Sialic acid acetylesterase (SIAE) is an enzyme that negatively regulates B lymphocyte antigen receptor signaling and is required for the maintenance of immunological tolerance in mice1, 2. Heterozygous loss-of-function germline rare variants and a homozygous defective polymorphic variant of SIAE were identified in 24/923 Caucasian subjects with relatively common autoimmune disorders and in 2/648 Caucasian controls. All heterozygous loss-of-function SIAE mutations tested were capable of functioning in a dominant negative manner. A homozygous secretion-defective polymorphic variant of SIAE was catalytically active, lacked the ability to function in a dominant negative manner, and was seen in 8 autoimmune subjects but in no control subjects. The Odds Ratio for inheriting defective SIAE alleles was 8.6 in all autoimmune subjects, 8.3 in subjects with rheumatoid arthritis, and 7.9 in subjects with type I diabetes. Functionally defective SIAE rare and polymorphic variants represent a strong genetic link to susceptibility in relatively common human autoimmune disorders.

Mature recirculating B cells are generally assumed to exist in follicular niches in secondary lymphoid organs, and these cells mediate T-dependent humoral immune responses. We show here that a large proportion of mature B lymphocytes occupy an anatomically and functionally distinct perisinusoidal niche in the bone marrow. Perisinusoidal B cells circulate freely, as revealed by parabiosis studies. However, unlike their counterparts in the follicular niche, these cells are capable of being activated in situ by blood-borne microbes in a T-independent manner to generate specific IgM antibodies. The bone marrow represents a unique type of secondary lymphoid organ in which mature B cells are strategically positioned in the path of circulating microbes.

Generation of Siae ⌬ 2/ ⌬ 2 mice Exon 2 of the Siae gene is unique to Lse . An engineered inframe deletion of exon 2 in a murine Lse complementary DNA resulted in a protein that lacked esterase activity ( Fig. 1 A ). Genomic deletion of exon 2 was achieved as described in the Materials and methods ( Fig. 1 B ). After germline transmission, homozygous mutant mice were generated and were found to be viable. Truncated exon 2 -defi cient Siae mRNA could be detected in KO mice (Fig. S3, available at http://www .jem.org/cgi/content/full/jem.20081399/DC1). Cytosolic esterase mRNA continues to be transcribed in these mutant mice. We refer to these KO animals as Siae ⌬ 2/ ⌬ 2 mice.Enhanced B lymphocyte antigen receptor signaling in Siae ⌬ 2/ ⌬ 2 mice Because Siae has the potential to remove 9-O -acetyl residues from ␣ 2 -6-linked sialic acid containing Siglec ligands, we predicted that B cells from mice lacking this esterase might exhibit enhanced BCR signaling similar to that noted in CD22-null mice ( 13 -16 ). Mice were fi rst bred into the C57BL/6 background for 10 generations. B cells from WT and Siae ⌬ 2/ ⌬ 2 mice were gated on, and the accumulation of cytoplasmic calcium after ligation of the BCR was analyzed using fl ow cytometry. As seen in Fig. 2 , BCR cross-linking resulted in an accelerated and enhanced calcium fl ux. A similar result was seen when purifi ed splenic B cells from mutant and WT mice were analyzed (Fig. S4, available at http://www.jem.org/cgi/content/ full/jem.20081399/DC1). These data suggested that in the absence of functional Siae, BCR signal strength is markedly enhanced and that this alteration in signal strength is an intrinsic property of mutant B lymphocytes.Defective CD22 signaling and hyperacetylation of ␣ 2 -6-linked sialic acid moieties on Siae mutant B cells The defect in Siae could result in the increased acetylation of ␣ 2 -6-linked sialic acid on N -glycans in B cells and, thus, attenuate the ability of glycoproteins on B cells to ligate CD22 and generate inhibitory signals. We sought to examine if there was a defect in CD22 signaling in Siae ⌬ 2/ ⌬ 2 mice. After BCR cross-linking, CD22 was isolated by immunoprecipitation, and immunoprecipitates were examined for CD22 tyrosine phosphorylation and for associated SHP-1 using Western blot assays. As seen in Fig. 3 (left, experiment 1; and right, experiment 2), tyrosine phosphorylation of CD22 after BCR signaling was reduced in Siae ⌬ 2/ ⌬ 2 mice in spite of similar levels of surface CD22 expression in WT and mutant mice ( It remained to be demonstrated whether a defect in Siae would result in enhanced 9-O-acetylation of ␣ 2 -6-linked sialic acid in Siae ⌬ 2/ ⌬ 2 mice. 9-O -acetylation of sialic acid has not

IntroductionNewly formed B cells emigrate from the bone marrow and home initially to the spleen 1 where they mature via transitional stages primarily into long-lived follicular B cells, and where they also differentiate into marginal zone (MZ) B cells. [2][3][4][5][6][7] Naïve follicular B cells express high levels of surface IgD and CD23 and have the ability to recirculate, entering and exiting follicular niches in secondary lymphoid organs in search of antigen. 8,9 Although mature B cells have long been described in the bone marrow, 10,11 our recent studies suggest that the bone marrow may provide an alternative perisinusoidal niche for follicular phenotype IgD hi B cells, wherein they have ready access to blood-borne pathogens and can thus contribute to T-independent IgM responses directed against such organisms. 12 These studies describing bone marrow antibody-secreting cells generated in response to blood-borne pathogens were performed on splenectomized mice, but it remained formally possible that the IgM plasma cells in the bone marrow observed in these circumstances could have migrated to the bone marrow from some other secondary lymphoid organ.Immature B cells in the bone marrow acquire the ability to migrate via the blood specifically to the spleen, presumably because these cells receive inhibitory signals that attenuate their ability to respond to chemokines and to thus home to lymph nodes or inflammatory sites. 13 The unique architectural organization of the spleen is important for MZ B-cell development and survival, 5,14 and the evolutionary requirement for immature B cells to home to this organ may be linked to the need for such cells to differentiate into MZ B cells. However, the teleologic rationale for mature follicular B-cell maturation to apparently occur only in the spleen, as is generally assumed to be the case, is unclear. It is reasonable to entertain the possibility that B-cell maturation might also occur in the bone marrow, particularly because immature B cells are generated there, and because the perisinusoidal bone marrow niche does contain a large number of B cells with an IgD hi phenotype. 12 Indeed, in splenectomized animals and humans, mature follicular phenotype B cells are abundant in lymph nodes and in the perisinusoidal bone marrow niche, and they presumably mature at some site distinct from the spleen, most likely within the bone marrow compartment itself.Another reason to explore the maturation of IgD hi B cells in the bone marrow relates to the poorly understood issue of the loss of these cells in certain mutant mice. Mice deficient in Aiolos, CD22, and CD72, for instance, all lose mature B cells in the bone marrow but preserve their phenotypic counterparts in other conventional secondary lymphoid organs. [15][16][17][18][19] These null mice lack distinct negative regulators of the BCR. Mice lacking VCAM-1 also lack B cells in the bone marrow, 20,21 suggesting that VCAM-1 may contribute to entry into or retention within this compartment. Although it has been suggested that CD...

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Disparate models for the development of peripheral B cells may reflect significant heterogeneity in recirculating long-lived B cells that have not been previously accounted for. We show in this study that the murine recirculating B cell pool contains two distinct, long-lived, posttransitional, follicular B cell populations. Follicular Type I IgMlow B cells require Ag-derived and Btk-dependent signals for their development and make up the majority of cells in the recirculating follicular B cell pool. Follicular type II B cells do not require Btk- or Notch-2-derived signals, make up about a third of the long-lived recirculating B cell pool, and can develop in the absence of Ag. These two follicular populations exhibit differences in basal tyrosine phosphorylation and in BCR-induced proliferation, suggesting that they may represent functionally distinct populations of long-lived recirculating B cells.

NF-κB1 and Notch2 are both required for the development of marginal zone (MZ) B cells. Analysis of B lymphocyte development in mice that are doubly heterozygous at the Notch2 and NF-κB1 loci revealed synergism between Notch2 and NF-κB1 during MZ B cell development. Two known transcriptional targets of the Notch pathway, Hes-5 and Deltex-1, were found to be preferentially expressed in MZ B cells and regulated by NF-κB1. These studies provide in vivo evidence for a genetic interaction between the Notch and NF-κB pathways.

Background: Bactericidal capacity, durable inhibition of biofilm formation, and a threedimensional (3D) porous structure are the emphases of infected bone defect (IBD) treatment via local scaffold implantation strategy. Purpose: In this study, silver nanoparticle (AgNP)-loaded nano-hydroxyapatite (nHA)@ reduced graphene oxide (RGO) 3D scaffolds (AHRG scaffolds) were designed to alleviate bone infection, inhibit biofilm formation, and promote bone repair through the synergistic effects of AgNPs, RGO, and nHA. Materials and Methods: AHRGs were prepared using a one-step preparation method, to create a 3D porous scaffold to facilitate a uniform distribution of AgNPs and nHA. Methicillin-resistant Staphylococcus aureus (MRSA) was used as a model-resistant bacterium, and the effects of different silver loadings on the antimicrobial activity and cytocompatibility of materials were evaluated. Finally, a rabbit IBD model was used to evaluate the therapeutic effect of the AHRG scaffold in vivo. Results: The results showed successful synthesis of the AHRG scaffold. The ideal 3D porous structure was verified using scanning electron microscopy and transmission electron microscopy, and X-ray photoelectron spectroscopy and selected area electron diffraction measurements revealed uniform distributions of AgNP and nHA. In vitro antibacterial and cytocompatibility indicated that the 4% AHRG scaffolds possessed the most favorable balance of bactericidal properties and cytocompatibility. In vivo evaluation of the IBD model showed promising treatment efficacy of AHRG scaffolds. Conclusion: The as-fabricated AHRG scaffolds effectively eliminated infection and inhibited biofilm formation. IBD repair was facilitated by the bactericidal properties and 3D porous structure of the AHRG scaffold, suggesting its potential in the treatment of IBDs.