Patients with tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) are hypersensitive to Toll-like receptor 9 stimulation.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder characterized by recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNFRSF1A, which encodes tumour necrosis factor receptor 1 (TNF-R1). Our aim was to understand the influence of TRAPS mutations on the response to stimulation of the pattern recognition Toll-like receptor (TLR)-9. Peripheral blood mononuclear cells (PBMCs) and serum were isolated from TRAPS patients and healthy controls: serum levels of 15 proinflammatory cytokines were measured to assess the initial inflammatory status. Interleukin (IL)-1ß, IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), interferon (IFN)-γ, monocyte chemoattractant protein 1 (MCP-1) and transforming growth factor (TGF)-ß were significantly elevated in TRAPS patients' sera, consistent with constitutive inflammation. Stimulation of PBMCs with TLR-9 ligand (ODN2006) triggered significantly greater up-regulation of proinflammatory signalling intermediates [TNF receptor-associated factor (TRAF 3), IL-1 receptor-associated kinase-like 2 (IRAK2), Toll interacting protein (TOLLIP), TRAF6, phosphorylated transforming growth factor-ß-activated kinase 1 (pTAK), transforming growth factor-ß-activated kinase-binding protein 2 (TAB2), phosphorylated TAK 2 (pTAB2), IFN-regulatory factor 7 (IRF7), receptor interacting protein (RIP), nuclear factor kappa B (NF-κB) p65, phosphorylated NF-κB p65 (pNF-κB p65) and mitogen-activated protein kinase kinase (MEK1/2)] in TRAPS patients' PBMCs. This up-regulation of proinflammatory signalling intermediates and raised serum cytokines occurred despite concurrent anakinra treatment and no overt clinical symptoms at time of sampling. These novel findings further demonstrate the wide-ranging nature of the dysregulation of innate immune responses underlying the pathology of TRAPS and highlights the need for novel pathway-specific therapeutic treatments for this disease.

Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

The application of protein microarray assays in psychoneuroimmunology.

Protein microarrays are miniaturized multiplex assays that exhibit many advantages over the commonly used enzyme-linked immunosorbent assay (ELISA). This article aims to introduce protein microarrays to readers of Brain, Behavior, and Immunity and demonstrate its utility and validity for use in psychoneuroimmunological research. As part of an ongoing investigation of psychological and behavioral influences on influenza vaccination responses, we optimized a novel protein microarray to quantify influenza-specific antibody levels in human sera. Reproducibility was assessed by calculating intra- and inter-assay coefficients of variance on serially diluted human IgG concentrations. A random selection of samples was analyzed by microarray and ELISA to establish validity of the assay. For IgG concentrations, intra-assay and inter-assay precision profiles demonstrated a mean coefficient of variance of 6.7% and 11.5% respectively. Significant correlations were observed between microarray and ELISA for all antigens, demonstrating the microarray is a valid alternative to ELISA. Protein microarrays are a highly robust, novel assay method that could be of significant benefit for researchers working in psychoneuroimmunology. They offer high throughput, fewer resources per analyte and can examine concurrent neuro-immune-endocrine mechanisms.

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

BACKGROUND: It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. METHODS: A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. RESULTS: KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. CONCLUSIONS: This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome.

The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1ß, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1ß and other pro-inflammatory cytokines in BS patients carrying p.E383K.