Background Cell surface glycosylation serves as a binding platform for endogenous lectin families such as galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs) with well-known immunoregulatory functions. Several studies, including our own work [1], have shown that Gals play an important role in pathogenesis of systemic lupus erythematosus (SLE) in animal models but their contribution to human SLE must be better elucidated. For these lectins to act, the appropriate glycan structures should be available on the target cells and should not be masked by alpha-2,6-sialylation which prevents binding of Gal-1, but not Gal-3. Although cancerous diseases are well-characterized by an altered glycome, limited information regarding cell surface glycosylation is gathered for autoimmune diseases like SLE. Objectives In the current study, we aim to analyse the ability of certain immune subsets to bind Gal-1, Gal-3, sialic acid binding C-type lectin (Siglec-1), the fucose binding Aleuria aurantia lectin (AAL), the sialic acid binding Sambucus nigra Agglutinin (SNA) lectin in SLE patients versus healthy controls. Methods We collected peripheral blood mononuclear cells (PBMCs) from 19 new or relapsing SLE patients with active disease (mean age: 47.5, SLEDAI-2K:15.8, anti-dsDNA level: 76.4 IU/ml) with no or minimal immunosuppressive therapy and 19 healthy age-matched controls. Multicolor antibody panel was designed to identify the main peripheral immune subsets: CD4+T-cells, CD8+ T-cells, CD4-/CD8- T-cells, CD4+/CD8+ T-cells; CD4+NKT-cells, CD8+NKT-cells, CD4-/CD8-NKT-cells, CD4+/CD8+NKT-cells; CD56+ NK cells; naive B-cells, memory B-cells, plasmablasts; classical monocytes, non-classical monocytes and intermediate monocytes. The five lectins (Gal-1, Gal-3, Siglec-1, AAL, SNA) were conjugated with different fluorophores for FACS. The binding of fluorochrome labelled lectins to each of these populations was assessed simultaneously by flow cytometry directly after thawing (resting state) and following activation. In case of T-cells, activation was achieved by a polyclonal activator (CytoStim), while B-cells and monocytes were activated by LPS. Results In our experiments with control PBMCs, titration and compensation of both antibodies and lectins were carried out and gating strategies were built to identify the above-mentioned immune subsets and their lectin binding characteristics. There were remarkable differences in lectin binding among different immune populations, e.g. classical and intermediate monocytes outperformed non-classical monocytes and lymphocytes in all lectin binding. Interestingly, naive B-cells were more sialylated (Median Fluorescence Intensity(MFI)SNA:2737) and bound less Gal-1 (MFIGal-1:5730) than both memory B-cells (MFISNA:2368; MFIGal-1:16189) or plasmablasts (MFISNA:1263; MFIGal-1:16515). Similarly, highest SNA (MFISNA:144) and lowest Gal-1 (MFIGal-1:3658) binding was observed for CD4+ T-cells among the analysed T-cell subsets (MFISNA range:12-120; MFIGal-1 range: 4201-5460) or NK cells (MFISNA not detectable; MFIGal-1:14288) or NKT-cells (MFISNA range: not detectable-108; MFIGal-1 range:3580-6327). Conclusion The focus of this study is to identify relevant changes in the cell surface glycosylation of SLE immune subsets which is a unique approach and could contribute to a better understanding of the glycobiology of lupus. Reference [1]Deak M,Hornung A et al. Immunobiology. 2014;220(4):483-489 Acknowledgements: NIL. Disclosure of Interests None Declared.

For decades the burn patients have been treated at dermatological institutions worldwide, due to the similarities of the burns to certain blistering dermatological conditions. Another explanation was the routine of these clinics in the conservative treatment of wounds and general care of patients with extensive skin defects. Later on, together with more knowledge about the pathomechanism of burns and as the operative treatment became dominant, complex burn care was provided by specialized burn departments. Thanks to these developments, most burn care institutions nowadays are either autonomous centers or departments closely associated with traumatology, intensive care, surgical, pediatric surgical, or plastic surgery clinics. In Debrecen, the second profile is dermatologic surgery, which is now a unique combination, so the burn service is provided by the Department of Dermatology. Since its inauguration in 1983, the department introduced an active, operational burns treatment strategy. In the past forty years it pioneered several ground breaking therapeutic approaches, such as fluidization therapy, negative pressure wound therapy (NPWT), biotechnological wound coverage with keratinocyte suspension and sheets, dermal substitution with Alloderm®, Integra®, etc. Thanks to the continuous improvement of the instrumentation of the burn ward and the specific intensive care unit, it became possible to successfully treat patients with extensive deep burn wounds as well as dermatological crisis situations requiring intensive support. The department has remarkable international connections and scientific publication activities. Despite unfavorable tendencies of the most recent years, reducing the work force of both doctors and related professionals, the burn unit in Debrecen is keen on providing continuous, up to date, high quality professional service to burn patients of our region.

Migraine is one of the most prevalent neurological disorders which is suggested to be associated with dysfunctions of the central nervous system. The purpose of the present study was to detect the altered functional connectivity architecture in the large-scale network of the whole brain in migraine without aura (MWoA). Meanwhile, the brain functional hubs which are targeted by MWoA could be identified. A new voxel-based method named functional connectivity density (FCD) mapping was applied to resting-state functional magnetic resonance imaging data of 55 female MWoA patients and 44 age-matched female healthy controls (HC). Comparing to HC, MWoA patients showed abnormal short-range FCD values in bilateral hippocampus, bilateral insula, right amygdale, right anterior cingulate cortex, bilateral putamen, bilateral caudate nucleus and the prefrontal cortex. The results suggested decreased intraregional connectivity of these pain-related brain regions in female MWoA. In addition, short-range FCD values in left prefrontal cortex, putamen and caudate nucleus were significantly negatively correlated with duration of disease in MWoA group, implying the repeated migraine attacks over time may consistently affect the resting-state functional connectivity architecture of these brain hubs. Our findings revealed the dysfunction of brain hubs in female MWoA, and suggested the left prefrontal cortex, putamen and caudate nucleus served as sensitive neuroimaging markers for reflecting the disease duration of female MWoA. This may provide us new insights into the changes in the organization of the large-scale brain network in MWoA.

Migraine is a chronic dysfunction characterized by recurrent pain, but its pathogenesis is still unclear. As a result, more and more methods have been focused on the study of migraine in recent years, including functional magnetic resonance imaging (fMRI), which is a mainstream technique for exploring the neural mechanisms of migraine. In this paper, we systematically investigated the fMRI functional connectivities (FCs) between large-scale brain networks in migraine patients from the perspective of multi-channel hierarchy, including static and dynamic FCs of group and individual levels, where the brain networks were obtained using group independent component analysis. Meanwhile, the corresponding topology properties of static and dynamic FCs networks in migraine patients were statistically compared with those in healthy controls. Furthermore, a graph metrics based method was used to detect the potential brain functional connectivity states in dynamic FCs at individual and group levels, and the corresponding topology properties and specificity of these brain functional connectivity states in migraine patients were explored compared with these in healthy controls. The results showed that the dynamic FCs and corresponding global topology properties among nine large-scale brain networks involved in this study have significant differences between migraine patients and healthy controls, while local topological properties and dynamic fluctuations were easily affected by window-widths. Moreover, the implicit dynamic functional connectivity patterns in migraine patients presented specificity and consistency under different window-widths, which suggested that the dynamic changes in FCs and topology structure between them played a key role in the brain functional activity of migraine. Therefore, it may be provided a new perspective for the clinical diagnosis of migraine.

The idea of one, unified, global internet is under threat. How much should we care, asks Douglas Heaven

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.

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

Pain interventions with no active ingredient, placebo, are sometimes effective in treating chronic pain conditions. Prior studies on the neurobiological underpinnings of placebo analgesia indicate endogenous opioid release and changes in brain responses and functional connectivity during pain anticipation and pain experience in healthy subjects. Here, we investigated placebo analgesia in healthy subjects and in interictal migraine patients (n = 9) and matched healthy controls (n = 9) using 11C-diprenoprhine Positron Emission Tomography (PET) and simultaneous functional Magnetic Resonance Imaging (fMRI). Intravenous saline injections (the placebo) led to lower pain ratings, but we did not find evidence for an altered placebo response in interictal migraine subjects as compared to healthy subjects.

Background and objectives Several recent studies highlighted the importance of special gene loci related to gout and hyperuricaemia. However, it is remarkable that only a minor fraction of hyperuricemic population produces the symptoms of gout. Discussing the pathomechnism of gouty inflammation a potential autoinflammatory explanation has emerged recently. Induced by MSU crystals, the pathologic activity of a multi-protein complex (NLRP3 inflammasome) might be responsible for the increased activation of interleukin 1-beta (IL-1β), a cytokine playing a central role in inflamation. Toll-like receptors (TLR2–4) have also been studied for the stepped-up production of pre- IL-1β, the pre form of the active metabolit. Human erythrocytes express numerous membrane proteins, several of them already identified as homologue proteins to those with specific function in other tissue, such as urate transporters. Our objective was to verify genetical variations, single nucleotid polymorphisms (SNP) leading to hyperuricemia and gout. We also analysed these gene loci coded proteins linked to elevated urate level, using a unique technique (EryTest) based on the presence of special membrane proteins on human erythrocytes. Materials and methods We studied the results of three groups. Patients with gout, control patients with hyperuricemia but with no arthritic event and patients with normal serum uric acid level. Specific monoclonal antibodies and IgG control were used for quantitative flow cytometry to determine proteins. Polymorphism specific and control primers were used for polymerase chain reaction (PCR) detecting SNPs. Results Increased level of CARD8 polymorphism was found in the gout group compared to the two control groups. Decreased ABCG2 protein expression was detected in heterozygous individuals in gouty and hyperuricemic groups compared to normouricemic controls. Conclusions We presume that depending on complex genetic and environmental factors, patients with CARD8 polymorphisms may have increased inflammatory response and those with decreased ABCG2 protein expression may have higher serum urate levels. From the clinical introduction of EryTest, a simple and rapid clinical method in the evaluation of hyperuricemia is expected. To confirm recent results of genetic variability and the clinical use of EryTest, further investigation is planned.

The major goals of this present study were 1) to further clarify which parasympathetic ganglion sends postganglionic fibers to the lower gingiva and lip that may be involved in the inflammatory processes besides the local factors; 2) to separately examine the central pathways regulating sympathetic and parasympathetic innervation; and 3) to examine the distribution of central premotor neurons on both sides. A retrogradely transported green fluorescent protein conjugated pseudorabies virus was injected into the lower gingiva and lip of intact and sympathectomized adult female rats. Some animals received virus in the adrenal medulla which receive only preganglionic sympathetic fibers to separately clarify the sympathetic nature of premotor neurons. After 72–120h of survival and perfusion, the corresponding thoracic part of the spinal cord, brainstem, hypothalamus, cervical, otic, submandibular and trigeminal ganglia were harvested. Frozen sections were investigated under a confocal microscope. Green fluorescence indicated the presence of the virus. The postganglionic sympathetic neurons related to both organs are located in the three cervical ganglia, the preganglionic neurons in the lateral horn of the spinal cord on ipsilateral side; premotor neurons were found in the ventrolateral medulla, locus ceruleus, gigantocellular and paraventricular nucleus and perifornical region in nearly the same number on both sides. The parasympathetic postganglionic neurons related to the gingiva are present in the otic and related to the lip are present in the otic and submandibular ganglia and the preganglionic neurons are in the salivatory nuclei. Third order neurons were found in the gigantocellular reticular and hypothalamic paraventricular nuclei and perifornical area.

Background and objectives Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation; however its participation in T-cell functions has remained largely elusive. To determine the function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. Methods Activated T-cells from healthy humans, Gal-1 knock-out and wild-type mice, and Gal-1 transgenic Jurkat (JGal) cells, established in our laboratory, were studied. Furthermore, T-cells were isolated from SLE patients during active disease (n = 20), in remission (n = 10), and healthy controls (n = 20). The expression and localisation of Gal-1 were examined with QPCR, Western blotting, cytofluorimetry or confocal microscopy. Apoptosis was studied in co-culture of PHA-activated T-cells with Gal-1-expressing HeLa tumour cells using fluorescent microscopy. Results Both JGaland activated T-cells produced Gal-1. The protein remained intracellularly without secretion. Wild-type mouse and Gal-1 transgenic Jurkat T-cells were significantly more susceptible to the apoptotic effect of exGal-1 than Gal-1 deficient mouse and wild type (Gal-1 non-expressing) Jurkat T-cells. Accordingly, activated T-cells from SLE patients which produced significantly less inGal-1 than the healthy control cells or T-cells from patients in remission after successful immunosuppressive therapy, responded poorly to the apoptotic signal by exGal-1 compared to those of healthy and inactive-lupus T-cells. Conclusions We have demonstrated in three independent experimental systems that the de novo expressed Gal-1 in activated T-cells remained intracellularly, confirming that the function of T-cell derived Gal-1 was not the regulation of T-cell viability via an autocrine fashion. Rather, the inGal-1 effectively regulated T-cell apoptosis triggered by environmental exGal-1. Moreover, we showed that low expression of Gal-1 in SLE T-cells resulted in the diminished down regulation of T-cell activity by exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus.