Microglia cells, the immune cells residing in the brain, express immune regulatory molecules that have a central role in the manifestation of age-related brain characteristics. Our hypothesis suggests that galectin-1, an anti-inflammatory member of the beta-galactoside-binding lectin family, regulates microglia and neuroinflammation in the aging brain. Through our in-silico analysis, we discovered a subcluster of microglia in the aged mouse brain that exhibited increased expression of galectin-1 mRNA. In our Western blotting experiments, we observed a decrease in galectin-1 protein content in our rat primary cortical cultures over time. Additionally, we found that the presence of lipopolysaccharide, an immune activator, significantly increased the expression of galectin-1 protein in microglial cells. Utilizing flow cytometry, we determined that a portion of the galectin-1 protein was localized on the surface of the microglial cells. As cultivation time increased, we observed a decrease in the expression of activation-coupled molecules in microglial cells, indicating cellular exhaustion. In our mixed rat primary cortical cell cultures, we noted a transition of amoeboid microglial cells labeled with OX42(CD11b/c) to a ramified, branched phenotype during extended cultivation, accompanied by a complete disappearance of galectin-1 expression. By analyzing the transcriptome of a distinct microglial subpopulation in an animal model of aging, we established a correlation between chronological aging and galectin-1 expression. Furthermore, our in vitro study demonstrated that galectin-1 expression is associated with the functional activation state of microglial cells exhibiting specific amoeboid morphological characteristics. Based on our findings, we identify galectin-1 as a marker for microglia activation in the context of aging.

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/β-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

In article number 1902621, Tamás A. Martinek and co-workers develop a pentapeptidic tag, which reads the glycan code of ganglioside GM1 and triggers lipid raft-mediated endocytosis, avoiding lysosomal entrapment. This carrier molecule can deliver macromolecular cargoes (e.g., IgG complexes) into live cells with the possibility to escape to the cytosol.

Mesenchymal stem cells (MSC) are emerging as an effective therapeutic tool in treating canine osteoarthritis (OA). In this report, we focused on the questions of whether MSC transplantation has long-term beneficial effects for the improvement in motion and also evaluated the safety of MSC injection. Visceral adipose tissue, a surgical waste obtained during routine ovariectomy served as a source of allogeneic MSCs and used to treat OA. Altogether, fifty-eight dogs were transplanted in the study suffering from OA in the elbow (42 animals), hip (5), knee (8), ankle (2), and hock (1). The effect of MSC transplantation was evaluated by the degree of lameness at a 4-5-years follow-up period based on the owners' subjective observations. The results showed that 83% of the OA patients improved or retained improvement in lameness. Clinical safety of the treatment was assessed by evaluating the coincidence of tumors or other diseases and other adverse reactions (such as local inflammation) after MSC cell therapy. Two incidences of local inflammation for <1 week at the site of injection were reported. No other adverse reactions were detected post-treatment. Sixteen dogs died during the study, 4 due to cancer and 12 due to other diseases, diagnosed by veterinarians. Overall, our survey suggests that MSC transplantation has long-term beneficial effects in reducing lameness. Moreover, no enrichment in a specific cause of death was observed in the transplanted animals, compared to reported literature. Our data suggest that MSC treatment could be an effective and safe long-term therapy for canine OA.

The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein-protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.

There is a pressing need to develop ways to deliver therapeutic macromolecules to their intracellular targets. Certain viral and bacterial proteins are readily internalized in functional form through lipid raft-mediated/caveolar endocytosis, but mimicking this process with protein cargoes at therapeutically relevant concentrations is a great challenge. Targeting ganglioside GM1 in the caveolar pits triggers endocytosis. A pentapeptide sequence WYKYW is presented, which specifically captures the glycan moiety of GM1 (K D = 24 nm). The WYKYW-tag facilitates the GM1-dependent endocytosis of proteins in which the cargo-loaded caveosomes do not fuse with lysosomes. A structurally intact immunoglobulin G complex (580 kDa) is successfully delivered into live HeLa cells at extracellular concentrations ranging from 20 to 160 nm, and escape of the cargo proteins to the cytosol is observed. The short peptidic WYKYW-tag is an advantageous endocytosis routing sequence for lipid raft-mediated/caveolar cell delivery of therapeutic macromolecules, especially for cancer cells that overexpress GM1.

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

Glycosylation (sugar code) is frequently altered on the surface of various cells in patients with immune-mediated diseases resulting in changes in signal transduction and metabolic control. However, alteration of glycosylation patterns of T cells has not yet been characterised in autoimmune disorders. We have previously demonstrated that T-cells of patients with systemic lupus erythematosus (SLE) are resistant to the apoptotic effects of galectin-1 (Gal-1), an immunoregulatory lectin. T-cells from patients with active SLE (n=18), rheumatoid arthritis (RA) (n=14) or Sjögren’s syndrome (SS) (n=14) and from healthy controls (n=19) were examined. Cell surface glycosylation was analysed with lectin-binding assay, and the expression of glycosyltransferase and glycosidase enzymes participating in the construction of the glycan chains was measured with reverse transcriptase polymerase chain reaction (RT-PCR). Resting SLE T-cells bound significantly higher amounts of several lectins, while RA and SS resting T-cells were similar to controls in their glycosylation pattern. Activated T-cells from all autoimmune groups bound significantly less Gal-1 than controls, while other lectins bound similarly. Gene expression including alpha mannosidases (MAN1A1, MAN1A2, MAN2A1 and MAN2A2) and beta-N-acetylglucosaminyltransferases (MGAT1–5) in autoimmune activated T-cells did not differ from controls with the only exception of MAN1A2 in SS. However, we found a significant increase in the mRNA ratios of certain sialyltransferases and neuraminidases, specifically in ST6GAL1/NEU1 in SLE and SS, and in ST3GAL6/NEU1 in SLE and RA patients compared to controls. Treatment of cells with neuraminidase resulted in a remarkable increase in Gal-1 binding The glycosylation pattern of resting SLE T-cells was consistent with a pre-activated phenotype. Decreased Gal-1 binding found in all three diseases can be explained with an increased terminal sialylation, which may be a consequence of an altered expression of sialyltransferase and neuraminidase genes, whose concerted action is responsible for the degree of sialylation of glycan structures. Accordingly, neuraminidase treatment resulted in remarkably increased Gal-1 binding. We propose that increased sialylation may at least partially explain the previously found resistance to the immunoregulatory effects of Gal-1 in SLE, hence contributing to the pathomechanism of the examined diseases.

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia.

Background We have previously shown that activated T-cells from systemic lupus erythematosus (SLE) patients are resistant to the apoptotic effects of galectin-1 (Gal-1), an endogenous immunoregulatory lectin. The expression of de novo synthesised, intracellular Gal-1 (icGal-1) was also deficient in SLE activated T-cells, and we have demonstrated that icGal-1 levels affect apoptosis induction by extracellular Gal-1 (ecGal-1). However, production of icGal-1 may not be the only regulatory factor in this process, since binding of ecGal-1 to T-cells and its apoptotic activity depends on the surface glycosylation of T-cells. We have hypothesised that the cell-surface glycosylation pattern, and consequently, lectin-binding ability in SLE T-cells is altered, and that the abnormal expression of glycosylation enzymes may account for these changes. Materials and methods Lectin-binding assays were performed using a set of plant lectins and human recombinant Gal-1 on resting and activated T-cells with multi-colour flow-cytometry in 9 patients with active SLE and 15 healthy controls. mRNA levels of thirteen glycosylation enzymes involved in the development of N-glycan structures on T-cells were measured with qPCR, and were correlated with lectin binding ability. Results As compared with the resting state, the increase in Gal-1 binding during activation was significantly lower in SLE T-cells than in controls, and the level of Gal-1 binding maximum was significantly reduced in SLE activated T-cells than in controls. Binding maximum of plant lectins that recognise high complexity N-glycans also increased less in SLE T-cells than in controls during activation. mRNA level of sialyltransferase ST3GAL6 was increased and neuraminidase Neu1 was decreased in active SLE patients as compared to controls. The ST6GAL1/NEU1 ratio in SLE patients positively correlated with the SLEDAI disease activity index. Conclusion SLE T-cells show decreased complexity of N-glycan structures. Increased ST3GAL6 and decreased Neu1 expression result in an increased density of terminal sialic acids, and this may explain the impaired Gal-1 binding. In addition to the previously described deficiency in icGal-1 expression upon activation, our present findings of an attenuated glycan complexity and a shift toward terminal sialylation provide a further mechanism of pathological T-cell activation and regulation of T-cell viability in SLE. Reference Deák M, Hornung Á, Novák J, Demydenko D, Szabó E, Czibula Á, Fajka-Boja R, Kriston-Pál É, Monostori É, Kovács L. Novel role for galectin-1 in T-cells under physiological and pathological conditions. Immunobiology. 2015 220(4):483–9.

Galectin-1 is an endogenous immunoregulatory lectin-type protein. Its most important effects are the inhibition of the differentiation and cytokine production of Th1 and Th17 cells, and the induction of apoptosis of activated T-cells. Galectin-1 has been identified as a key molecule in antitumor immune surveillance, and data are accumulating about the pathogenic role of its deficiency, and the beneficial effects of its administration in various autoimmune disease models. Initial animal and human studies strongly suggest deficiencies in both galectin-1 production and responsiveness in systemic lupus erythematosus (SLE) T-cells. Since lupus features widespread abnormalities in T-cell activation, differentiation and viability, in this review the authors wished to highlight potential points in T-cell signalling processes that may be influenced by galectin-1. These points include GM-1 ganglioside-mediated lipid raft aggregation, early activation signalling steps involving p56Lck, the exchange of the CD3 ζ-ZAP-70 to the FcRγ-Syk pathway, defective mitogen-activated protein kinase pathway activation, impaired regulatory T-cell function, the failure to suppress the activity of interleukin 17 (IL-17) producing T-cells, and decreased suppression of the PI3K-mTOR pathway by phosphatase and tensin homolog (PTEN). These findings place galectin-1 into the group of potential pathogenic molecules in SLE.

Background We have previously found that activated T-cells from systemic lupus erythematosus (SLE) patients express lower amount of intracellular galectin-1 (icGal-1) than those of healthy controls and are resistant to the apoptotic effect of extracellular galectin-1 (ecGal-1), an endogenous immunoregulatory lectin. We also demonstrated that the de novo synthesized icGal-1 level affects apoptosis of T-cells induced by ecGal-1, since low icGal-1 expression resulted in reduced sentsitivity to ecGal-1 (Deák M et al). We have therefore proposed the defective icGal-1 production to be an explanation to the insufficient regulatory effects of ecGal-1 in SLE. However, altered binding of ecGal-1 to T-cells due to changes in surface glycosylation may also regulate the apoptotic activity of ecGal-1. Objectives We have herein hypothesized that the cell-surface glycosylation pattern, and consequently, lectin-binding ability in SLE T-cells is altered, and that an abnormal expression of glycosylation enzymes may account for these changes. Methods In order to analyse the glycosylation pattern of cell surface glycoproteins, lectin-binding assays were performed using 5 different plant lectins and human recombinant Gal-1 on resting and activated T-cells from patients with active SLE (n=8) with multi-colour flow-cytometry, and were compared with 15 healthy controls. mRNA levels of 13 glycosylation enzymes involved in the development of N-glycan structures on T-cells were measured with qPCR, and were correlated with the specific lectin binding data. Results As compared with the resting state, the increase in Gal-1 binding during activation was significantly lower in SLE T-cells than in controls, and the level of Gal-1 binding maximum was significantly reduced in SLE activated T-cells than in controls. Binding maximum of plant lectins that recognise high complexity N-glycans also increased less in SLE T-cells than in controls during activation. mRNA level of sialyltransferase ST3GAL6 was increased and neuraminidase Neu1 was decreased in active SLE patients as compared to controls. The ST6GAL1/NEU1 ratio in SLE patients positively correlated with the SLEDAI disease activity index. Conclusions SLE T-cells show decreased complexity of N-glycan structures. Increased ST3GAL6 and decreased Neu1 expression result in an increased density of terminal sialic acids, and this may explain the impaired Gal-1 binding. In addition to the previously described deficiency in icGal-1 expression upon activation, our present findings of an attenuated glycan complexity and a shift toward terminal sialylation provide a further mechanism of pathological T-cell activation and regulation of T-cell viability in SLE. References Deák M, Hornung Ά, Novák J, Demydenko D, Szabό E, Czibula Ά, Fajka-Boja R, Kriston-Pál Έ, Monostori Έ, Kovács L. Novel role for galectin-1 in T-cells under physiological and pathological conditions. Immunobiology. 2015 Apr;220(4):483–9. Disclosure of Interest None declared

Mesenchymal stem or stromal cells (MSCs) act on different components of the immune response including macrophages (MΦs). Therefore this study has been committed to explore how MSCs may modify the effect of MΦ polarization upon an inductive environment using mouse bone marrow (BM)-derived "naïve", unpolarized MΦs. Phagocytosis of various MΦ subtypes was different since M1 and M2b showed poorer, while M2a higher rate of phagocytosis. MSCs significantly promoted yeast ingestion by M1 and M2b and diminished it by M2a cells. Under polarizing conditions, MSCs profoundly affected the TNFα production of MΦ subtypes since M1 and M2b MΦs produced less and M2a produced higher amount of TNFα while the amount of IL-10 was not affected. The most striking effect of MSCs was registered on M2b cells since the inflammatory TNFα dominance remarkably shifted to the immunosuppressive IL-10. Prepolarized M1 cells readily converted to M2a and M2b states when polarizing conditions changed from M1 to M2a or M2b induction, respectively. Repolarizing from M1 to M2a resulted in the decline of IL-10 and TNFα and defined elevation of Ym1 similar to levels characteristic to M2a primarily polarized from naïve BM-MΦs. Similarly, polarization of M1 to M2b MΦs was successful showing increase in IL-10 and reduction in TNFα levels characteristic to M2b cells. However, when co-culturing with MSCs, M1-M2a or M1-M2b transition was not affected. Crosstalk between MΦs and MSCs depended on PGE-2 since COX-2 inhibition reduced the effect of MSCs to establish an IL-10-dominant cytokine production by MΦs.

BACKGROUND AIMS:Mesenchymal stromal cells (MSCs) have powerful immunosuppressive activity. This function of MSCs is attributed to plethora of the expressed immunosuppressive factors, such as galectin-1 (Gal-1), a pleiotropic lectin with robust anti-inflammatory effect. Nevertheless, whether Gal-1 renders or contributes to the immunosuppressive effect of MSCs has not been clearly established. Therefore, this question was the focus of a complex study.METHODS:MSCs were isolated from bone marrows of wild-type and Gal-1 knockout mice and their in vitro anti-proliferative and apoptosis-inducing effects on activated T cells were examined. The in vivo immunosuppressive activity was tested in murine models of type I diabetes and delayed-type hypersensitivity.RESULTS:Both Gal-1-expressing and -deficient MSCs inhibited T-cell proliferation. Inhibition of T-cell proliferation by MSCs was mediated by nitric oxide but not PD-L1 or Gal-1. In contrast, MSC-derived Gal-1 triggered apoptosis in activated T cells that were directly coupled to MSCs, representing a low proportion of the T-cell population. Furthermore, absence of Gal-1 in MSCs did not affect their in vivo immunosuppressive effect.CONCLUSIONS:These results serve as evidence that Gal-1 does not play a role in the systemic immunosuppressive effect of MSCs. However, a local contribution of Gal-1 to modulation of T-cell response by direct cell-to-cell interaction cannot be excluded. Notably, this study serves a good model to understand how the specificity of a pleiotropic protein depends on the type and localization of the producing effector cell and its target.

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein–ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Background The increasing number of patients treated with biologic therapies, the extending duration and costs of these treatments necessitate the development of strategies to the tapering or discontinuation of biologics in rheumatoid arthritis (RA) patients with long-standing remission on these agents. As flares occur frequently in patients in whom biologics are stopped, the identification of predictive markers for the preservation of long-standing remission after discontinuation is an important need. Several microRNA-s (miRNA) and SuPar (soluble urokinase plasminogen activator receptor) have been found to correlate with disease activity, and to be sensitive biomarkers of treatment response. Objectives The authors' aim was to prospectively follow the disease course after the permanent cessation of biologics, and to identify clinical and biochemical predictors of sustained biologic-free remission. Methods In this prospective, multicentre, controlled trial, eligible RA patients were treated with anti-TNF or IL6R-blocker, had a minimum of one-year remission (DAS-28<2.6), and no corticosteroid therapy. After discontinuation of the biologics, DAS28, HAQ, ESR and CRP were recorded at baseline, month 1, 3 and every 3 months thereafter. SuPar and soluble miRNA146a, miRNA16, miRNA155, miRNA223 and miRNA346 levels were measured by ELISA or QRT-PCR, respectively at baseline, month 1, 3, 6 and at relapse, if occurred. In the control group (n=5), patients had sustained remission on biologics, and had similar demographic and serological parameters, but the biologic therapy was not stopped. Results At present 23 patients with RA have been enrolled (average age: 59,95/35–79/ years).The mean duration of biologic therapy was 54,04 (25–84) months, and was started an average of 62,3 months (1–240 months) after the diagnosis. After the discontinuation, 14 patients are in biologic-free remission for an average of 5,21 (1–16 months). Relapse occurred in 9 cases an average of 5,20 (2–12) months after the discontinuation. miRNA146a levels at stop were higher in relapsers than in those with sustained remission (relative expression to cel miR39: -3,08 vs -7,87, p=0.009) (n=8 stopped patients, n=1 control). Circulating miRNA223, miRNA16, miRNA155 and miRNA346 levels were comparable in the two drug-cessation groups. SuPar levels were not different in the 12 patients in remission and 7 relapsers, in whom the measurements have been performed so far. Conclusions In RA patients with long-term remission on anti-TNF or IL6R-blocker, elevated level of miRNA146a may indicate a higher risk of relapse after stopping biologics. Further follow-up and the involvement of more patients are currently underway, and this may identify further predictive markers. References Toldi G, Kádár G et al. Soluble urokinase plasminogen activator receptor (suPAR) in the asessment of inflammatory activity of rheumatoid arthritis in patients in remission. ClinChem Lab Med 51(2):327–332. 2013 Disclosure of Interest None declared

Apoptotic cell death is an integral part of development and cell turnover in multicellular organisms. Since early 1970’s, when apoptosis was defined on morphological basis, plethora of genes has been identified participating in initiation, execution and regulation of cell death. This article reviews these latest advances and describes our present understanding of the sequential events of apoptotic cell death, from the early steps of death receptor initiated and mitochondrial pathways to activation of caspases, and finally, the proper corpse clearance. It also discusses dysregulation of apoptosis, leading to various pathologies, such as cancer, autoimmune disease and neurodegenerative disorders. Acta Biol Szeged 59(Suppl.1):143-156 (2015) Key woRdS apoptosis caspases death receptors mitochondrial pathway programmed cell death Submitted March 8, 2015; Accepted June 5, 2015 *Corresponding author. E-mail: fajka_boja.roberta@brc.mta.hu