PI3K beta Plays a Critical Role in Neutrophil Activation by Immune Complexes

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (Fc gamma Rs). Here, we used genetic and pharmacological approaches to define a selective role for the beta isoform of phosphoinositide 3-kinase (PI3K beta) in Fc gamma R-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3K beta alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3K beta and PI3K delta, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3K beta by immune complexes involved cooperation between Fc gamma Rs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B-4. Coincident activation by a tyrosine kinase-coupled receptor (Fc gamma R) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the beta isoform of PI3K. PI3K beta-deficient mice were highly protected in an Fc gamma R-dependent model of autoantibody-induced skin blistering and were partially protected in an Fc gamma R-dependent model of inflammatory arthritis, whereas combined deficiency of PI3K beta and PI3K delta resulted in near-complete protection in the latter case. These results define PI3K beta as a potential therapeutic target in inflammatory disease.

Glycoprotein VI/Fc receptor gamma chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRgamma (Fc receptor gamma) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRgamma chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cgamma2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (similar to 10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRgamma chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti(alpha2 integrin) antibodies Ha1/29 and HMalpha2, but not by blockade of alphaIIbbeta3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI-FcRgamma chain complex, and is facilitated by binding of collagen to integrin alpha2beta1.

The 2,5 Oligoadenylate Synthetase/RNaseL pathway is a novel effector of BRCA1 - and interferon-gamma-mediated apoptosis

I discovered that 2,5OAS family of proteins was transcriptionally upregulated by BRCA1 and interferon gamma in a synergistic manner. This correlated with synergistically induced apoptosis and both the induction of 2,5OAS and the accompanying apoptosis could be inhibited by 2,5OAS specific siRNA proving 2,5OAS was the apoptotic effector.

Peripheral markers of the gamma-aminobutyric acid (GABA)ergic system in Angelman's syndrome.

It has recently been demonstrated that patients with Angelman's syndrome who exhibited a deletion on cytogenetic tests show more severe clinical pictures with drug-resistant epilepsy than patients with Angelman's syndrome not carrying the deletion. To verify if this difference in clinical severity can be attributed to genes for the three gamma-aminobutyric acid (GABA)A receptor subunits (GABRB3, GABRA5, GABRG3) located in the deleted region, a possible modification of peripheral markers of the GABAergic system was investigated in 12 subjects with Angelman's syndrome and 20 age-matched subjects (8 with idiopathic epilepsy and 12 not affected by neurologic diseases). The results confirmed a more severe clinical picture, and epilepsy syndrome in particular, in Angelman's syndrome patients with deletions versus patients without deletions. In contrast, biochemical study (based on dosage of plasma levels of GABA and diazepam binding inhibitor, an endogenous ligand of GABAA and peripheral benzodiazepine receptors, showed contradictory results: patients with Angelman's syndrome showed significantly higher levels of GABA and diazepam binding inhibitor than patients without neurologic impairment but significantly lower levels than epileptic controls.

Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Granulomatous Diseases Review Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells. (C) 2008 by Radiation Research Society.

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors.

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.