume-sensitive chloride currents are differentially regulated by agonists and store-operated Ca2+ entry.

Using patch-clamp and calcium imaging techniques, we characterized the effects of ATP and histamine on human keratinocytes. In the HaCaT cell line, both receptor agonists induced a transient elevation of [Ca2+]i in a Ca2+-free medium followed by a secondary [Ca2+]i rise upon Ca2+ readmission due to store-operated calcium entry (SOCE). In voltage-clamped cells, agonists activated two kinetically distinct currents, which showed differing voltage dependences and were identified as Ca2+-activated (ICl(Ca)) and volume-regulated (ICl, swell) chloride currents. NPPB and DIDS more efficiently inhibited ICl(Ca) and ICl, swell, respectively. Cell swelling caused by hypotonic solution invariably activated ICl, swell while regulatory volume decrease occurred in intact cells, as was found in flow cytometry experiments. The PLC inhibitor U-73122 blocked both agonist- and cell swelling–induced ICl, swell, while its inactive analogue U-73343 had no effect. ICl(Ca) could be activated by cytoplasmic calcium increase due to thapsigargin (TG)-induced SOCE as well as by buffering [Ca2+]i in the pipette solution at 500 nM. In contrast, ICl, swell could be directly activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a cell-permeable DAG analogue, but neither by InsP3 infusion nor by the cytoplasmic calcium increase. PKC also had no role in its regulation. Agonists, OAG, and cell swelling induced ICl, swell in a nonadditive manner, suggesting their convergence on a common pathway. ICl, swell and ICl(Ca) showed only a limited overlap (i.e., simultaneous activation), although various maneuvers were able to induce these currents sequentially in the same cell. TG-induced SOCE strongly potentiated ICl(Ca), but abolished ICl, swell, thereby providing a clue for this paradox. Thus, we have established for the first time using a keratinocyte model that ICl, swell can be physiologically activated under isotonic conditions by receptors coupled to the phosphoinositide pathway. These results also suggest a novel function for SOCE, which can operate as a "selection" switch between closely localized channels.

Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes.

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Differential effects of the CCKA receptor ligands PD-140, 548 and A-71623 on latent inhibition in the rat.

Latent inhibition (LI) is a behavioural paradigm in which repeated exposure to a stimulus without consequence inhibits the formation of any new associations with that stimulus. To the extent that LI reflects a process of learning to ignore irrelevant stimuli, disrupted LI has been suggested as an animal model for the attentional deficits observed in schizophrenia. The antipsychotic potential of cholecystokinin (CCK) stems from its colocalization with dopamine (DA) in the mesolimbic pathway, where it demonstrates both excitatory and inhibitory effects on dopaminergic activity. This may be explained by mediation through different receptor subtypes. A variety of hypotheses has emerged regarding the potential clinical application of subtype-selective CCK-based drugs. The present experiments examined the effects on LI of two selective CCKA ligands: PD-140,548 (a CCKA antagonist, Experiment 1: 0.001, 0.01, and 0.1 mg/kg) and A-71623 (a CCKA agonist, Experiment 2: 0.02, 0.05, and 0.1 mg/kg). In both experiments, the effects of haloperidol (0.1 mg/kg) were also investigated. Animals receiving 0.1 mg/kg of haloperidol or 0.001 or 0.1 mg/kg (but not 0.01 mg/kg) of PD-140,548 treated the preexposed stimulus as irrelevant after a low number of preexposures. In contrast, no facilitatory effect on LI was detectable at any of the A-71623 doses. The finding that A-71623 failed to enhance LI indicates that it is unlikely that this compound would have any antipsychotic effect within the clinical setting. Considering the facilitatory effect exerted by PD-140,548 on LI, it is probable that the inhibition of CCK activity might prove a more promising strategy for the pharmacological treatment of schizophrenia.

Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

An early and critical event in beta2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent beta2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) beta2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3´,5´-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected beta2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in beta2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.