Platelet FcγRIIA-induced serotonin release exacerbates the severity of Transfusion-Related Acute Lung Injury in mice

Transfusion-related acute lung injury (TRALI) remains a major cause of transfusion-related fatalities. The mechanism of human antibody-mediated TRALI, especially the involvement of the Fcγ receptors, is not clearly established. Contrary to mice, human platelets are unique in their expression of the FcγRIIA/CD32A receptor, suggesting that our understanding of the pathogenesis of antibody-mediated TRALI is partial, as the current murine models incompletely recapitulate the human immunology. We evaluated the role of FcγRIIA/CD32A in TRALI using a humanized mouse model expressing the FcγRIIA/CD32A receptor. When challenged with a recombinant chimeric human immunoglobulin G1/mouse anti–major histocompatibility complex class I monoclonal antibody, these mice exhibited exacerbated alveolar edema and higher mortality compared with wild-type (WT) mice. Unlike in WT mice, monocytes/macrophages in CD32A(+) mice were accessory for TRALI initiation, indicating the decisive contribution of another cell type. Platelet activation was dramatically increased in CD32A(+) animals, resulting in their increased consumption and massive release of their granule contents. Platelet depletion prevented the exacerbation of TRALI in CD32A(+) mice but did not affect TRALI in WT animals. By blocking platelet serotonin uptake with fluoxetine, we showed that the severity of TRALI in CD32A(+) mice resulted from the serotonin released by the activated platelets. Furthermore, inhibition of 5-hydroxytryptamine 2A serotonin receptor with sarpogrelate, before or after the induction of TRALI, abolished the aggravation of lung edema in CD32A(+) mice. Our findings show that platelet FcγRIIA/CD32A activation exacerbates antibody-mediated TRALI and provide a rationale for designing prophylactic and therapeutic strategies targeting the serotonin pathway to attenuate TRALI in patients

Production and characterization of Orpinomyces mutant xylanases with improved temperature and pH stabilities

The error-prone PCR technique has been widely used in order to obtain thermostable enzymes more suitable for industrial conditions. The Orpinomyces xynA mutant library allowed the selection of four thermostable mutants (M1-M4). Molecular dynamics (MD) predicted an N-terminal tail as being a destabilizing structural region and allowed further enhancing of the mutant xylanases thermostability. Thus, removal of the 27 amino acid residues enabled an increase in the enzyme half-life values (t1/2). However, besides the improved thermostability, the large enzyme production and high catalytic performance are also relevant for the biotechnological application of enzymes. During the mutant enzymes production in E. coli, the IPTG induction protocol allowed high expression levels of soluble and active xylanases. The mutant xylanases without the 27 amino acid residues showed improved thermostability and the shorter versions of M2 and M4 (named as SM2 and SM4) also presented a good performance in more extreme pH conditions

The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3-kinase-dependent mechanism

AbstractHigh concentrations of adenosine-5′-diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor-deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor-deficient platelets in response to 100 μM ADP or 1 μM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of 32P-labeled platelets showed that P2Y12-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor-independent inside-out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3-kinase

Severe bleeding and absent ADP-induced platelet aggregation associated with inherited combined CalDAG-GEFI and P2Y12 deficiencies

--

The ATP-gated P2X1 ion channel contributes to the severity of antibody-mediated Transfusion-Related Acute Lung Injury in mice

The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X1 cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses

Cooperation between NMDA-Type Glutamate and P2 Receptors for Neuroprotection during Stroke: Combining Astrocyte and Neuronal Protection

Excitotoxicity is the principle mechanism of acute injury during stroke. It is defined as the unregulated accumulation of excitatory neurotransmitters such as glutamate within the extracellular space, leading to over-activation of receptors, ionic disruption, cell swelling, cytotoxic Ca2+ elevation and a feed-forward loop where membrane depolarisation evokes further neurotransmitter release. Glutamate-mediated excitotoxicity is well documented in neurons and oligodendrocytes but drugs targeting glutamate excitotoxicity have failed clinically which may be due to their inability to protect astrocytes. Astrocytes make up ~50% of the brain volume and express high levels of P2 adenosine triphosphate (ATP)-receptors which have excitotoxic potential, suggesting that glutamate and ATP may mediate parallel excitotoxic cascades in neurons and astrocytes, respectively. Mono-cultures of astrocytes expressed an array of P2X and P2Y receptors can produce large rises in [Ca2+]i; mono-cultured neurons showed lower levels of functional P2 receptors. Using high-density 1:1 neuron:astrocyte co-cultures, ischemia (modelled as oxygen-glucose deprivation: OGD) evoked a rise in extracellular ATP, while P2 blockers were highly protective of both cell types. GluR blockers were only protective of neurons. Neither astrocyte nor neuronal mono-cultures showed significant ATP release during OGD, showing that cell type interactions are required for ischemic release. P2 blockers were also protective in normal-density co-cultures, while low doses of combined P2/GluR blockers where highly protective. These results highlight the potential of combined P2/GluR block for protection of neurons and glia.</jats:p

The P2Y1 receptor is involved in the maintenance of glucose homeostasis and in insulin secretion in mice

Pancreatic β cells express several P2 receptors including P2Y1 and the modulation of insulin secretion by extracellular nucleotides has suggested that these receptors may contribute to the regulation of glucose homeostasis. To determine whether the P2Y1 receptor is involved in this process, we performed studies in P2Y1 mice. In baseline conditions, P2Y1-mice exhibited a 15% increase in glycemia and a 40% increase in insulinemia, associated with a 10% increase in body weight, pointing to a role of the P2Y1 receptor in the control of glucose metabolism. Dynamic experiments further showed that P2Y1-mice exhibited a tendency to glucose intolerance. These features were associated with a decrease in the plasma levels of free fatty acid and triglycerides. When fed a lipids and sucrose enriched diet for 15 weeks, the two genotypes no longer displayed any significant differences. To determine whether the P2Y1 receptor was directly involved in the control of insulin secretion, experiments were carried out in isolated Langerhans islets. In the presence of high concentrations of glucose, insulin secretion was significantly greater in islets from P2Y1-mice. Altogether, these results show that the P2Y1 receptor plays a physiological role in the maintenance of glucose homeostasis at least in part by regulating insulin secretion