Protein glycosylation in bacterial and viral infections / Glikozylacja bialek w infekcjach bakteryjnych i wirusowych.

Glycosylated proteins play a key role in the various stages of bacterial and viral invasions. Glycosylation is a common process across all domains of life. Initially, this process was attributed only to eukaryotic organisms, in which the synthesis takes place in the rough endoplasmic reticulum and the Golgi apparatus. Over time, it has been shown that many bacteria and viruses express N-glycans and O-glycans on their surface. Prokaryotes are able to synthesize glycans, while virions take over the host's cellular machinery to produce glycans. Pathogens use glycoproteins to regulate adhesion to infected cells (Ebola virus), protect receptor-binding epitopes (HIV) and evade the immune system detection by molecular mimicry (Helicobacter pylori, Haemophilus influenzae). Successful infection also depends on the host surface glycans, mainly in determining the tissue tropism of viruses (Influenza A viruses) and the sliding motility of bacteria (Mycoplasma sp.). Modification of glycan structures, important at various levels of the infectious cycle, creates new therapeutic possibilities that gives a chance to limit the spread of infectious diseases.

The role of N-glycosylation in B-cell biology and IgG activity. The aspects of autoimmunity and anti-inflammatory therapy.

The immune system is strictly regulated by glycosylation through the addition of highly diverse and dynamically changing sugar structures (glycans) to the majority of immune cell receptors. Although knowledge in the field of glycoimmunology is still limited, numerous studies point to the key role of glycosylation in maintaining homeostasis, but also in reflecting its disruption. Changes in oligosaccharide patterns can lead to impairment of both innate and acquired immune responses, with important implications in the pathogenesis of diseases, including autoimmunity. B cells appear to be unique within the immune system, since they exhibit both innate and adaptive immune activity. B cell surface is rich in glycosylated proteins and lectins which recognise glycosylated ligands on other cells. Glycans are important in the development, selection, and maturation of B cells. Changes in sialylation and fucosylation of cell surface proteins affect B cell signal transduction through BCRs, CD22 inhibitory coreceptor and Siglec-G. Plasmocytes, as the final stage of B cell differentiation, produce and secrete immunoglobulins (Igs), of which IgGs are the most abundant N-glycosylated proteins in human serum with the conserved N-glycosylation site at Asn297. N-oligosaccharide composition of the IgG Fc region affects its secretion, structure, half-life and effector functions (ADCC, CDC). IgG N-glycosylation undergoes little change during homeostasis, and may gradually be modified with age and during ongoing inflammatory processes. Hyperactivated B lymphocytes secrete autoreactive antibodies responsible for the development of autoimmunity. The altered profile of IgG N-glycans contributes to disease progression and remission and is sensitive to the application of therapeutic substances and immunosuppressive agents. In this review, we focus on the role of N-glycans in B-cell biology and IgG activity, the rearrangement of IgG oligosaccharides in aging, autoimmunity and immunosuppressive therapy.

Cytotoxic and Antioxidant Activity of Hypericum perforatum L. Extracts against Human Melanoma Cells from Different Stages of Cancer Progression, Cultured under Normoxia and Hypoxia.

Oxidative stress and the hypoxic microenvironment play a key role in the progression of human melanoma, one of the most aggressive skin cancers. The aim of our study was to evaluate the effect of Hypericum perforatum extracts of different origins (both commercially available (HpEx2) and laboratory-prepared from wild grown (HpEx12) and in vitro cultured (HpEx13) plants) and hyperforin salt on WM115 primary and WM266-4 lymph node metastatic human melanoma cells cultured under normoxic and hypoxic conditions. The polyphenol content, radical scavenging activity, and hyperforin concentration were determined in the extracts, while cell viability, apoptosis, ROS production, and expression of NRF2 and HO-1, important oxidative stress-related factors, were analyzed after 24 h of cell stimulation with HpExs and hyperforin salt. We found that cytotoxic, pro-apoptotic and antioxidant effects depend on the extract composition, the stage of melanoma progression, and the oxygen level. Hyperforin salt showed lower activity than H. perforatum extracts. Our study for the first time showed that the anticancer activity of H. perforatum extracts differs in normoxia and hypoxia. Importantly, the composition of extracts of various origins, including in vitro cultured, resulting in their unique properties, may be important in the selection of plants for therapeutic application.

Effects of Piptoporus betulinus Ethanolic Extract on the Proliferation and Viability of Melanoma Cells and Models of Their Cell Membranes.

Piptoporus betulinus is a fungus known for its medicinal properties. It possesses antimicrobial, anti-inflammatory, and anti-cancer activity. In this study, several tests were performed to evaluate the cytotoxic effect of the ethanolic extract of Piptoporus betulinus on two melanoma human cell lines, WM115 primary and A375 metastatic cell lines, as well as Hs27 human skin fibroblasts. The extract proved to affect cancer cells in a dose-dependent manner, and at the same time showed a low cytotoxicity towards the normal cells. The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method (F-C), and the potential antioxidant activity was measured by ferric-reducing antioxidant power (FRAP) assay. One of the active compounds in the extract is betulin. It was isolated and then its cytotoxic activity was compared to the results obtained from the Piptoporus betulinus extract. To further understand the mechanism of action of the extract's anticancer activity, tests on model cell membranes were conducted. A model membrane of a melanoma cell was designed and consisted of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, disialoganglioside-GD1a and cholesterol: DMPC:GD1a:chol (5:2:3 mole ratio). Changes in a Langmuir monolayer were observed and described based on Π-Amol isotherm and compressibility modulus changes. LB lipid bilayers were deposited on a hydrophilic gold substrate and analyzed by IR and X-ray photoelectron spectroscopy. Our study provides new data on the effect of Piptoporus betulinus extract on melanoma cells and its impact on the model of melanoma plasma membranes.

Changes of IgG N-Glycosylation in Thyroid Autoimmunity: The Modulatory Effect of Methimazole in Graves' Disease and the Association With the Severity of Inflammation in Hashimoto's Thyroiditis.

The N-glycome of immunoglobulin G (IgG), the most abundant glycoprotein in human blood serum, reflects pathological conditions of autoimmunity and is sensitive to medicines applied in disease therapy. Due to the high sensitivity of N-glycosylation, the IgG N-glycan profile may serve as an indicator of an ongoing inflammatory process. The IgG structure and its effector functions are strongly dependent on the composition of N-glycans attached to the Fc fragment, and the binding of antigens is regulated by Fab sugar moieties. Because of the crucial role of N-glycans in IgG function, remodeling of its N-oligosaccharides can induce pathological changes that ultimately contribute to the development of autoimmunity; restoration of their physiological structure is critical to the reduction of disease symptoms. Our recently published data have shown that the pathology of autoimmune thyroid diseases (AITDs), including Hashimoto's thyroiditis (HT) and Graves' disease (GD), is accompanied by alterations of the composition of IgG N-glycans. The present study is a more in-depth investigation of IgG glycosylation in both AITDs, designed to determine the relationship between the severity of thyroid inflammation and IgG N-glycan structures in HT, and to assess the impact of immunosuppressive therapy on the N-glycan profile in GD patients. The study material consisted of human serum samples collected from donors with elevated anti-thyroglobulin (Tg) and/or anti-thyroperoxidase (TPO) IgGs without symptoms of hypothyroidism (n=68), HT patients characterized by high autoantibody titers and advanced destruction of the thyroid gland (n=113), GD patients with up-regulated IgG against thyroid-stimulating hormone receptor (TSHR) before (n=62) and after (n=47) stabilization of TSH level as a result of methimazole therapy (study groups), and healthy donors (control group, n=90). IgG was isolated from blood serum using protein G affinity chromatography. N-glycans were released from IgG by PNGase F digestion and analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) after 2-aminobenzamide (2-AB) labeling. UPLC-MS chromatograms were integrated into 25 peaks (GP) in the Waters UNIFI Scientific Information System, and N-glycans were assigned based on the glucose unit values and mass-to-charge ratios (m/z) of the detected ions. The Kruskal-Wallis non-parametric test was used to determine the statistical significance of the results (p<0.05). The obtained results suggest that modifications of IgG sialylation, galactosylation and core-fucosylation are associated with the severity of HT symptoms. Methimazole therapy implemented in GD patients affected the IgG N-glycan profile; as a result, the content of the sialylated and galactosylated oligosaccharides with core fucose differed after treatment. Our results suggest that N-glycosylation of IgG undergoes dynamic changes during the intensification of thyroiditis in HT, and that in GD autoimmunity it is affected significantly by immunosuppressive therapy.

Glycosylation in Autoimmune Diseases.

Autoimmune diseases are accompanied by changes in protein glycosylation, in both the immune system and target tissues. The best-studied alteration in autoimmunity is agalactosylation of immunoglobulin G (IgG), characterized primarily in rheumatoid arthritis (RA), and then detected also in systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and multiple sclerosis (MS). The rebuilding of IgG N-glycans in RA correlates with the relapses and remissions of the disease, is associated with physiological states such as pregnancy but also depends on applied anti-inflammatory therapy. In turn, a decreased core fucosylation of the whole pool of IgG N-glycans is a serum glycomarker in autoimmune thyroid diseases (AITD) encompassing Hashimoto's thyroiditis (HT) and Grave's disease (GD). However, fucosylation of anti-thyroglobulin IgG (an immunological marker of HT) was elevated in HT serum. Core fucosylation of IgG oligosaccharides was also lowered in MS and SLE. In AITD and IBD, chronic inflammation T lymphocytes showed the reduced expression of MGAT5 gene encoding ß1,6-N-acetylglucosaminyltransferase V (GnT-V) responsible for ß1,6-branching of N-glycans, which is important for T cell receptor activation. Structural changes of glycans have a profound effect on the pro-inflammatory activity of immune cells and serum immune proteins, including IgG in autoimmunity.

The Time-Course of Antioxidant Irisin Activity: Role of the Nrf2/HO-1/HMGB1 Axis.

The production of free radicals is one of the basic mechanisms giving rise to the antimicrobial activity of macrophages; however, excessive accumulation of reactive oxygen species (ROS) can lead to cell damage, cell death, and release of the highly proinflammatory alarmin high-mobility group box 1 (HMGB1). This study aimed to evaluate the kinetics of antioxidant properties of the adipomyokine irisin administered shortly before or after macrophage activation to assess its effect on the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/HMGB1 pathway. The studies were performed on RAW 264.7 mouse macrophages treated with irisin (0, 25, and 50 nM) 2 h before or after lipopolysaccharide (LPS) stimulation. The effectiveness of respiratory burst and the expression of key factors of the antioxidant pathway, such as HO-1, Nrf2, superoxide dismutase 1 (SOD-1), SOD-2, glutathione peroxidase (GPx), catalase-9 (Cat-9), and HMGB1, were assessed. Irisin (50 nM) effectively reduced the free-radical production by macrophages. Furthermore, in both models, irisin altered the kinetics of expression of key factors of the downstream Nrf2/HO-1/HMGB1 pathway, leading to the increased production of Nrf2 and HO-1 and significantly reduced expression and release of HMGB1. In conclusion, irisin is a modulator of the Nrf2/HO-1/HMGB1 pathway and shows antioxidative and anti-inflammatory effects when administered both before and shortly after the activation of inflammatory mechanisms in mouse macrophages.

Decreased Immunoglobulin G Core Fucosylation, A Player in Antibody-dependent Cell-mediated Cytotoxicity, is Associated with Autoimmune Thyroid Diseases.

Autoimmune thyroid diseases (AITD) are the most common group of autoimmune diseases, associated with lymphocyte infiltration and the production of thyroid autoantibodies, like thyroid peroxidase antibodies (TPOAb), in the thyroid gland. Immunoglobulins and cell-surface receptors are glycoproteins with distinctive glycosylation patterns that play a structural role in maintaining and modulating their functions. We investigated associations of total circulating IgG and peripheral blood mononuclear cells glycosylation with AITD and the influence of genetic background in a case-control study with several independent cohorts and over 3,000 individuals in total. The study revealed an inverse association of IgG core fucosylation with TPOAb and AITD, as well as decreased peripheral blood mononuclear cells antennary α1,2 fucosylation in AITD, but no shared genetic variance between AITD and glycosylation. These data suggest that the decreased level of IgG core fucosylation is a risk factor for AITD that promotes antibody-dependent cell-mediated cytotoxicity previously associated with TPOAb levels.

The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto's Thyroiditis: An in Vitro Model of Thyroid Autoimmunity.

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto's thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.

Altered N-glycan profile of IgG-depleted serum proteins in Hashimoto's thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is an autoimmune disease characterized by chronic inflammation of thyroid gland. Although HT is the most common cause of hypothyroidism, the pathogenesis of this disease is not fully understood. Glycosylation of serum proteins was examined in HT only to a limited extent. The study was designed to determine the glycosylation pattern of IgG-depleted sera from HT patients. METHODS: Serum N-glycans released by N-glycosidase F (PNGase F) digestion were analyzed by normal-phase high-performance liquid chromatography (NP-HPLC). N-glycan structures in each collected HPLC fraction were determined by liquid chromatography-mass spectrometry (LC-MS) and exoglycosidase digestion. Fucosylation and sialylation was also analyzed by lectin blotting. RESULTS: The results showed an increase of monosialylated tri-antennary structure (A3G3S1) and disialylated diantennary N-glycan with antennary fucose (FA2G2S2). Subsequently, we analyzed the serum N-glycan profile by lectin blotting using lectins specific for fucose and sialic acid. We found a significant decrease of Lens culinaris agglutinin (LCA) staining in HT samples, which resulted from the reduction of α1,6-linked core fucose in HT serum. We also observed an increase of Maackia amurensis II lectin (MAL-II) reaction in HT due to the elevated level of α2,3-sialylation in HT sera. CONCLUSIONS: The detected alterations of serum protein sialylation might be caused by chronic inflammation in HT. The obtained results complete our previous IgG N-glycosylation analysis in autoimmune thyroid patients and show that the altered N-glycosylation of serum proteins is characteristic for autoimmunity process in HT. General Significance Thyroid autoimmunity is accompanied by changes of serum protein sialylation.

Irisin as a Multifunctional Protein: Implications for Health and Certain Diseases.

Sedentary life style is considered to be an independent risk factor for many disorders, including development of type 2 diabetes, obesity, immune dysfunction, asthma, and neurological or coronary heart disease. Irisin is released from myocytes during physical activity, and acts as a link between muscles and other tissues and organs. This myokine is produced as a result of proteolytic cleavage of FNDC5 protein present in the membrane of myocytes. Secretion of irisin is regulated by N-linked oligosaccharides attached to the protein molecule. The two N-glycan molecules, which constitute a significant part of the irisin glycoprotein, regulate the browning of adipocytes, which is the most important function of irisin. A receptor specific for irisin has still not been discovered. In some tissues irisin probably acts via integrins, which are widely expressed transmembrane receptors. Many studies have confirmed the multifunctional role of irisin and the beneficial effects of this molecule on body homeostasis. Irisin reduces systemic inflammation, maintains the balance between resorption and bone formation, and modulates metabolic processes and the functioning of the nervous system. It suppresses the expression and release of pro-inflammatory cytokines in obese individuals and attenuates inflammation in adipose tissue. The impact of irisin on cancer cell proliferation, migration, and invasion has also been demonstrated in numerous studies, which proves its role in carcinogenesis. Owing to these pleiotropic and beneficial properties, irisin may be a potential option to prevent and treat civilization-related diseases which are, nowadays, considered to be the major health problems in Western societies.

Glycosylation of thyroid-stimulating hormone receptor.

Thyroid-stimulating hormone receptor (TSHR) is a typical membrane receptor with 7-transmembrane helix domain (7TMR), coupled to the G protein. The mature receptor, present in the cell membrane, is composed of the A subunit comprising a large extracellular domain, and the B subunit, which consists of a short extracellular fragment anchored in the cell membrane and an intracellular part. The TSH receptor is subject to numerous post-translational modifications that determine its final structure and significantly affect its activity. One of them is glycosylation. TSHR is abundantly N-glycosylated, due to the presence of six N-glycosylation sites in the extracellular domain (Asn77, Asn99, Asn113, Asn177, Asn198, Asn302), mostly evolutionarily conserved. N-glycans constitute 30-40% of the receptor molecular weight. The glycans are necessary for the receptor trafficking to the plasma membrane and binding of TSH to the receptor. Fucosylated and sialylated N-oligosaccharides were found on TSHR molecules. The increased sialylation of TSHR glycans correlates positively with the receptor binding ability and prolongs the time of receptor incorporation into the cell membrane. TSHR is the main autoantigen in Graves' disease (GD), one of the thyroid autoimmune diseases. One hypothesis assumes that the higher N-glycosylation of THSR in human compared to animals influences the breaking of autotolerance and GD development. N-oligosaccharides are the important part of THSR molecule, necessary for the proper functioning of receptors and probably involved in thyroid autoimmunity in GD.

Glycosylation in the Thyroid Gland: Vital Aspects of Glycoprotein Function in Thyrocyte Physiology and Thyroid Disorders.

The key proteins responsible for hormone synthesis in the thyroid are glycosylated. Oligosaccharides strongly affect the function of glycosylated proteins. Both thyroid-stimulating hormone (TSH) secreted by the pituitary gland and TSH receptors on the surface of thyrocytes contain N-glycans, which are crucial to their proper activity. Thyroglobulin (Tg), the protein backbone for synthesis of thyroid hormones, is a heavily N-glycosylated protein, containing 20 putative N-glycosylated sites. N-oligosaccharides play a role in Tg transport into the follicular lumen, where thyroid hormones are produced, and into thyrocytes, where hyposialylated Tg is degraded. N-glycans of the cell membrane transporters sodium/iodide symporter and pendrin are necessary for iodide transport. Some changes in glycosylation result in abnormal activity of the thyroid and alteration of the metabolic clearance rate of hormones. Alteration of glycan structures is a pathological process related to the progression of chronic diseases such as thyroid cancers and autoimmunity. Thyroid carcinogenesis is accompanied by changes in sialylation and fucosylation, ß1,6-branching of glycans, the content and structure of poly-LacNAc chains, as well as O-GlcNAcylation, while in thyroid autoimmunity the main processes affected are sialylation and fucosylation. The glycobiology of the thyroid gland is an intensively studied field of research, providing new data helpful in understanding the role of the sugar component in thyroid protein biology and disorders.

Vitamin B2 deficiency enhances the pro-inflammatory activity of adipocyte, consequences for insulin resistance and metabolic syndrome development.

AIMS: Adipose tissue is an endocrine organ important for regulation of such physiological processes as energy metabolism or lipids homeostasis. In an obesity state, it participates in the induction of chronic systemic inflammation accompanied by pro-inflammatory cytokines and fatty acid elevation. For this reasons, adipose tissue is involved in, e.g., insulin resistance, type 2 diabetes or hyperlipidemia development. In our previous study, we have shown that riboflavin deficiency induces a pathological pro-inflammatory response of macrophages, the main component of adipose tissue. Therefore, in the current study, we investigated the alteration of the pro-inflammatory activity of adipocytes. MAIN METHODS: The study was conducted on mouse 3T3 L1 preadipocytes differentiated to adipocyte and culture in the state of riboflavin deficiency (3.1nM) or control condition (10.4nM). The cell viability, adiposity and glucose uptake was assessed. Moreover, mRNA expression, as well as crucial pro-inflammatory cytokines (TNFα, IL-6) and adipokines (adiponectin, leptin, resistin) release and NFκB activation, were evaluated. KEY FINDINGS: Results showed that riboflavin deprivation induced a significant elevation in adipocyte lipolysis and enhance obesity-related apoptosis of adipocytes. The generation of reactive oxygen species was enhanced in riboflavin-deficient adipocytes by 43%. Moreover, NFκB phosphorylation and the expression and release of both TNFα, IL-6 as well as leptin were elevated in a deficient group what was accompanied by a reduction of adiponectin level. CONCLUSION: Our study shows that riboflavin deficiency can promote the intensification of pro-inflammatory activity of adipocyte cells, leading consequently to the severity of chronic inflammation that accompanies obesity state.

Anti-Inflammatory Properties of Irisin, Mediator of Physical Activity, Are Connected with TLR4/MyD88 Signaling Pathway Activation.

Irisin, an adipomiokine known as a mediator of physical activity, induces the browning of adipose tissue and it has potentially protective properties in the development of obesity-related states, such as insulin resistance, arteriosclerosis, and type 2 diabetes. Despite numerous studies conducted on this factor, still little is known about its impact on the functioning of immunocompetent cells, but its potential anti-inflammatory properties were previously suggested. In the current study we investigated the role of irisin (0-100 nM) in the downstream pathway activation of Toll-like receptor 4 (TLR4) in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS; 100 ng/mL). The results have shown that irisin in high concentrations (50, 100 nM) significantly decreased the TLR4 and MyD88 protein levels, as well as the phosphorylation of nuclear factor-κB (NF-κB), consequently leading to the reduction in the release of crucial pro-inflammatory cytokines. The above was confirmed for interleukin 1ß (IL-1ß), tumor necrosis factor α (TNFα), interleukin 6 (IL-6), keratinocyte chemoattractant (KC), monocyte chemotactic protein 1 (MCP-1), as well as for high mobility group box 1 (HMGB1). Moreover, our results indicate that this effect is connected with irisin's impact on the phosphorylation of mitogen-activated protein kinases (MAPKs), where a significant reduction in p-JNK and p-ERK but not p-p38 was observed. In conclusion, these data suggest that irisin has potentially anti-inflammatory properties connected with the downregulation of downstream pathways of TLR4/MyD88.

On the trail of the glycan codes stored in cancer-related cell adhesion proteins.

Changes in the profile of protein glycosylation are a hallmark of ongoing neoplastic transformation. A unique set of tumor-associated carbohydrate antigens expressed on the surface of malignant cells may serve as powerful diagnostic and therapeutic targets. Cell-surface proteins with altered glycosylation affect the growth, proliferation and survival of those cells, and contribute to their acquisition of the ability to migrate and invade. They may also facilitate tumor-induced immunosuppression and the formation of distant metastases. Deciphering the information encoded in these particular glycan portions of glycoconjugates may shed light on the mechanisms of cancer progression and metastasis. A majority of the related review papers have focused on overall changes in the patterns of cell-surface glycans in various cancers, without pinpointing the molecular carriers of these glycan structures. The present review highlights the ways in which particular tumor-associated glycan(s) coupled with a given membrane-bound protein influence neoplastic cell behavior during the development and progression of cancer. We focus on altered glycosylated cell-adhesion molecules belonging to the cadherin, integrin and immunoglobulin-like superfamilies, examined in the context of molecular interactions.

Riboflavin Reduces Pro-Inflammatory Activation of Adipocyte-Macrophage Co-culture. Potential Application of Vitamin B2 Enrichment for Attenuation of Insulin Resistance and Metabolic Syndrome Development.

Due to the progressive increase in the incidence of obese and overweight individuals, cardiometabolic syndrome has become a worldwide pandemic in recent years. Given the immunomodulatory properties of riboflavin, the current study was performed to investigate the potency of riboflavin in reducing obesity-related inflammation, which is the main cause of insulin resistance, diabetes mellitus 2 or arteriosclerosis. We determined whether pretreatment with a low dose of riboflavin (10.4-1000 nM) affected the pro-inflammatory activity of adipocyte-macrophage co-culture (3T3 L1-RAW 264.7) following lipopolysaccharide stimulation (LPS; 100 ng/mL) which mimics obesity-related inflammation. The apoptosis of adipocytes and macrophages as well as tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), interleukin 1beta (IL-1ß), monocyte chemotactic protein 1 (MCP-1), high-mobility group box 1 (HMGB1), transforming growth factor-beta 1 (TGFß), interleukin 10 (IL-10), inducible nitric oxide synthase (iNOS), nitric oxide (NO), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1) expression and release, macrophage migration and adipokines (adiponectin and leptin) were determined. Our results indicated an efficient reduction in pro-inflammatory factors (TNFα, IL-6, MCP-1, HMGB1) upon culture with riboflavin supplementation (500-1000 nM), accompanied by elevation in anti-inflammatory adiponectin and IL-10. Moreover, macrophage migration was reduced by the attenuation of chemotactic MCP-1 release and degradation of the extracellular matrix by MMP-9. In conclusion, riboflavin effectively inhibits the pro-inflammatory activity of adipocyte and macrophage co-cultures, and therefore we can assume that its supplementation may reduce the likelihood of conditions associated with the mild inflammation linked to obesity.

ß1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells.

Integrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of ß1,6-acetylglucosaminyltransferase V, we showed that ß1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvß3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvß3 caring overexpressed ß1,6-branched structures, important for FAK upregulation.

Gal-3 does not suppress cisplatin-induced apoptosis in A-375 melanoma cells.

Melanoma is the most aggressive of all skin cancers and is exceptionally resistant to therapies. During melanoma progression, cancer cells reprogram their proliferation and survival pathways and achieve resistance to treatment-induced apoptosis. Galectin-3 (gal-3) is a member of the lectin family and is involved in such biological processes as cell adhesion, growth and differentiation, the cell cycle, and apoptosis. Gal-3 also plays an important role in tumor development and metastasis. The relationship between gal-3 expression and these processes is specific to the tumor type and the stage of cancer progression. The biological functions of gal-3 depend on its localization in the cell. In the present study, human metastatic melanoma A-375 cells, characterized by weak endogenous expression of gal-3, were transfected with gal-3 cDNA and cisplatin-induced apoptosis was measured. Data from AnnexinV and mitochondrial membrane potential analysis revealed that gal-3 did not protect the A-375 melanoma cells against cisplatin. This result probably is associated with its nuclear localization in the cells.

HMGB1 Inhibition During Zymosan-Induced Inflammation: The Potential Therapeutic Action of Riboflavin.

Sepsis, also known as systemic inflammatory response syndrome, is a life-threatening condition caused by a pathogenic agent and leading to multiple organ dysfunction syndrome. One of the factors responsible for the excessive intensification of the inflammatory response in the course of inflammation is high-mobility group protein B1 (HMGB1). HMG-1 is a nuclear protein which, after being released to the intercellular space, has a highly pro-inflammatory effect and acts as a late mediator of lethal damage. The purpose of this study was to examine whether the anti-inflammatory action of riboflavin is accompanied by inhibition of HMGB1 release during peritoneal inflammation and zymosan stimulation of macrophages. Peritonitis was induced in male BALB/c and C57BL/6J mice via intraperitoneal injection of zymosan (40 mg/kg). RAW 264.7 macrophages were activated with zymosan (250 µg/ml). Riboflavin (mice, 50 mg/kg; RAW 264.7, 25 µg/ml) was administered 30 min before zymosan, simultaneously with, or 2, 4, 6 h after zymosan. Additionally, mRNA expression of HMGB1 and its intracellular and serum levels were evaluated. The research showed that riboflavin significantly reduces both the expression and the release of HMGB1; however, the effect of riboflavin was time-dependent. The greatest efficacy was found when riboflavin was given 30 min prior to zymosan, and also 2 and 4 h (C57BL/6J; RAW 264.7) or 4 and 6 h (BALB/c) after zymosan. Research showed that riboflavin influences the level of HMGB1 released in the course of inflammation; however, further study is necessary to determine its mechanisms of action.