Interactions of T cells with fibroblast-like synoviocytes: role of the B7 family costimulatory ligand B7-H3

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-alpha, IFN-gamma, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.     

Morphological, Chemical, and Genetic Diversity of Tropical Marine Cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales)

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L. majuscula Gomont, Lyngbya spp., and Symploca spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp. from Symploca spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L. majuscula. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L. majuscula showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L. majuscula, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species.     

Temperature-dependence of rapid axonal transport in sympathetic nerves of the rabbit.

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-beta-hydroxylase (DBH) activity in rabbit sciatic nerves in vitro. These nerves were cooled locally to 2 degrees C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration at various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13 degrees C and 42 degrees C. Over this range of temperatures, the results were well described by the equation: V=0.546(1.09)T, where V is velocity in mm/hr, and T is temperature in degrees centigrade. The Q10 between 13 degrees and 42 degrees C was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47 degrees C, although only about half of the DBH activity disappeared during incubation at this temperature. Another transition occurred at 13 degrees C; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13 degrees C.     

Myeloid dendritic cells display downregulation of C-type lectin receptors and aberrant lectin uptake in systemic lupus erythematosus

Introduction  

There is a growing body of evidence implicating aberrant dendritic cell function as a crucial component in the immunopathogenesis of systemic lupus erythematosus. The purpose of the present study was to characterize the phagocytic capacity and expression of receptors involved in pathogen recognition and self-nonself discrimination on myeloid dendritic cells from patients with lupus.     

Passive mechanical properties of the lumbar multifidus muscle support its role as a stabilizer

The purpose of this study was to compare the passive mechanical properties and titin isoform sizes of the multifidus, longissimus, and iliocostalis muscles. Given our knowledge of each muscle's architecture and the multifidus’ operating range, we hypothesized that multifidus would have higher elastic modulus with corresponding smaller titin isoforms compared to longissimus or iliocostalis muscles. Single-fiber and fiber-bundle material properties were derived from passive stress–strain tests of excised biopsies (n=47). Titin isoform sizes were quantified via sodium dodecyl sulfate-vertical agarose gel electrophoresis (SDS-VAGE) analysis. We found that, at the single-fiber level, all muscles had similar material properties and titin isoform sizes. At the fiber-bundle level, however, we observed significantly increased stiffness (～45%) in multifidus compared to longissimus and iliocostalis muscles. These data demonstrate that each muscle may have a different scaling relationship between single-fiber and fiber-bundle levels, suggesting that the structures responsible for higher order passive mechanical properties may be muscle specific. Our results suggest that divergent passive material properties are observed at size scales larger than the single cell level, highlighting the importance of the extracellular matrix in these muscles. In addition to architectural data previously reported, these data further support the unique stabilizing function of the multifidus muscle. These data will provide key input variables for biomechanical modeling of normal and pathologic lumbar spine function and direct future work in biomechanical testing in these important muscles.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

Commonality and biosynthesis of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.     

Photoluminescence of Dy3+ activator sensitized by Ce3+ in KNaSO4 microphosphor

The KNaSO₄ microphosphor doped with Ce or Ce and Dy prepared by a wet chemical method was studied by scanning electron microscopy (SEM) and characterized by photoluminescence (PL). KNaSO₄ has a 5‐µm particle size detected by SEM. The KNaSO₄:Ce³⁺ spectrum shows a single emission band at 327 nm for an excitation of 269 nm due to 5d → 4f transition of the Ce³⁺ ion, indicating weak spin orbiting coupling of the Ce³⁺ ground state. Efficient energy transfer takes place from Ce³⁺ → Dy³⁺ sublattices indicating that Ce³⁺ could effectively sensitize Dy³⁺ (orange emission) and that the Ce³⁺ emission weakens significantly in KNaSO₄. The powder form of prepared KNaSO₄ show negligible change in morphologies and hence no effect on the particle size. The characteristics of this powder could provide improved luminescence properties. The development and understanding of this photoluminescence and the effect of Dy³⁺ on KNaSO₄: Ce³⁺ are discussed. Copyright © 2014 John Wiley & Sons, Ltd.