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The human gut symbiont Lacticaseibacillus (L.) casei (previously Lactobacillus casei) is under intense research due to its wide range of immunomodulatory effects on the human host. Dendritic cells (DCs) are crucial players in the direct and indirect communication with lactobacilli in the gastrointestinal tract. Here, we demonstrate that human monocyte-derived DCs (moDCs) are able to engulf L. casei BL23, in which the intact bacterial cell wall and morphology have a key role. The absence of the bacterial cell-wall-degrading enzyme, Lc-p75, in L. casei cells causes remarkable morphological changes, which have important consequences in the phagocytosis of L. casei by moDCs. Our results showed that the Lc-p75 mutation induced defective internalization and impaired proinflammatory and T-cell-polarizing cytokine secretion by bacteria-exposed moDCs. The T helper (Th) 1 and Th17 cell activating capacity of moDCs induced by the mutant L. casei was consequently reduced. Moreover, inhibition of the phagocytosis of wild-type bacteria showed similar results. Taken together, these data suggested that formation of short bacterial chains helps to exert the potent immunomodulatory properties of L. casei BL23.

The human gut symbiont

Summary Mesenchymal stromal cell-like (MSCl) cells generated from human embryonic stem cells are considered to be an eligible cell line to model the immunomodulatory behavior of mesenchymal stromal cells (MSCs) in vitro. Dendritic cells (DCs) are essential players in the maintenance and restoration of the sensitive balance between tolerance and immunity. Here, the effects of MSCl cells on the in vitro differentiation of human monocytes into DCs were investigated. MSCl cells promote the differentiation of CTLA-4 expressing DCs via the production of all-trans retinoic acid (ATRA) functioning as a ligand of RARα, a key nuclear receptor in DC development. These semi-matured DCs exhibit an ability to activate allogeneic, naive T cells and polarize them into IL-10 + IL-17 + double-positive T helper cells in a CTLA-4-dependent manner. Mapping the molecular mechanisms of MSC-mediated indirect modulation of DC differentiation may help to expand MSCs' clinical application in cell-free therapies., Graphical abstract, Highlights • Mesenchymal stromal cell-like cells alter moDC differentiation via RARα activation • Mesenchymal stromal cell-like cells express genes known to play role in ATRA synthesis • MoDCs, differentiated in the presence of MSCl-derived factors, express CTLA-4 • CTLA-4+ moDCs are able to induce polarization of IL-10- and IL-17-producing helper T cells, Molecular Biology; Immunology; Components of the Immune System; Stem Cells Research

Purpose. This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). Methods. SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNFα, IL-1β, IFNγ), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. Results. The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNFα, or IL-1β, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. Conclusion. Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT’s (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow–derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin–rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.—Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

In this study, we examined the relationship between levels of lactoferrin (LF) and IL-17 in human serum and breast milk and the development of allergy in children. LF and IL-17 levels were determined by ELISA in healthy (n=19) and allergic mothers (n=21) on the 5th day after delivery. Two years later, information on breastfeeding and allergic outcomes was collected by questionnaires from parents of both groups and district child care nurses. Significantly higher concentrations of LF were found in the breast milk of allergic mothers compared to the healthy controls. At 2 years of age, only those three infants became allergic from the atopic group in whose starting breast milk samples a very high LF level (306 μg mg–1 protein) or simultaneously elevated concentrations of LF and IL-17 were measured. These findings indicate that the very early measurement of LF and IL-17 levels in the breast milk of allergic mothers may help to predict the allergy development in their infants.

Background BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. Methods Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. Results Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5ʹppp-dsRNA leads to increased activity of the IRF3-IFNβ pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/β, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. Conclusion RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNβ production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.

The cytoplasmic nucleotide oligomerization domain 2 (NOD2) receptor recognizes the bacterial cell wall component muramyl dipeptide (MDP). NOD2 ligation initiates the nuclear factor kappa B and the mitogen-activated protein kinase cascades. However, administering MDP alone is insufficient to elicit strong cytokine responses in various immune cells, including dendritic cells (DCs). Because the simultaneous presence of various microbial products and cytokines in inflamed tissues modulates DC function, we initiated this study to examine how interferon gamma (IFNγ), a central modulator of inflammation, affects the NOD2-mediated signaling pathway in human conventional DCs (cDCs). Synergistic stimulation of DCs with MDP and IFNγ increased the expression of CD40, CD80, CD83, CD86, and human leukocyte antigen DQ proteins and significantly elevated the production of pro-inflammatory cytokines IL-1β, IL-6, IL-12, and tumour necrosis factor (TNF), as well as anti-inflammatory cytokine IL-10. Furthermore, the simultaneous presence of MDP and IFNγ was necessary to decrease IkBα protein levels. By investigating various mechanisms implicated in MDP- and IFNγ-mediated signaling pathways, we revealed that the increased production of pro-inflammatory cytokines is highly dependent on the X-linked inhibitor of apoptosis protein (XIAP) but not on cellular IAP1 and IAP2. We also found that the NOD2 signaling pathway is regulated by the mammalian target of rapamycin (mTOR) but is not affected by phosphatidylinositol-3 kinase or signal transducer and activator of transcription 1 inhibition. Our results demonstrate, for the first time, that IFNγ positively affects NOD2-mediated signaling in human cDCs, in a manner considerably dependent on XIAP and partially dependent on mTOR.

Serotonin is a monoamine neurotransmitter that signals through a wide array of receptors (5-HT1–7) many of which are also involved in immune processes. Dendritic cells (DCs) are crucial players in immune defense by bridging innate and adaptive immune responses via their vast repertoire of pattern recognition receptors and antigen-presenting capability. Although serotonin is known to influence immunity at many levels, cell type-specific expression and function of its receptors remains poorly understood. Here we aimed to study 5-HT1–7 expression and function in CD1a− and CD1a+ human monocyte-derived DCs (moDCs). We found that the 5-HT2B receptor-subtype is solely expressed by the inflammatory CD1a+ moDC subset. Specific 5-HT2B activation potently inhibited TLR2, TLR3, and TLR7/8-induced proinflammatory cytokine and chemokine (TNF-α, IL-6, IL-8, IP-10, IL-12) but not type I interferon-β responses. 5-HT2B agonism also interfered with the polarization of CD1a+ moDC-primed CD4+ T cells towards inflammatory Th1 and Th17 effector lymphocytes. Here we report the subset-specific expression and immunomodulatory function of 5-HT2B in human moDCs. Our results expand the biological role of 5-HT2B which may act not only as a neurotransmitter receptor, but also as an important modulator of both innate and adaptive immune responses.

Rationale A targeted proteomics method based on selected reaction monitoring (SRM) is a relevant approach for the analysis of multiple analytes in biological samples. Defensins are phylogenetically conserved small antimicrobial peptides contributing to innate host defense and exhibiting low immunogenicity, resistance to proteolysis and a broad range of antimicrobial activities. The goal of the present study was to develop and optimize SRM-based targeted proteomics methods for the detection of human β-defensins 1–4 in various biological fluids. Methods An SRM-based targeted proteomics method was developed and validated for the detection of human β-defensins 1–4. The supernatants of resting and IL-1β-stimulated Caco2, HT-29 and SW-1116 colonic epithelial cells (CEC), cell lysates of CECs and tear samples of human healthy individuals were analyzed and the feasibility of the developed method was validated by ELISA and dot-blot analysis complemented by RT-qPCR. Results Our results demonstrate that the developed SRM method offers an alternative approach for the cost-effective and rapid analysis of human β-defensins in samples with biological relevance. Conclusions A semi-quantitative targeted mass spectrometry method was developed and validated for the relative quantification of β-defensins 1–4 in cell culture supernatants and body fluid analyses. Copyright © 2015 John Wiley & Sons, Ltd.

Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using anin vitromodel system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116) derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1βor TNF-αthis effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4+T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses.

Dendritic cells (DCs) are considered as the main coordinators of both mucosal and systemic immune responses thus playing a determining role in shaping the outcome of effector cell responses. However, it is still uncovered how primary human monocyte-derived DC populations (moDC) drive the polarization of helper T (Th) cells in the presence of commensal bacteria harboring unique immunomodulatory properties. Furthermore, the individual members of the gut microbiota have the potential to modulate the outcome of immune responses and shape the immunogenicity of differentiating moDCs via the activation of retinoic-acid receptor alpha (RARα). Here we report that moDCs are able to mediate robust Th1 and Th17 responses upon stimulation by Escherichia coli Schaedler or Morganella morganii, while the probiotic Bacillus subtilis strain limits this effect. Moreover, physiological concentrations of all-trans retinoic-acid (ATRA) are able to re-program the differentiation of moDCs resulting in altered gene expression profiles of the master transcription factors RARα and interferon-regulatory factor (IRF) 4, and concomitantly regulate the cell surface expression levels of CD1 proteins and also the mucosa-associated CD103 integrin to different directions. It was also demonstrated that the ATRA-conditioned moDCs exhibited enhanced pro-inflammatory cytokine secretion, while reduced their co-stimulatory and antigen presenting capacity thus reducing Th1 and presenting undetectable Th17 type responses against the tested microbiota strains. Importantly, these regulatory circuits could be prevented by the selective inhibition of RARα functionality. These results altogether demonstrate that selected commensal bacterial strains are able to drive strong effector immune responses by moDCs, while in the presence of ATRA they support the development of both tolerogenic and inflammatory moDC in a RARα dependent manner.

The vertebrate gut symbiont Lactobacillus reuteri exhibits strain-specific adhesion and health-promoting properties. Here, we investigated the role of the mucus adhesins, CmbA and MUB upon interaction with the L. reuteri strains ATCC PTA 6475 and ATCC 53608 in human monocyte-derived DCs (moDCs). We showed that mucus adhesins increased the capacity of L. reuteri strains to interact with moDCs and promoted phagocytosis. Our data also suggest that mucus adhesins mediate anti- and pro-inflammatory effects by the induction of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), IL-1s, IL-6, and IL-12 cytokines. L. reuteri ATCC PTA 6475 and ATCC 53608 strains were exclusively able to induce moDC-mediated Th1 and Th17 immune responses. We further showed that purified MUB activates moDCs and induces Th1 polarized immune responses associated with increased IFNγ production. MUB appeared to mediate these effects via binding to C-type lectin receptors (CLRs), as shown using cell reporter assays. Blocking moDCs with antibodies against dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) or Dectin-2 did not affect the uptake of the MUB-expressing strain, but reduced the production of TNF-α and IL-6 by moDCs significantly in line with the Th1 polarizing capacity of moDCs. The direct interaction between MUB and CLRs was further confirmed by force spectroscopy. Taken together these data suggest that mucus adhesins expressed at the cell surface of L. reuteri strains may exert immunoregulatory effects in the gut through modulating the Th1-promoting capacity of DCs upon interaction with C-type lectins.

Skin dendritic cells of patients with atopic dermatitis (AD) are well characterized, but less is known about their peripheral blood precursors. The aim of this study was to investigate the phenotypic features and chemokine production of myeloid pre-dendritic cells of patients with AD ex vivo and after stimulation with Staphylococcus enterotoxin B and thymic stromal lymphopoietin, representing an AD-like microenvironment. The expression of cell surface markers was measured by flow cytometry, while chemokine production was monitored with chemokine antibody array and confirmed by enzyme-linked immunoassays. AD pre-dendritic cells expressed higher levels of Fc?RI and the maturation and activation markers tended to be altered. They produced both AD (CCL17/18/22) and maturation-related (CCL3/4/5) chemokines at higher level than controls. The production of CCL3/4 and CCL18 were significantly higher even without AD-specific stimulation, while the production of CCL17 and CCL22 were significantly higher only after stimulation. These results indicate that circulating AD pre-dendritic cells are premature and bear atopic characteristics even without tissue-specific stimulation, suggesting that their development is not only influenced by the skin microenvironment, but even earlier by the local milieu in the blood.

Type I and III IFNs are crucial, soluble components of potent antiviral responses. It has been explored recently that mTOR is involved in the regulation of IFN-α/β production by pDCs, albeit its role in the induction of IFN responses in cDCs remained unrevealed. In this study, we demonstrate that the PI3K/mTOR pathway is indispensable for eliciting intact type I and III IFN responses in moDCs stimulated with polyI:C. The inhibition of mTOR functionality by rapamycin impairs the pIRF3 and also a few members of the MAPK family, suggesting that mTOR contributes to the activation of multiple signaling pathways in the presence of viral antigens. Furthermore, rapamycin-treated moDCs show decreased capacity to prime IFN-γ secretion by naive CD8+ T-lymphocytes. As in moDCs, mTOR-mediated regulation is also essential for the production of type I and III IFNs in circulating CD1c+ DCs. To our best knowledge, these results demonstrate for the first time that mTOR has an impact on the functional activities of cDCs via modulating the outcome of IFN secretion.

OBJECTIVE In this study the alteration of endothelial function, arterial stiffness and autoantibodies was investigated in patients with UCTD. METHODS Thirty-one patients with UCTD were included in this prospective study. All the patients remained in the UCTD stage during the average 3.8 years follow-up period. The onset of UCTD was denoted as UCTD1, while the end of the follow-up period was called UCTD2. Flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT), autoantibodies [such as anti-SSA, anti-SSB, anti-DNA, anti-RNP, anti-CCP, aCL, anti-oxidized low-density lipoprotein (oxLDL) and AECA], von Willebrand factor antigen, thrombomodulin (TM), endothelin 1 (ET-1) and lipid parameters were measured. RESULTS In the UCTD1 stage, high-sensitivity CRP (hsCRP) and endothelial cell activation and/or damage markers such as TM, ET-1 and AECA levels were significantly higher compared with controls (controls vs UCTD1: hsCRP, P < 0.0001; TM, P = 0.001; ET-1, P < 0.0001). In the UCTD2 stage, the carotid IMT increased (UCTD1 vs UCTD2, P = 0.01) and FMD further deteriorated (UCTD1 and UCTD2, P = 0.001). In UCTD2 there was a close correlation between the carotid IMT, and duration of the disease (r = 0.612, P < 0.001), the level of TM (r = 0.673, P < 0.001) and anti-oxLDL (r = 0.800, P < 0.001). CONCLUSION Our data suggest that the presence of inflammation and autoantibodies provoke endothelial cell activation and/or injury in UCTD patients. The persistent endothelial dysfunction may provoke the development of atherosclerosis. FMD was found to be the most sensitive marker for arterial stiffness, and the increase of IMT clearly indicated the existence of preclinical atherosclerosis in UCTD patients.

Plasmacytoid dendritic cells (pDCs) are professional type I interferon (IFN)-producing cells that play an essential role in antiviral immunity. In many cell types, detection of intracellular pathogens is mostly dependent on endosomal Toll-like receptors (TLRs) and cytosolic sensors, such as retinoic acid-inducible gene I (RIG-I). However, the possible interplay between these two systems has not yet been elucidated. Here we aimed to study the collaboration of endosomal TLRs and RIG-I in primary human pDCs. We found that under steady-state conditions, pDCs express RIG-I at very low level, but the expression of this receptor is rapidly and dramatically upregulated upon stimulation by the TLR7 ligand imiquimod or the TLR9 ligand type A CpG. We also demonstrated that pDCs are able to sense and respond to 5'-triphosphate double-stranded RNA (5'-ppp-dsRNA) only following activation by endosomal TLRs. Experiments on primary pDCs with functionally blocked IFN-α/β receptor 1 (IFNAR1) and those on human pDC leukemia (pDC-L) cells defective in type I IFN secretion indicated that the upregulation of RIG-I expression in pDCs upon stimulation by endosomal TLR occurs in a type I IFN-independent manner. Selective phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 could be identified as an early signaling event in this process. Our results show that in contrast to many other cell types, where RIG-I expression is induced by type I IFN, in pDCs a disparate mechanism is responsible for the upregulation of RIG-I. Our findings also indicate that along with autophagy, an additional mechanism is operating in pDCs to promote the detection of replicating viruses.

Purpose: To study the association between different types of mediators in the tear fluid and topographic indices characterizing the severity of keratoconus (KC). Methods: In this study, nonstimulated tear fluid samples were collected from 14 eyes of 11 patients with KC. The following indices were measured by corneal topography: maximum K value, average K value, Klyce/Maeda keratoconus index (KCI), Smolek/Klyce keratoconus severity index, opposite sector index, center/surround index, keratoconus prediction index and standard deviation of corneal power. The concentrations of interleukin (IL)-6, IL-13, CXCL8 (IL-8), chemokine (C-C motif) ligand 5 (CCL5, regulated and normal T cell expressed and secreted), matrix metalloproteinase-9 (MMP-9), MMP-13, tissue inhibitor of metalloproteinase-1, nerve growth factor (NGF) and epidermal growth factor were measured by cytometric bead array technology. Release of mediators was calculated from their concentrations and the volume of tears collected over 2 min. Results: Significant positive associations were found between CCL5, MMP-13 and NGF and several topographic indices. Significant negative correlations were found between IL-6 and KCI. Age-dependent associations were observed between IL-13, CXCL8, CCL5 and MMP-13 and the topographic data. Conclusion: Several correlations were observed between the mediators and the topographic indices, suggesting possible roles in the pathophysiology of KC. Our data indicate that some mediators have different effects on the severity of disease in an age-dependent manner.

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration–dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration–dependent pathway was independent of peroxisome proliferator–activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid–mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.

No abstract available Keywords: monocyte-derived dendritic cells; monocyte-derived macrophages; monocyte–macrophage precursor cells; neuroimmune interactions; neuroimmunomodulation.

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.

In the last three decades, the incidence of melanoma has increased worldwide and no effective treatment modalities have been developed yet. All-trans retinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) are strong inducers of toll-like receptor 3 (TLR3) and MDA5 expression, and polyI:C-induced TLR3 and MDA5 signaling specifically causes cell death in melanoma cells in vitro. We addressed the question of whether ATRA pretreatment could enhance the efficacy of polyI:C and, if so, would ATRA have any additional effects on this process. We found that the combined treatment of human melanoma cells with ATRA and polyI:C strongly increased the expression of TLR3 and MDA5 in both WM35 and WM983A cells associated with significantly higher mRNA and secreted levels of interferon β (IFNβ), CXCL1, CXCL8/IL-8, CXCL9, and CXCL10 than cells treated with either ATRA or polyI:C. Silencing of MDA5 by siRNA moderately affected IFNβ secretion, whereas TLR3 knockdown interfered with both CXCL chemokine and IFNβ production. Furthermore, the supernatants of ATRA+polyI:C-activated cultures increased the migration of both human monocyte-derived macrophages and CD1a dendritic cells significantly as compared with the supernatants of cells treated with either ATRA or polyI:C, and this effect occurred in a TLR3-dependent manner. In conclusion, consecutive treatment with ATRA and polyI:C results in strong, TLR3/MDA5-mediated chemokine and IFN responses in cultured human melanoma cells, which triggers a functional migratory response in professional antigen-presenting cells. This novel mode of concomitant activation may represent a more efficient treatment option for future melanoma therapy.

Identification of the CD1a+ moDC as an inflammatory subset with specialized activation of RLR and associated cytokine and functional activities. Cytosolic RIG-I-like helicases (RLR) are PRRs involved in type I IFN production and antiviral immunity. This study focuses to the comparison of the expression, function, and signaling cascades associated to RLR in the previously identified CD14−DC-SIGN+PPARγlowCD1a+ and CD14lowDC-SIGN+PPARγhighCD1a− human moDC subsets. Our results revealed that the expression of RLR genes and proteins as well as the activity of the coupled signaling pathways are significantly higher in the CD1a+ subset than in its phenotypically and functionally distinct counterpart. Specific activation of RLR in moDCs by poly(I:C) or influenza virus was shown to induce the secretion of IFN-β via IRF3, whereas induction of proinflammatory cytokine responses were predominantly controlled by TLR3. The requirement of RLR-mediated signaling in CD1a+ moDCs for priming naïve CD8+ T lymphocytes and inducing influenza virus-specific cellular immune responses was confirmed by RIG-I/MDA5 silencing, which abrogated these functions. Our results demonstrate the subset-specific activation of RLR and the underlying mechanisms behind its cytokine secretion profile and identify CD1a+ moDCs as an inflammatory subset with specialized functional activities. We also provide evidence that this migratory DC subset can be detected in human tonsil and reactive LNs.

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.

GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.

Eur J Clin Invest 2012; 42 (1): 49–60 Abstract Background Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2−/−) mice. Materials and methods The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures. Results PLCγ2−/− mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2−/− mice was similar to, or even more robust than, that in wild-type animals. Conclusions Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency–induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis.

Stem cell therapy (SCT) is a promising and prospective approach in the treatment of patients with severe peripheral arterial disorders, primarily with Buerger’s disease. However, very little is known about the duration of the effect of SCT, and to our best knowledge no data are available on the efficacy and safety of repeated SCT in patients with Buerger’s disease. Here we report on two patients with severe Buerger’s disease, who received repeated autologous bone marrow-derived stem cell therapy. Our results show that a second SCT, applied to a previously treated leg 30 or 36 months after the first treatment was efficient in both cases. After twelve months, the clinical state of the repeatedly treated lower limb improved spectacularly and non-healing ulcers healed more rapidly than after the first SCT. No severe adverse events were detected. Thus repeated SCT offers a safe and efficient treatment option for relapsing patients at the advanced stage of Buerger’s disease.

Type I interferon (IFN) production is a key event during innate immune responses. This early and prompt mechanism is mediated by multiple factors that involve various pattern recognition receptors (PRRs), adaptor proteins, kinases and transcription factors. Stimulator of IFN genes (STING) is an important transmembrane adaptor protein that plays a role in the activation of downstream transcription factors, such as IFN regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6, via TANK-binding kinase 1 (TBK1), which has been considered the main innate immune defense weaponry against viruses and intracellular pathogens. In a current issue of Nature Immunology, You et al. showed that, after viral infection, the ETS-related transcription factor ELF4 (previously known as MEF) interacts with STING and is consequently activated by TBK1. This process leads to the nuclear translocation of ELF4 and its binding to IFN promoters (Figure 1). The group also demonstrated that ELF4 acts as an IFN transcription factor, as it has the potential to increase the binding affinity of IRF3 and IRF7 through cooperative binding to newly identified enhancer elements (EICE) in the IFN promoters.1 These new findings not only extend our knowledge about the details of the fine-tuning of the innate IFN responses, but also represent a new milestone by describing the role of EICE elements in this process.

PPARγ expression and activation in CD1a– monocyte derived dendritic cells modulates the engulfment of and the cytokine and T cell response to apoptotic neutrophils. The means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by human monocyte-derived DCs is slow and less efficient than that of macrophages, and CD1a– DCs are more active in the engulfment of apoptotic neutrophils than CD1a+ DCs. Blocking DC-SIGN function partially interferes with the uptake of apoptotic cells, and long-term interaction of apoptotic neutrophils with DCs makes them prone to proinflammatory cytokine responses. Engulfment of apoptotic cells sensitizes CD1a– DCs for high IL-8, TNF-α, IL-6, and CD1a+ cells for IL-12 and IL-10 cytokine secretion elicited by additional inflammatory stimuli, which also result in the polarization of autologous T lymphocytes to Th1 effector cells. Ligand-induced activation of PPARγ by RSG results in enhanced phagocytosis, but the proinflammatory response and the capacity to trigger Th1 cell activation of CD1a– DCs are not enhanced. These results demonstrate that DCs are able to respond to allogeneic, apoptotic neutrophils with inflammatory cytokines and T cell responses in a subtype-specific manner that is modulated by the anti-inflammatory effects of PPARγ.

It has been demonstrated that pollen grains contain NAD(P)H oxidases that induce oxidative stress in the airways, and this oxidative insult is critical for the development of allergic inflammation in sensitized mice. On the basis of this observation, we have examined whether pollen grain exposure triggers oxidative stress in dendritic cells (DCs), altering their functions. To test this hypothesis, human monocyte-derived DCs were treated with ragweed pollen grains. Our findings show that exposure to pollen grains induces an increase in the intracellular levels of reactive oxygen species in DCs. Our data also indicate that besides the NAD(P)H oxidases, other component(s) of pollen grains contributes to this phenomenon. Elevated levels of intracellular reactive oxygen species triggered the production of IL-8 as well as proinflammatory cytokines, such as TNF-α and IL-6. Treatment with pollen grains initiated the maturation of DCs, strongly upregulated the membrane expression of CD80, CD86, CD83, and HLA-DR, and caused only a slight increase in the expression of CD40. The pollen-treated DCs induced the development of naive T lymphocytes toward effector T cells with a mixed profile of cytokine production. Antioxidant inhibited both the phenotypic and functional changes of DCs, underlining the importance of oxidative stress in these processes. Collectively, these data show that pollen exposure-induced oxidative stress may contribute to local innate immunity and participate in the initiation of adaptive immune responses to pollen Ags.

Modulation of the expression and activity of plasma membrane ion channels is one of the mechanisms by which immune cells can regulate their intracellular Ca2+ signaling pathways required for proliferation and/or differentiation. Voltage-gated K+ channels, inwardly rectifying K+ channels, and Ca2+-activated K+ channels have been described to play a major role in controlling the membrane potential in lymphocytes and professional APCs, such as monocytes, macrophages, and dendritic cells (DCs). Our study aimed at the characterization and identification of ion channels expressed in the course of human DC differentiation from monocytes. We report in this study for the first time that immature monocyte-derived DCs express voltage-gated Na+ channels in their plasma membrane. The analysis of the biophysical and pharmacological properties of the current and PCR-based cloning revealed the presence of Nav1.7 channels in immature DCs. Transition from the immature to a mature differentiation state, however, was accompanied by the down-regulation of Nav1.7 expression concomitant with the up-regulation of voltage-gated Kv1.3 K+ channel expression. The presence of Kv1.3 channels seems to be common for immune cells; hence, selective Kv1.3 blockers may emerge as candidates for inhibiting various functions of mature DCs that involve their migratory, cytokine-secreting, and T cell-activating potential.

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-beta1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow—derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. A total of 5 patients with SPAD were treated by ABMSCT using isolated CD34+ cells with characterized phenotype administered by intramuscular injections. The follow-up before and 1, 3, 6, 9, and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, nonhealing ulcers, ankle-brachial index [ABI]) and laboratory (angiography, duplex and laser ultrasonography, TcPO2) parameters. Significant improvement of pain and walking distance was observed in all patients. Nonhealing ulcers disappeared in 3 patients and became smaller and thinner in 1 patient. The average of ABI improved significantly on the treated limb but did not change on the contralateral limb. New collaterals were detected by angiography in 3 patients, but duplex ultrasonography detected improvement in one patient only. Laser ultrasonography showed a mild significant change, TcPO2 values improved mainly on the foot. Severe adverse events were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, and results in sustained clinical benefit for patients with SPAD.

Preventive vaccination is the most successful approach against infectious diseases and has a great impact on world health. Vaccines operate through the activation of innate immunity that helps to stimulate antigen-specific T- and B-lymphocytes. These events are orchestrated by dendritic cells (DCs) that are able to sample foreign structures and concomitantly sense ‘danger signals’. Thus, DCs provide a functional link between innate and acquired immunity, and due to their regulatory potential are referred to as natural adjuvants. Human conventional and plasmacytoid DCs express different sets of well-characterized Toll-like membrane receptors (TLRs) that recognize a broad range of conserved molecular patterns of pathogens. The recently discovered cytosolic Nod-like receptors (NLRs) and RIG-like helicases (RLHs) also turned out to participate in pathogen recognition and modulation of immune responses through interacting signaling pathways. As a result of their collaboration, the TLR, NLR and RLH recognition systems induce the secretion of different combinations of cytokines that play a fundamental role in T-cell activation and instruction. Ligands of the innate recognition systems emerge as new adjuvants for vaccine design, whereas manipulation of the signaling pathways mediated by these receptors offers new avenues for fine tuning immune responses and optimizing immunotherapies.

Súlyos perifériás artériás érbetegségekben a gyógyszeres és/vagy érsebészeti beavatkozások kimerülését követően a tűrhetetlen fájdalom, kiterjedt végtagi fekélyek, gangraenák megszüntetésének egyetlen módja a végtag amputációja. Betegek és módszerek: A szerzők – hazánkban elsőként – 5 előrehaladott perifériás artériás érbetegben (1 arteriosclerosis obliterans és 4 thromboangiitis obliterans) autológ csontvelői eredetű őssejtterápiát végeztek. A csontvelői őssejteket (CD34+ sejtek) crista biopsia végzésével nyerték. Mágneses sejtszeparálással CD34+ sejtszuszpenziót állítottak elő. Meghatározták a CD34+, CD133+ és CD45± sejtek számát és arányát. Az őssejtszuszpenziót intramuscularis injekció formájában a beteg végtagba juttatták vissza. Betegenként 0,37–1,14 × 10 5 /kg őssejt visszaadására került sor. Betegeiket 12 hónapig követték. Vizsgálatok történtek a beavatkozás előtt és után (1, 3, 6, 9 és 12 hónappal). Klinikai vizsgálatok: nyugalmi fájdalom, dysbasiás távolság, ischaemiás fekélyek gyógyhajlama, boka-kar index. Laboratóriumi vizsgálatok: angiográfia (az őssejtterápia előtt és után 1 és 6 hónappal), duplex ultrahang- és lézer-Doppler-scan, transcutan oxigéntenzió mérése, az endothelfunkciók vizsgálata. Eredmények: A nyugalmi fájdalom mind az öt betegük esetében megszűnt. A dysbasiás távolság szignifikánsan nőtt (36/440 m). Három beteg végtagi ischaemiás fekélye begyógyult, egy beteg nagyméretű fekélye lényegesen kisebbé és felületesebbé vált, egy betegben a végtagi fekély nem változott. A kezelt oldalon a boka-kar index szignifikánsan nőtt (0,41/0,83) tizenkét hónappal az őssejtterápiát követően, s nem változott az ellenoldalon. Három betegben tapasztaltak számottevő változást angiográfiával az őssejtterápia után hat hónappal. Csak szerény javulást észleltek color-Doppler- és lézer-Doppler-vizsgálatokkal. Az őssejtterápia előtt és után 1, 6 és 12 hónappal a transcutan oxigéntenzió-értékek a lábháton 18,10/16,78/23,83/37,50 Hgmm-re, míg a lábszáron 36,66/31,25/45,00/37,30 Hgmm-re változtak. A makro- és mikrocirkulációs paraméterek nem mutattak javulást az őssejtterápiát követően 1 hónappal, azonban az őssejtterápia után 3, 6, 9 és 12 hónappal már mérhető javulást tapasztaltak. Szövődményt, mellékhatást nem észleltek. Következtetések: Klinikai eredményeik alapján az autológ csontvelői eredetű őssejtterápiát hatásosnak, tartósnak és biztonságosnak tartják előrehaladott perifériás artériás érbetegségben. Szükség van további klinikai tapasztalatgyűjtésre.

We define mAb proteomics as the global generation of disease specific antibodies that permit mass screening of biomarkers. An integrated, high-throughput, disease-specific mAb-based biomarker discovery platform has been developed. The approach readily provided new biomarker leads with the focus on large-scale discovery and production of mAb-based, disease-specific clinical assay candidates. The outcome of the biomarker discovery process was a highly specific and sensitive assay, applicable for testing of clinical validation paradigms, like response to treatment or correlation with other clinical parameters. In contrast to MS-based or systems biology-based strategies, our process produced prevalidated clinical assays as the outcome of the discovery process. By re-engineering the biomarker discovery paradigm, the encouraging results presented in this paper clearly demonstrate the efficiency of the mAb proteomics approach, and set the grounds for the next steps of studies, namely, the hunt for candidate biomarkers that respond to drug treatment.

A targeted proteomics method based on selected reaction monitoring (SRM) is a relevant approach for the analysis of multiple analytes in biological samples. Defensins are phylogenetically conserved small antimicrobial peptides contributing to innate host defense and exhibiting low immunogenicity, resistance to proteolysis and a broad range of antimicrobial activities. The goal of the present study was to develop and optimize SRM-based targeted proteomics methods for the detection of human β-defensins 1-4 in various biological fluids.An SRM-based targeted proteomics method was developed and validated for the detection of human β-defensins 1-4. The supernatants of resting and IL-1β-stimulated Caco2, HT-29 and SW-1116 colonic epithelial cells (CEC), cell lysates of CECs and tear samples of human healthy individuals were analyzed and the feasibility of the developed method was validated by ELISA and dot-blot analysis complemented by RT-qPCR.Our results demonstrate that the developed SRM method offers an alternative approach for the cost-effective and rapid analysis of human β-defensins in samples with biological relevance.A semi-quantitative targeted mass spectrometry method was developed and validated for the relative quantification of β-defensins 1-4 in cell culture supernatants and body fluid analyses.

The major reservoir of human multipotent mesenchymal stem/stromal cells (MSCs) is the bone marrow (BM) with the capability to control hematopoietic stem cell development. The regenerative potential of MSCs is associated with enhanced endogenous repair and healing mechanisms that modulate inflammatory responses. Our previous results revealed that MSC-like (MSCl) cells derived from pluripotent human embryonic stem cells resemble BM-derived MSCs in morphology, phenotype, and differentiating potential. In this study, we investigated the effects of MSCl cells on the phenotype and functions of dendritic cells (DCs). To assess how antiviral immune responses could be regulated by intracellular pattern recognition receptors of DCs in the presence of MSCl cells, we activated DCs with the specific ligands of retinoic acid-inducible gene-I (RIG-I) helicases and found that activated DCs cocultured with MSCl cells exhibited reduced expression of CD1a and CD83 cell surface molecules serving as phenotypic indicators of DC differentiation and activation, respectively. However, RIG-I-mediated stimulation of DCs through specific ligands in the presence of MSCl cells resulted in significantly higher expression of the costimulatory molecules, CD80 and CD86, than in the presence of BM-MSCs. In line with these results, the concentration of IL-6, IL-10, and CXCL8 was increased in the supernatant of the DC-MSCl cocultures, while the secretion of TNF-α, CXCL10, IL-12, and IFNγ was reduced. Furthermore, the concerted action of mechanisms involved in the regulation of DC migration resulted in the blockade of cell migration, indicating altered DC functionality mediated by MSCl cell-derived signals and mechanisms resulting in a suppressive microenvironment.

The antifungal protein AFP is a small, cystein-rich protein secreted by the imperfect ascomycete Aspergillus giganteus. The protein efficiently inhibits the growth of filamentous fungi, including a variety of serious human and plant pathogens mainly of the genera Aspergillus and Fusarium, whereas AFP does not affect the growth of yeast and bacteria. This restricted susceptibility range makes it very attractive for medical or biotechnological use to combat fungal infection and contamination. We, therefore, analyzed whether AFP affects the growth or function of a number of mammalian cells. Here we show that the protein neither provokes any cytotoxic effects on human endothelial cells isolated from the umbilical vein nor activates the immune system. Moreover, potassium currents of neurons and astrocytes do not change in the presence of AFP and neither excitatory processes nor the intracellular calcium homeostasis of cultured skeletal muscle myotubes are affected by AFP. Our data, therefore, suggest that AFP is indeed a promising candidate for the therapeutic or biotechnological use as a potential antifungal agent.