Genetic diversity of Lactobacillus delbrueckii isolated from raw milk in Hokkaido, Japan.

Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii, one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products.

IgG1 production by sIgD+ splenic B cells and peritoneal B-1 cells in response to IL-5 and CD38 ligation.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induces proliferation, IL-5 receptor alpha chain expression and tyrosine phosphorylation of Bruton's tyrosine kinase. Furthermore, stimulation of splenic B cells with IL-5 together with CS/2 induces Blimp-1 expression and differentiation into Ig-producing cells. Here we examined the role of IL-5 in IgG1 and IgA production by B cells isolated from the spleen and peritoneal cavity. CD38 recognized by CS/2 was expressed in the follicular mantle B cells surrounding the germinal center, sIgD+ splenic B cells and peritoneal B cells. IL-5 induced IgG1 production in splenic sIgD+ B cells stimulated with CS/2, while it was ineffective to induce IgA production. Among the various cytokines tested, only IL-5 had a synergistic effect on IgG1 production with CS/2. IL-5 could induce the generation of S micro-Sgamma1 reciprocal recombination DNA products in CS/2-stimulated B cells. IL-4 was ineffective to induce either micro-gamma1 switch recombination or IgG1 secretion with CS/2, demonstrating that IL-5 promotes both micro-gamma1 switch recombination and IgG1 secretion in an IL-4-independent manner. The peritoneal B-2 cells exhibited both IgG1 and IgA production in response to IL-5 plus CS/2, while B-1 cells produced IgG1. These results imply that the pattern of differentiation to Ig-producing cells seen with peritoneal B cells is not identical to the pattern seen with splenic B cells and that peritoneal B-2 cells contain precursors of IgA-producing cells responding to IL-5 plus CS/2.

IL-5 induces IgG1 isotype switch recombination in mouse CD38-activated sIgD-positive B lymphocytes.

Mouse B cells express CD38, whose ligation by anti-CD38 Ab induces their proliferation and protection from apoptosis. We previously showed that stimulation of mouse splenic B cells with IL-5 together with CS/2, an anti-mouse CD38 mAb, induces production of IgG1 and IgM. Here we examined the role of IL-5 and CS/2 in the expression of germline gamma1 transcripts and the generation of reciprocal products forming DNA circles as byproducts of mu-gamma1 switch recombination. By itself, CS/2 induced significant expression of germline gamma1 transcripts in splenic naive B cells, whereas IL-5 neither induced nor enhanced germline gamma1 expression. Increased cellular content of reciprocal product, which is characteristic of mu-gamma1 recombination, was not observed after culturing B cells with CS/2, but increased reciprocal product, along with high levels of lgG1 secretion, was found when B cells were cultured with CS/2 plus IL-5. Although IL-4 did not, by itself, induce mu-gamma1 recombination in B cells stimulated with CS/2, in conjunction with CS/2 plus IL-5, IL-4 dramatically enhanced sterile gamma1 transcription and IgG1 production. These results demonstrate that CD38 ligation induces only germline gamma1 transcription and that IL-5 promotes both mu-gamma1 switch recombination and lgG1 secretion in an IL-4-independent manner.

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5.

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor alpha chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn-/-) and lyn-deficient (Lyn-/-) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor alpha chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn-/-) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn-/- mice, and it was severely impaired in B cells from Lyn-/- and Fyn/Lyn-/- mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn-/- and Fyn/Lyn-/- mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.