Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.     

Simulated brain tumor growth dynamics using a three-dimensional cellular automaton

We have developed a novel and versatile three-dimensional cellular automaton model of brain tumor growth. We show that macroscopic tumor behavior can be realistically modeled using microscopic parameters. Using only four parameters, this model simulates Gompertzian growth for a tumor growing over nearly three orders of magnitude in radius. It also predicts the composition and dynamics of the tumor at selected time points in agreement with medical literature. We also demonstrate the flexibility of the model by showing the emergence, and eventual dominance, of a second tumor clone with a different genotype. The model incorporates several important and novel features, both in the rules governing the model and in the underlying structure of the model. Among these are a new definition of how to model proliferative and non-proliferative cells, an isotropic lattice, and an adaptive grid lattice.     

Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as a selective and potent agonist of IL-15 action through IL-15R beta/gamma. Hyperagonist IL-15 x IL-15R alpha fusion proteins

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.     

Interleukin-15 plays a central role in human kidney physiology and cancer through the γc signaling pathway

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation).     

Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation

Background

Since the first fungal genome sequences became available, investigators have been employing comparative genomics to understand how fungi have evolved to occupy diverse ecological niches. The secretome, i.e. the entirety of all proteins secreted by an organism, is of particular importance, as by these proteins fungi acquire nutrients and communicate with their surroundings.

Results

It is generally assumed that fungi with similar nutritional lifestyles have similar secretome compositions. In this study, we test this hypothesis by annotating and comparing the soluble secretomes, defined as the sets of proteins containing classical signal peptides but lacking transmembrane domains of fungi representing a broad diversity of nutritional lifestyles. Secretome size correlates with phylogeny and to a lesser extent with lifestyle. Plant pathogens and saprophytes have larger secretomes than animal pathogens. Small secreted cysteine-rich proteins (SSCPs), which may comprise many effectors important for the interaction of plant pathogens with their hosts, are defined here to have a mature length of ≤ 300 aa residues, at least four cysteines, and a total cysteine content of ≥5%. SSCPs are found enriched in the secretomes of the Pezizomycotina and Basidiomycota in comparison to Saccharomycotina. Relative SSCP content is noticeably higher in plant pathogens than in animal pathogens, while saprophytes were in between and closer to plant pathogens. Expansions and contractions of gene families and in the number of occurrences of functional domains are largely lineage specific, e.g. contraction of glycoside hydrolases in Saccharomycotina, and are only weakly correlated with lifestyle. However, within a given lifestyle a few general trends exist, such as the expansion of secreted family M14 metallopeptidases and chitin-binding proteins in plant pathogenic Pezizomycotina.

Conclusions

While the secretomes of fungi with similar lifestyles share certain characteristics, the expansion and contraction of gene families is largely lineage specific, and not shared among all fungi of a given lifestyle.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-722) contains supplementary material, which is available to authorized users.     

Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif.

Plasmid pSC101 harbors a 28-bp sequence which is homologous to dif, the target site of the XerC/XerD-dependent recombination system in Escherichia coli. Using a technique which allows very sensitive detection of plasmid loss, we show that recombination at this site, termed psi for pSC101 stabilized inheritance, causes a moderate increase in pSC101 stability. The role of the psi sequence in site-specific recombination has been explored in two other contexts. It was cloned in a derivative of plasmid p15A and inserted into the chromosome in place of dif. In the first situation, psi activity requires accessory sequences and results in multimer resolution; in the second situation, it suppresses the effects of the dif deletion and can promote intermolecular exchanges. Thus, psi is a site whose recombinational activity depends on the context, the first in the cer/dif family known to exhibit such flexibility.     

Posttranslational Modification of the NH2-terminal Region of CXCL5 by Proteases or Peptidylarginine Deiminases (PAD) Differently Affects Its Biological Activity

Posttranslational modifications, e.g. proteolysis, glycosylation, and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1–78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2–78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg⁹ in CXCL5 to citrulline, generating [Cit⁹]CXCL5(1–78). Compared with CXCL5(1–78), [Cit⁹]CXCL5(1–78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2, and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8–78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH₂-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin α-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to up-regulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8–78) and CXCL5(9–78) induced a more pronounced neutrophil influx in vivo compared with CXCL5(1–78). Administration of 300 pmol of either CXCL5(1–78) or [Cit⁹]CXCL5(1–78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9–78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.     

Future climate‐driven shifts in distribution of Calanus finmarchicus

Calanus finmarchicus is a key‐structural species of the North Atlantic polar biome. The species plays an important trophic role in subpolar and polar ecosystems as a grazer of phytoplankton and as a prey for higher trophic levels such as the larval stages of many fish species. Here, we used a recently developed ecological niche model to assess the ecological niche (sensu Hutchinson) of C. finmarchicus and characterize its spatial distribution. This model explained about 65% of the total variance of the observed spatial distribution inferred from an independent dataset (data of the continuous plankton recorder survey). Comparisons with other types of models (structured population and ecophysiological models) revealed a clear similarity between modeled spatial distributions at the scale of the North Atlantic. Contemporary models coupled with future projections indicated a progressive reduction of the spatial habitat of the species at the southern edge and a more pronounced one in the Georges Bank, the Scotian Shelf and the North Sea and a potential increase in abundance at the northern edge of its spatial distribution, especially in the Barents Sea. These major changes will probably lead to a major alteration of the trophodynamics of North Atlantic ecosystems affecting the trophodynamics and the biological carbon pump.     

Carrier screening for cystic fibrosis in a prenatal setting

Maternal prenatal cystic fibrosis (CF) screening was offered from September, 1997, to April, 1999, at the Ghent University Hospital, to couples undergoing prenatal diagnosis (amniocentesis) for reasons not related to CF. Fifteen minutes were devoted to explaining CF, CF screening, and the study protocol. The purpose was to assess the short- and long-term knowledge of CF, the attitude towards carrier screening, and carriership. A total of 314 couples entered the pilot study; 13 female CF carriers were identified. None of their partners carried an identifiable mutation. Our survey results show that information about CF and CF screening can be given effectively as part of antenatal care because most couples recalled important medical and genetic issues, valued the genetic test for CF, and seemed to cope well with the results. Risk estimates and actual numbers were more difficult to process and recall. From the small number of couples in which the woman alone was found to be a carrier, there was little or no evidence of marked distress.