Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae

The fimbriae of Porphyromonas gingivalis, the causative agent of periodontitis, have been implicated in various aspects of pathogenicity, such as colonization, adhesion and aggregation. Porphyromonas gingivalis ATCC 33277 has two adhesins comprised of the FimA and Mfa1 fimbriae. We characterized the PGN0289 (Mfa3) protein, which is one of the three accessory proteins of Mfa1 fimbriae in P. gingivalis. The Mfa3 protein was present in two different sizes, 40 and 43 kDa, in the cell. The 43‐kDa and 40‐kDa Mfa3 were detected largely in the inner membrane and the outer membrane, respectively. Purified Mfa1 fimbriae contained the 40‐kDa Mfa3 alone. Furthermore, the 40‐kDa Mfa3 started with the Ala⁴⁴ residue of the deduced amino acid sequence, indicating that the N‐terminal region of the nascent protein expressed from the mfa3 gene is processed in the transport step from the inner membrane into fimbriae. Immuno‐electron microscopy revealed that Mfa3 localized at the tip of the fimbrial shaft. Interestingly, deletion of the mfa3 gene resulted in the absence of other accessory proteins, PGN0290 and PGN0291, in the purified Mfa1 fimbriae, suggesting that Mfa3 is required for integration of PGN0290 and PGN0291 into fimbriae. A double mutant of mfa3 and fimA genes (phenotype Mfa1 plus, FimA minus) showed increased auto‐aggregation and biofilm formation similar to a double mutant of mfa1 and fimA genes (phenotype Mfa1^–, FimA^–). These findings suggest that the tip protein Mfa3 of the Mfa1 fimbriae may function in the integration of accessory proteins and in the colonization of P. gingivalis.     

Peptidoglycan recognition proteins in Drosophila immunity

Innate immunity is the front line of self-defense against infectious non-self in vertebrates and invertebrates. The innate immune system is mediated by germ-line encoding pattern recognition molecules (pathogen sensors) that recognize conserved molecular patterns present in the pathogens but absent in the host. Peptidoglycans (PGN) are essential cell wall components of almost all bacteria, except mycoplasma lacking a cell wall, which provides the host immune system an advantage for detecting invading bacteria. Several families of pattern recognition molecules that detect PGN and PGN-derived compounds have been indentified, and the role of PGRP family members in host defense is relatively well-characterized in Drosophila. This review focuses on the role of PGRP family members in the recognition of invading bacteria and the activation and modulation of immune responses in Drosophila.     

Emerging roles of basophils in allergic inflammation

Basophils have long been neglected in immunological studies because they were regarded as only minor relatives of mast cells. However, recent advances in analytical tools for basophils have clarified the non-redundant roles of basophils in allergic inflammation. Basophils play crucial roles in both IgE-dependent and -independent allergic inflammation, through their migration to the site of inflammation and secretion of various mediators, including cytokines, chemokines, and proteases. Basophils are known to produce large amounts of IL-4 in response to various stimuli. Basophil-derived IL-4 has recently been shown to play versatile roles in allergic inflammation by acting on various cell types, including macrophages, innate lymphoid cells, fibroblasts, and endothelial cells. Basophil-derived serine proteases are also crucial for the aggravation of allergic inflammation. Moreover, recent reports suggest the roles of basophils in modulating adaptive immune responses, particularly in the induction of Th2 differentiation and enhancement of humoral memory responses. In this review, we will discuss recent advances in understanding the roles of basophils in allergic inflammation.     

Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells

The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10–200 μM ZEA showed efficacious metabolism of the toxin. α-zearalenol and β-zearalenol were the measured preponderant metabolites (respectively 40.7 ± 3.1% and 31.9 ± 4.9% of total metabolites, after a 3 h exposure to 10 μM ZEA), whereas ZEA-glucuronide and α-zearalenol glucuronide were less produced (respectively 8.2 ± 0.9% and 19.1 ± 1.3% of total metabolites, after a 3 h exposure to 10 μM ZEA). Cell production of reduced metabolites was strongly inhibited by α-and β-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited α-hydroxysteroid dehydrogenase type II and β-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, α-zearalenol was preponderantly found at the basal side, whereas β-zearalenol and both glucuronides were preferentially excreted at the apical side. As α-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.     

TLR2在小鼠肿瘤细胞表面的表达

目的检测TLR2在多种组织来源小鼠肿瘤细胞表面的表达，观察TLR2配基PCN对肿瘤细胞增殖的影响。方法RT-PCR及流式细胞仪检测肿瘤细胞THL2的表达，并用肽聚糖（PCN）刺激膜表面表达TLR2的肿瘤细胞，观察其增殖情况。结果EMT-6、RM-1、SP2／0、YAC-1，FBL3、NS-1、RAW254．7均检出TLR2 mRNA，并且除YAC-1外均检出TLR2蛋白表达。而高剂量的PGN能够明显的抑制细胞膜表面表达TLR2的肿瘤细胞的增殖。结论小鼠肿瘤细胞可表达TLR2，并介导TLR2配基PGN的抑制肿瘤生长作用。     

Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastacus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP.     

Natural products and the search for novel vaccine adjuvants

Vaccines that protect against intracellular infections such as malaria, Leishmania and Chlamydia require strong cellular responses based on CD4⁺ T cells and CD8⁺ T cells in addition to antibodies. Such cell-mediated responses can be potentiated with adjuvants. However, very few adjuvants have been licensed for use in humans; thus there is an urgent need for the discovery of new non-toxic adjuvants in order to produce more efficacious vaccines. Until recently, the mechanisms of how adjuvants worked remained largely unknown, but, it is becoming clearer that many function via host germline-encoded pattern recognition receptors (PRRs) expressed by most immune and non-immune cells. Most PRRs sense infection and transmit a series of signals that ultimately lead to the development of immunity. PRR mediated signalling can be harnessed to search for new vaccine adjuvants. Dendritic cells (DCs) express many PRRs and are remarkably effective at directing T cell immunity. Natural products (NPs) have been the basis of many drugs and are a rich source of immune activators and/or regulators of the immune response. Here we review PRRs in the context of NPs and propose the use of DCs as biological probes to help identify novel immune type molecules and adjuvants within collections of NPs.     

Renal NF-κB activation impairs uric acid homeostasis to promote tumor-associated mortality independent of wasting

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The phosphatidylinositol 3-kinase/Akt pathway negatively regulates Nod2-mediated NF-kappaB pathway

Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways, including NF-kappaB pathway. Here, we show that Nod2 agonist muramyldipeptide (MDP) induces Akt phosphorylation in time and dose-dependent manner. The pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin) and dominant-negative forms of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active forms of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF-kappaB activation and the target gene interleukin (IL)-8 induced by MDP. In addition, the pharmacological inhibitors of PI3K (wortmannin and LY294002) enhance phosphorylation of NF-kappaB p65 on Ser529 and Ser536 residues, which result in enhanced p65 transactivation activity. Furthermore, we show that the inhibition of PI3K by the pharmacological inhibitors prevent the inactivation of glycogen synthase kinase (GSK)-3beta, suggesting that the negative regulation of PI3K/Akt on MDP-induced NF-kappaB activation is at least in part mediated through inactivation of GSK-3beta. Taken together, our results demonstrate that PI3K/Akt pathway is activated by Nod2 agonist MDP and negatively regulates NF-kappaB pathway downstream of Nod2 activation. Our results suggest that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation.