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The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack.

Over 35% of children in a region of malaria endemicity are infected with Epstein-Barr virus (EBV) by 6 months of age. This susceptibility may be linked to impaired transplacental transfer of antibodies. In this study, we determined the effect of malaria exposure during pregnancy on the transfer of EBV-specific maternal antibodies in a region of western Kenya that experiences endemic malaria. Pregnant mothers were recruited and followed up until delivery to determine levels of neonatal malaria exposure. Levels of EBV lytic (viral capsid antigen [VCA], Z transcriptional activator [Zta], and early diffuse antigen complex [EAd]) and EBV latent (EBV nuclear antigen-1 (EBNA1]) and tetanus-specific IgG antibodies were measured in 70 paired maternal and cord blood samples using a Luminex-bead-based assay. A high proportion (63%) of the infants were exposed to malaria in utero . Levels of EBV- and tetanus-specific antibodies were similar in malaria-infected mothers and in mothers who had no detectable malaria infection. Malaria-exposed neonates had significantly lower levels of anti-EBNA1, anti-Zta, and anti-EAd antibodies than were seen in their mothers. In utero malaria exposure resulted in significant reductions in transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies of 13% and 22%, respectively. Neonates received significantly low levels of anti-Zta and anti-EAd antibodies irrespective of malaria exposure levels. In multivariate analysis, in utero malaria exposure was associated with a significant reduction in the transfer of anti-VCA-p18 and anti-EBNA1 antibodies to the neonates ( P = 0.0234 and P = 0.0017, respectively). Malaria during pregnancy results in differential levels of transfer of EBV-specific antibodies from the mother to the fetus. The impaired transplacental transfer of some antibodies may lead to the malaria-exposed neonates being susceptible to early EBV infection.

The individual role of pathogen-binding Toll-like receptors (TLRs) and antibody-binding Fc receptors (FcRs) during pathogenic infections has been studied extensively. However, combined activation of these different receptor classes has received little attention, even though they are triggered simultaneously when immune cells bind antibody-opsonized pathogens. In the last few years, it has become evident that joined activation of TLRs and FcRs substantially tailors inflammatory immune responses, which is an efficient and controlled mechanism of the host to act upon invading pathogens. In this review, we discuss the mechanisms of cross-talk between different TLRs and FcRs and the resulting inflammatory immune responses. Furthermore, we propose how chronic activation via this cross-talk might be detrimental in inflammatory (auto) immune diseases. We conclude with the potential exploitation of the interplay between TLRs and FcRs for monoclonal antibody therapy to target tumors. Future interests in this field of research include establishing a more detailed and mechanistic understanding of the mode of action of TLR and FcR cross-talk and exploration of its physiological importance in health and disease. This may furthermore open up novel therapeutic options for intervention in inflammatory diseases or cancer.

Functional impairment of dendritic cells (DCs) is part of a survival strategy evolved by Leishmania and Plasmodium parasites to evade host immune responses. Here, the effects of co-exposing human monocyte-derived DCs to Leishmania donovani promastigotes and Plasmodium falciparum-infected erythrocytes were investigated. Co-stimulation resulted in a dual, dose-dependent effect on DC differentiation which ranged from semi-mature cells, secreting low interleukin(-12p70 levels to a complete lack of phenotypic maturation in the presence of high parasite amounts. The effect was mainly triggered by the Leishmania parasites, as illustrated by their ability to induce semi-mature, interleukin-10-producing DCs, that poorly responded to lipopolysaccharide stimulation. Conversely, P. falciparum blood-stage forms failed to activate DCs and only slightly interfered with lipopolysaccharide effects. Stimulation with high L. donovani concentrations triggered phosphatidylserine translocation, whose onset presented after initiating the maturation impairment process. When added in combination, the two parasites could co-localize in the same DCs, confirming that the leading effects of Leishmania over Plasmodium may not be due to mutual exclusion. Altogether, these results suggest that in the presence of visceral leishmaniasis-malaria co-infections, Leishmania-driven effects may overrule the more silent response elicited by P. falciparum, shaping host immunity towards a regulatory pattern and possibly delaying disease resolution.

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-β-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.

CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN.

After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a+ MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a−/CD14+/CD68+ which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets.

Dendritic cells (DC) vaccination is a potent therapeutic approach for inducing tumor-directed immunity, but challenges remain. One of the particular interest is the induction of an immune response targeting multiple (unknown) tumor-associated antigens (TAA), which requires a polyvalent source of TAA. Previously, we described the preferred use of apoptotic cell-derived blebs over the larger apoptotic cell remnants, as a source of TAA for both in situ loading of skin-resident DC and in vitro loading of monocyte-derived DC (MoDC). Recent reports suggest that MoDC cultured in the presence of GM-CSF supplemented with IFNα (IFNα MoDC), as compared to IL-4 (IL-4 MoDC), have an increased capacity to cross-present antigen to CD8(+) T cells. As culture conditions, maturation methods and antigen sources differ between the conducted studies, we analyzed the functional differences between IL-4 MoDC and IFNα MoDC, loaded with blebs, in a head-to-head comparison using commonly used protocols. Our data show that both MoDC types are potent (cross-) primers of CD8(+) T cells. Whereas IFNα MoDC were more potent in their capacity to cross-present a 25-mer MART-1 synthetic long peptide (SLP) to a MART-1aa26-35 recognizing CD8(+) T cell line, IL-4 MoDC proved more potent cross-primers of antigen-specific CD8(+) T cells when loaded with blebs. The latter is likely due to the observed greater capacity of IL-4 MoDC to ingest apoptotic blebs. In conclusion, our data indicate the use of IFNα MoDC over IL-4 MoDC in the context of DC vaccination with SLP, whereas IL-4 MoDC are preferred for vaccination with bleb-derived antigens.

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.

Summary Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of type 1 and type 2 von Willebrand disease (VWD). More than 20 different VWF point mutations have already been reported in patients with enhanced clearance. These include the VWD-Vicenza variant, which is characterized by an Arg1205His substitution in the VWF D3 domain. Critically, however, the molecular mechanisms through which single amino acid substitutions in VWF result in enhanced clearance of this complex multimeric glycoprotein have not been defined. Objectives In this study, we have investigated the biological basis underlying the enhanced clearance of the VWF-R1205H variant. Methods Using VWF−/− mice, in vivo clearance rates were determined for a series of full-length and truncated recombinant VWF variants. In addition, the role of macrophages in modulating enhanced VWD-Vicenza clearance was investigated using clodronate liposome administration. Results Our findings demonstrate that substitutions of R1205 with histidine, cysteine or serine all result in markedly reduced survival of full-length recombinant VWF. Importantly, D'A3 fragments containing these same R1205 substitutions also demonstrated significantly enhanced clearance. In contrast to the reduced in vivo survival observed with R1205H, clearance of R1204H was not enhanced. Recent studies have demonstrated that hepatic and splenic macrophages play key roles in regulating VWF clearance. Importantly, macrophage-depletion also served to markedly attenuate the enhanced clearance phenotypes associated with VWF-R1205H, VWF-R1205S and VWF-R1205C. Conclusions Collectively, these novel findings demonstrate a specific and critical role for the R1205 residue in modulating macrophage-mediated clearance of VWF in vivo.

Ang W, Wolfs TFW. Diagnostiek van lymeborreliose. Huisarts Wet 2015;58(5):264-8. Serologie is de geeigende microbiologische diagnostiek voor het aantonen van lymeborreliose. Naast de traditionele serologische tests zijn er enkele andere diagnostische modaliteiten ontwikkeld die worden ingezet bij de diagnostiek van Borrelia-infecties. Sommige van deze tests voert men uit in niet officieel geaccrediteerde laboratoria, soms in het buitenland. Dit artikel biedt een overzicht van enkele veel gebruikte tests en geeft per test de waarde en beperking voor de diagnostiek van lymeborreliose aan.

Objective Recent data suggest the involvement of dectin-1 in atherosclerosis through regulation of local reactive oxygen species production. The aim of the current study was to assess the effect of dectin-1 deficiency on atherosclerotic plaque development. Methods Using immunohistochemistry dectin-1 expression was observed on foamy macrophages in atherosclerotic lesions in mice. Following lethal irradiation LDLR −/− mice were reconstituted with bone marrow from either wild type or dectin-1 −/− mice. After recovery, mice were fed a high fat diet for 9 weeks and atherosclerotic lesions were analyzed. Results and conclusion Overall, we found no significant differences in plaque size or severity between the groups. Also no differences were observed in granulocyte or macrophage composition of the plaques or in the ability to produce reactive oxygen species by macrophages from both groups. Dectin-1 is dispensable for the development of atherosclerotic lesions in mice.

Aim Various types of cholinergic receptors are expressed on intestinal epithelia. Their function is not completely understood. We hypothesize that cholinergic receptor activation on epithelium may serve a protective function in cytokine-induced barrier dysfunction. Methods The effect of cholinergic receptor activation on cellular barrier function in epithelial cells was assessed by measuring electrical impedance, and by determining para-cellular transport in transwell experiments. Cell lysates treated with cytokine and/or cholinergic agonists were analysed for cyto- and chemokine production, and tight junction (TJ) protein rearrangement was assessed. Primary colonic epithelial cells were isolated from surgically resected colon tissue of patients with inflammatory bowel disease. Results IL-1β induced production of chemokines (CXCL-1, CXCL-10, IL-8, CCL-7) and led to a rearrangement of TJ proteins (occludin and ZO-1). This response was inhibited by pre-treatment with muscarinic, rather than nicotinic, acetylcholine receptor agonists. Treatment with IL-1β enhanced paracellular permeability (4kD dextran) and reduced impedance across the monolayer, which was counteracted by pre-incubation with acetylcholine, or muscarinic receptor agonist bethanechol. The protective effect of acetylcholine was antagonized by atropine, underscoring muscarinic receptor involvement. IL-1β induced transcription of myosin light chain kinase and phosphorylation of myosin light chain, and this cytokine-induced phosphorylation of MLC was inhibited by muscarinic receptor agonists. Furthermore, in epithelial cells from resection material of patients with Crohn's disease and ulcerative colitis, high expression of CXCL-8 was associated with a reduced choline acetyl transferase expression, suggesting an aberrant epithelial production of ACh in inflammatory context. Conclusion Acetylcholine acts on muscarinic receptors on epithelial cells to maintain epithelial barrier function under inflammatory conditions.

Based on their ability to balance tolerance and inflammation, antigen presenting cells, such as dendritic cells and macrophages contribute to the maintenance of immune homeostasis as well as the instigation of immune activation. Acting as key sensors of tissue integrity and pathogen invasion, they are well equipped with a wide variety of pattern recognition receptors, to which the C-type lectin family also belongs. C-type lectins are glycan-binding receptors that mediate cell-cell communication and pathogen recognition, besides participating in the endocytosis of antigens for presentation to T cells and the fine-tuning of immune responses. Here we review the current state-of-the-art on the dendritic cell and macrophage-expressed C-type lectin macrophage galactose-type lectin (MGL), highlighting the binding specificities, signaling properties and modulation of innate and adaptive immunity by its human and murine orthologues.

This study improves our understanding of the pathogenesis of tuberculous meningitis (TBM) and may direct future strategies for the prevention of immunopathology associated with TBM. Our data suggest that host biomarker signatures in the cerebrospinal fluid have promising diagnostic applications and require urgent investigation. Background. Tuberculous meningitis (TBM) is a severe complication of tuberculosis predominantly affecting young children. Early treatment is vital to prevent morbidity and mortality, emphasizing the importance of early diagnosis. The lack of sensitive methods for early diagnosis is the most common cause of delay. Attempts have been made to develop simplified tests for tuberculosis, but their diagnostic power remains poor. The clinical picture of TBM is mainly driven by the host's immune response to Mycobacterium tuberculosis; therefore, identification of disease-specific biomarkers may have diagnostic and therapeutic value and improve our understanding of its pathogenesis. Methods. We investigated disease-specific biomarkers of childhood TBM in a cohort of children aged 3 months–13 years with symptoms and signs suggestive of meningitis. Cerebrospinal fluid (CSF) and serum from 56 patients with and 55 patients without TBM were assessed for 28 soluble mediators. Results. Unsupervised hierarchical clustering analysis revealed a disease-specific pattern of biomarkers for TBM relative to other types of meningitis. A biomarker-based diagnostic prediction model for childhood TBM based on CSF concentrations of interleukin 13 (cutoff value, 37.26 pg/mL), vascular endothelial growth factor (cutoff value, 42.92 pg/mL), and cathelicidin LL-37 (cutoff value, 3221.01 pg/mL) is presented with a sensitivity of 0.52 and a specificity of 0.95. Conclusions. These data highlight the potential of biosignatures in the host's CSF for diagnostic applications and for improving our understanding of the pathogenesis of TBM to discover strategies to prevent immunopathological sequelae.

Soft-tissue augmentation with permanent fillers can lead to severe granulomatous foreign-body reactions (GFBRs), but the immune pathomechanism of this complication is still unknown. We performed conventional histologic examination and immunostaining for plasmacytoid dendritic cells (pDCs) in skin sections from patients with GFBR to 4 permanent filler agents, which have been widely used in recent decades.Twenty-one skin biopsies were studied from 19 patients with GFBR to polyalkylimide 4% gel (PAIG, n = 10), polyacrylamide 2.5% gel (PAAG, n = 2), hydroxyethyl methacrylate/ethyl methacrylate in hyaluronic acid (HEMA/EMA, n = 4), or liquid injectable silicone (n = 5). GFBRs were analyzed in hematoxylin and eosin stained sections and pDCs detected using CD123 antibodies. Anti-CD11c immunostaining was performed for comparison.Grading of the inflammatory infiltrates observed histologically did not correlate with the clinical features of inflammation. Immunostaining for CD123 did not detect pDCs in 8 of 10 polyalkylimide gel, 1 of 2 polyacrylamide gel, and the 5 liquid injectable silicone biopsies. In contrast, all 4 HEMA/EMA biopsies contained collections of pDCs in lymphocytic infiltrates close to filler particles and adjacent sarcoidal granulomas.Our data suggest that pDCs contribute to the sarcoidal granulomas associated with injected HEMA/EMA. Recruited pDCs may exert their pro-inflammatory effects by the release of interferon-α at the site of these filler deposits.

The T-synthase (core 1 β3-galactosyltransferase) and its molecular chaperone Cosmc regulate the biosynthesis of mucin type O-glycans on glycoproteins, and evidence suggests that both T-synthase and Cosmc are transcriptionally suppressed in several human diseases, although the transcriptional regulation of these two genes is not understood. Here, we characterized the promoters essential for human Cosmc and T-synthase transcription. The upstream regions of the genes lack a conventional TATA box but contain CpG islands, cCpG-I and cCpG-II for Cosmc and tCpG for T-synthase. Using luciferase reporter assays, site-directed mutagenesis, ChIP assays, and mithramycin A treatment, we identified the core promoters within cCpG-II and tCpG, which contain two binding sites for Krüppel-like transcription factors, including SP1/SP3, respectively. Methylome analysis of Tn4 B cells, which harbor a silenced Cosmc, confirmed the hypermethylation of the Cosmc core promoter but not for T-synthase. These results demonstrate that Cosmc and T-synthase are transcriptionally regulated at a basal level by the specificity protein/Krüppel-like transcription factor family of members, which explains their ubiquitous and coordinated expression, and also indicate that they are differentially epigenetically regulated beyond X chromosome imprinting. These results are important in understanding the regulation of these genes that have roles in human diseases, such as IgA nephropathy and cancer.

Urogenital Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the most prevalent bacterial STIs worldwide. Molecular tests are the standard for the detection of CT and NG, as these are difficult to culture. The recently introduced CE-IVD marked GMT Presto assay promises to be a valuable addition in CT and NC diagnostics. The advantage of the Presto assay is that it works on many PCR systems and the DNA can be isolated by any system. We compared the Presto assay to the widely used Roche cobas (R) 4800 CT/NC test for the detection of CT and NC in 612 vaginal and rectal dry collected swabs. Discrepant samples were tested by the TIB MOLBIOL Lightmix Kit 480 HT CT/NC assay. The alloyed gold standard was defined as two concurring Presto and cobas (R) 4800 results, or, with discrepant Presto and cobas (R) results, two concurring results of either test together with the Lightmix Kit 480 HT CT/NC assay. For the Presto assay, we observed 77 CT positive (13%) and 22 NC positive (3,6%) vaginal samples, and 41 CT positive (6,7%) and 11 NC positive (1,8%) rectal samples. For the cobas (R) 4800 assay, we observed 77 CT positive (13%) and 21 NC positive (3,4%) vaginal samples, and 39 CT positive (6,4%) and 11 NC positive (1,8%) rectal samples. Ten CT samples were discrepant between Presto and cobas (R) 4800 CT/NC assays, while two NC samples were discrepant. CT sensitivity in both assays was 100% compared to the alloyed gold standard. The sensitivity was 100% for both vaginal and rectal dry swabs, underlining the suitability of these sample types for detection of CT and NC. The Presto assay is therefore valuable for molecular detection of CT and NC in dry vaginal and rectal swabs.

BackgroundRecently, sodium tetrachloropalladate (Na2PdCl4) was found to be a more sensitive palladium patch test allergen than palladium dichloride (PdCl2).ObjectivesTo determine the optimal test concentration of Na2PdCl4, to evaluate the prevalence of palladium sensitization with Na2PdCl4 and PdCl2, and to compare the results with nickel sensitization in a European multicentre study.Materials and methodsIn addition to the European or national baseline series including NiSO4·6H20 5% pet., consecutive patients were tested with PdCl2 and Na2PdCl4 2%, 3% and 4% pet. in eight European dermatology clinics. The age and sex distributions were also evaluated in patients sensitized to nickel and palladium.ResultsIn total, 1651 patients were tested. Relative to 3% Na2PdCl4, 4% Na2PdCl4 did not add any information. Two per cent Na2PdCl4 resulted in more doubtful reactions and showed suboptimal reactivity. The prevalence of palladium sensitization doubled with Na2PdCl4 and was similar to that of nickel. Interestingly, in contrast to nickel sensitization, palladium sensitization was not related to female sex.ConclusionsThree per cent Na2PdCl4 pet. is the most suitable patch test concentration. Sensitization to palladium is almost as prevalent as sensitization to nickel. The sex distribution is different between nickel-sensitized and palladium-sensitized patients, suggesting different sources of exposure.

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar (HTscar) and not to normotrophic scar (NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix (ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes (TNF, IL-1, IL-1RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL-10) and an extended increased expression of ECM-related genes (PLAU, Col3A1, TGF3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti-inflammatory scar therapies.

OBJECTIVERisk factors for rectal carriage of ESBL-E and transmission were investigated in an outbreak of extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E).DESIGNRectal carriage of ESBL-E was determined in a cross-sectional survey by culture of perianal swabs or fecal samples. Both phenotypical and genotypical methods were used to detect the production of ESBL. Nosocomial transmission was defined as the presence of genotypically related strains in ≥2 residents within the NH. Patient characteristics and variables in infection control practices were registered to investigate risk factors for transmission.SETTINGA nursing home (NH) in the southern Netherlands.PARTICIPANTSOf 189 residents, 160 residents (84.7%) were screened for ESBL-E carriage. Of these 160 residents, 33 (20.6%) were ESBL-E positive. ESBL carriage rates varied substantially between wards (range, 0–47%). Four different ESBL-E clusters were observed. AblaCTX-M1-15positiveE. coliST131 constituted the largest cluster (n=21) and was found in multiple wards (n=7).RESULTSOur investigation revealed extensive clonal dissemination ofblaCTX-M1-15-positiveE. coliST131 in a nursing home. Unexplained differences in ESBL prevalence were detected among the wards.CONCLUSIONSAs NHs constitute potential sources of multidrug-resistant bacteria, it is important to gain a better understanding of the risks factors and routes of transmission of ESBL-E.Infect Control Hosp Epidemiol 2014;00(0): 1–7

Dendritic cells (DC) are attractive targets for cancer immunotherapy as they initiate strong and long-lived tumour-specific T cell responses. DC can be effectively targeted in vivo with tumour antigens by using nanocarriers such as liposomes. Cross-presentation of tumour antigens is enhanced with strong adjuvants such as TLR ligands. However, often these adjuvants have off-target effects, and would benefit from a DC-specific targeting strategy, similar to the tumour antigen. The goal of this study was to develop a strategy for specifically targeting DC with tumour antigen and adjuvant by using glycoliposomes. We have generated liposomes containing the glycan Lewis(Le)(X) which is highly specific for the C-type lectin receptor DC-SIGN expressed by DC. Le(X)-modified liposomes were taken up by human monocyte-derived DC in a DC-SIGN-specific manner. As adjuvants we incorporated the TLR ligands Pam3CySK4, Poly I:C, MPLA and R848 into liposomes and compared their adjuvant capacity on DC. Incorporation of the TLR4 ligand MPLA into glycoliposomes induced DC maturation and production of pro-inflammatory cytokines, in a DC-SIGN-specific manner, and DC activation was comparable to administration of soluble MPLA. Incorporation of MPLA into glycoliposomes significantly enhanced antigen cross-presentation of the melanoma tumour antigen gp100280-288 peptide to CD8(+) T cells compared to non-glycosylated MPLA liposomes. Importantly, antigen cross-presentation of the gp100280-288 peptide was significantly higher using MPLA glycoliposomes compared to the co-administration of soluble MPLA with glycoliposomes. Taken together, our data demonstrates that specific targeting of a gp100 tumour antigen and the adjuvant MPLA to DC-SIGN-expressing DC enhances the uptake of peptide-containing liposomes, the activation of DC, and induces tumour antigen-specific CD8(+) T cell responses. These data demonstrate that adjuvant-containing glycoliposome-based vaccines targeting DC-SIGN(+) DC represent a powerful new approach for CD8(+) T cell activation.

Background and Purpose— Secondary intracerebral hemorrhage (sICH) is a potentially serious complication of ischemic stroke, in particular under concomitant oral anticoagulation. Previous studies in murine stroke models defined a novel vascular repair function of hematogenous monocytes/macrophages (MO/MP), which proved essential for the prevention of oral anticoagulation–associated sICH. Here, we addressed the question whether hyperglycemia as a clinically relevant prohemorrhagic risk factor and peroxisome proliferator-activated receptor gamma (PPARγ) activation affect MO/MP differentiation and the risk of sICH after ischemic stroke. Methods— Oral anticoagulation–associated sICH was induced by phenprocoumon feeding to mice undergoing transient middle cerebral artery occlusion. Hyperglycemia was induced by streptozotocin treatment. The role of PPARγ-dependent MO/MP differentiation was addressed in mice with myeloid cell–specific PPARγ-knockout (LysM-PPARγ(KO)). Pharmacological PPARγ activation via pioglitazone was tested as a treatment option. Results— Hyperglycemic mice and normoglycemic LysM-PPARγ(KO) mice exhibited abnormal proinflammatory skewing of their hematogenous MO/MP response and abnormal vascular remodeling in the infarct border zone, leading to an increased rate of oral anticoagulation–associated sICH. Pharmacological PPAR γ activation in hyperglycemic mice corrected the inflammatory response toward an anti-inflammatory profile, stabilized neovessels in the infarct border zone, and reduced the rate of sICH. This preventive effect was dependent on the presence of macrophages, but independent from effects on blood glucose levels. Conclusions— Hyperglycemia and macrophage-specific PPARγ activation exert opposing effects on MO/MP polarization in ischemic stroke lesions and, thereby, critically determine the risk of hemorrhagic infarct transformation.

In patients with celiac disease, gluten consumption causes inflammation of the duodenum, and, to a lesser extent, the proximal jejunum. Immune-dominant gluten peptides are modified by the enzyme TG2, leading to their high-affinity binding to HLA-DQ2 or HLA-DQ8 molecules, present in people with a predisposition to celiac disease. Gluten peptide−loaded HLA-DQ2 or HLA-DQ8 molecules are recognized by highly conserved receptors on CD4 + T cells in the lamina propria. B cells specific for TG2 and modified gluten peptides are also abundant in the lamina propria of patients with celiac disease. In the epithelium, interleukin-15 activates intraepithelial lymphocytes that promote destruction of epithelial cells. However, it is not clear how the immune responses in the lamina propria and the epithelium, separated by a basement membrane, are linked. We review the immune processes that occur in the lamina propria and their potential effects on epithelial pathology in celiac disease.

ObjectiveDepending on the location of injury, wounds can heal with different outcomes. In addition foetal wounds heal fast without scar formation, while scars are a common feature of regular skin repair. Since inflammation is very limited in these wounds reduced numbers or even absence of immune cells might be responsible for scarless foetal wound healing. It is thought that various immune cells, such as macrophages, neutrophils and T-cells, play a role in aberrant wound healing and the fibrotic process seen in scar formation in the adult skin. Similar to the foetus, oral wounds show comparable healing properties by means of accelerated reepithelialization and negligible scar formation. It is possible that reduced inflammatory reaction as a result of lower numbers of immune cells are present in oral wounds compared to skin wounds.DesignHere we investigated the presence of various immune cells in human skin and oral mucosa, with or without scars. The presence or absence of these cells may play a role in the different modes of healing observed between the two types of tissue. Mast cells, neutrophils, M1/M2 macrophages, T-cells and blood vessels were localized in healthy and scarred skin and oral mucosa (scars >1 year old).ResultsOral mucosa had significantly fewer neutrophils, macrophages, mannose receptor-positive M2 macrophages, but more blood vessels. Scars contained similar numbers of immune cells compared to healthy tissues.ConclusionsLess immune cells in the healthy oral mucosa may induce a diminished immune reaction when wounding occurs, and could explain the better healing capacity of the oral mucosa.

Microflora-induced TLR signaling in the intestinal epithelium is essential for a proper intestinal barrier function. Because of the close interactions of this epithelial layer with underlying mononuclear phagocytes, we hypothesized that epithelial TLR signaling may affect the differentiation of myeloid cell populations. In in vitro cultures we observed that colonic epithelial monolayers actively transported TLR2 ligands towards their basolateral side. The transported TLR2 ligands strongly stimulated the development of Ly6C(+) monocytes, while dendritic cell differentiation was inhibited. The TLR2 effect on monocyte and dendritic cell differentiation was mediated by the production of G-CSF. Mice lacking TLR signaling and mice that were treated with antibiotics showed decreased numbers of Ly6C(+) monocytes in bone marrow and spleen, which points to a role for microbial derived TLR-ligands in the homeostasis of Ly6C(+) monocytes. In conclusion, our results indicate that TLR ligands that are transported by intestinal epithelial cells stimulate Ly6C(+) monocyte development and suggest that this process may be involved in the maintenance of systemic Ly6C(+) monocyte numbers.

After mucosal damage or gingival inflammation, complement proteins leak into the oral cavity and mix with salivary proteins such as salivary agglutinin (SAG/gp-340/DMBT1). This protein is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1), and it aggregates bacteria, viruses and fungi, and activates the lectin pathway of the complement system. In the lectin pathway, carbohydrate structures on pathogens or altered self cells are recognized. SAG is highly glycosylated, partly on the basis of the donor’s blood group status. Whereas secretors express Lewis b, Lewis y, and antigens from the ABO-blood group system on SAG, non-secretors do not. Through mannose-binding lectin (MBL) binding and C4 deposition assays, we aimed to identify the chemical structures on SAG that are responsible for complement activation. The complement-activating properties of SAG were completely abolished by oxidation of its carbohydrate moiety. SAG-mediated activation of complement was also inhibited in the presence of saccharides such as fucose and Lewis b carbohydrates, and also after pretreatment with the fucose-binding lectin, Anguilla anguilla agglutinin. Complement activation was significantly (p

The Fox system of Pseudomonas aeruginosa is a cell-surface signaling (CSS) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor σFoxI. In the absence of ferrioxamine, the activity of σFoxI is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of σFoxI. Interestingly, we have recently shown that FoxR undergoes self-cleavage between the periplasmic Gly-191 and Thr-192 residues independent of the perception of ferrioxamine. This autoproteolytic event, which is widespread among CSS anti-sigma factors, produces two distinct domains that interact and function together to transduce the presence of the signal. In this work, we provide evidence that the self-cleavage of FoxR is not an enzyme-dependent process but is induced by an N-O acyl rearrangement. Mutation analysis showed that the nucleophilic side chain of the Thr-192 residue at +1 of the cleavage site is required for an attack on the preceding Gly-191, after which the resulting ester bond is likely hydrolyzed. Because the cleavage site is well preserved and the hydrolysis of periplasmic CSS anti-sigma factors is widely observed, we hypothesize that cleavage via an N-O acyl rearrangement is a conserved feature of these proteins. Background: Many transmembrane anti-sigma factors, including P. aeruginosa FoxR, undergo periplasmic self-cleavage by an unknown mechanism. Results: Presence of an OH or SH group at +1 of the cleavage site is essential for FoxR self-cleavage. Conclusion: FoxR self-cleavage is mediated by an N-O acyl rearrangement. Significance: The complex proteolytic network that modulates alternative sigma factor activity in Gram-negative bacteria also includes an enzyme-independent step.

Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le(Y)), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le(Y)-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN(+) DCs and mediated efficient antigen presentation to CD4(+) and CD8(+) T cells. Surprisingly, although langerin bound to Le(Y)-modified liposomes, LCs exposed to Le(Y)-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le(Y)-modified synthetic long peptides. In contrast, Le(Y)-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration.

Cholangiocarcinoma (CC) is typically diagnosed at an advanced stage and is refractory to surgical intervention and chemotherapy. Despite a global increase in the incidence of CC, little progress has been made toward the development of treatments for this cancer. Here we utilized human tissue; CC cell xenografts; a p53-deficient transgenic mouse model; and a non-transgenic, chemically induced rat model of CC that accurately reflects both the inflammatory and regenerative background associated with human CC pathology. Using these systems, we determined that the WNT pathway is highly activated in CCs and that inflammatory macrophages are required to establish this WNT-high state in vivo. Moreover, depletion of macrophages or inhibition of WNT signaling with one of two small molecule WNT inhibitors in mouse and rat CC models markedly reduced CC proliferation and increased apoptosis, resulting in tumor regression. Together, these results demonstrate that enhanced WNT signaling is a characteristic of CC and suggest that targeting WNT signaling pathways has potential as a therapeutic strategy for CC.

CD1d-restricted invariant natural killer T (iNKT) cells constitute an important immunoregulatory T cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and initiate antitumor immune responses. As cancer patients are frequently treated with aminobisphosphonates (NBP), it is relevant to determine possible effects of NBP on CD1d-restricted glycolipid Ag-presentation to iNKT cells. We report a striking reduction of α-GalCer-induced iNKT cell activation by monocyte derived dendritic cells (moDC) upon their exposure to NBP during maturation. We found that production of apolipoprotein E (apoE), which is a known facilitator of trans-membrane transport of exogenously derived glycolipids, was significantly diminished in moDC exposed to NBP. As the inhibitory effect of NBP on iNKT cell activation was alleviated by exogenous apoE, our data indicate that reduced apoE production by antigen presenting cells (APC) through NBP limits glycolipid-induced iNKT cell activation. This should be taken into account in the design of iNKT cell-based anti-cancer therapies.

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt. ispartof: Cell Metabolism vol:21 issue:2 pages:286-297 ispartof: location:United States status: published

In HLA-DQ8–associated celiac disease (CD), the pathogenic T cell response is directed toward an immunodominant α-gliadin–derived peptide (DQ8-glia-α1). However, our knowledge of TCR gene usage within the primary intestinal tissue of HLA-DQ8+ CD patients is limited. We identified two populations of HLA-DQ8-glia-α1 tetramer+ CD4+ T cells that were essentially undetectable in biopsy samples from patients on a gluten-free diet but expanded rapidly and specifically after antigenic stimulation. Distinguished by expression of TRBV9, both T cell populations displayed biased clonotypic repertoires and reacted similarly against HLA-DQ8-glia-α1. In particular, TRBV9 paired most often with TRAV26-2, whereas the majority of TRBV9− TCRs used TRBV6-1 with no clear TRAV gene preference. Strikingly, both tetramer+/TRBV9+ and tetramer+/TRBV9− T cells possessed a non–germline-encoded arginine residue in their CDR3α and CDR3β loops, respectively. Comparison of the crystal structures of three TRBV9+ TCRs and a TRBV9− TCR revealed that, as a result of distinct TCR docking modes, the HLA-DQ8-glia-α1 contacts mediated by the CDR3-encoded arginine were almost identical between TRBV9+ and TRBV9− TCRs. In all cases, this interaction centered on two hydrogen bonds with a specific serine residue in the bound peptide. Replacement of serine with alanine at this position abrogated TRBV9+ and TRBV9− clonal T cell proliferation in response to HLA-DQ8-glia-α1. Gluten-specific memory CD4+ T cells with structurally and functionally conserved TCRs therefore predominate in the disease-affected tissue of patients with HLA-DQ8–mediated CD.

Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes.

Galectin-1 (Gal-1), an endogenous glycan-binding protein, is widely distributed at sites of inflammation and microbial invasion. Despite considerable progress regarding the immunoregulatory activity of this lectin, the role of endogenous Gal-1 during acute parasite infections is uncertain. In this study, we show that Gal-1 functions as a negative regulator to limit host-protective immunity following intradermal infection with Trypanosoma cruzi. Concomitant with the upregulation of immune inhibitory mediators, including IL-10, TGF-β1, IDO, and programmed death ligand 2, T. cruzi infection induced an early increase of Gal-1 expression in vivo. Compared to their wild-type (WT) counterpart, Gal-1–deficient (Lgals1−/−) mice exhibited reduced mortality and lower parasite load in muscle tissue. Resistance of Lgals1−/− mice to T. cruzi infection was associated with a failure in the activation of Gal-1–driven tolerogenic circuits, otherwise orchestrated by WT dendritic cells, leading to secondary dysfunction in the induction of CD4+CD25+Foxp3+ regulatory T cells. This effect was accompanied by an increased number of CD8+ T cells and higher frequency of IFN-γ–producing CD4+ T cells in muscle tissues and draining lymph nodes as well as reduced parasite burden in heart and hindlimb skeletal muscle. Moreover, dendritic cells lacking Gal-1 interrupted the Gal-1–mediated tolerogenic circuit and reinforced T cell–dependent anti-parasite immunity when adoptively transferred into WT mice. Thus, endogenous Gal-1 may influence T. cruzi infection by fueling tolerogenic circuits that hinder anti-parasite immunity.

While genetic polymorphisms play a paramount role in tuberculosis (TB), less is known about their contribution to the severity of diseases caused by other intracellular bacteria and fastidious microorganisms. We searched electronic databases for observational studies reporting on host factors and genetic predisposition to infections caused by intracellular fastidious bacteria published up to 30 May 2014. The contribution of genetic polymorphisms was documented for TB. This includes genetic defects in the mononuclear phagocyte/T helper cell type 1 (Th1) pathway contributing to disseminated TB disease in children and genome-wide linkage analysis (GWAS) in reactivated pulmonary TB in adults. Similarly, experimental studies supported the role of host genetic factors in the clinical presentation of illnesses resulting from other fastidious intracellular bacteria. These include IL-6 -174G/C or low mannose-binding (MBL) polymorphisms, which are incriminated in chronic pulmonary conditions triggered by C. pneumoniae, type 2-like cytokine secretion polymorphisms, which are correlated with various clinical patterns of M. pneumoniae infections, and genetic variation in the NOD2 gene, which is an indicator of tubal pathology resulting from Chamydia trachomatis infections. Monocyte/macrophage migration and T lymphocyte recruitment defects are corroborated to ineffective granuloma formation observed among patients with chronic Q fever. Similar genetic polymorphisms have also been suggested for infections caused by T. whipplei although not confirmed yet. In conclusion, this review supports the paramount role of genetic factors in clinical presentations and severity of infections caused by intracellular fastidious bacteria. Genetic predisposition should be further explored through such as exome sequencing.

The role of the macrophage in the immunopathology of periodontitis has not been well defined. In this study, we show that intraoral inoculation of mice with Porphyromonas gingivalis resulted in infection, alveolar bone resorption, and a significant increase in F4/80+ macrophages in gingival and submandibular lymph node tissues. Macrophage depletion using clodronate-liposomes resulted in a significant reduction in F4/80+ macrophage infiltration of gingival and submandibular lymph node tissues and significantly (p < 0.01) less P. gingivalis–induced bone resorption compared with controls in BALB/c and C57BL/6 mice. In both mouse strains, the P. gingivalis–specific IgG Ab subclass and serum cytokine [IL-4, IL-10, IFN-γ, and IL-12 (p70)] responses were significantly (p < 0.01) lower in the macrophage-depleted groups. Macrophage depletion resulted in a significant reduction in the level of P. gingivalis infection, and the level of P. gingivalis infection was significantly correlated with the level of alveolar bone resorption. M1 macrophages (CD86+), rather than M2 macrophages (CD206+), were the dominant macrophage phenotype of the gingival infiltrate in response to P. gingivalis infection. P. gingivalis induced a significant (p < 0.01) increase in NO production and a small increase in urea concentration, as well as a significant increase in the secretion of IL-1β, IL-6, IL-10, IL-12 (p70), eotaxin, G-CSF, GM-CSF, macrophage chemoattractant protein-1, macrophage inflammatory protein-α and -β, and TNF-α in isolated murine macrophages. In conclusion, P. gingivalis infection induced infiltration of functional/inflammatory M1 macrophages into gingival tissue and alveolar bone resorption. Macrophage depletion reduced P. gingivalis infection and alveolar bone resorption by modulating the host immune response.

Macrophages form a heterogeneous cell population displaying multiple functions, and can be polarized into pro- (M1) or anti-inflammatory (M2) macrophages, by environmental factors. Their activation status reflects a beneficial or detrimental role in various diseases. Currently several in vitro maturation and activation protocols are used to induce an M1 or M2 phenotype. Here, the impact of different maturation factors (NHS, M-CSF, or GM-CSF) and activation methods (IFN-γ/LPS, IL-4, dexamethason, IL-10) on the macrophage phenotype was determined. Regarding macrophage morphology, pro-inflammatory (M1) activation stimulated cell elongation, and anti-inflammatory (M2) activation induced a circular appearance. Activation with pro-inflammatory mediators led to increased CD40 and CD64 expression, whereas activation with anti-inflammatory factors resulted in increased levels of MR and CD163. Production of pro-inflammatory cytokines was induced by activation with IFN-γ/LPS, and TGF-β production was enhanced by the maturation factors M-CSF and GM-CSF. Our data demonstrate that macrophage marker expression and cytokine production in vitro is highly dependent on both maturation and activation methods. In vivo macrophage activation is far more complex, since a plethora of stimuli are present. Hence, defining the macrophage activation status ex vivo on a limited number of markers could be indecisive. From this study we conclude that maturation with M-CSF or GM-CSF induces a moderate anti- or pro-inflammatory state respectively, compared to maturation with NHS. CD40 and CD64 are the most distinctive makers for human M1 and CD163 and MR for M2 macrophage activation and therefore can be helpful in determining the activation status of human macrophages ex vivo.

Minor histocompatibility antigens are highly immunogeneic polymorphic peptides playing crucial roles in the clinical outcome of HLA-identical allogeneic stem cell transplantation. Although the introduction of genome-wide association-based strategies significantly has accelerated the identification of minor histocompatibility antigens over the past years, more efficient, rapid and robust identification techniques are required for a better understanding of the immunobiology of minor histocompatibility antigens and for their optimal clinical application in the treatment of hematologic malignancies. To develop a strategy that can overcome the drawbacks of all earlier strategies, we now integrated our previously developed genetic correlation analysis methodology with the comprehensive genomic databases from the 1000 Genomes Project. We show that the data set of the 1000 Genomes Project is suitable to identify all of the previously known minor histocompatibility antigens. Moreover, we demonstrate the power of this novel approach by the identification of the new HLA-DP4 restricted minor histocompatibility antigen UTDP4-1, which despite extensive efforts could not be identified using any of the previously developed biochemical, molecular biological or genetic strategies. The 1000 Genomes Project-based identification of minor histocompatibility antigens thus represents a very convenient and robust method for the identification of new targets for cancer therapy after allogeneic stem cell transplantation.

Objective Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn9s disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium. Design Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems. Results Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-κB pathway and upregulation of the novel NF-κB inhibitor SLPI but no other known NF-κB inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-κB activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs. Conclusions We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is an important virulence factor for this yeast-like fungus. Previously, we had shown that disruption of the CAP10 gene, encoding a putative xylosyltransferase, results in mutant cells that lack most of the capsular polysaccharides on the cell surface, but do not show a typical acapsular phenotype. In contrast to the acapsular cap59 mutant, cap10 did not induce maturation of dendritic cells when exposed to components of the immune system. In order to gain further insight into the causes of this phenotype displayed by the cap10 mutant, we performed a more in-depth phenotypic analysis of the cell wall and surface structures of this mutant compared to the wild type strain and acapsular mutant cap59. Moreover, we analyzed the cap10 mutant and the wild type strain for differential gene expression of, amongst others, enzymes that are involved in biogenesis of cell wall and capsule components. We conclude that a mutation in the CAP10 gene results in a pleiotropic phenotype with effects on different cellular processes affecting, amongst others, cell size, expression of virulence factors and size of extracellular vesicles.

Current concepts regarding the pathogenesis of Parkinson's disease support a model whereby environmental factors conspire with a permissive genetic background to initiate the disease. The identity of the responsible environmental trigger has remained elusive. There is incontrovertible evidence that aggregation of the neuronal protein alpha-synuclein is central to disease pathogenesis. A novel hypothesis of Parkinson's pathogenesis, articulated by Braak and colleagues, implicates a pathogen acting in the olfactory mucosa and gastrointestinal tract as the inciting agent. In this point-of-view article, we hypothesize that α-synuclein aggregation in Parkinson's disease is an Epstein-Barr virus (EBV)-induced autoimmune phenomenon. Specifically, we have shown evidence for molecular mimicry between the C-terminal region of α-synuclein and a repeat region in the latent membrane protein 1 encoded by EBV. We hypothesize that, in genetically-susceptible individuals, anti-EBV latent membrane protein antibodies targeting the critical repeat region cross react with the homologous epitope on α-synuclein and induce its oligomerization. Consistent with the Braak's proposed pattern of spread, we contend that axon terminals in the lamina propria of the gut are among the initial targets, with subsequent spread of pathology to the CNS. While at this time, we can only provide evidence from the literature and preliminary findings from our own laboratory, we hope that our hypothesis will stimulate the development of tractable experimental systems that can be exploited to test it. Further support for an EBV-induced immune pathogenesis for Parkinson's disease could have profound therapeutic implications.

Biobanks are invaluable resources in genomic research of both the infectious diseases and their hosts. This article examines the role of biobanks in basic research of infectious disease genomics, as well as the relevance and applicability of biobanks in the translation of impending knowledge and the clinical uptake of knowledge of infectious diseases. Our research identifies potential fields of interaction between infectious disease genomics and biobanks, in line with global trends in the integration of genome-based knowledge into clinical practice. It also examines various networks and biobanks that specialize in infectious diseases (including HIV, HPV and Chlamydia trachomatis), and provides examples of successful research and clinical uptake stemming from these biobanks. Finally, it outlines key issues with respect to data privacy in infectious disease genomics, as well as the utility of adequately designed and maintained electronic health records. We maintain that the public should be able to easily access a clear and detailed outline of regulations and procedures for sample and data utilization by academic or commercial investigators, and also should be able to understand the precise roles of relevant governing bodies. This would ultimately facilitate uptake by researchers and clinics. As a result of the efforts and resources invested by several networks and consortia, there is an increasing awareness of the prospective uses of biobanks in advancing infectious disease genomic research, diagnostics and their clinical management., Highlights • The role of biobanks in research of host genomic factors and infectious diseases. • Examples of translation of HIV, HPV and Chlamydia research results into clinics. • Lack of published overviews of infectious disease biobanks, result is low visibility. • Regulations and sample utilization procedures should be more easily accessible.

Background & Aims: In most cases celiac disease (CD) is successfully treated with a gluten-free diet (GFD). However, some patients become refractory to the GFD. Refractory CD (RCD) patients have an increased risk for developing enteropathy associated T-cell lymphoma and early diagnosis is therefore of importance. Currently, RCD diagnosis relies on endoscopy and adequate serological markers are lacking. Antibodies against glycoprotein-2 (GP2A) were described in Crohn's disease (CrD) and active CD but not in ulcerative colitis (UC), suggesting this is a specific marker for small intestinal lesions.Methods: Sera obtained from patients visiting our outpatient clinic for routine serological tests for diagnosis and/or follow-up of inflammatory bowel disease (n=78), active CD (n=45), GFD (n=34) and RCD (n=15) were analysed for GP2A titres.Results: Increased GP2A-IgA levels in CrD and active CD as compared to controls (p

Background & Aims Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. Methods First-generation Ad vectors encoding reporter genes (luciferase or β-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4+, CD8+ T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. Results The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100–1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. Conclusions Transient CD4+ T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.

Summary The cell-envelope of Mycobacterium tuberculosis plays a key role in bacterial virulence and antibiotic resistance. Little is known about the molecular mechanisms of regulation of cell-envelope formation. Here, we elucidate functional and structural properties of RNase AS, which modulates M. tuberculosis cell-envelope properties and strongly impacts bacterial virulence in vivo. The structure of RNase AS reveals a resemblance to RNase T from Escherichia coli , an RNase of the DEDD family involved in RNA maturation. We show that RNase AS acts as a 3′-5′-exoribonuclease that specifically hydrolyzes adenylate-containing RNA sequences. Also, crystal structures of complexes with AMP and UMP reveal the structural basis for the observed enzyme specificity. Notably, RNase AS shows a mechanism of substrate recruitment, based on the recognition of the hydrogen bond donor NH2 group of adenine. Our work opens a field for the design of drugs able to reduce bacterial virulence in vivo.

CD169-positive macrophages in the marginal zone of the spleen and subcapsular sinus of lymph nodes play an important role as gatekeepers, strategically located to capture pathogens. Here we identified a population of CD169-positive macrophages in the colon and investigated which factors influenced their development. Murine colonic CD115+ F4/80(lo) CD11c(lo) macrophages expressing CD169 were present in the lamina propria, mainly surrounding the crypts. In spite of the high levels of bacterial flora in the colon and the importance of Toll-like receptor signalling in mucosal homeostasis, the presence of CD169+ macrophages was not affected in mice that were deficient in MyD88-mediated Toll-like receptor signalling and in mice in which the bacterial flora was eradicated. Whereas the development of splenic CD169+ macrophages was dependent on lymphotoxin α, colonic CD169+ macrophages were present in normal numbers in lymphotoxin α-deficient mice. In contrast, reduced numbers of CD169+ macrophages were found in the colon of mice deficient in vitamin A, whereas CD169+ macrophages in the spleen were unaffected. In conclusion, we identified a new macrophage subset in the lamina propria of the colon characterized by the expression of CD169. Its differentiation, unlike CD169+ macrophages in lymphoid organs, is independent of lymphotoxin α signalling, but requires vitamin A.

Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8–11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcβ1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections.

Vitamin A and its active metabolite retinoic acid are essential for the development and function of many tissues including the immune system. The induction of mucosal homing receptors on T and B cells by mucosal dendritic cells (DC) depends on the presence of vitamin A. Recent studies indicate that also the differentiation of CD11b+ DC subsets in the mucosa as well as the spleen depend on vitamin A signalling. As CD11b+ DC subsets exert non-redundant functions in anti-bacterial and anti-fungal immune responses, defects in CD11b+ DC differentiation will contribute to the clinical problems observed during vitamin A deficiency.

BACKGROUND: Methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI) are the active ingredients in commonly used preservative systems (e.g. Kathon CG(®) ). MCI/MI is present in the European baseline patch test series at 100 ppm aq. Since 1986, 200 ppm (dose 0.006 mg/cm(2) ) has been used in Sweden without causing skin irritation. Centres in Spain, the United Kingdom and Ireland have also used 200 ppm in their baseline series.OBJECTIVES: To find the optimal patch test concentration for MCI/MI.MATERIALS AND METHODS: MCI/MI 100 ppm aq. and MCI/MI 200 ppm aq. were simultaneously patch tested in 3300 consecutively tested dermatitis patients at eight European patch test clinics and one US patch test clinic. With the Finn Chambers(®) technique (diameter 8 mm), 15 µl was micropipetted on to the filter paper in the chamber. The corresponding volume for Van der Bend(®) chambers was 20 µl, and that for IQ Chambers(®) was 25 µl.RESULTS: Contact allergy to MCI/MI at 100 and 200 ppm was found in 1.2% and 2.1% of patients, respectively (p CONCLUSIONS: MCI/MI 200 ppm aq. (dose 0.006 mg/cm(2) ) diagnoses significantly more contact allergy than the presently used concentration of 100 ppm (dose 0.003 mg/cm(2) ), without resulting in more adverse reactions. MCI/MI at 200 ppm should therefore be considered for inclusion in the European baseline test series.