Genetic characterisation and structural analysis of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:1c

Many, but not all, of the current 21 serotypes of Yersinia pseudotuberculosis have been investigated with regard to the chemical structures of their O-specific polysaccharide (OPS) and the genetic basis of their biosynthesis. Completion of the genetics and structures of the remaining serotypes will enhance our understanding of the emerging relationship between genetics and structures within this species. Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:1c OPS. Our results showed that this OPS has the same backbone as Y. pseudotuberculosis O:2b, but with a 3,6-dideoxy-D-ribo-hexofuranose (paratofuranose, Parf) side-branch instead of a 3,6-dideoxy-D-xylo-hexopyranose (abequopyranose, Abep). The 3'-end of the gene cluster is the same as for O:2b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5'-end of the cluster consists of the same genes as O:1b for synthesis of Parf and a related gene for its transfer to the repeating unit backbone.

Different correlation of Sphingosine-1-Phosphate receptor 1 with receptor activator of nuclear factor kappa B ligand and regulatory T cells in rat periapical lesions

Introduction Sphingosine-1-phosphate receptor 1 (S1P1) is crucial for regulation of immunity and bone metabolism. This study aimed to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and regulatory T (Treg) cells. Methods Periapical lesions were induced by pulp exposure in the first lower molars of 55 Wistar rats. Thirty rats were killed on days 0, 7, 14, 21, 28, and 35, and their mandibles were harvested for x-ray imaging, micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. The remaining 25 rats were killed on days 0, 14, 21, 28, and 35, and mandibles were harvested for flow cytometry. Results The volume and area of the periapical lesions increased from day 0 to day 21 and then remained comparably stable after day 28. S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14 and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells but was negatively correlated with that of Treg cells. Conclusions S1P1 expression was differentially correlated with RANKL and Treg cell infiltration in the periapical lesions and is therefore a contributing factor to the pathogenesis of such lesions.

Molecular analysis of asymptomatic bacteriuria escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

The role of inflammation in cutaneous repair

Inflammation is a fundamental component of the normal adult wound healing response occurring even in the absence of infection. It performs many beneficial roles such as the clearing of damaged cells and extracellular matrix (ECM), the removal of pathogens that might other wise multiply and spread, and the secretion of mediators that regulate other aspects of wound healing such as proliferation, re-epithelialisation and wound remodelling. Yet, excess and/or prolonged inflammation is detrimental to wound healing and leads to increased fibrosis and scarring, which can be disfiguring and, in cases such as contractures, can lead to disability. Furthermore, excessive inflammation is a major contributing factor to the persistence of chronic non-healing wounds, which are “stuck” in the inflammatory phase of healing and fail to reepithelialise. Current research suggest that the type of immune cells, their timing and the level of inflammation in a wound could have dramatic effect on whether a wound heals in a timely fashion and the final quality of the repaired tissue. Studies suggest that altering the level of inflammation might be beneficial in terms of reducing scarring and improving the rate of healing in chronic wounds. This review looks at the role of the major immune cells in normal and impaired wound healing and strategies that might be used to reduce inflammation in wounds.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Comparison of stool microbiota compositions, stool alpha1-antitrypsin and calprotectin concentrations, and diarrhoeal morbidity of Indonesian infants fed breast milk or probiotic/prebiotic-supplemented formula

Aim The composition of faecal microbiota of babies is known to be influenced by diet. Faecal calprotectin and α1-antitrypsin concentrations may be associated with mucosal permeability and inflammation. We aimed to assess whether there was any difference after consumption of a probiotic/prebiotic formula on faecal microbiota composition, calprotectin and α1-antitrypsin levels, and diarrhoea in comparison with breast milk-fed Indonesian infants. Methods One hundred sixty infants, 2 to 6 weeks old, were recruited to the study. They were either breastfed or formula fed (80 per group). Faecal samples were collected at recruitment and 3 months later. Bacterial groups characteristic of the human faecal microbiota were quantified in faeces by quantitative polymerase chain reaction. Calprotectin and α1-antitrypsin concentrations were measured using commercial kits. Details of diarrhoeal morbidity were documented and rated for severity. Results The compositions of the faecal microbiota of formula-fed compared with breast milk-fed children were similar except that the probiotic strain Bifidobacterium animalis subsp. lactisâ€...DR10 was more abundant after 3 months consumption of the formula. Alpha1-antitrypsin levels were higher in breastfed compared with formula-fed infants. The occurrence of diarrhoea did not differ between the groups of babies. Conclusion Feeding Indonesian babies with a probiotic/prebiotic formula did not produce marked differences in the composition of the faecal microbiota in comparison with breast milk. Detrimental effects of formula feeding on biomarkers of mucosal health were not observed.

Conserved gene structure and function of interleukin-10 in teleost fish

Interleukin-10 (IL-10) is an important immunoregulatory cytokine produced by various types of cells. Researchers describe here the isolation and characterization of olive flounder IL-10 (ofIL-10) cDNA and genomic organization. The ofIL-10 gene encodes a 187 amino acid protein and is composed of a five exon/four intron structure, similar to other known IL-10 genes. The ofIL-10 promoter sequence analysis shows a high level of homology in putative binding sites for transcription factors which are sufficient for transcriptional regulation ofIL-10. Important structural residues are maintained in the ofIL-10 protein including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10 and two extra cysteine residues that exist only in fish species. The phylogenetic analysis clustered ofIL-10 with other fish IL-10s and apart from mammalian IL-10 molecules. Quantitative real-time Polymerase Chain Reaction (PCR) analysis demonstrated ubiquitous ofIL-10 gene expression in the 13 tissues examined. Additionally, the induction of ofIL-10 gene expression was observed in the kidney tissue from olive flounder infected with bacteria (Edawardsiella tarda) or virus (Viral Hemorrhagic Septicemia Virus; VHSV). These data indicate that IL-10 is an important immune regulator that is conserved strictly genomic organization and function during the evolution of vertebrate immunity.

A "Candidate-Interactome" Aggregate Analysis of Genome-Wide Association Data in Multiple Sclerosis

Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a "candidate interactome" (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms. © 2013 Mechelli et al.

Interleukin-23 mediates the intestinal response to microbial β-1,3-glucan and the development of spondyloarthritis pathology in SKG mice

Objective Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70W163C-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan). Methods Eight weeks after curdlan injection in wild-type or IL-17A-/- SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs. Results In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22. Conclusion In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance.

Budesonide and Formoterol Reduce Early Innate Anti-Viral Immune Responses In Vitro

Asthma is a chronic inflammatory airways disease in which respiratory viral infections frequently trigger exacerbations. Current treatment of asthma with combinations of inhaled corticosteroids and long acting beta2 agonists improves asthma control and reduces exacerbations but what impact this might have on innate anti-viral immunity is unclear. We investigated the in vitro effects of asthma drugs on innate anti-viral immunity. Peripheral blood mononuclear cells (PBMC) from healthy and asthmatic donors were cultured for 24 hours with the Toll-like receptor 7 agonist, imiquimod, or rhinovirus 16 (RV16) in the presence of budesonide and/or formoterol. Production of proinflammatory cytokines and expression of anti-viral intracellular signalling molecules were measured by ELISA and RT-PCR respectively. In PBMC from healthy donors, budesonide alone inhibited IP-10 and IL-6 production induced by imiquimod in a concentration-dependent manner and the degree of inhibition was amplified when budesonide and formoterol were used in combination. Formoterol alone had little effect on these parameters, except at high concentrations (10−6 M) when IL-6 production increased. In RV16 stimulated PBMC, the combination of budesonide and formoterol inhibited IFNα and IP-10 production in asthmatic as well as healthy donors. Combination of budesonide and formoterol also inhibited RV16-stimulated expression of the type I IFN induced genes myxovirus protein A and 2′, 5′ oligoadenylate synthetise. Notably, RV16 stimulated lower levels of type Myxovirus A and oligoadenylate synthase in PBMC of asthmatics than control donors. These in vitro studies demonstrate that combinations of drugs commonly used in asthma therapy inhibit both early pro-inflammatory cytokines and key aspects of the type I IFN pathway. These findings suggest that budesonide and formoterol curtail excessive inflammation induced by rhinovirus infections in patients with asthma, but whether this inhibits viral clearance in vivo remains to be determined.

Microbes, the gut and ankylosing spondylitis

It is increasingly clear that the interaction between host and microbiome profoundly affects health. There are 10 times more bacteria in and on our bodies than the total of our own cells, and the human intestine contains approximately 100 trillion bacteria. Interrogation of microbial communities by using classic microbiology techniques offers a very restricted view of these communities, allowing us to see only what we can grow in isolation. However, recent advances in sequencing technologies have greatly facilitated systematic and comprehensive studies of the role of the microbiome in human health and disease. Comprehensive understanding of our microbiome will enhance understanding of disease pathogenesis, which in turn may lead to rationally targeted therapy for a number of conditions, including autoimmunity.

Clinical factors associated with the humoral immune response to influenza vaccination in chronic obstructive pulmonary disease

Background and objective Individuals with chronic obstructive pulmonary disease (COPD) are at a high risk of developing significant complications from infection with the influenza virus. It is therefore vital to ensure that prophylaxis with the influenza vaccine is effective in COPD. The aim of this study was to assess the immunogenicity of the 2010 trivalent influenza vaccine in persons with COPD compared to healthy subjects without lung disease, and to examine clinical factors associated with the serological response to the vaccine. Methods In this observational study, 34 subjects (20 COPD, 14 healthy) received the 2010 influenza vaccine. Antibody titers at baseline and 28 days post-vaccination were measured using the hemagglutination inhibition assay (HAI) assay. Primary endpoints included seroconversion (≥4-fold increase in antibody titers from baseline) and the fold increase in antibody titer after vaccination. Results Persons with COPD mounted a significantly lower humoral immune response to the influenza vaccine compared to healthy participants. Seroconversion occurred in 90% of healthy participants, but only in 43% of COPD patients (P=0.036). Increasing age and previous influenza vaccination were associated with lower antibody responses. Antibody titers did not vary significantly with cigarette smoking, presence of other comorbid diseases, or COPD severity. Conclusion The humoral immune response to the 2010 influenza vaccine was lower in persons with COPD compared to non-COPD controls. The antibody response also declined with increasing age and in those with a history of prior vaccination.

Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Mimicry of 21. 5 KDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKRPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TPC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogenesis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Does the microbiome play a causal role in spondyloarthritis?

The purpose of this study is to review the potential causal role of the microbiome in the pathogenesis of spondyloarthritis. The method used for the study is literature review. The microbiome plays a major role in educating the immune response. The microbiome is strongly implicated in inflammatory bowel disease which has clinical and genetic overlap with spondyloarthritis. The microbiome also plays a causal role in bowel and joint disease in HLA B27/human beta 2 microglobulin transgenic rats. The mechanism(s) by which HLA B27 could influence the microbiome is unknown but theories include an immune response gene selectivity, an effect on dendritic cell function, or a mucosal immunodeficiency. Bacteria are strongly implicated in the pathogenesis of spondyloarthritis. Studies to understand how HLA B27 affects bacterial ecosystems should be encouraged.