Comparison of the incidence of intra-operative haemorrhage from ovarian vessels during ovariectomy and ovariohysterectomy of dogs and cats performed by inexperienced surgeons using surgeon's and constrictor knots.

AIMS: To compare the incidence of intra-operative haemorrhage from ovarian vessels during ovariectomy (OV) and ovariohysterectomy (OVH) in dogs and cats performed by inexperienced surgeons using surgeon's and constrictor knots to ligate the ovarian pedicles. METHODS: All client-owned female dogs (n = 51) and cats (n = 102) that presented for OV or OVH at two referral clinics in Belgium and France from January to June 2019 were included. Surgeries were performed by inexperienced surgeons under the supervision of diplomates or residents of the European College of Veterinary Surgeons. During routine and emergency OV or OVH one pedicle of each animal was ligated using a constrictor knot and the other with a surgeon's knot, the side assigned randomly. Time taken to perform each knot and the number of ligatures that resulted in intraoperative haemorrhage from the ovarian vessels were recorded. Body condition score (BCS) was recorded for 33 dogs and 22 cats. RESULTS: The median time taken to perform constrictor knots in dogs (81 seconds), was longer than for surgeon's knots (69 seconds; p = 0.005). In cats, the median time taken to perform constrictor knots (109 seconds) was also longer than for surgeon's knots (90.5 seconds; p = 0.02).The number of dogs that had intra-operative haemorrhages from pedicles ligated with constrictor knots (2/102; 3.9%) was less than those ligated with surgeon's knots (10/102; 19.6%; p = 0.027). The median BCS of dogs that experienced haemorrhage (n = 8; median 5/9, min 3/9, max 7/9) was higher (p = 0.019) than that of dogs that did not experience haemorrhage (n = 25; median 4/9, min 3/9, max 8/9).There was no evidence (p = 1.0) of a difference in the proportion of cats that experienced intra-operative haemorrhage from pedicles ligated with constrictor knots (3/44; 6.8%) and those ligated with surgeon's knots (2/44; 4.5%). Two-thirds of the haemorrhages with constrictor knots were caused by pedicle rupture during tightening. CONCLUSIONS: The rate of intraoperative haemorrhage from the ovarian pedicle was lower when a constrictor knot was used compared to a surgeon's knot in dogs but not in cats. While the constrictor knot was more time consuming to tie this is not likely to have a clinical impact. Use of this knot by inexperienced surgeons in dogs may help to reduce the risk of intraoperative haemorrhage during OVH/OV.

Gut microbiota and immunity in common variable immunodeficiency: crosstalk with pro-inflammatory cytokines.

In recent years, gut microbiota (GM) has emerged as a key factor in shaping the pathogenesis of a vast array of immune-mediated diseases, as well as in the response to immune-based treatments such as anti PD-1 and anti-CTLA4 therapy or influenza vaccination. In addition, GM has a significant role in the immune system development and is fundamental in developing mucosal immunity. Recent data suggest that GM plays an important role in the immune system of immune deficient patients. GM status has a remarkable impact on the immune system and in immune deficient patients; this can lead to important consequences. Prebiotics are indeed a promising candidate in restoring GM homeostasis and improving immunity. Antibiotics are also capable of altering the microbial equilibrium.

ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival.

The ATP-gated receptor P2X7 is expressed in multiple malignant tumours including neuroblastoma, melanoma, prostate, lung and breast. P2X7 has a significant role in mediating diverse cell responses, which upon dysregulation are associated with tumour initiation and development. The rapid, ATP-mediated activation of P2X7 induces a fast-inward cation current in cells. However, prolonged ATP-mediated activation of P2X7 leads to formation of a pore that increases membrane permeability and eventually causes cell death. This presents a potential paradox, as the tumour microenvironment contains extracellular ATP at levels sufficient to activate the P2X7 pore and trigger cell death. However, P2X7 expression is associated with enhanced cancer cell survival, proliferation and metastatic potential. At least one distinct conformational form of P2X7, termed non-pore functional P2X7 (nfP2X7), has been described, which is not able to form a functional pore. We demonstrate for the first time in this study that exposure to a high ATP concentration, equivalent to those measured in the tumour microenvironment, drives nfP2X7 expression and also that nfP2X7 is essential for tumour cell survival. We show that monoclonal antibodies raised against a P2X7 amino acid sequence (200-216), whose conformation is distinct from that of wild-type (WT) P2X7, bind specifically to nfP2X7 expressed on the surface of tumour cells. We also show that nfP2X7 is broadly expressed in patient-derived tumour sections from a wide range of cancers. Therefore, antibodies raised against E200 provide tools that can differentiate between forms of the P2X7 receptor that have a key role in cancer.

Extracellular purines, purinergic receptors and tumor growth.

Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host-tumor interaction and highlights novel therapeutic options stemming from recent advances in this field.

ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment.

Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b(+)/Gr-1(+) cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1(+) population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-ß1 (TGF-ß1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-ß1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.

The P2X7 receptor is a key modulator of aerobic glycolysis.

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

Nimodipine inhibits IL-1ß release stimulated by amyloid ß from microglia.

BACKGROUND AND PURPOSE: There is growing evidence that inflammation plays a major role in the pathogenesis of neural damage caused by deposition of amyloid ß (Aß) in the brain. Nimodipine has received attention as a drug that might improve learning and reduce cognitive deficits in Alzheimer's disease, but the mechanism of action is poorly known. In this study, we tested the hypothesis that nimodipine inhibited Aß-stimulated IL-1ß release from microglia. EXPERIMENTAL APPROACH: Cultures of N13 microglia cells or primary mouse microglia were treated with nimodipine, and intracellular accumulation and release of IL-1ß in response to Aß or to the P2 receptor agonists ATP and benzoyl ATP (BzATP) were measured. Accumulation of IL-1ß was measured in vivo after intrahippocampal inoculation of Aß in the absence or presence of nimodipine. The effect of nimodipine on Aß-triggered cytotoxicity was also investigated. KEY RESULTS: We show here that nimodipine dose-dependently inhibited Aß-stimulated IL-1ß synthesis and release from primary microglia and microglia cell lines. Furthermore, nimodipine also inhibited Aß-induced IL-1ßin vivo accumulation at concentrations known to be reached in the CNS. Finally, nimodipine protected microglia from Aß-dependent cytotoxicity. CONCLUSION AND IMPLICATIONS: These data suggest that alleviation of symptoms of Alzheimer's disease following nimodipine administration might be due to an anti-inflammatory effect and point to a novel role for nimodipine as a centrally acting anti-inflammatory drug.

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation.

BACKGROUND: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. METHODS: In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. RESULTS: Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. CONCLUSIONS: In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.

P2X7 gene polymorphisms do not appear to be a susceptibility gene locus in sporadic cases of systemic lupus erythematosus.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. A loss-of-function single nucleotide polymorphism (SNP) at position 1513 (1513 A-->C) of the P2X(7) gene has recently been identified in both healthy and chronic lymphocytic leukemia (CLL) B-cells, translating into a loss of P2X(7)-mediated apoptosis in these cells. This antiapoptotic effect results in increased B-cell numbers, thereby potentially contributing to the survival of B-CLL clones. It was hypothesized that prolonged cell survival may also predispose to induction of autoimmunity. The objective of this study is to analyze the role of the P2X(7) receptor and its loss-of-function 1513 A-->C polymorphism (SNP) in the development of systemic lupus erythematosus (SLE). DNA samples obtained from patients with sporadic SLE were analyzed for the presence of the 1513 A-->C polymorphism using polymerase chain reaction (PCR) amplification and then direct sequencing. No significant difference in allele frequencies (1513 A-->C polymorphism) between sporadic cases of SLE and controls was found. A loss-of-function SNP at position 1513 (1513 A-->C) of the P2X(7) gene does not appear to be a susceptibility gene locus for the development of sporadic SLE.

Modulation of P2X7 receptor functions by polymyxin B: crucial role of the hydrophobic tail of the antibiotic molecule.

BACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.

Adenosine 5'-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation.

Human health is under constant threat of a wide variety of dangers, both self and nonself. The immune system is occupied with protecting the host against such dangers in order to preserve human health. For that purpose, the immune system is equipped with a diverse array of both cellular and non-cellular effectors that are in continuous communication with each other. The naturally occurring nucleotide adenosine 5'-triphosphate (ATP) and its metabolite adenosine (Ado) probably constitute an intrinsic part of this extensive immunological network through purinergic signaling by their cognate receptors, which are widely expressed throughout the body. This review provides a thorough overview of the effects of ATP and Ado on major immune cell types. The overwhelming evidence indicates that ATP and Ado are important endogenous signaling molecules in immunity and inflammation. Although the role of ATP and Ado during the course of inflammatory and immune responses in vivo appears to be extremely complex, we propose that their immunological role is both interdependent and multifaceted, meaning that the nature of their effects may shift from immunostimulatory to immunoregulatory or vice versa depending on extracellular concentrations as well as on expression patterns of purinergic receptors and ecto-enzymes. Purinergic signaling thus contributes to the fine-tuning of inflammatory and immune responses in such a way that the danger to the host is eliminated efficiently with minimal damage to healthy tissues.

New pathways for reactive oxygen species generation in inflammation and potential novel pharmacological targets.

Oxidative agents are generated in large amounts during inflammation. These highly reactive intermediates interact with several extracellular and intracellular molecules and with each other, thus generating a complex network of responses culminating in an outcome that may be detrimental or beneficial for the host. Alongside with the well known systems involved in production of reactive oxygen species or reactive nitrogen species, such as the NADPH oxidase or the nitric oxide synthase, novel enzymatic pathways have been discovered. This has unveiled new targets and functions for oxidant species, and has prompted the development of innovative anti-inflammatory drugs. In the new integrate scenario stemming from these studies oxidant species are increasingly recognized as true messengers, and even their toxic effects are viewed as the result of the perversion of an otherwise physiological extra/intra cellular signaling.

P2X7 receptors and apoptosis in tuberculosis infection.

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. Evidence accumulated has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. Triggering of apoptosis, in response to intracellular infection, has been identified for a wide range of pathogens and host organisms, and there is now emerging interest about mechanism mediating host cell death and its role in pulmonary tuberculosis. The physiological meaning of P2X receptor-dependent cell death is not completely understood, but and involvement in immune-mediated reactions is postulated.

Functional characterization of P2Y and P2X receptors in human eosinophils.

Activation of purinoceptor by ATP induces in eosinophils various cell responses including calcium transients, actin polymerization, production of reactive oxygen metabolites, CD11b-expression, and chemotaxis. Here, the effect of ion channel-gated P2X and/or G protein-coupled P2Y receptor agonists ATP, ATPgammaS, alpha,beta-meATP, 2-MeSATP, BzATP, ADP, CTP, and UTP on the intracellular Ca(2+)-mobilization, actin polymerization, production of reactive oxygen metabolites, CD11b expression and chemotaxis of human eosinophils were measured and the biological activity was analyzed. Although all tested nucleotides were able to induce all these cell responses, the biological activity of the analyzed nucleotides were distinct. Agonists of the G protein-coupled P2Y receptors such as 2-MeSATP, UTP, and ADP have a higher biological activity for production of reactive oxygen metabolites, actin polymerization and chemotaxis in comparison to the ion channel-gated P2X agonists alphabeta-meATP, BzATP, and CTP. In contrast, P2Y and P2X agonist showed similar potencies in respect to intracellular calcium transient and CD11b up-regulation. This conclusion was further supported by experiments with receptor iso-type antagonist KN62, EGTA or with the G(i) protein-inactivating pertussis toxin. These findings indicate participation of different purinorecptors in the regulation of cell responses in eosinophils.

Proinflammatory cytokines inhibit secretion in rat bile duct epithelium.

BACKGROUND AND AIMS: Cholestatic disorders often are associated with portal inflammation, but whether or how inflammation contributes to cholestasis is unknown. Thus we studied the effects of proinflammatory cytokines on bile duct epithelia secretory mechanisms. METHODS: Isolated bile duct units (IBDUs) were cultured with interleukin (IL)-6, interferon gamma, tumor necrosis factor (TNF)-alpha, and IL-1 alone or in combination. Ductular secretion was measured using video-optical planimetry. Bicarbonate and Cl(-) transport were assessed microfluorimetric measuring pH(i) (BCECF) and [Cl(-)](i) transients (MEQ). Expression of Cl(-)/HCO(3)(-) exchanger (AE-2), cystic fibrosis transmembrane conductance regulator (CFTR), and the secretin receptor (SR) were assessed by ribonuclease protection assay. Cellular cyclic adenosine monophosphate (cAMP) levels were studied by enzymatic immunoassay. Paracellular permeability was assessed using fluorescein-labeled dextrans (FD) in cholangiocyte monolayers (NRC-1). RESULTS: Although not effective when given alone, each combination of IL-6, interferon gamma, IL-1, and TNF-alpha inhibited secretion in IBDU. Cytokines inhibited cAMP formation, AE-2 activity, and cyclic AMP-dependent Cl(-) efflux, but not that induced by purinergic agonists. AE-2 gene expression was unaffected by proinflammatory cytokines, whereas CFTR and SR expression was increased. In addition, paracellular transit of FD across NRC-1 monolayers was increased. CONCLUSIONS: Inflammatory cytokines inhibit cAMP-dependent fluid secretion in cholangiocytes and impair the barrier functions of biliary epithelia. These changes may represent the molecular mechanisms by which inflammation leads to ductular cholestasis in vivo.

The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action.

The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) by increasing the passive Ca2+ leak from the organelle. To investigate whether this Ca2+ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca2+]er by different pharmacological and molecular approaches. All conditions that lowered [Ca2+]er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca2+]er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca2+ from the endoplasmic reticulum and thus [Ca2+] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca2+]er levels, as well as the buffering of cytoplasmic [Ca2+] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca2+]er is an important component of the anti-apoptotic program controlled by this oncogene.

A defect in glycogen synthesis characterizes insulin resistance in hypertensive patients with type 2 diabetes.

A subgroup of patients with type 2 diabetes shows a clustering of abnormalities such as peripheral insulin resistance, hypertension, and microalbuminuria. To evaluate whether these traits reflect intrinsic disorders of cell function rather than in vivo environmental effects, we studied a group of 7 nondiabetic hypertensive subjects with an altered albumin excretion rate (AER) (HyMA+) and 3 groups of patients with type 2 diabetes: 7 with normal blood pressure and normal AER (DH-MA-), 7 with high blood pressure and normal AER (DH+MA-), and 7 with both high blood pressure and altered AER (DH+MA+). Glucose disposal was measured during an hyperinsulinemic clamp (40 mU. m(2)(-1). min(-1)) with primed deuterated [6.6 (2)H(2)] glucose infusion. In the same subjects, a skin biopsy was performed and the following parameters were investigated: glucose transport (as determined by [(3)H]2-deoxyglucose uptake); glycogen synthase activity (as determined by [(14)C] glucose incorporation from UDP-[U-(14)C] glucose into glycogen); glycogen phosphorylase activity (as measured by the incorporation of [U-(14)C]glucose 1-phosphate into glycogen); and total glycogen content. In vivo glucose disposal was significantly reduced in DH+MA- and DH+MA+, with respect to DH-MA-, HyMA+, and controls. Insulin-stimulated glucose transport was similar in the 3 groups of patients with diabetes. A significant reduction of intracellular glycogen content was observed in DH+MA- and DH+MA+ compared with DH-MA- in both basal and insulin-stimulated conditions, probably because of a major impairment of glycogen synthase activity. Glycogen phosphorylase activity did not show differences between the groups. These results suggest that (1) the combination of type 2 diabetes with hypertension and altered AER is associated with impaired insulin sensitivity, and (2) intrinsic, possibly genetic, factors may account for increased peripheral insulin resistance in hypertensive microalbuminuric patients with type 2 diabetes, pointing to the reduction of glycogen synthase activity as a shared common defect.