Probiotics beneficial effects on the host are associated with regulation of the intestinal microbial homeostasis and with modulation of inflammatory immune responses in the gut and in periphery. In this study, we investigated the clinical efficacy of two lactobacillus and two bifidobacterium probiotic strains in experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE) models, induced in Lewis rats. Treatment with probiotics led to less severe disease manifestation in both models; ex vivo analyses showed preservation of neuromuscular junction in EAMG and myelin content in EAE spinal cord. Immunoregulatory transcripts were found differentially expressed in gut associated lymphoid tissue and in peripheral immunocompetent organs. Feeding EAMG animals with probiotics resulted in increased levels of Transforming Growth Factor-β (TGFβ) in serum, and increased percentages of regulatory T cells (Treg) in peripheral blood leukocyte. Exposure of immature dendritic cells to probiotics induced their maturation toward an immunomodulatory phenotype, and secretion of TGFβ. Our data showed that bifidobacteria and lactobacilli treatment effectively modulates disease symptoms in EAMG and EAE models, and support further investigations to evaluate their use in autoimmune diseases.

Integrin ␣2␤1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3K␤, but not of PI3K␥ or PI3K␣. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3K␤ (PI3K␤ KD ), but occurred normally in PI3K␥ KD platelets. Integrin ␣2␤1 failed to stimulate PI3K␤ in platelets from phospholipase C␥2 (PLC␥2)-knockout mice, and we found that intracellular Ca 2؉ linked PLC␥2 to PI3K␤ activation. Integrin ␣2␤1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLC␥2 and intracellular Ca 2؉ . Whereas activation of Pyk2 occurred normally in PI3K␤ KD platelets, stimulation of PI3K␤ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3K␤ was required for ␣2␤1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin ␣IIb␤3 were reduced after inhibition of PI3K␤ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3K␤ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3K␤ downstream of integrin ␣2␤1, and document a novel role for Pyk2 and PI3K␤ in integrin ␣2␤1 promoted inside-out activation of integrin ␣IIb␤3 and thrombus formation. (Blood. 2012;119(3):847-856) IntroductionClass I PI3Ks are key signaling enzymes that phosphorylate the inositol ring of membrane phospholipids and generate different 3-phosphoinositides, important intracellular messengers that regulate several cellular processes through the downstream activation of the protein Ser/Thr kinase Akt. 1 Circulating blood platelets express all members of the class I PI3K family, which includes the PI3K␣, PI3K␤, PI3K␦, and PI3K␥ isoforms. PI3K activity is essential for platelet aggregation and thrombus formation, 1,2 and therefore these enzymes are potential novel targets for antithrombotic agents. For this reason, it is essential to recognize the precise contribution of every PI3K isoform in platelet activation induced by different extracellular agonists.Pharmacologic and genetic evidence indicates that PI3K␤ plays a predominant role in the regulation of platelet function. [3][4][5][6][7] Selective inactivation of PI3K␤ completely prevents platelet aggregation induced by the collagen receptor glycoprotein VI (GPVI) and reduces occlusive thrombus formation. 4,5 PI3K␤ is also implicated in the platelet response to agonists that stimulate G-protein coupled receptors (GPCRs) such as ADP or thromboxane A 2 (TxA 2 ). 3,4,8,9 Whereas PI3K␦ has been demonstrated to play a minor role in platelet activation, 10 PI3K␣ was recently proposed to be as important as PI3K␤ in GPVI signaling. 7 Similarly, several reports have documented that, in addition to PI3K␤, PI3K␥ is also implicated in GPCR-mediated platelet activation. 4,9,11,12 These observations are indicative of a still poorly appreciated interplay...

Key Points• The tyrosine kinase Pyk2 regulates a p38MAPK-cPLA2 pathway required for thrombin-induced thromboxane A2 production and platelet aggregation.• Pyk2 deletion in mice confers protection against pulmonary thromboembolism and arterial thrombosis.In the present study, we used a knockout murine model to analyze the contribution of the Ca 2؉ -dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin ␣ IIb ␤ 3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A 2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA 2 IntroductionPyk2, also known as RAFTK or CAD␤, is a nonreceptor tyrosine kinase that is highly homologous to the focal adhesion kinase FAK and is predominantly expressed in the CNS and hematopoietic cells. 1-3 Like FAK, Pyk2 does not possess SH2 or SH3 domains, but has a centrally located catalytic domain flanked by an N-terminal FERM domain and a C-terminal focal adhesion targeting FAT domain. Pyk2 can be tyrosine phosphorylated and activated by a variety of different cellular stimuli, including cytokines, growth factors, agonists of G-protein-coupled receptors (GPCRs), integrin ligands, and stress stimuli. [3][4][5][6] Typically, Pyk2 activation is mediated by both Src kinase-and cytosolic Ca 2ϩ -dependent pathways. Src kinases phosphorylate Pyk2 at Tyr579, Tyr580, and Tyr881, increasing the catalytic activity of the kinase and promoting autophosphorylation on Tyr402 in the FERM domain. 5,6 Even in the absence of Src-mediated phosphorylation, Pyk2 can be activated on increase of the cytosolic Ca 2ϩ concentration. [4][5][6][7] It has been shown that Ca 2ϩ and calmodulin bind to the N-terminal FERM domain of Pyk2, triggering Pyk2 dimerization, activation, and autophosphorylation at Tyr402. 8 Phosphorylated Tyr402 is a binding site for the SH2 domain of Src. 5,6 Therefore, Pyk2 is a kinase that can link Ca 2ϩ -based signaling pathways to protein tyrosine phosphorylation.Some observations reported over the past 2 decades have implicated Pyk2 in platelet activation. Pyk2 is highly expressed in megakaryocytes and platelets and is rapidly phosphorylated on stimulation with se...

Calcitonin gene related peptide (CGRP) and adrenomedullin are potent biologically active peptides that have been proposed to play an important role in vascular and inflammatory diseases. Their function in the central nervous system is still unclear since they have been proposed as either pro-inflammatory or neuroprotective factors. We investigated the effects of the two peptides on astrocytes and microglia, cells of the central nervous system that exert a strong modulatory activity in the neuroinflammatory processes. In particular, we studied the ability of CGRP and adrenomedullin to modulate microglia activation, i.e. its competence of producing and releasing pro-inflammatory cytokines/chemokines that are known to play a crucial role in neuroinflammation. In this work we show that the two neuropeptides exert a potent inhibitory effect on lipopolysaccharide-induced microglia activation in vitro, with strong inhibition of the release of pro-inflammatory mediators (such as NO, cytokines and chemokines). Both CGRP and adrenomedullin are known to promote cAMP elevation, this second messenger cannot fully account for the observed inhibitory effects, thereby suggesting that other signaling pathways are involved. Interestingly, the inhibitory effect of CGRP and adrenomedullin appears to be stimulus specific, since direct activation with pro-inflammatory cytokines was not affected.Our findings clarify aspects of microglia activation, and contribute to the comprehension of the switch from reparative to detrimental function that occurs when glia is exposed to different conditions. Moreover, they draw the attention to potential targets for novel pharmacological intervention in pathologies characterized by glia activation and neuroinflammation.

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.