Nucleosides Present on Phlebotomine Saliva Induce Immunossuppression and Promote the Infection Establishment

Background

Sand fly saliva plays a crucial role in establishing Leishmania infection. We identified adenosine (ADO) and adenosine monophosphate (AMP) as active pharmacologic compounds present in Phlebotomus papatasi saliva that inhibit dendritic cell (DC) functions through a PGE₂/IL 10-dependent mechanism.

Methodology/Principal Findings

Herein, we prepared a mixture of ADO and AMP in equimolar amounts similar to those present in the salivary-gland extract (SGE) form one pair of salivary glands of P. papatasi and co-injected it with Leishmania amazonensis or L. major into mouse ears. ADO+AMP mimicked exacerbative effects of P. papatasi saliva in leishmaniasis, increasing parasite burden and cutaneous lesions. Enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect associated with IL-10 enhancement. Immunosuppressive factors COX2 and IL-10 were upregulated and failed to enhance ear lesion and parasite burden in IL 10^-/- infected mice. Furthermore, nucleosides increased regulatory T cell (Treg) marker expression on CD4⁺CD25^- cells, suggesting induction of Tregs on effector T cells (T eff). Treg induction (iTreg) was associated with nucleoside-induced tolerogenic dendritic cells (tDCs) expressing higher levels of COX₂ and IL-10. In vitro generation of Tregs was more efficient in DCs treated with nucleosides. Suppressive effects of nucleosides during cutaneous leishmaniasis were mediated through an A2_AR-dependent mechanism. Using BALB/c mice deficient in A2A ADO receptor (A2_AR^–/–), we showed that co-inoculated mice controlled infection, displaying lower parasite numbers at infection sites and reduced iTreg generation.

Conclusion/Significance

We have demonstrated that ADO and AMP in P. papatasi saliva mediate exacerbative effects of Leishmania infection by acting preferentially on DCs promoting a tolerogenic profile in DCs and by generating iTregs in inflammatory foci through an A2_AR mechanism.     

Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca

The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species.     

Inosine triphosphatase allele frequency and association with ribavirin-induced anaemia in Brazilian patients receiving antiviral therapy for chronic hepatitis C

Inosine triphosphatase (ITPA) single nucleotide polymorphisms (SNPs) are stronglyassociated with protection against ribavirin (RBV)-induced anaemia in European,American and Asian patients; however, there is a paucity of data for Brazilianpatients. The aim of this study was to evaluate the ITPA SNP (rs7270101/rs1127354)frequency in healthy and hepatitis C virus (HCV)-infected patients from Brazil andthe association with the development of severe anaemia during antiviral therapy. ITPASNPs were determined in 200 HCV infected patients and 100 healthy individuals bysequencing. Biochemical parameters and haemoglobin (Hb) levels were analysed in 97patients who underwent antiviral therapy. A combination of AArs7270101+CCrs1127354(100% ITPase activity) was observed in 236/300 individuals. Anaemia was observed in87.5% and 86.2% of treated patients with AA (rs7270101) and CC genotypes (rs1127354),respectively. Men with AA (rs7270101) showed a considerable reduction in Hb at week12 compared to those with AC/CC (p = 0.1475). In women, there was no influence ofgenotype (p = 0.5295). For rs1127354, men with the CC genotype also showed a suddenreduction in Hb compared to those with AC. Allelic distribution of rs7270101 andrs1127354 shows high rates of the genotypes AA and CC, respectively, suggesting thatthe study population had a great propensity for developing RBV-induced anaemia. Aprogressive Hb reduction during treatment was observed; however, this reduction wasgreater in men at week 12 than in women.     

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.     

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ_XAC1133, this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ_XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ_XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX_XAC2398, which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX_XAC2398in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ_XAC1133. Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ_XAC1133 interactions with FimX_XAC2398 and PilB_XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of “degenerate” GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.     

Brain changes in BDNF and S100B induced by ketogenic diets in Wistar rats

AimsWe investigated the effects of ketogenic diet (KD) on levels of tumor necrosis factor alpha (TNF-α, a classical pro-inflammatory cytokine), BDNF (brain-derived neurotrophic factor, commonly associated with synaptic plasticity), and S100B, an astrocyte neurotrophic cytokine involved in metabolism regulation.Main methodsYoung Wistar rats were fed during 8 weeks with control diet or two KD, containing different proportions of omega 6 and omega 3 polyunsaturated fatty acids. Contents of TNF-α, BDNF and S100B were measured by ELISA in two brain regions (hippocampus and striatum) as well as blood serum and cerebrospinal fluid.Key findingsOur data suggest that KD was able to reduce the levels of BDNF in the striatum (but not in hippocampus) and S100B in the cerebrospinal fluid of rats. These alterations were not affected by the proportion of polyunsaturated fatty acids offered. No changes in S100B content were observed in serum or analyzed brain regions. Basal TNF-α content was not affected by KD.SignificanceThese findings reinforce the importance of this diet as an inductor of alterations in the brain, and such changes might contribute to the understanding of the effects (and side effects) of KD in brain disorders.     

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.