Abstract Background Endometritis is an inflammatory condition of the uterus leading to complications with reproductive organs, miscarriage and infertility. The first-line treatment of endometritis in pigs are antibiotics, which do not always resolve inflammation and contribute to antibiotic resistance. There is an urgent need for the development of alternative treatments for endometritis. Here we propose and validate the first porcine tissue explant model for uterine inflammation and test naturally derived compounds for their effectiveness as anti-inflammatory therapies in porcine endometrial tissue. Methods Uterine endometrial explants were harvested from uteri of sows sent to slaughter. Tissue biopsies were collected at follicular and luteal phases of the oestrous cycle. Biopsies were washed and these tissue ‘explants’ were cultured. Explants were treated at 24 hr with oxytocin (to stimulate PGF2α production) or LPS (to mimic infection and induce inflammation. Explants were retained for RT-PCR analysis. Supernatants were harvested and concentrations of markers were measured by ELISA and Luminex. Results The data generated thus far show a dose dependent response of IL-6 secretion to LPS stimulation, which demonstrates the validity of our model. Inflammation can be stimulated in an in vitro setting to mimic porcine endometritis and we present data to show the change in cytokine levels in response to treatment with naturally derived compounds. Conclusions This data shows that inflammation can be stimulated in an in vitro setting to mimic porcine endometritis This model is a tool for testing of novel compounds and their potential anti-inflammatory effects on the porcine endometrium. Supported by an Innovate UK Grant (Project Reference: 98015) (https://gtr.ukri.org/projects?ref=98015)

The most common world-wide cause of antibiotic-associated infectious diarrhea and colitis is the toxin producing bacterium, Clostridioides difficile (C. difficile). Here we review the background and characteristics of the bacterium and the toxins produced together with the epidemiology and the complex pathogenesis that leads to a broad clinical spectrum of disease. The review describes the difficulties faced in obtaining a quick and accurate diagnosis despite the range of sensitive and specific diagnostic tools available. We also discuss the problem of disease recurrence and the importance of disease prevention. The high rates of infection recurrence mean that treatment strategies are constantly under review and we outline the diverse treatment options that are currently in use and explore the emerging treatment options of pulsed antibiotic use, microbial replacement therapies and the use of monoclonal antibodies. We summarize the future direction of treatment strategies which include the development of novel antibiotics, the administration of oral polyclonal antibody formulations, the use of vaccines, the administration of competitive non-toxigenic spores and the neutralization of antibiotics at the microbiota level. Future successful treatments will likely involve a combination of therapies to provide the most effective and robust approach to C. difficile management.

The rheumatological diseases are a group of chronic, painful, degenerative and debilitating conditions with an increasing prevalence across the globe. The pathogenesis of these disorders is complex, overlapping and not fully understood. As such, it is difficult and time consuming to achieve correct diagnosis and complete remission for an individual patient. In this review we describe the most common forms of inflammatory arthritis and discuss how the management and treatment options for these rheumatic diseases have developed over time. We outline the successes and the limitations of current treatment regimens and discuss the economic burden of the current options. With advancements in understanding of disease mechanisms, we discuss the importance of the biologics revolution in the context of rheumatological disease and how the development of biosimilars and small molecule inhibitors will impact current treatment options in order to alleviate some of the cost burden of biological therapies. The ideal treatment strategy for the future would involve personalized and predictive medicine where by treatments can be tailored to an individual patient's needs in order to achieve fast and successful remission with no adverse events.

Clostridioides difficileinfection (CDI) is a toxin-mediated infection in the gut and a major burden on healthcare facilities worldwide. We rationalized that it would be beneficial to design an antibody therapy that is delivered to, and is active at the site of toxin production, rather than neutralizing the circulating and luminal toxins after significant damage of the layers of the intestines has occurred. Here we describe a highly potent therapeutic, OraCAb, with high antibody titers and a formulation that protects the antibodies from digestion/inactivation in the gastrointestinal tract. The potential of OraCAb to prevent CDI in anin vivohamster model and anin vitrohuman colon model was assessed. In the hamster model we optimized the ratio of the antibodies against each of the toxins produced byC. difficile(Toxins A and B). The concentration of immunoglobulins that is effective in a hamster model of CDI was determined. A highly significant difference in animal survival for those given an optimized OraCAb formulation versus an untreated control group was observed. This is the first study testing the effect of oral antibodies for treatment of CDI in anin vitrogut model seeded with a human fecal inoculum. Treatment with OraCAb successfully neutralized toxin production and did not interfere with the colonic microbiota in this model. Also, treatment with a combination of vancomycin and OraCAb prevented simulated CDI recurrence, unlike vancomycin therapy alone. These data demonstrate the efficacy of OraCAb formulation for the treatment of CDI in pre-clinical models.

In response to complement activation, the membrane attack complex (MAC) assembles from fluid-phase proteins to form pores in lipid bilayers. MAC directly lyses pathogens by a ‘multi-hit’ mechanism; however, sublytic MAC pores on host cells activate signalling pathways. Previous studies have described the structures of individual MAC components and subcomplexes; however, the molecular details of its assembly and mechanism of action remain unresolved. Here we report the electron cryo-microscopy structure of human MAC at subnanometre resolution. Structural analyses define the stoichiometry of the complete pore and identify a network of interaction interfaces that determine its assembly mechanism. MAC adopts a ‘split-washer’ configuration, in contrast to the predicted closed ring observed for perforin and cholesterol-dependent cytolysins. Assembly precursors partially penetrate the lipid bilayer, resulting in an irregular β-barrel pore. Our results demonstrate how differences in symmetric and asymmetric components of the MAC underpin a molecular basis for pore formation and suggest a mechanism of action that extends beyond membrane penetration.

Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families.Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa.We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1-4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa. (C) 2014 The Authors. Published by Elsevier Ltd.

PMNs incubated with JSLE serum had 11% ( 12.46%) less phagocytosis compared with control serum, see Table 1. Disease activity of each of the 10 clinical episodes was measured using British Isles Lupus Assessment (BILAG) index. There was moderate negative correlation (Spearman’s rank correlation coefficient, r1⁄4 –0.45, P1⁄40.18) between the disease activity scores and the phagocytic activity; 28% of variation in phagocytosis could be explained by BILAG scores, coefficient of determination, r21⁄40.28. Conclusion: JSLE serum reduces the phagocytic ability of healthy neutrophils, whilst patient disease activity had a trend towards moderate negative correlation with this reduction in phagocytosis. To date there is a paucity of data examining the role of neutrophils as phagocytic effectors of E coli in JSLE. As previous work has demonstrated significantly increased levels of apoptotic cells in JSLE patients, future investigation will analyse whether JSLE serum has an effect on the ability of neutrophils to phagocytose apoptotic cells. Funding: This work was supported by Alder Hey NHS Foundation Trust and Lupus UK. Disclosure statement: The author has declared no conflicts of interest.

Objectives. HLA-B*27: 05 is associated with AS whereas HLA-B*27: 09 is not associated. We hypothesized that different interactions with KIR immune receptors could contribute to the difference in disease association between HLA-B*27: 05 and HLAB*27: 09. Thus, the objective of this study was to compare the formation of b2m-free heavy chain (FHC) including B27 dimers (B272) by HLA-B*27: 05 and HLA-B*27: 09 and their binding to KIR immunoreceptors.Methods. We studied the formation of HLA-B*27:05 and HLA-B*27:09 heterotrimers and FHC forms including dimers in vitro and in transfected cells. We investigated HLA-B*27:05 and HLA-B*27:09 binding to KIR3DL1, KIR3DL2 and LILRB2 by FACS staining with class I tetramers and by quantifying interactions with KIR3DL2CD3 epsilon-reporter cells and KIR3DL2-expressing NK cells. We also measured KIR expression on peripheral blood NK and CD4 T cells from 18 HLA-B*27:05 AS patients, 8 HLA-B27 negative and 12 HLA-B*27:05+ and HLA-B*27:09+ healthy controls by FACS staining.Results. HLA-B*27:09 formed less B27(2) and FHC than HLA-B*27:05. HLA-B*27:05-expressing cells stimulated KIR3DL2CD3 epsilon-reporter T cells more effectively. Cells expressing HLA-B*27:05 promoted KIR3DL2+ NK cell survival more strongly than HLA-B*27:09. HLA-B*27:05 and HLA-B*27:09 dimer tetramers stained KIR3DL1, KIR3DL2 and LILRB2 equivalently. Increased proportions of NK and CD4 T cells expressed KIR3DL2 in HLA-B*27:05+ AS patients compared with HLA-B*27:05+, HLA-B*27:09+ and HLA-B27- healthy controls.Conclusion. Differences in the formation of FHC ligands for KIR3DL2 by HLA-B*27:05 and HLA-B*27:09 could contribute to the differential association of these alleles with AS.

OBJECTIVE:Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with β(2) -microglobulin (β(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form β(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS:We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS:Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION:These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.

Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with β(2)-microglobulin (β2m) and peptide and (β2m free) free H chain (FHC) forms including B27 dimers (termed B27(2)) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K(D) of 5.3 ± 1.5 μM but did not bind B27 FHC. LILRB2 bound to B27(2) and B27 FHC and B27 heterotrimers with K(D)s of 2.5, 2.6, and 22 ± 6 μM, respectively. Domain exchange experiments showed that B27(2) bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K(D)s of 15.0 ± 0.8 and 16.0 ± 2.0 μM, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.