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1.  Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration 
eLife  null;5:e13149.
Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment.
DOI: http://dx.doi.org/10.7554/eLife.13149.001
eLife digest
The blood vessels in the brain help to control the entry of nutrients, cells and waste products into and out of the brain. In doing so, they create a protective barrier between the blood and the brain known as the blood-brain barrier. However, this barrier loses its protective function in individuals with multiple sclerosis or other disorders that affect the brain. Multiple sclerosis patients develop inflammation and their immune cells become able to enter the brain. These immune cells may then attack layers of insulation called myelin that surround nerve cells. Myelin helps nerve cells to work properly so the loss of this insulation can lead to tissue damage and cognitive problems. When immune cells called T cells enter the brain they can become primed to recognize myelin and attack it in the same way that they would attack viruses or bacteria. However, it is not clear precisely how these T cells develop the ability to cross the blood-brain barrier and attack myelin.
Now, Lopes Pinheiro et al. show that “endothelial” cells in the blood-brain barrier are able to present fragments of myelin to T cells, which enables the T cells to identify myelin and move into the brain. First, the blood-brain barrier cells absorb and break down proteins in the myelin, and then they present fragments of these proteins on their surfaces with the help of protein clusters called major histocompatibility complexes (MHCs). Other protein fragments that can also activate T cells in other parts of the body did not affect the blood-brain barrier when they were presented by MHCs, which suggests that the effect could be specific to myelin proteins.
The experiments also show that it is possible to stop T cells from crossing the blood-brain barrier by preventing them from interacting with myelin fragments presented by MHCs. This suggests that therapies that interfere with the ability of blood-brain barrier cells to break down myelin proteins and present them to T cells might help to protect the brains of patients with multiple sclerosis.
DOI: http://dx.doi.org/10.7554/eLife.13149.002
doi:10.7554/eLife.13149
PMCID: PMC4959842  PMID: 27336724
encephalitogenic T cells; blood-brain barrier; multiple sclerosis; Human; Mouse
2.  The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10 
PLoS Pathogens  2016;12(6):e1005696.
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.
Author Summary
Mycobacteria are well protected from effectors of the immune system and from antibiotics by their cell envelope. The mycobacterial capsule constitutes the outer layer of this cell envelope. This capsule consists of glucan-like polysaccharides, proteins and glycolipid molecules and is thought to interact with the immune system of the host. In this study, we show that one of the protein secretion systems of slow-growing mycobacteria, called ESX-5, is important in maintaining the structure of this capsule. Furthermore, we identified PPE10, a protein secreted via ESX-5, as the main protein responsible for capsular integrity. We show that disturbed capsule integrity affects the ability of the mycobacteria to disrupt membranes. The bacterial mutants in esx-5 or ppe10 were also attenuated in a zebrafish model for tuberculosis, indicating that the capsular integrity is important for mycobacteria to establish disease. Knowledge on the mycobacterial capsule is only recently emerging and these results could open up new avenues in tuberculosis vaccine or treatment design.
doi:10.1371/journal.ppat.1005696
PMCID: PMC4900558  PMID: 27280885
3.  Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells 
eLife  null;5:e11765.
Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies.
DOI: http://dx.doi.org/10.7554/eLife.11765.001
eLife digest
Immune cells called dendritic cells play a crucial role in defending the body against tumor cells and invading viruses. The dendritic cells take up molecules called antigens from these threats and then display them on their surface. This enables the antigens to be identified by other immune cells that are capable of killing the viruses and the tumor cells. The dendritic cells recognize the antigens with the help of receptor proteins called C-type lectin receptors (CLRs). These receptors can bind to sugar molecules that are naturally found on many antigens. For example, a C-type lectin receptor called MGL1 can bind to sugars known as LewisX and Lewisa on tumor and virus proteins. However, it is not clear how important these receptors are in triggering immune responses.
An antigen called Ovalbumin – which is found in chicken egg white – can trigger immune responses in mammals and so researchers often use it to study the immune system. Although this antigen has several sugar molecules attached to it, quite a large amount of Ovalbumin is needed to trigger strong immune responses. Now, Streng-Ouwehand et al. examine whether attaching LewisX to Ovalbumin can results in stronger immune responses in mice.
The experiments show that injecting mice with Ovalbumin-LewisX triggers a much stronger immune response than normal Ovalbumin does. This enhanced response was not observed in mice that lacked the MGL1 receptor, which suggests that this receptor is involved in detecting Ovalbumin-LewisX. Furthermore, the dendritic cells store the altered Ovalbumin for longer than they store normal Ovalbumin, which gives the cells more time to present the altered Ovalbumin to other immune cells.
Vaccines and some other therapies help to boost immune responses to viruses and tumors by exposing the body to antigens. However, these therapies often use antigens that don’t have sugar molecules attached to them, or are missing the sugar molecules they would normally have. Streng-Ouwehand et al.’s findings suggest that adding specific sugars to antigens in immune therapies might help to make these therapies more effective.
DOI: http://dx.doi.org/10.7554/eLife.11765.002
doi:10.7554/eLife.11765
PMCID: PMC4811763  PMID: 26999763
dendritic cells; cross-presentation; T cell priming; glycosylation; MGL1; Mouse
4.  Moderate hyperoxic versus near-physiological oxygen targets during and after coronary artery bypass surgery: a randomised controlled trial 
Critical Care  2016;20:55.
Background
The safety of perioperative hyperoxia is currently unclear. Previous studies in patients undergoing coronary artery bypass surgery suggest reduced myocardial damage when avoiding extreme perioperative hyperoxia (>400 mmHg). In this study we investigated whether an oxygenation strategy from moderate hyperoxia to a near-physiological oxygen tension reduces myocardial damage and improves haemodynamics, organ dysfunction and oxidative stress.
Methods
This was a single-blind, single-centre, open-label, randomised controlled trial in patients undergoing elective coronary artery bypass surgery. Fifty patients were randomised to a partial pressure of oxygen in arterial blood (PaO2) target of 200–220 mmHg during cardiopulmonary bypass and 130–150 mmHg during intensive care unit (ICU) admission (control group) versus lower targets of 130–150 mmHg during cardiopulmonary bypass and 80–100 mmHg at the ICU (conservative group). Primary outcome was myocardial injury (CK-MB and Troponin-T) at ICU admission and 2, 6 and 12 hours thereafter.
Results
Weighted PaO2 during cardiopulmonary bypass was 220 mmHg (interquartile range (IQR) 211–233) vs. 157 (151–162) in the control and conservative group, respectively (P < 0.0001). During ICU admission, weighted PaO2 was 107 mmHg (86–141) vs. 90 (84–98) (P = 0.03), respectively. Area under the curve of CK-MB was median 23.5 μg/L/h (IQR 18.4–28.1) vs. 21.5 (15.8–26.6) (P = 0.35) and 0.30 μg/L/h (0.25–0.44) vs. 0.39 (0.24–0.43) (P = 0.81) for Troponin-T. Cardiac index, systemic vascular resistance index, creatinine, lactate and F2-isoprostane levels were not different between groups.
Conclusions
Compared to moderate hyperoxia, a near-physiological oxygen strategy does not reduce myocardial damage in patients undergoing coronary artery bypass surgery. Conservative oxygen administration was not associated with increased lactate levels or hypoxic events.
Trial registration
Netherlands Trial Registry NTR4375, registered on 30 January 2014
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-016-1240-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13054-016-1240-6
PMCID: PMC4788916  PMID: 26968380
Hyperoxia; Oxygen; Intensive care unit; Cardiac surgery; CABG
5.  Analysis of the role of the LH92_11085 gene of a biofilm hyper-producing Acinetobacter baumannii strain on biofilm formation and attachment to eukaryotic cells 
Virulence  2016;7(4):443-455.
ABSTRACT
Acinetobacter baumannii is a nosocomial pathogen that has a considerable ability to survive in the hospital environment partly due to its capacity to form biofilms. The first step in the process of establishing an infection is adherence of the bacteria to target cells. Chaperone-usher pili assembly systems are involved in pilus biogenesis pathways that play an important role in adhesion to host cells and tissues as well as medically relevant surfaces. After screening a collection of strains, a biofilm hyper-producing A. baumannii strain (MAR002) was selected to describe potential targets involved in pathogenicity. MAR002 showed a remarkable ability to form biofilm and attach to A549 human alveolar epithelial cells. Analysis of MAR002 using transmission electron microscopy (TEM) showed a significant presence of pili on the bacterial surface. Putative protein-coding genes involved in pili formation were identified based on the newly sequenced genome of MAR002 strain (JRHB01000001/2 or NZ_JRHB01000001/2). As assessed by qRT-PCR, the gene LH92_11085, belonging to the operon LH92_11070-11085, is overexpressed (ca. 25-fold more) in biofilm-associated cells compared to exponential planktonic cells. In the present work we investigate the role of this gene on the MAR002 biofilm phenotype. Scanning electron microscopy (SEM) and biofilm assays showed that inactivation of LH92_11085 gene significantly reduced bacterial attachment to A549 cells and biofilm formation on plastic, respectively. TEM analysis of the LH92_11085 mutant showed the absence of long pili formations normally present in the wild-type. These observations indicate the potential role this LH92_11085 gene could play in the pathobiology of A baumannii.
doi:10.1080/21505594.2016.1145335
PMCID: PMC4871663  PMID: 26854744
acinetobacter baumannii; attachment; biofilm; pathogenicity; pili; virulence
6.  N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression*  
Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.
doi:10.1074/mcp.M115.051235
PMCID: PMC4762531  PMID: 26537799
7.  Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells 
PLoS ONE  2015;10(11):e0143519.
Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.
doi:10.1371/journal.pone.0143519
PMCID: PMC4659545  PMID: 26605924
8.  The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation 
PLoS ONE  2015;10(10):e0140454.
The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08–0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients.
doi:10.1371/journal.pone.0140454
PMCID: PMC4608671  PMID: 26473355
9.  Galectin-2 Induces a Proinflammatory, Anti-Arteriogenic Phenotype in Monocytes and Macrophages 
PLoS ONE  2015;10(4):e0124347.
Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.
doi:10.1371/journal.pone.0124347
PMCID: PMC4401781  PMID: 25884209
10.  The Consequences of Multiple Simultaneous C-Type Lectin–Ligand Interactions: DCIR Alters the Endo-Lysosomal Routing of DC-SIGN 
Antigen-presenting cells (APCs) are equipped with multiple receptors to allow proper pathogen recognition and capture. C-type lectin receptors (CLRs) recognize glycan structures on pathogens and endogenous glycoproteins for internalization and antigen processing and presentation. Often, the glycan specificity of these receptors is overlapping and/or pathogens are decorated with ligands for multiple CLRs, posing the question whether interference or cooperativity within the CLR family exists. Here, we used imaging flow cytometry to investigate the internalization properties of four different CLRs [mannose receptor, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), macrophage galactose-type lectin, and dendritic cell immunoreceptor (DCIR)] on different APCs, as well as their intracellular routing. Although the internalization score of the investigated CLRs was similar on monocytes, macrophages, and dendritic cells (DCs), DCIR internalization rates were lower compared to the other CLRs. Upon triggering, DCIR routed to intracellular compartments outside of the classical endo-lysosomal pathway, resulting in poor CD4+ T-cell stimulation. Although DC maturation reduced CLR expression levels, it did not affect their internalization rates. Although CLR internalization appeared to be independently regulated, DC-SIGN routing was affected when DCIR was triggered simultaneously. In conclusion, our results provide new insights for the design of DC-based immunotherapeutic strategies and suggest that DCIR is an inferior target in this respect.
doi:10.3389/fimmu.2015.00087
PMCID: PMC4354414  PMID: 25806031
mannose receptor; dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; dendritic cell immunoreceptor; macrophage galactose-type lectin; dendritic cells; antigen uptake; intracellular routing
11.  Understanding the Biology of Antigen Cross-Presentation for the Design of Vaccines Against Cancer 
Antigen cross-presentation, the process in which exogenous antigens are presented on MHC class I molecules, is crucial for the generation of effector CD8+ T cell responses. Although multiple cell types are being described to be able to cross-present antigens, in vivo this task is mainly carried out by certain subsets of dendritic cells (DCs). Aspects such as the internalization route, the pathway of endocytic trafficking, and the simultaneous activation through pattern-recognition receptors have a determining influence in how antigens are handled for cross-presentation by DCs. In this review, we will summarize new insights in factors that affect antigen cross-presentation of human DC subsets, and we will discuss the possibilities to exploit antigen cross-presentation for immunotherapy against cancer.
doi:10.3389/fimmu.2014.00149
PMCID: PMC3986565  PMID: 24782858
cross-presentation; dendritic cells; antigen processing and presentation; anti-cancer vaccine; CD8+ T cells
12.  Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain 
PLoS ONE  2013;8(6):e66266.
C-type lectins are innate receptors expressed on antigen-presenting cells that are involved in the recognition of glycosylated pathogens and self-glycoproteins. Upon ligand binding, internalization and/or signaling often occur. Little is known on the glycan specificity and ligands of the Dendritic Cell Immunoreceptor (DCIR), the only classical C-type lectin that contains an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM). Here we show that purified DCIR binds the glycan structures Lewisb and Man3. Interestingly, binding could not be detected when DCIR was expressed on cells. Since DCIR has an N-glycosylation site inside its carbohydrate recognition domain (CRD), we investigated the effect of this glycan in ligand recognition. Removing or truncating the glycans present on purified DCIR increased the affinity for DCIR-binding glycans. Nevertheless, altering the glycosylation status of the DCIR expressing cell or mutating the N-glycosylation site of DCIR itself did not increase glycan binding. In contrast, cis and trans interactions with glycans induced DCIR mediated signaling, resulting in a decreased phosphorylation of the ITIM sequence. These results show that glycan binding to DCIR is influenced by the glycosylation of the CRD region in DCIR and that interaction with its ligands result in signaling via its ITIM motif.
doi:10.1371/journal.pone.0066266
PMCID: PMC3679074  PMID: 23776650
13.  A Polymorphism in the Coding Region of Il12b promotes IL-12p70 and IL-23 Heterodimer Formation 
Interleukin (IL)-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12 p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid (TNBS)-induced colitis. In addition, this region determines strain differences in LPS induced IL-12 responses.
Here we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to either IL-12p40 from the colitis-resistant C57Bl/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than constructs harboring the C57Bl/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion implicating a higher affinity of SJL/J derived IL-12p40 for its IL-12p35 subunit. This higher affinity is also associated with increased IL-23 synthesis. In addition, C57Bl/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 and were more prone to develop colonic inflammation than did C57Bl/6 mice transgenic for the C57Bl/6-p40 variant upon LPS challenge. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism.
The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid pro-inflammatory skewing of immune responses and distortion of the homeostatic balance underlying the higher susceptibility for colitis.
doi:10.4049/jimmunol.1001330
PMCID: PMC3673698  PMID: 21321105
14.  Glycan-based DC-SIGN targeting to enhance antigen cross-presentation in anticancer vaccines 
Oncoimmunology  2013;2(2):e23040.
In vivo dendritic-cell targeting constitutes a promising strategy for anticancer vaccination. Here, we discuss the usage of multivalent DC-SIGN-targeting glycan platforms that allow for the efficient routing of antigens to the endo-lysosomal pathway as well as to a yet uncharacterized cross-presentation mechanism inducing CD4+ and CD8+ T-cell responses.
doi:10.4161/onci.23040
PMCID: PMC3601176  PMID: 23525136
cell surface molecules; dendritic cells; DC-SIGN; glycans; human; rodent
15.  Skin-Resident Antigen-Presenting Cells: Instruction Manual for Vaccine Development 
The induction of antigen-specific effector T cells is driven by proper antigen presentation and co-stimulation by dendritic cells (DCs). For this reason strategies have been developed to instruct DCs for the induction of CD4+ and CD8+ T cell responses. Since DCs are localized, amongst other locations, in peripheral tissues such as the skin, new vaccines are aiming at targeting antigens to DCs in situ. Optimal skin-DC targeting in combination with adequate adjuvant delivery facilitates DC maturation and migration to draining lymph nodes and enhances antigen cross-presentation and T cell priming. In this review we describe what DC subsets populate the human skin, as well as current vaccination strategies based on targeting strategies and alternative administration for the induction of robust long-lived anti-cancer effector T cells.
doi:10.3389/fimmu.2013.00157
PMCID: PMC3687254  PMID: 23801994
skin; antigen-presenting cells; vaccination; microneedles; C-type lectin receptors; glycans
16.  Analytical Tools for the Study of Cellular Glycosylation in the Immune System 
It is becoming increasingly clear that glycosylation plays important role in intercellular communication within the immune system. Glycosylation-dependent interactions are crucial for the innate and adaptive immune system and regulate immune cell trafficking, synapse formation, activation, and survival. These functions take place by the cis or trans interaction of lectins with glycans. Classical immunological and biochemical methods have been used for the study of lectin function; however, the investigation of their counterparts, glycans, requires very specialized methodologies that have been extensively developed in the past decade within the Glycobiology scientific community. This mini-review intends to summarize the available technology for the study of glycan biosynthesis, its regulation and characterization for their application to the study of glycans in immunology.
doi:10.3389/fimmu.2013.00451
PMCID: PMC3858669  PMID: 24376449
glycan analysis; glycosyltransferases; glycans; lectins; immune cells
17.  A novel Kluyveromyces marxianus strain with an inducible flocculation phenotype 
AMB Express  2012;2:38.
Flocculation is a very useful phenotype for industrial yeast strains, since it facilitates cell harvest and represents an easy way of cell immobilization in continuous fermentation processes. The present work represents the first time that an inducible flocculation phenotype has been generated in a non flocculent strain of Kluyveromyces marxianus. This was accomplished by expressing Saccharomyces cerevisiae FLO5 gene in K. marxianus CECT 11769 strain. The FLO 5 gene was placed under the control of an EPG promoter, not repressed by glucose and induced by anoxia. Our experimental approach successfully generated two novel K. marxianus flocculent phenotypes: one inducible and one constitutive. The constitutive phenotype originated from deletions in the FLO5 promoter region, indicating the existence of putative upstream repressor site involved in oxygen regulation of the EPG1 promoter. The novel strains here generated had a unique set of characteristics that provided an advantage, over the wild-type strain, for the industrial co-production of ethanol and polygalacturonase.
doi:10.1186/2191-0855-2-38
PMCID: PMC3431993  PMID: 22838397
Kluyveromyces marxianus; Inducible flocculent phenotype; Polygalacturonase; Ethanol
18.  Construction of a novel Pichia pastoris strain for production of xanthophylls 
AMB Express  2012;2:24.
In this study, we used the yeast carotenogenic producer Pichia pastoris Pp-EBIL strain, which has been metabolically engineered, by heterologously expressing β-carotene-pathway enzymes to produce β-carotene, as a vessel for recombinant astaxanthin expression. For this purpose, we designed new P. pastoris recombinant-strains harboring astaxanthin-encoding genes from carotenogenic microorganism, and thus capable of producing xanthophyllic compounds. We designed and constructed a plasmid (pGAPZA-WZ) containing both the β-carotene ketolase (crtW) and β-carotene hydroxylase (crtZ) genes from Agrobacterium aurantiacum, under the control of the GAP promoter and containing an AOX-1 terminator. The plasmid was then integrated into the P. pastoris Pp-EBIL strain genomic DNA, producing clone Pp-EBILWZ. The recombinant P. pastoris (Pp-EBILWZ) cells exhibited a strong reddish carotenoid coloration and were confirmed, by HPLC, to produce not only the previous described carotenoids lycopene and β-carotene, but also de novo synthesized astaxanthin.
doi:10.1186/2191-0855-2-24
PMCID: PMC3485114  PMID: 22534340
Pichia pastoris; Carotenoids; β-carotene; Astaxanthin
19.  Campylobacter jejuni Lipooligosaccharides Modulate Dendritic Cell-Mediated T Cell Polarization in a Sialic Acid Linkage-Dependent Manner ▿ 
Infection and Immunity  2011;79(7):2681-2689.
Carbohydrate mimicry between Campylobacter jejuni lipooligosaccharides (LOS) and host neural gangliosides plays a crucial role in the pathogenesis of Guillain-Barré syndrome (GBS). Campylobacter jejuni LOS may mimic various gangliosides, which affects the immunogenicity and the type of neurological deficits in GBS patients. Previous studies have shown the interaction of LOS with sialic acid-specific siglec receptors, although the functional consequences remain unknown. Cells that express high levels of siglecs include dendritic cells (DCs), which are crucial for initiation and differentiation of immune responses. We confirm that α2,3-sialylated GD1a/GM1a mimic and α2,8-sialylated GD1c mimic LOS structures interact with recombinant Sn and siglec-7, respectively. Although the linkage of the terminal sialic acid of LOS did not regulate expression of DC maturation markers, it displayed clear opposite expression levels of interleukin-12 (IL-12) and OX40L, molecules involved in DC-mediated Th cell differentiation. Accordingly, targeting DC-expressed siglec-7 with α2,8-linked sialylated LOS resulted in Th1 responses, whereas Th2 responses were induced by targeting with LOS containing α2,3-linked sialic acid. Thus, our data demonstrate for the first time that depending on the sialylated composition of Campylobacter jejuni LOS, specific Th differentiation programs are initiated, possibly through targeting of distinct DC-expressed siglecs.
doi:10.1128/IAI.00009-11
PMCID: PMC3191980  PMID: 21502591
20.  Detecting Unknown Attacks in Wireless Sensor Networks That Contain Mobile Nodes 
Sensors (Basel, Switzerland)  2012;12(8):10834-10850.
As wireless sensor networks are usually deployed in unattended areas, security policies cannot be updated in a timely fashion upon identification of new attacks. This gives enough time for attackers to cause significant damage. Thus, it is of great importance to provide protection from unknown attacks. However, existing solutions are mostly concentrated on known attacks. On the other hand, mobility can make the sensor network more resilient to failures, reactive to events, and able to support disparate missions with a common set of sensors, yet the problem of security becomes more complicated. In order to address the issue of security in networks with mobile nodes, we propose a machine learning solution for anomaly detection along with the feature extraction process that tries to detect temporal and spatial inconsistencies in the sequences of sensed values and the routing paths used to forward these values to the base station. We also propose a special way to treat mobile nodes, which is the main novelty of this work. The data produced in the presence of an attacker are treated as outliers, and detected using clustering techniques. These techniques are further coupled with a reputation system, in this way isolating compromised nodes in timely fashion. The proposal exhibits good performances at detecting and confining previously unseen attacks, including the cases when mobile nodes are compromised.
doi:10.3390/s120810834
PMCID: PMC3472860  PMID: 23112632
wireless sensor networks; mobility; unknown attacks; clustering algorithms; reputation systems
21.  Improving Social Odometry Robot Networks with Distributed Reputation Systems for Collaborative Purposes 
Sensors (Basel, Switzerland)  2011;11(12):11372-11389.
The improvement of odometry systems in collaborative robotics remains an important challenge for several applications. Social odometry is a social technique which confers the robots the possibility to learn from the others. This paper analyzes social odometry and proposes and follows a methodology to improve its behavior based on cooperative reputation systems. We also provide a reference implementation that allows us to compare the performance of the proposed solution in highly dynamic environments with the performance of standard social odometry techniques. Simulation results quantitatively show the benefits of this collaborative approach that allows us to achieve better performances than social odometry.
doi:10.3390/s111211372
PMCID: PMC3251988  PMID: 22247671
collaborative robots; robot networks; social odometry; collective decision; reputation systems; trust algorithms; unsupervised techniques
22.  Interaction of Polysialic Acid with CCL21 Regulates the Migratory Capacity of Human Dendritic Cells 
PLoS ONE  2009;4(9):e6987.
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs). Immature DCs (iDCs) are situated in the periphery where they capture pathogen. Subsequently, they migrate as mature DCs (mDCs) to draining lymph nodes to activate T cells. CCR7 and CCL21 contribute to the migratory capacity of the DC, but it is not completely understood what molecular requirements are involved. Here we demonstrate that monocyte-derived DCs dramatically change ST8Sia IV expression during maturation, leading to the generation of polysialic acid (polySia). PolySia expression is highly upregulated after 2 days Toll-like receptor-4 (TLR4) triggering. Surprisingly, only immunogenic and not tolerogenic mDCs upregulated polySia expression. Furthermore, we show that polySia expression on DCs is required for CCL21-directed migration, whereby polySia directly captures CCL21. Corresponding to polySia, the expression level of CCR7 is maximal two days after TLR4 triggering. In contrast, although TLR agonists other than LPS induce upregulation of CCR7, they achieve only a moderate polySia expression. In situ we could detect polySia-expressing APCs in the T cell zone of the lymph node and in the deep dermis. Together our results indicate that prolonged TLR4 engagement is required for the generation of polySia-expressing DCs that facilitate CCL21 capture and subsequent CCL21-directed migration.
doi:10.1371/journal.pone.0006987
PMCID: PMC2737307  PMID: 19750015
23.  Improving Security for SCADA Sensor Networks with Reputation Systems and Self-Organizing Maps 
Sensors (Basel, Switzerland)  2009;9(11):9380-9397.
The reliable operation of modern infrastructures depends on computerized systems and Supervisory Control and Data Acquisition (SCADA) systems, which are also based on the data obtained from sensor networks. The inherent limitations of the sensor devices make them extremely vulnerable to cyberwarfare/cyberterrorism attacks. In this paper, we propose a reputation system enhanced with distributed agents, based on unsupervised learning algorithms (self-organizing maps), in order to achieve fault tolerance and enhanced resistance to previously unknown attacks. This approach has been extensively simulated and compared with previous proposals.
doi:10.3390/s91109380
PMCID: PMC3260646  PMID: 22291569
SCADA control system; cyber security; critical infrastructure; reputation system; countermeasure; security framework
24.  Using Reputation Systems and Non-Deterministic Routing to Secure Wireless Sensor Networks 
Sensors (Basel, Switzerland)  2009;9(5):3958-3980.
Security in wireless sensor networks is difficult to achieve because of the resource limitations of the sensor nodes. We propose a trust-based decision framework for wireless sensor networks coupled with a non-deterministic routing protocol. Both provide a mechanism to effectively detect and confine common attacks, and, unlike previous approaches, allow bad reputation feedback to the network. This approach has been extensively simulated, obtaining good results, even for unrealistically complex attack scenarios.
doi:10.3390/s90503958
PMCID: PMC3297150  PMID: 22412345
security; reputation system; wireless sensor networks; routing protocol; sybil attack; countermeasure
25.  Convergent Actions of IκB Kinase β and Protein Kinase Cδ Modulate mRNA Stability through Phosphorylation of 14-3-3β Complexed with Tristetraprolin†  
Molecular and Cellular Biology  2005;25(15):6454-6463.
Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The β1,4-galactosyltransferase 1 (β4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3β is required to bind β4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Iκβ kinase and protein kinase Cδ is involved in the phosphorylation of 14-3-3β on two serine residues, paralleled by release of binding of TTP and 14-3-3β from β4GalT1 mRNA, nuclear sequestration of TTP, and β4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3β.
doi:10.1128/MCB.25.15.6454-6463.2005
PMCID: PMC1190353  PMID: 16024783

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