bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

A rearranged T cell receptor (TCR) V␣ and J␣ gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic J␣ region. The introduced TCR ␣ chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR ␣ chain. This decrease is caused by a rearrangement of TCR ␣ chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR ␣ chain and the transgenic TCR ␤ chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR ␣ chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR ␣␤ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR ␣ chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR ␣ chain gene play an important role in the formation of the T cell repertoire.A productive VDJ rearrangement of T cell antigen receptor (TCR) ␤ chain inhibits a secondary TCR ␤ gene rearrangement (1), a process called ''allelic exclusion.'' This process ensures that each T cell expresses only one TCR ␤ chain. In contrast, VJ rearrangement of the TCR ␣ chain is not subject to allelic exclusion, and the expression of a functional TCR ␣ chain does not inhibit another TCR ␣ gene rearrangement (2-4). A pre-existing, functionally rearranged V␣-J␣ segment can be deleted by a secondary rearrangement on the same chromosome (5). These results clearly demonstrate that TCR ␣ and ␤ chain genes are regulated differently for the initiation of rearrangement and for allelic exclusion. The mechanisms controlling these differences are not known, and the biological significance of this differential regulation is not understood.To study the regulation of TCR ␣ chain rearrangement and expression during T cell maturation in the thymus, we established a genetically modified mouse line. In this knock-in (KI) mouse, the rearranged TCR V␣J␣ gene from a cytochrome c (Cyt c)-specific T cell hybridoma, 2B4 (6), is inserted in the 5Ј region of the germ-line J␣ locus by using homologous recombination in an embryonic stem (ES) cell. Analysis of the TCR ␣ chain KI mice revealed a rapid deletion of the introduced TCR ␣ gene in the CD4 CD8 double-positive (DP) stage by rearrangement of endogenous TCR ␣ chains. This finding indicates that the TCR ␣ chain locus continuously rearranges and deletes pre-existing functionally rearranged VJ segments in vivo. By crossing the KI mouse to the 2B4 TCR ␤ chain transgenic mouse, we demonstrate that the positive selection of T cells efficiently terminated secondary rearrangements and prevented deletion of the introduced TCR ␣ chain gene. Furthermore, we demonstrate that T cells expressing the KI...

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Peptidoglycan (PGN), as the major components of the bacterial cell wall, is known to cause excessive proinflammatory cytokine production. Toll-like receptor 2 (TLR2) is abundantly expressed on immune cells and has been shown to be involved in PGN-induced signaling. Although more and more evidences have indicated that PGN is recognized by TLR2, the role of TLR2 PGN recognition is controversial. Mannan-binding lectin (MBL), a plasma C-type lectin, plays a key role in innate immunity. More and more evidences show that MBL could suppress the amplification of inflammatory signals. Whether MBL can alter PGN-elicited cellular responses through TLR2 in macrophages is still unknown, and possible mechanism underlying it should be investigated. In this study, we found that MBL significantly attenuated PGN-induced inflammatory cytokine production, including TNF-α and IL-6, in PMA-stimulated THP-1 cells at both mRNA and protein levels. The expression of TLR2 was strongly induced by PGN stimulation. Furthermore, the administration of TLR2-neutralized antibody effectively suppressed PGN-induced TNF-α and IL-6 expression. These results supplied the evidence that PGN from Saccharomyces cerevisiae could be recognized by TLR2. In addition, we also found that MBL decreased PGN-induced TLR2 expression and suppressed TLR2-mediated downstream signaling, including the phosphorylation of IκBα, nuclear translocation of NF-κBp65, and phosphorylation of MAPK p38 and ERK1/2. Administration of MBL alone did not have an effect on the expression of TLR2. Finally, our data showed that PGN-mediated immune responses were more severely suppressed by preincubation with MBL and indicated that MBL can combine with both TLR2 and PGN to block the inflammation cytokine expression induced by PGN. All these data suggest that MBL could downregulate inflammation by modulating PGN/TLR2 signaling pathways. This study supports an important role for MBL in immune regulation and signaling pathways involved in inflammatory responses.

Background Candida albicans (C. albicans), the most common human fungal pathogen, can cause fatal systemic infections under certain circumstances. Mannan-binding lectin (MBL),a member of the collectin family in the C-type lectin superfamily, is an important serum component associated with innate immunity. Toll-like receptors (TLRs) are expressed extensively, and have been shown to be involved in C. albicans-induced cellular responses. We first examined whether MBL modulated heat-killed (HK) C. albicans-induced cellular responses in phorbol 12-myristate 13-acetate (PMA)-activated human THP-1 macrophages. We then investigated the possible mechanisms of its inhibitory effect.Methodology/Principal FindingEnzyme-linked immunosorbent assay (ELISA) and reverse transcriptasepolymerase chain reaction (RT-PCR) analysis showed that MBL at higher concentrations (10–20 µg/ml) significantly attenuated C. albicans-induced chemokine (e.g., IL-8) and proinflammatory cytokine (e.g., TNF-α) production from PMA-activated THP-1 cells at both protein and mRNA levels. Electrophoretic mobility shift assay (EMSA) and Western blot (WB) analysis showed that MBL could inhibit C. albicans-induced nuclear factor-κB (NF-κB) DNA binding and its translocation in PMA-activated THP-1 cells. MBL could directly bind to PMA-activated THP-1 cells in the presence of Ca2+, and this binding decreased TLR2 and TLR4 expressions in C. albicans-induced THP-1 macrophages. Furthermore, the binding could be partially inhibited by both anti-TLR2 monoclonal antibody (clone TL2.1) and anti-TLR4 monoclonal antibody (clone HTA125). In addition, co-immunoprecipitation experiments and microtiter wells assay showed that MBL could directly bind to the recombinant soluble form of extracellular TLR2 domain (sTLR2) and sTLR4.Conclusions/SignificanceOur study demonstrates that MBL can affect proinflammatory cytokine and chemokine expressions by modifying C. albicans-/TLR-signaling pathways. This study supports an important role for MBL on the regulation of C. albicans-induced cellular responses.