A novel inflammatory pathway involved in leukocyte recruitment: role for the kinin B1 receptor and the chemokine CXCL5

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine IL-1beta. However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1beta-induced leukocyte rolling, adherence, and emigration in mesenteric venules of wild-type (WT) mice, associated with an increase in B1 receptor mRNA expression, were substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down-regulation of IL-1beta-induced CXCL5 mRNA and protein expression compared with WT mice. Furthermore a selective neutralizing CXCL5 Ab caused profound suppression of leukocyte emigration in IL-1beta-treated WT mice. Finally, treatment of human endothelial cells with IL-1beta enhanced mRNA expression of the B1 receptor and the human (h) CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by approximately 50% when cells were pretreated with the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin while treatment with des-Arg9-bradykinin, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a proinflammatory pathway centered on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.     

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.     

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8+ T cells with detection by ELISPOT and HLA-multimer staining

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R ² = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.     

Convergence and Divergence in Direct and Indirect Life-History Traits of Closely Related Parasitoids (Braconidae: Microgastrinae)

Closely related species in nature often show similarities in suites of direct and indirect traits that reveal aspects of their phylogenetic history. Here we tested how common descent affects trait evolution in several closely related parasitoid species in the genera Cotesia and Microplitis (Hymenoptera: Braconidae: Microgastrinae) by comparing development, resource use and allocation into reproduction and maintenance. Parasitoids in these genera exhibit traits, like haemolymph feeding as larvae and external pupation that are rare in most parasitoid lineages. The growth of parasitized hosts was reduced by 90 % compared with healthy hosts, and maximum host size depended to a large extent on adult parasitoid size. Development time was longer in the more generalist parasitoids than in the specialists. Adult body mass was sexually dimorphic in all Cotesia species, with females being larger, but not in Microplitis spp. In contrast, in one of the Microplitis species males were found to be the larger sex. Egg load dynamics during the first 6 days after emergence were highly variable but egg number was typically higher in Cotesia spp. compared to Microplitis spp. Longevity in the various species was only greater in female than in male wasps in two Microplitis sp. There was a clear inverse relationship between resource use and allocation, e.g. maximum egg load and longevity, in these parasitoids. Our results reveal that adaptation to constraints imposed by host quality and availability has resulted in trait convergence and divergence at the species, genus and subfamily level.     

The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells

We have shown that the microtopography (mT) underlying colon cancer changes as a tumor de-differentiates. We distinguish the well-differentiated mT based on the increasing number of "pits" and poorly differentiated mT on the basis of increasing number of "posts." We investigated Rho A as a mechanosensing protein using mT features derived from those observed in the ECM of colon cancer. We evaluated Rho A activity in less-tumorogenic (Caco-2 E) and more tumorigenic (SW620) colon cancer cell-lines on microfabricated pits and posts at 2.5 μm diameter and 200 nm depth/height. In Caco-2 E cells, we observed a decrease in Rho A activity as well as in the ratio of G/F actin on surfaces with either pits or posts but despite this low activity, knockdown of Rho A led to a significant decrease in confined motility suggesting that while Rho A activity is reduced on these surfaces it still plays an important role in controlling cellular response to barriers. In SW620 cells, we observed that Rho A activity was greatest in cells plated on a post microtopography which led to increased cell motility, and an increase in actin cytoskeletal turnover.     

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin

Introduction

B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers.

Methods

A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples.

Results

The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC₅₀, nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels.

Conclusions

Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases.     

TRPM channels mediate zinc homeostasis and cellular growth during Drosophila larval development

TRPM channels have emerged as key mediators of diverse physiological functions. However, the ionic permeability relevant to physiological function in vivo remains unclear for most members. We report that the single Drosophila TRPM gene (dTRPM) generates a conductance permeable to divalent cations, especially Zn(2+) and in vivo a loss-of-function mutation in dTRPM disrupts intracellular Zn(2+) homeostasis. TRPM deficiency leads to profound reduction in larval growth resulting from a decrease in cell size and associated defects in mitochondrial structure and function. These phenotypes are cell-autonomous and can be recapitulated in wild-type animals by Zn(2+) depletion. Both the cell size and mitochondrial defect can be rescued by extracellular Zn(2+) supplementation. Thus our results implicate TRPM channels in the regulation of cellular Zn(2+) in vivo. We propose that regulation of Zn(2+) homeostasis through dTRPM channels is required to support molecular processes that mediate class I PI3K-regulated cell growth.     

Stochastic processes are key determinants of short-term evolution in influenza a virus

Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.     

Corneodesmosin, a component of epidermal corneocyte desmosomes, displays homophilic adhesive properties.

Corneodesmosomes, the modified desmosomes of the uppermost layers of the epidermis, play an important role in corneocyte cohesion. Corneodesmosin is a secreted glycoprotein located in the corneodesmosomal core and covalently linked to the cornified envelope of corneocytes. Its glycine- and serine-rich NH(2)-terminal domain may fold to give structural motifs similar to the glycine loops described in epidermal cytokeratins and loricrin and proposed to display adhesive properties. A chimeric protein comprising human corneodesmosin linked to the transmembrane and cytoplasmic domains of mouse E-cadherin was expressed in mouse fibroblasts to test the ability of corneodesmosin to promote cell-cell adhesion. Classic aggregation assays indicated that corneodesmosin mediates homophilic cell aggregation. Moreover, Ca(2+) depletion showed a moderate effect on aggregation. To assess the involvement of the glycine loop domain in adhesion, full-length corneodesmosin, corneodesmosin lacking this domain, or this domain alone were expressed as glutathione S-transferase fusion proteins and tested for protein-protein interactions by overlay binding assays. The results confirmed that corneodesmosin presents homophilic interactions and indicated that its NH(2)-terminal glycine loop domain is sufficient but not strictly necessary to promote binding. Altogether, these results provide the first experimental evidence for the adhesive properties of corneodesmosin and for the involvement of its glycine loop domain in adhesion.     

Soil CO2 venting as one of the mechanisms for tolerance of Zn deficiency by rice in flooded soils

We sought to explain rice (Oryza sativa) genotype differences in tolerance of zinc (Zn) deficiency in flooded paddy soils and the counter‐intuitive observation, made in earlier field experiments, that Zn uptake per plant increases with increasing planting density. We grew tolerant and intolerant genotypes in a Zn‐deficient flooded soil at high and low planting densities and found (a) plant Zn concentrations and growth increased with planting density and more so in the tolerant genotype, whereas the concentrations of other nutrients decreased, indicating a specific effect on Zn uptake; (b) the effects of planting density and genotype on Zn uptake could only be explained if the plants induced changes in the soil to make Zn more soluble; and (c) the genotype and planting density effects were both associated with decreases in dissolved CO₂ in the rhizosphere soil solution and resulting increases in pH. We suggest that the increases in pH caused solubilization of soil Zn by dissolution of alkali‐soluble, Zn‐complexing organic ligands from soil organic matter. We conclude that differences in venting of soil CO₂ through root aerenchyma were responsible for the genotype and planting density effects.