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Background We have previously shown that 5-fluorouracil (5-FU) selectively kills myeloid-derived suppressor cells (MDSCs) and activates NLRP3 (NOD-leucine rich repeat and pyrin containing protein 3) inflammasome. NLRP3 activation leads to caspase-1 activation and production of IL-1β, which in turn favors secondary tumor growth. We decided to explore the effects of either a heat shock (HS) or the deficiency in heat shock protein (HSP) 70, previously shown to respectively inhibit or increase NLRP3 inflammasome activation in macrophages.Methods Caspase-1 activation was detected in vitro in MSC-2 cells by western blot and in vivo or ex vivo in tumor and/or splenic MDSCs by flow cytometry. The effects of HS, HSP70 deficiency and anakinra (an IL-1 inhibitor) on tumor growth and mice survival were studied in C57BL/6 WT or Hsp70−/− tumor-bearing mice. Finally, Th17 polarization was evaluated by qPCR (Il17a, Rorc) and angiogenic markers by qPCR (Pecam1, Eng) and immunohistochemistry (ERG).Results HS inhibits 5-FU-mediated caspase-1 activation in vitro and in vivo without affecting its cytotoxicity on MDSCs. Moreover, it enhances the antitumor effect of 5-FU treatment and favors mice survival. Interestingly, it is associated to a decreased Th17 and angiogenesis markers in tumors. IL-1β injection is able to bypass HS+5-FU antitumor effects. In contrast, in Hsp70−/− MDSCs, 5-FU-mediated caspase-1 activation is increased in vivo and in vitro without effect on 5-FU cytotoxicity. In Hsp70−/− mice, the antitumor effect of 5-FU was impeded, with an increased Th17 and angiogenesis markers in tumors. Finally, the effects of 5-FU on tumor growth can be restored by inhibiting IL-1β, using anakinra.Conclusion This study provides evidence on the role of HSP70 in tuning 5-FU antitumor effect and suggests that HS can be used to improve 5-FU anticancer effect.

Background Pharmacological studies using selective P2Y1 antagonists, such as MRS2500, and studies with P2Y1−/− knockout mice have demonstrated that purinergic neuromuscular transmission is mediated by P2Y1 receptors in the colon. The aim of the present study was to test whether P2Y1 receptors are involved in purinergic neurotransmission in the antrum and cecum. Methods Microelectrode recordings were performed on strips from the antrum and cecum of wild type animals (WT) and P2Y1−/− mice. Key Results In the antrum, no differences in resting membrane potential and slow wave activity were observed between groups. In WT animals, electrical field stimulation elicited a MRS2500-sensitive inhibitory junction potential (IJP). In P2Y1−/− mice, a nitrergic IJP ( Nω-nitro- l-arginine-sensitive), but not a purinergic IJP was recorded. This IJP was equivalent to the response obtained in strips from WT animals previously incubated with MRS2500. Similar results were obtained in the cecum: 1- the purinergic IJP (MRS2500-sensitive) recorded in WT animals was absent in P2Y1−/− mice 2- nitrergic neurotransmission was preserved in both groups. Moreover, 1- spontaneous IJP (MRS2500-sensitive) could be recorded in WT, but not in P2Y1−/− mice 2- MRS2365 a P2Y1 agonist caused smooth muscle hyperpolarization in WT, but not in P2Y1−/− animals, and 3- β-NAD caused smooth muscle hyperpolarization both in WT and P2Y1−/− animals. Conclusions & Inferences 1- P2Y1 receptor is the general mechanism of purinergic inhibition in the gastrointestinal tract, 2- P2Y1−/− mouse is a useful animal model to study selective impairment of purinergic neurotransmission and 3- P2Y1−/− mouse might help in the identification of purinergic neurotransmitter(s). [ABSTRACT FROM AUTHOR]

Obtaining vascular access by catheterisation is a good option, especially in patients with vascular system fragility. In the authors' department, there was an increase in Gram Negative Bacillus (GNB) infection in patients with long term catheters (LTC). An objective was set to design an action plan and a new working methodology in order to eradicate the infection and the cause. Three periods were established in the prospective follow-up of LTC patients: the pre-epidemic period (01/94 to 03/99), with a bacteraemia every 144 days per patient, the epidemic period (04/99 to 12/00) with a bacteraemia every ten days per patient, and the post-epidemic period (01/01 to 04/02). A multidisciplinary working group was established, which produced action plans for nursing and technical staff. The working methodology of the service was studied and analysed by means of a review. The deionised water cultures at the entrance to the haemodialysis ward were negative. The dialysis and connector cultures were positive for GNB, confirming that they were of the same genetic origin. An evaluation of the periods was carried out, studying the working methodology, to which no changes were made between the pre-epidemic and epidemic period. In the post-epidemic period, a number of changes were made to the care dynamic, with no other bacteraemia arising to date. [ABSTRACT FROM AUTHOR]

P2Y 1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (K ca 2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y 1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y 1 receptor and openers (CyPPA) and blockers (apamin) of K ca 2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5') tetraphosphate ammonium salt (Ap 4 A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap 4 A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100 μM Ap 4 A response was antagonised by MRS2500 (1 μM), BPTU (3 μM) and apamin (1 μM). CyPPA (10 μM) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap 4 A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap 4 A but not PolyP activates P2Y 1 receptors causing smooth muscle hyperpolarization and relaxation. Ap 4 A signalling causes activation of K ca 2 channels through activation of P2Y 1 receptors. In contrast, CyPPA acts directly on K ca 2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The enteric nervous system (ENS) is responsible for the genesis of motor patterns ensuring an appropriate intestinal transit. Enteric neurons are classified into afferent, interneuron, and motoneuron types, with the latter two being further categorized as excitatory or inhibitory, which cause smooth muscle contraction or inhibition, respectively. Muscle relaxation mechanisms are key for the understanding of physiological processes such as sphincter relaxation, gastric accommodation, or descending peristaltic reflex. Nitric oxide (NO) and ATP or a related purine represent the primary inhibitory neurotransmitters. Nitrergic neurons synthesize NO through nNOS enzyme activity. NO diffuses across the cell membrane to bind its receptor, namely, guanylyl cyclase, and then activates a number of intracellular mechanisms that ultimately result in muscle relaxation. ATP acts as an inhibitory neurotransmitter together with NO, and the purinergic P2Y1 membrane receptor has been identified as a key item in order to understand how ATP may relax intestinal smooth muscle. Although, probably, no clinician doubts the significance of NO in the pathophysiology of digestive motility, the relevance of purinergic neurotransmission is apparently much lower, as ATP has not been associated with any specific motor dysfunction yet. The goal of this review is to discuss the function of both relaxation mechanisms in order to establish the physiological grounds of potential motor dysfunctions arising from impaired intestinal relaxation.

P2Y1 receptors mediate nerve mediated purinergic inhibitory junction potentials (IJP) and relaxations in the gastrointestinal (GI) tract in a wide range of species including rodents and humans. A new P2Y1 antagonist, with a non-nucleotide structure, BPTU, has recently been described using X-ray crystallography as the first allosteric G-protein-coupled receptor antagonist located entirely outside of the helical bundle. In this study, we tested its effect on purinergic responses in the gastrointestinal tract of rodents using electrophysiological and myographic techniques. BPTU concentration dependently inhibited purinergic inhibitory junction potentials and inhibition of spontaneous motility induced by electrical field stimulation in the colon of rats (EC50 = 0.3 μM) and mice (EC50 = 0.06 μM). Mechanical inhibitory responses were also concentration-dependently blocked in the stomach of both species. Compared to MRS2500, BPTU displays a lower potency. In the rat colon nicotine induced relaxation was also blocked by BPTU. BPTU also blocked the cessation of spontaneous contractility elicited by ADPβS and the P2Y1 agonist MRS2365. We conclude that BPTU is a novel antagonist with different structural and functional properties than nucleotidic antagonists that is able to block the P2Y1 receptor located at the neuromuscular junction of the GI tract., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Interaction of different neuromyogenic mechanisms determines colonic motility. In rats, cyclic depolarizations and slow waves generate myogenic contractions of low frequency (LF) and high frequency (HF), respectively. Interstitial cells of Cajal (ICC) located near the submuscular plexus (SMP) generate slow waves. Inhibitory junction potential (IJP) consists on a purinergic fast (IJPf) followed by a nitrergic slow (IJPs) component leading to relaxation. In the present study, we characterized (1) the dynamics of purinergic-nitrergic inhibitory co-transmission and (2) its contribution on prolonged inhibition of myogenic activity. Different protocols of electrical field stimulation (EFS) under different pharmacological conditions were performed to characterize electrophysiological and mechanical responses. Smooth muscle cells (SMCs) in tissue devoid of ICC-SMP had a resting membrane potential (RMP) of -40.7 ± 0.7 mV. Single pulse protocols increased purinergic and nitrergic IJP amplitude in a voltage-dependent manner (IJPfMAX = -26.4 ± 0.6 mV, IJPsMAX = -6.7 ± 0.3 mV). Trains at increasing frequencies enhanced nitrergic (k = 0.8 ± 0.2 s, IJPs∞ = -15 ± 0.5 mV) whereas they attenuated purinergic responses (k = 3.4 ± 0.6 s,IJPf∞ = -8.9 ± 0.6 mV). In tissues with intact ICC-SMP, the RMP was -50.0 ± 0.9 mV and nifedipine insensitive slow waves (10.1 ± 2.0 mV, 10.3 ± 0.5 cpm) were recorded. In these cells, (1) nitrergic and purinergic responses were reduced and (2) slow waves maintained their intrinsic frequency and increased their amplitude under nerve-mediated hyperpolarization. Based on the co-transmission process and consistent with the expected results on RMP, prolonged EFS caused a progressive reduction of LF contractions whereas HF contractions were partially insensitive. In conclusion, inhibitory neurons modulate colonic spontaneous motility and the principles determining post-junctional responses are (1) the frequency of firing that determines the neurotransmitter/receptor involved, (2) the transwall gradient and (3) the origin and nature of each myogenic activity

The purine receptor involved in inhibitory responses in the gastrointestinal tract has been recently identified. P2Y1 receptor activation mediates the fast component of the inhibitory junction potential (IJPf) and the non-nitrergic relaxation. The aim of the present work has been to investigate which purinergic agonist better mimics endogenous responses. We used different agonist and antagonist of P2 receptors. Contractility and microelectrode experiments were used to compare the effects of exogenously added purines and electrical field stimulation (EFS)-induced nerve mediated effects in rat and human colonic strips. In rat colon, the IJPf and EFS-induced inhibition of contractions were concentration-dependently inhibited by the P2Y1 antagonist MRS2500 but not by iso-PPADS or NF023 (P2X antagonists) up to 1 μM. In samples from human colon, EFS-induced inhibition of contractions was inhibited by either MRS2500 or apamin (1 μM) but not by iso-PPADS. In both species, α,β-meATP, a stable analog of ATP, caused inhibition of spontaneous contractions. α,β-meATP effect was concentration-dependent (EC50: 2.7 μM rat, 4.4 μM human) and was antagonized by either MRS2500 or apamin but unaffected by P2X antagonists. ATP, ADP, β-NAD and ADP-ribose inhibited spontaneous contractions but did not show the same sensitivity profile to purine receptor antagonists as EFS-induced inhibition of contractions. The effect of α,β-meATP is due to P2Y1 receptor activation leading the opening of sKca channels. Accordingly, α,β-meATP mimics the endogenous purinergic mediator. In contrast, exogenously added putative neurotransmitters do not exactly mimic the endogenous mediator. Quick degradation by ecto-nuclease or different distribution of receptors (junctionally vs extrajunctionally) might explain these results., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purinergic and nitrergic co-transmission is the dominant mechanism responsible for neural-mediated smooth muscle relaxation in the gastrointestinal tract. The aim of the present paper was to test whether or not P2Y(1) receptors are involved in purinergic neurotransmission using P2Y(1)(−/−) knock-out mice. Tension and microelectrode recordings were performed on colonic strips. In wild type (WT) animals, electrical field stimulation (EFS) caused an inhibitory junction potential (IJP) that consisted of a fast IJP (MRS2500 sensitive, 1 μm) followed by a sustained IJP (N(ω)-nitro-L-arginine (L-NNA) sensitive, 1 mm). The fast component of the IJP was absent in P2Y(1)(−/−) mice whereas the sustained IJP (L-NNA sensitive) was recorded. In WT animals, EFS-induced inhibition of spontaneous motility was blocked by the consecutive addition of L-NNA and MRS2500. In P2Y(1)(−/−) mice, EFS responses were completely blocked by L-NNA. In WT and P2Y(1)(−/−) animals, L-NNA induced a smooth muscle depolarization but ‘spontaneous' IJP (MRS2500 sensitive) could be recorded in WT but not in P2Y(1)(−/−) animals. Finally, in WT animals, 1 μm MRS2365 caused a smooth muscle hyperpolarization that was blocked by 1 μm MRS2500. In contrast, 1 μm MRS2365 did not modify smooth muscle resting membrane potential in P2Y(1)(−/−) mice. β-Nicotinamide adenine dinucleotide (β-NAD, 1 mm) partially mimicked the effect of MRS2365. We conclude that P2Y(1) receptors mediate purinergic neurotransmission in the gastrointestinal tract and β-NAD partially fulfils the criteria to participate in rodent purinergic neurotransmission. The P2Y(1)(−/−) mouse is a useful animal model to study the selective loss of purinergic neurotransmission.

According to previous reviews, hemoperitoneum episodes appear in 6.1-8.4% of the peritoneal dialysis patients, and they are severe in a 20% of them. Due to the absence of severe hemoperitoneum in our peritoneal dialysis program, we retrospectively reviewed hemoperitoneum non-related with abdominal surgery or catheter placing. We analyzed its incidence, etiology, prognostic and clinical outcome, as well as the possible effect of recurrent hemoperitoneum on peritoneal function. A total of 132 patients were treated in our centre during a period of 173 months. Mean age at the beginning of peritoneal dialysis was 59+/-17.1 years, 43.2% were females, and 22.8% of them were menstruating women. Twenty-two patients had at least one hemoperitoneum episode during follow-up, with an incidence of 17%. The mean time interval between the start of peritoneal dialysis and the first hemoperitoneum episode was 0.66+/-0.94 years (range: 0.01-3.20 years). 73% were women. Most cases (59%) were due to menstruation. Remarkably, all the menstruating women presented hemoperitoneum at least once with a high incidence of recurrent episodes. The other hemoperitoneum episodes were mainly of unknown etiology (32% of patients), being this one the main cause in males. We only observed two more cases: a male who presented hemoperitoneum related to dicumarinic overdose and a female who presented hemoperitoneum due to mesenteric ischemia. All the 22 patients had a favourable outcome, except for the woman with mesenteric ischemia, what represented an incidence of 4.5% of severe hemoperitoneum. No significant association was found between episodes of hemoperitoneum and aspirin treatment, dicumarinic treatment or the presence of coagulopathy. There was no association either between recurrent hemoperitoneum and the number of peritonitis episodes, peritoneal function or technique survival. In conclusion, hemoperitoneum is a common and usually benign problem in peritoneal dialysis patients, frequently due to retrograde menstruation, and no deleterious long-term effects were found in patients with recurrent hemoperitoneum.

Vascular access through a venous catheter for haemodialysis is associated with increased risk of thrombosis, central venous stenosis, short access survival and inadequate dialysis. The most important catheter-related complications, which determine method survival, are infection and dysfunction. In particular, infectious episodes are in some studies the leading cause for untimely catheter removal and for catheter-related morbidity but also for morbidity in dialysis patients. Double-lumen central venous catheters used for haemodialysis, are common causes of septicaemia. Most cases are caused by staphylococci. Episodes of gram-negative bacteriemia have been traced to bacterial contamination of water and/or dialysate, errors in dialyzer reprocessing, and improper setup procedures. In this paper, we describe and outbreak of gram-negative bacteremia, firstly E. cloacae, in an outpatients haemodialysis unit, in the patients with long-term tunnelled haemodialysis catheters. We describe the epidemic investigation that we achieved to identify the source of contaminating bacteria and the route by which bacteria gained access to the bloodstream. We prove the contamination by gram-negative bacterium of the water-distribution lines and haemodialysis machines. Moreover, E. cloacae strains isolated from the lines and machines are genotypically identical to the isolated from the patients. Also, we prove that the hands of health care personnel are unintentional carriers. The outbreak was finished when decontamination of dialysis machines was enhanced and dialyzer-priming fluid was modified.

This study looked at the incidence of infection complications, in relation to central vein catheterisation as a provisional HD access, by means of the establishment of a nursing protocol for the handling of these catheters. Central vein catheterisation is a classical technique in Nephrology.