Heat-aggregated noninfectious influenza virus induces a more balanced CD8(+)-T-lymphocyte immunodominance hierarchy than infectious virus

CD8(+)-T-cell (T(CD8+)) responses to infectious viruses are characterized by an immunodominance hierarchy in which the majority of T(CD8+) respond to one or a few immunodominant determinants, with a minority of T(CD8+) responding to a number of subdominant determinants. It is now well established that exogenous antigens are capable of inducing T(CD8+) to such immunodominant determinants, but the diversity of the response and the nature of the immunodominance hierarchy have not been examined. We addressed this issue by characterizing T(CD8+) responses to influenza virus preparations rendered inert by incubation for 10 min at 100 degrees C, as first reported by Speidel et al. (Eur. J. Immunol. 27:2391-2399, 1997). Extending these findings, we show that the primary T(CD8+) response to boiled virus can be sufficiently robust to be detected ex vivo by intracellular cytokine staining and that the response encompasses many of the peptides recognized by T(CD8+) induced by infectious virus. Importantly, the immunodominance hierarchy elicited was leveled, and we were unable to detect T(CD8+) that were specific for boiled virus. We used peritoneal exudate cells as antigen-presenting cells in vitro, and a number of observations indicated that boiled virus is processed via a phagocytic route that is likely to be endosomal in nature. These findings suggest that the repertoires of immunogenic peptides generated by endosomes and cytosolic processes overlap to a surprising degree. Furthermore, they demonstrate that the form of antigen administered can influence immunodominance hierarchies and that exogenous-antigen vaccines can induce broad and balanced T(CD8+) responses.     

IL-27 enhances LPS-induced proinflammatory cytokine production via upregulation of TLR4 expression and signaling in human monocytes

IL-27, which is produced by activated APCs, bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed, proinflammatory and anti-inflammatory activities are attributed to IL-27, and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however, the molecular mechanism behind this phenomenon is not understood. In this study, we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6, TNF-α, MIP-1α, and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming, we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore, IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge, this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections.     

Dependence of neurotrophic factor activation of Trk tyrosine kinase receptors on cellular sialidase

A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.     

Metabolomics Profiling of Tunisian Sonchus oleraceus L. Extracts and Their Antioxidant Activities

Sonchus oleraceus (L.) L. (Asteraceae) is an edible wild plant, known for its uses in traditional medicine. The aim of this study is to explore the phytochemical composition of the aerial parts (AP) and roots (R) of aqueous extracts of Sonchus oleraceus L. growing in Tunisia, using liquid chromatography-tandem mass spectrometry(LC/MS/MS), and determine the content of polyphenols and antioxidant activities. Results showed that aqueous extracts of AP and R contained, respectively, 195.25±33 μg/g and 118.66±14 μg/g gallic acid equivalent (GAE), and 52.58±7 μg/g and 3.2±0.3μg/g quercetin equivalent. AP and R extracts also contained tannins, 581.78±33 μg/g and 948.44±19 μg/g GAE. The AP extract in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activities, hydroxyl radical scavenging (OH−) and in cupric reducing antioxidant activity (CUPRAC) assays were respectively 0.325±0.036 mg/mL, 0.053±0.018 mg/mL, 0.696±0.031 mg/mL and 60.94±0.004 μMTE/g, while the R extract using the same assays showed, 0.209±0.052 mg/mL, 0.034±0.002 mg/mL, 0.444±0.014 mg/mL and 50.63±0.006 μM Trolox equivalent/g, respectively. A total of 68 compounds were tentatively identified by LC/MS/MS in both extracts in which quinic acid, pyrogallol, osthrutin, piperine, gentisic acid, fisetin, luteolin, caffeic acid, gingerol, were the most abundant in the LC/MS/MS spectrum. Many of these metabolites were found for the first time in Tunisian Sonchus oleraceus L. which may take account for the antioxidant activities exhibited by the plant.     

GEMC1 is a critical regulator of multiciliated cell differentiation

The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1‐deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.     

Kröhnke pyridines: Rapid and facile access to Mcl-1 inhibitors

The tumorigenic activity of upregulated Mcl-1 is manifested by binding the BH3 α-helical death domains of opposing Bcl-2 family members, neutralizing them and preventing apoptosis. Accordingly, the development of Mcl-1 inhibitors largely focuses on synthetic BH3 mimicry. The condensation of α-pyridinium methyl ketone salts and α,β-unsaturated carbonyl compounds in the presence of a source of ammonia, or the Kröhnke pyridine synthesis, is a simple approach to afford highly functionalized pyridines. We adapted this chemistry to rapidly generate low-micromolar inhibitors of Mcl-1 wherein the 2,4,6-substituents were predicted to mimic the i, i?+?2 and i?+?7 side chains of the BH3 α-helix.     

Analysis of two susceptibility SNPs in HLA region and evidence of interaction between rs6457617 in <Emphasis Type="BoldItalic">HLA-DQB1</Emphasis> and <Emphasis Type="BoldItalic">HLA-DRB1</Emphasis>*04 locus on Tunisian rheumatoid arthritis

Previous genomewide association studies (GWAS) and meta-analyses have enumerated several genes/loci in major histocompatibility complex region, which are consistently associated with rheumatoid arthritis (RA) in different ethnic populations. Given the genetic heterogeneity of the disease, it is necessary to replicate these susceptibility loci in other populations. In this case, we investigate the analysis of two SNPs, rs13192471 and rs6457617, from the human leukocyte antigen (HLA) region with the risk of RA in Tunisian population. These SNPs were previously identified to have a strong RA association signal in several GWAS studies. A case–control sample composed of 142 RA patients and 123 healthy controls was analysed. Genotyping of rs13192471 and rs6457617 was carried out using real-time PCR methods by TaqMan allelic discrimination assay. A trend of significant association was found in rs6457617 TT genotype with susceptibility to RA (\(P = 0.04\), \(p_{c} = 0.08\), \(\hbox {OR} = 1.73\)). Moreover, using multivariable analysis, the combination of rs6457617*TT–HLA-DRB1*\(04^{+}\) increased risk of RA (\(\hbox {OR} = 2.38\)), which suggest a gene–gene interaction event between rs6457617 located within the HLA-DQB1 and HLA-DRB1. Additionally, haplotypic analysis highlighted a significant association of rs6457617*T–HLA-DRB1*\(04^{+}\) haplotype with susceptibility to RA (\(P = 0.018\), \(p_{c} = 0.036\), \(\hbox {OR} = 1.72\)). An evidence of association was shown subsequently in \(\hbox {antiCCP}^{+}\) subgroup with rs6457617 both in T allele and TT genotype (\(P = 0.01\), \(p_{c} = 0.03\), \(\hbox {OR} = 1.66\) and \(P = 0.008\), \(p_{c} = 0.024\), \(\hbox {OR} = 1.28\), respectively). However, no association was shown for rs13192471 polymorphism with susceptibility and severity to RA. This study suggests the involvement of rs6457617 locus as risk variant for susceptibility/severity to RA in Tunisian population. Secondly, it highlights the gene–gene interaction between HLA-DQB1 and HLA-DRB1.     

Analogs design,synthesis and biological evaluation of peptidomimetics with potential anti-HCV activity

Two series of peptidomimetics were designed, prepared and evaluated for their anti-HCV activity. One series possesses a C-terminal carboxylate functionality. In the other series, the electrophilic vinyl sulfonate moiety was introduced as a novel class of HCV NS3/4A protease inhibitors. In vitro based studies were then performed to evaluate the efficacies of the inhibitors using Human hepatoma cells, with the vinyl sulfonate ester (10) in particular, found to have highly potent anti-HCV activity with an EC₅₀ = 0.296 μM. Finally, molecular modeling studies were performed through docking of the synthesized compounds in the HCV NS3/4A protease active site to assess their binding modes with the enzyme and gain further insight into their structure–activity relationships.