Isolation of segmented filamentous bacteria from complex gut microbiota.

Segmented filamentous bacteria (SFB) modulate the ontogeny of the immune system, and their presence can significantly affect mouse models of disease. Until recently, the inability to successfully culture SFB has made controlled studies on the mechanisms by which these bacteria exert their influence problematic. Here, we report a new method for selecting SFB from complex microbial mixtures, providing researchers a simple and cost-effective means to prepare pure infective inocula for prospective studies and also to compare individual SFB isolates.

Common signaling pathways link activation of murine PAR-1, LPA, and S1P receptors to proliferation of astrocytes.

Receptors for the serine protease thrombin and for lysophospholipids are coupled to G proteins and control a wide range of cellular functions, including mitogenesis. Activators of these receptors are present in blood, and can enter the brain during central nervous system (CNS) injury. Reactive astrogliosis, a prominent component of CNS injury with potentially harmful consequences, may involve proliferation of astrocytes. In this study, we have examined the expression and activation of protease activated receptors (PARs), lysophosphatidic acid (LPA) receptors, and sphingosine-1-phosphate (S1P) receptors on murine astrocytes. We show that activation of these three receptor classes can lead to astrogliosis in vivo and proliferation of astrocytes in vitro. Cultured murine cortical astrocytes express mRNA for multiple receptor subtypes of PAR (PAR-1-4), LPA (LPA-1-3) and S1P (S1P-1, -3, -4, and -5) receptors. Comparison of the intracellular signaling pathways of glial PAR-1, LPA, and S1P receptors indicates that each receptor class activates multiple downstream signaling pathways, including Gq/11-directed inositol lipid/Ca2+ signaling, Gi/o activation of mitogen-activated protein kinases (MAPK) (extracellular signal-regulated kinase 1/2 and stress activated protein kinase/c-jun N-terminal kinase, but not p38), and activation of Rho pathways. Furthermore, activation of these different receptor classes can differentially regulate two transcription factor pathways, serum response element and nuclear factor of activated T cells. Blockade of Gi/o signaling with pertussis toxin, MAPK activation with 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene (U0126), or Rho kinase signaling with R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide (Y27632) can markedly reduce the proliferative response of glial cells to PAR-1, LPA, or S1P receptor activation, suggesting that each of these pathways is important in coupling of receptor activation to glial proliferation.