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Background and Purpose: ρ-Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α(1) (A) -adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ-Da1a on prostatic function, both in vivo and in vitro.Experimental Approach: ρ-Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α(1) (A) -adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ-Da1a and tamsulosin on phenylephrine (PHE)-induced increases in intra-urethral (IUP) and arterial pressures (AP) in anaesthetized rats, following i.v. or p.o. administration.Key Results: On COS cells expressing human α(1) (A) -adrenoceptors and on human prostatic strips, ρ-Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ-Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pK(B) values for tamsulosin and ρ-Da1a for this effect were similar. With regards to AP, ρ-Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 μg·kg(-1) ), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 μg·kg(-1) .Conclusions and Implications: ρ-Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ-Da1a is more uroselective than tamsulosin. ρ-Da1a is the most selective peptidic antagonist for α(1A) -adenoceptors identified to date and could be a new treatment for various urological diseases. [ABSTRACT FROM AUTHOR]

Background and Purpose ρ- Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α1 A-adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ- Da1a on prostatic function, both in vivo and in vitro. Experimental Approach ρ- Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α1 A-adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ- Da1a and tamsulosin on phenylephrine ( PHE)-induced increases in intra-urethral ( IUP) and arterial pressures ( AP) in anaesthetized rats, following i.v. or p.o. administration. Key Results On COS cells expressing human α1 A-adrenoceptors and on human prostatic strips, ρ- Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ- Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pKB values for tamsulosin and ρ- Da1a for this effect were similar. With regards to AP, ρ- Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 μg·kg−1), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 μg·kg−1. Conclusions and Implications ρ- Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ- Da1a is more uroselective than tamsulosin. ρ- Da1a is the most selective peptidic antagonist for α 1A-adenoceptors identified to date and could be a new treatment for various urological diseases. [ABSTRACT FROM AUTHOR]

1 In order to compare the β2- and β3-udrenocepior (β-AR) desensitisation process in human near-term myomelrium. we examined the influence of a pretreatment of myomelrial strips with either a β2 or a β3-AR agonist (salbutamol or SR 59119A. respectively, both at 10μM. for 5 and 15h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production indueed by these agonists. 2 To assess some of the mechanisms potentially implicated in the β-AR desensitisation process, we studied the influence of such treatment on the number of β2- and β3-AR binding siies. the β2- and β3,-AR transeripls expression and the phosphudie-uerase 4 (PDE4) activity. 3 Salbutamol. but not SR 59119A. concentration-response curve (CRC) was shifted by a 15h Milhiitamol preincubation, with a signillcant difference in -log EC20 (6.31±0.13 vs 5.58±0.24, for control and I5h salhutamol prelrealment, respectively P<0.05). Neither salbutamol nor SR 59119A CRCs were modified after a 15 h preincubation with SR 59119A. 4 A 15 h exposure of myometrial strips to salbutamol signitlcaiuK reduced the salbutamol-induced (0.60± 0.26 vs l.54±0.24pmolmg−1 prolein. <0.05). but not the SR 59t 19A-induced. cAMP production. No decrease in cAMP production was observed after a 15 h SR 59119A exposure. 5 A IS fa salbutamol exposure of myometrial strips significantly reduced the β2- but not the β3-AR binding site density, whereas no decrease in the number of β2 and β3-AR binding sites was observed after a 15h SR 59119A treatment. 6 Neither PDE4 activity nor the β2- and β3-AR niRNA expression levels were affected by salbulamol or SR 59119A treatments. 7 Our results indicate that β3-AR, but not β2-AR, are resistant to the agonist-induced desensitisation. In our model. β2-AR desensitisalion is mediated by a decreased number of β2-AR that was not explained by transcriptional regulation of the receptor. [ABSTRACT FROM AUTHOR]

Hypoxia, IL-1β production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1β production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1β released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.

Neuronal voltage-gated potassium channels, K V 7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K V 7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K V 7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K V 7 isoforms was investigated in human detrusor tissue using a panel of K V 7 openers with distinct activity profiles among K V 7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K V 7.4 did not reduce detrusor contraction. This may suggest that the homomeric K V 7.4 channel plays a less significant role in bladder contraction and further investigation is needed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

NRLP3 inflammasome is a protein complex involved in the maturation of IL1β. In the onset of type 1 diabetes as well as in islet transplantation, IL-1β is one of the cytokines involved in the recruitment of immune cells in islets and eventually in islet destruction. Whether IL-1β is produced by islet cells is still under debate and NLRP3 inflammasome-dependent IL-1β production has not been yet determined in human islets. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets. Human islets were stimulated with LPS and successively with ATP (LPS + ATP) in the presence or absence of the inflammasome inhibitor glyburide. Islets were also incubated in hypoxic or normoxic conditions for 24 h in the presence or absence of glyburide. Then, IL1B and NLRP3 expression was studied by real time PCR, protein expression by western blot, protein localization by immunofluorescence and protein secretion by ELISA. LPS + ATP increased gene expression of NRLP3 and IL1B. Glyburide partially prevented this effect. IL-1β protein was localized in β and non-β cells. Moreover, LPS + ATP increased IL-1β protein expression and production, which were prevented by glyburide. Hypoxia increased gene expression of NRLP3 and IL1B and induced IL-1β and caspase-1 production. Finally, hypoxia-induced cell death which was not prevented by inhibition of NLRP3 inflammasome. NRLP3 inflammasome is expressed and plays a role in IL-1β production by human islets. By contrast, NRLP3 inflammasome activation is not involved in islet cell death induced by hypoxia.

T lymphocyte alterations are central to sepsis pathophysiology, whereas related mechanisms remain poorly understood. We hypothesized that metabolic alterations could play a role in sepsis-induced T lymphocyte dysfunction. Samples from septic shock patients were obtained at day 3 and compared with those from healthy donors. T cell metabolic status was evaluated in the basal condition and after T cell stimulation. We observed that basal metabolic content measured in lymphocytes by nuclear magnetic resonance spectroscopy was altered in septic patients. Basal ATP concentration, oxidative phosphorylation (OXPHOS), and glycolysis pathways in T cells were decreased as well. After stimulation, T lymphocytes from patients failed to induce glycolysis, OXPHOS, ATP production, GLUT1 expression, glucose entry, and proliferation to similar levels as controls. This was associated with significantly altered mTOR, but not Akt or HIF-1α, activation and only minor AMPKα phosphorylation dysfunction. IL-7 treatment improved mTOR activation, GLUT1 expression, and glucose entry in septic patients' T lymphocytes, leading to their enhanced proliferation. mTOR activation was central to this process, because rapamycin systematically inhibited the beneficial effect of recombinant human IL-7. We demonstrate the central role of immunometabolism and, in particular, mTOR alterations in the pathophysiology of sepsis-induced T cell alterations. Our results support the rationale for targeting metabolism in sepsis with recombinant human IL-7 as a treatment option., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Septic patients develop immune dysfunctions, the intensities and durations of which are associated with deleterious outcomes. LILRB2 (leukocyte immunoglobulin-like receptors subfamily B, member 2), an inhibitory member of the LILR family of receptors, is known for its immunoregulatory properties. In a microarray study, we identified LILRB2 as an upregulated gene in septic shock patients. On monocytes primed with LPS ex vivo, LILRB2 mRNA and protein expressions were dose-dependently downregulated and subsequently highly upregulated versus non-stimulated cells. This is concordant with clinical data, since both LILRB2 mRNA and protein expressions were significantly increased in septic shock patients at day 3. In a cohort of more than 700 patients, only after septic shock were LILRB2 mRNA levels increased compared with non-infected or less severely infected patients. This was preceded by a phase of downregulated mRNA expression during the first hours after septic shock. Interestingly, the intensity of this decrease was associated with increased risk of death after septic shock. LILRB2 protein and mRNA expressions are deregulated on monocytes after septic shock and this can be reproduced ex vivo after LPS challenge. Considering LILRB2 inhibitory properties, we can hypothesize that LILRB2 may participate in the altered immune response after septic shock., (Copyright © 2017. Published by Elsevier Inc.)

Purpose: The aim of this study was to assess the potential involvement of a specific subtype of 5-hydroxytryptamine (5-HT), 5HT 2 receptors in neurally-induced contractions of the human detrusor., Methods: Contractile responses to electrical field stimulation (EFS) were examined in human isolated urinary bladder muscle strips. The potentiation of EFS-induced detrusor contraction was examined by adding cumulative concentrations of a 5-HT and 5-HT 2 receptor agonist, α-methyl-serotonin (α-Me-5-HT) (1nM-100μM) in the presence or absence of a 5-HT 2 antagonist, ketanserin (5-HT 2A >5-HT 2C ) or naftopidil (5-HT 2B >5-HT 2A ) (0.3-3μM)., Results: 5-HT and α-Me-5-HT potentiated EFS-induced contraction with a maximal effect (E max ) of 37.6% and 38.6%, respectively, and with pEC 50 (negative logarithm of the concentration required for a half-maximal response to an agonist) values of 8.3 and 6.8, respectively. Neither ketanserin nor naftopidil at any concentration produced a rightward displacement of the α-Me-5-HT concentration response curve. Instead, the E max of α-Me-5-HT increased in the presence of ketanserin at 0.3-1μM and in the presence of naftopidil at 1μM to 51% and 56%, respectively, while the E max in the presence of vehicle alone was 36%. The highest concentration (3μM) of either drug, however, fully reversed the enhancement., Conclusions: The potentiating effect of α-Me-5-HT on neurally-induced contraction of human urinary bladder muscle strips was not found to be mediated via any 5-HT 2 receptor subtypes. The underlying mechanism for the enhancement of the α-Me-5-HT potentiating effect on detrusor contractility by ketanserin and naftopidil remains unknown; however, our results suggest that these drugs may be useful for treating contractile dysfunction of the detrusor, as manifested in conditions such as underactive bladder.

Severe injuries such as severe sepsis, burn, trauma and major surgery lead to an overlapping development of pro- and anti- inflammatory responses. It is now well established that these injuries are associated with the secondary development of immune suppression, which results in significant morbidity and mortality. Recent data suggest that immunostimulatory drugs might prevent these complications. However, intensive care patients are heterogeneous, making patient stratification essential for a targeted treatment. In the present review, we discuss potential biomarkers of injury-induced immunoparalysis, mainly focusing on these that have been associated with poor outcome in various clinical settings. We namely present clinical data on monocyte human leukocyte antigen DR, lymphopenia, PD-1/PD-L1 and transcriptomic approach.

In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer. We also shortened the incubation time to 8h, and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation, the optimal dose of stimulant was defined to be 4μg/mL of phytohaemagglutinin. Lastly, we determined that the number of T cells needed for this measurement was as low as 50,000, which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the β-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed.

Activation of β3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of β3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of β3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of β3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αβ-methylene adenosine triphosphate (αβ-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the β3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1μM) were inhibited 25% by isoproterenol (3μM) while contractions to 10Hz in the same strip were inhibited 67%. The selective β3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αβ-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of β3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by β3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional β3-adrenoceptors may inhibit neurotransmitter release., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

Background: The clinical equivalence of plasma treated to reduce pathogen transmission and untreated plasma has not been extensively studied. A clinical trial was conducted in liver transplant recipients to compare the efficacy of three plasmas., Study Design and Methods: A randomized, equivalence, blinded trial was performed in four French liver transplantation centers. The three studied (fresh-frozen) plasmas were quarantine (Q-FFP), methylene blue (MB-FFP), and solvent/detergent (S/D-FFP) plasmas. The primary outcome was the volume of plasma transfused during transplantation. Secondary outcomes included intraoperative blood loss, hemostasis variables corrections, and adverse events., Results: One-hundred patients were randomly assigned in the MB-FFP, 96 in the S/D-FFP, and 97 in the Q-FFP groups, respectively. The median volumes of plasma transfused were 2254, 1905, and 1798 mL with MB-FFP, S/D-FFP, and Q-FFP, respectively. The three plasmas were not equivalent. MB-FFP was not equivalent to the two other plasmas, but S/D-FFP and Q-FFP were equivalent. The median numbers of transfused plasma units were 10, 10, and 8 units with MB-FFP, S/D-FFP, and Q-FFP, respectively. Adjustment on bleeding risk factors diminished the difference between groups: the excess plasma volume transfused with MB-FFP compared to Q-FFP was reduced from 24% to 14%. Blood loss and coagulation factors corrections were not significantly different between the three arms., Conclusion: Compared to both Q-FFP and S/D-FFP, use of MB-FFP was associated with a moderate increase in volume transfused, partly explained by a difference in unit volume and bleeding risk factors. Q-FFP was associated with fewer units transfused than either S/D-FFP or MB-FFP., (© 2012 American Association of Blood Banks.)

Fenoterol has been reported to be a potent and selective β(2)-adrenoceptor agonist and is currently used clinically to treat asthma. Electrical field stimulation (EFS) of isolated urinary bladder mimics the voiding contraction by stimulating parasympathetic nerves, resulting in neurogenic contractions. To determine if stimulation of β(2)-adrenoceptors can inhibit this response, fenoterol was tested against EFS-induced contractions in human isolated urinary bladder and compared with mouse and rat. Bladder strips were mounted in organ baths and reproducible contractions induced by EFS. Fenoterol was added cumulatively in the presence of the β(2)-adrenoceptor antagonist ICI118551 or the β(3)-adrenoceptor antagonist L-748337. Fenoterol inhibited neurogenic contractions in all three species in a concentration-dependent manner with pEC(50) values of 6.66 ± 0.11, 6.86 ± 0.06 and 5.71 ± 0.1 in human, mouse and rat respectively. In human bladder strips ICI118551 (100 nM) did not affect responses to fenoterol, while L-748337 (0.3-3 μM) produced rightward shifts of the concentration-response curves with a pA(2) value of 8.10. In mouse bladder strips ICI118551 (30 nM) blocked the inhibitory effect of fenoterol (pA(2)=8.80), while L-748337 (10 μM) inhibited the response with a pA(2) of 5.79. In rat bladder ICI118551 (30 nM) was without effect, while L-748,337 (10 μM) inhibited the response to fenoterol with a pA(2) of 5.40. From these results it is clear that fenoterol potently activates β(3)-adrenoceptors in human isolated urinary bladder to inhibit EFS-induced contractions. Fenoterol also activates β(3)-adrenoceptors in rat, but β(2)-adrenoceptors in mouse bladder to inhibit EFS-induced contractions., (Copyright © 2012 Elsevier B.V. All rights reserved.)