Analysis of the Specificity of IgA Antibodies Produced in the Mouse Small Intestine

Intestinal microbiota controls multiple aspects of body homeostasis. The microbiota composition changes easily in response to internal or external factors, which may result in dysbiosis and associated inflammatory reactions. Thus, maintaining the microbiota composition by the host immune system is crucial, and one of the main mechanisms for microbiota control is production of immunoglobulin A (IgA) at mucosal surfaces. The molecular mechanisms regulating the interactions between the immune system and microbiota remain obscure. A panel of hybridoma cell lines was constructed to produce monoclonal IgA antibodies specific to various commensal bacteria present in intestinal microbiota. The panel can be used to further understand the mechanisms whereby the adaptive immune system controls the microbiota composition.     

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a₃ components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a₃ was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.     

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.     

Marked perinatal lethality and cellular signaling deficits in mice null for the two sphingosine 1-phosphate (S1P) receptors,S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/EDG-3

Five cognate G protein-coupled receptors (S1P(1-5)) have been shown to mediate various cellular effects of sphingosine 1-phosphate (S1P). Here we report the generation of mice null for S1P(2) and for both S1P(2) and S1P(3). S1P(2)-null mice were viable and fertile and developed normally. The litter sizes from S1P(2)S1P(3) double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S1P signaling pathways. Wild-type MEFs were responsive to S1P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S1P(2)-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S1P(3)-null mice and indicate preferential coupling of the S1P(2) and S1P(3) receptors to Rho and PLC/Ca(2+) pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S1P signaling by these receptors.     

In vivo roles of lysophospholipid receptors revealed by gene targeting studies in mice

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are extracellular ligands for a family of G protein-coupled receptors (GPCRs), LPA1/2/3 and S1P1/2/3/4/5. Through coupling to multiple classes of G proteins and activating multiple signaling pathways, LPA/S1P receptors have been shown to be integral players for many essential cellular and physiological processes. Generation and analysis of mice deficient in each of LPA1, LPA2, S1P1, S1P2, and S1P3 have provided valuable information on the in vivo roles of these receptors. This review is focussed on expression patterns of each receptor gene in wild-type mice, targeted deletion approaches for generating mutant animals, main phenotypes of receptor-null mice, and alterations in signaling characteristics in receptor-deficient primary cells. Altogether, these data give insights to the importance of LPA/S1P receptors at the cellular and organismal level.     

Inefficient transmission of H5N1 influenza viruses in a ferret contact model

The abilities to infect and transmit efficiently among humans are essential for a novel influenza A virus to cause a pandemic. To evaluate the pandemic potential of widely disseminated H5N1 influenza viruses, a ferret contact model using experimental groups comprised of one inoculated ferret and two contact ferrets was used to study the transmissibility of four human H5N1 viruses isolated from 2003 to 2006. The effects of viral pathogenicity and receptor binding specificity (affinity to synthetic sialosaccharides with alpha2,3 or alpha2,6 linkages) on transmissibility were assessed. A/Vietnam/1203/04 and A/Vietnam/JP36-2/05 viruses, which possess "avian-like" alpha2,3-linked sialic acid (SA) receptor specificity, caused neurological symptoms and death in ferrets inoculated with 10(3) 50% tissue culture infectious doses. A/Hong Kong/213/03 and A/Turkey/65-596/06 viruses, which show binding affinity for "human-like" alpha2,6-linked SA receptors in addition to their affinity for alpha2,3-linked SA receptors, caused mild clinical symptoms and were not lethal to the ferrets. No transmission of A/Vietnam/1203/04 or A/Turkey/65-596/06 virus was detected. One contact ferret developed neutralizing antibodies to A/Hong Kong/213/03 but did not exhibit any clinical signs or detectable virus shedding. In two groups, one of two na?ve contact ferrets had detectable virus after 6 to 8 days when housed together with the A/Vietnam/JP36-2/05 virus-inoculated ferrets. Infected contact ferrets showed severe clinical signs, although little or no virus was detected in nasal washes. This limited virus shedding explained the absence of secondary transmission from the infected contact ferret to the other na?ve ferret that were housed together. Our results suggest that despite their receptor binding affinity, circulating H5N1 viruses retain molecular determinants that restrict their spread among mammalian species.     

LKB1 inhibition of NF‐κB in B cells prevents T follicular helper cell differentiation and germinal center formation

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (T_FH) cell differentiation and expansion to support a ∽100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of T_FH-cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control T_FH-cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity.