(1->3),(1->4)-{beta}-d-Glucans in the cell walls of the Poales (sensu lato): an immunogold labeling study using a monoclonal antibody

(1→3),(1→4)-?-Glucans had previously been detected in nonlignified cell wall preparations of only the Poaceae and five other families in the graminoid clade of the Poales (s.l.). Cell walls of vegetative organs of 12 species in nine families of the Poales (s.l.) were examined by immunogold labeling using a monoclonal antibody to (1→3),(1→4)-?-glucans. Three types of wall-labeling patterns were identified depending on the density of labeling of the nonlignified walls of epidermal and parenchyma cells and the lignified walls of sclerenchyma fibers and xylem tracheary elements: type 1 in Poaceae and Flagellariaceae, type 2 in Restionaceae and Xyridaceae, and type 3 in Cyperaceae and Juncaceae. Type 1 had the heaviest labeling of nonlignified walls and type 2 the heaviest labeling of lignified walls. Type 3 had the least wall labeling, with only very light labeling of nonlignified and lignified walls. No labeling was found over walls of Typhaceae, Sparganiaceae, or Bromeliaceae. The results are discussed in relation to Poales phylogeny.     

A high-density screen for linkage in multiple sclerosis

To provide a definitive linkage map for multiple sclerosis, we have genotyped the Illumina BeadArray linkage mapping panel (version 4) in a data set of 730 multiplex families of Northern European descent. After the application of stringent quality thresholds, data from 4,506 markers in 2,692 individuals were included in the analysis. Multipoint nonparametric linkage analysis revealed highly significant linkage in the major histocompatibility complex (MHC) on chromosome 6p21 (maximum LOD score [MLS] 11.66) and suggestive linkage on chromosomes 17q23 (MLS 2.45) and 5q33 (MLS 2.18). This set of markers achieved a mean information extraction of 79.3% across the genome, with a Mendelian inconsistency rate of only 0.002%. Stratification based on carriage of the multiple sclerosis–associated DRB1*1501 allele failed to identify any other region of linkage with genomewide significance. However, ordered-subset analysis suggested that there may be an additional locus on chromosome 19p13 that acts independent of the main MHC locus. These data illustrate the substantial increase in power that can be achieved with use of the latest tools emerging from the Human Genome Project and indicate that future attempts to systematically identify susceptibility genes for multiple sclerosis will have to involve large sample sizes and an association-based methodology.     

Cutting edge: antibody production to pneumococcal polysaccharides requires CD1 molecules and CD8+ T cells

T cell involvement in Ab responses to thymus-independent type 2 Ags is an immunologic enigma. The identity of these cells and the mechanisms of their TCR engagement to carbohydrate molecules remain unknown. We measured IgG Ab production after immunization with pneumococcal polysaccharides in mice with disruptions in selected genes of the T cell pathway. Nonclassical MHC class I-like CD1 molecules and MHC class I-dependent CD8+ cells were found to be essential. Our findings set forth a new paradigm for humoral responses in which CD1 expression as well as a subset of CD8+ cells are required to provide helper function for Ab production against thymus-independent type 2 polysaccharides, similar to MHC class II-restricted CD4+ cells for protein Ags.     

The src homology 2 domain-containing tyrosine phosphatase 2 regulates primary T-dependent immune responses and Th cell differentiation

The src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) plays an important role in development and in growth factor receptor signaling pathways, yet little is known of its role in the immune system. We generated mice expressing a dominant-negative version of the protein, SHP2(CS), specifically in T cells. In SHP2(CS) mice, T cell development appears normal with regard to both negative and positive selection. However, SHP2(CS) T cells express higher levels of activation markers, and aged mice have elevated serum Abs. This is associated with a marked increase in IL-4, IL-5, and IL-10 secretion by SHP2(CS) T cells in vitro. In addition, primary thymus-dependent B cell responses are deficient in SHP2(CS) mice. We show that whereas TCR-induced linker for activation of T cells phosphorylation is defective, CTLA-4 and programmed death-1 signaling are not affected by SHP2(CS) expression. Our results suggest that a key action of wild-type SHP2 is to suppress differentiation of T cells to the Th2 phenotype.     

Sphingosine kinase 1 is a negative regulator of CD4+ Th1 cells

CD4+ Th1 cells produce IFN-gamma, TNF-alpha, and IL-2. These Th1 cytokines play critical roles in both protective immunity and inflammatory responses. In this study we report that sphingosine kinase 1 (SPHK1), but not SPHK2, is highly expressed in DO11.10 Th1 cells. The expression of SPHK1 in Th1 cells requires TCR signaling and new protein synthesis. SPHK1 phosphorylates sphingosine to form sphingosine-1-phosphate. Sphingosine-1-phosphate plays important roles in inhibition of apoptosis, promotion of cell proliferation, cell migration, calcium mobilization, and activation of ERK1/2. When SPHK1 expression was knocked down by SPHK1 short interfering RNA, the production of IL-2, TNF-alpha, and IFN-gamma by Th1 cells in response to TCR stimulation was enhanced. Consistently, overexpression of dominant-negative SPHK1 increased the production of IL-2, TNF-alpha, and IFN-gamma in Th1 cells. Furthermore, overexpression of SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, TNF-alpha, and IFN-gamma. Several chemokines, including Th2 chemokines CCL17 and CCL22, were up-regulated by SPHK1 short interfering RNA and down-regulated by overexpression of SPHK1. We also showed that Th2 cells themselves express CCL17 and CCL22. Finally, we conclude that SPHK1 negatively regulates the inflammatory responses of Th1 cells by inhibiting the production of proinflammatory cytokines and chemokines.     

Induction of cell cycle arrest in lymphocytes by Actinobacillus actinomycetemcomitans cytolethal distending toxin requires three subunits for maximum activity

We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor encoded by the cytolethal distending toxin (cdt)B gene. In this study, we used rCdt peptides to study the contribution of each subunit to toxin activity. As previously reported, CdtB is the only Cdt subunit that is capable of inducing cell cycle arrest by itself. Although CdtA and CdtC do not exhibit activity alone, each subunit is able to significantly enhance the ability of CdtB to induce G2 arrest in Jurkat cells; these effects were dependent upon protein concentration. Moreover, the combined addition of both CdtA and CdtC increased the ED50 for CdtB >7000-fold. In another series of experiments, we demonstrate that the three Cdt peptides are able to form a functional toxin unit on the cell surface. However, these interactions first require that a complex forms between the CdtA and CdtC subunits, indicating that these peptides are required for interaction between the cell and the holotoxin. This conclusion is further supported by experiments in which both Jurkat cells and normal human lymphocytes were protected from Cdt holotoxin-induced G2 arrest by pre-exposure to CdtA and CdtC. Finally, we have used optical biosensor technology to show that CdtA and CdtC have a strong affinity for one another (10(-7) M). Furthermore, although CdtB is unable to bind to either CdtA or CdtC alone, it is capable of forming a stable complex with CdtA/CdtC. The implications of our results with respect to the function and structure of the Cdt holotoxin are discussed.     

Complete functional rescue of the ABCA1-/- mouse by human BAC transgenesis

Humanized mouse models are useful tools to explore the functional and regulatory differences between human and murine orthologous genes. We have combined a bioinformatics approach and an in vivo approach to assess the functional and regulatory differences between the human and mouse ABCA1 genes. Computational analysis identified significant differences in potential regulatory sites between the human and mouse genes. The effect of these differences was assessed in vivo, using a bacterial artificial chromosome transgenic humanized ABCA1 mouse model that expresses the human gene in the absence of mouse ABCA1. Humanized mice expressed human ABCA1 protein at levels similar to wild-type mice and fully compensated for cholesterol efflux activity and lipid levels seen in ABCA1-deficient mice. Liver X receptor agonist administration resulted in significant increases in HDL values associated with parallel increases in the hepatic ABCA1 protein and mRNA levels in the humanized ABCA1 mice, as seen in the wild-type animals. Our studies indicate that despite differences in potential regulatory regions, the human ABCA1 gene is able to functionally fully compensate for the mouse gene. Our humanized ABCA1 mice can serve as a useful model system for functional analysis of the human ABCA1 gene in vivo and can be used for the generation of potential new therapeutics that target HDL metabolism.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.