Gliko-immunológiai vizsgálatok autoimmun folyamatok kialakulásának felderítésére = Glycoimmunological studies in search of the mechanism of autoimmune processes

Munkánk során az autoimmunitás glikoimmunológiai vonatkozásait vizsgáltuk. Igazoltuk, hogy glükózaminoglikánnal reagáló természetes autoantitestek igen nagy mennyiségben vannak jelen a normál felnőtt szérumban, és szintjük rheumatoid arthritisben szignifikánsan emelkedett. A kondroitin szulfát C ellenes IgM természetes autoantitestet mint a rheumatoid arthritis új stádium markerét azonosítottuk. Igazoltuk, hogy az ízületekre jellemző legjelentősebb glikozidáz enzim a hexózaminidáz, melynek fő forrása a synoviális fibroblaszt mind rheumatoid arthritisben, mint osteoarthritisben. Ugyanezen sejtek csak igen kis mértékben járulnak hozzá az ízületekben a béta glükuronidáz termeléshez. A Toll like receptor ligand oligoszacharid oldalláncokat létrehozni képes endoglikozidáz enzimek ízületen belüli expressziója rendkívül alacsony, míg az ismeretlen funkciójú Hc gp 39 (CHI3L1) kiemelkedően magas ízületi expressziót mutat. Adatbázis elemzéssel kimutattuk, a kísérletesen igazolt T sejtek epitópok glikozilációja rendkívül alacsony a normál fehérjékéhez képest, az autoantigén jelleg összefügg a molekula N-glikozilációjával, valamint, hogy a bakteriális-humán megegyező szekvenciák N glikozilációja meglepően alacsony. Összefüggést mutattunk ki a glükuronidázt kódoló KLOTHO gén egy SNP-je, a lizoszomális GusB gén két SNP-jének együttes előfordulása, valamint a rheumatoid arthritis között. | In our work we investigated glycoimmunological aspects of autoimmunity. We have shown that natural autoantibodies reactive with glycosaminoglycans are present in large amounts in healthy adult serum, and the level is significantly elevated in rheumatoid arthritis. We identified the chondroitin sulphate C-specific IgM antibody as a novel disease stage marker in rheumatoid arthritis. We found that the major glycosidase enzyme in the joint is hexosaminidase, the primary source of which are synovial fibroblasts both in rheumatoid arthritis and osteoarthritis. The same cells are characterized by very small contribution to the beta-glucuronidase production of the joints. Endoglycosidases that generate TLR ligand oligosaccharides show very low expression within the joints. On the contrary, Hc gp 39 (CHIL3L1), a member of the chitinase family of proteins with unknown function shows a robust expression within the joints. Using database analysis we have found association between the autoantigenic nature of a given protein with its N-glycosylation, surprisingly low N-glycosylation of experimentally proven T cell epitopes as well as strikingly low expression of sequences shared by bacteria and human proteins. We found significant association of an SNP of the KLOTHO gene, the simultaneous presence of two GusB SNPs and rheumatoid arthritis

The emerging and diverse roles of Src-like adaptor proteins in health and disease

Although Src-like adaptor proteins (SLAP-1 and SLAP-2) were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins

Az allergia és az allergiás asztma pathomechanizmusának molekuláris biológiai és genetikai vizsgálata = Molecular biologic and genetic investigation of the pathomechanism of allergy and allergic asthma

Az asztmásokban szignifikánsan alacsonyabb volt az MCP-1 szint, mint az egészségesekben. Az asztmás csoporton belül az atópiásokban (IgE>100kU/l) szignifikánsan alacsonyabb volt az MCP-1 szint, mint a nem-atópiásokban. Az asztmások között szignifikánsan több olyan gyermeket találtunk, akik hordoztak legalább egy MBL mutációt és pozitívak voltak Chlamydophila pneumoniae (CP) ellenes IgG-re, mint a kontroll csoportban. Az asztmás csoporton belül szignifikánsan több allergiás-asztmás beteg volt pozitív CP-specifikus IgA-ra és IgA+IgG-re, mint nem-allergiás asztmás. A TNF? -308A allélja jelentősen megnöveli annak az esélyét, hogy a CP fertőzött gyermekekben asztma alakuljon ki. Az asztmás gyerekek között szignifikánsan magasabb volt a krónikus Mycoplasma pneumoniae (MP) fertőzöttek aránya (31.1% az asztmás gyermekekben és 18.1% az egészséges kontrollokban). A CCR5?32 allélfrekvenciája szignifikánsan magasabb volt a krónikus MP fertőzöttek között, mint a nem fertőzöttekben, illetve nem-krónikus fertőzöttekben. Ezzel szemben a krónikusan fertőzött asztmásokban alacsonyabb volt a CCR5?32 allélfrekvenciája, mint a krónikusan fertőzött kontrollokban. Az asztma biobankunkban 425 beteg DNS-ét és adatait gyűjtöttük össze. Emellett, 404 allergiás, de nem asztmás, illetve több mint 500 egészséges kontroll DNS-e, illetve adatai állnak rendelkezésünkre. Elkezdtük az asztma parciális genomszűrését a 11q13 genomrégióban 67 SNP genotipizálásával. | The serum levels of the MCP-1 protein were significantly lower in asthmatic children than in healthy controls. Furthermore, within the asthmatic group, the serum levels of MCP-1 were significantly lower in children with atopic phenotypes than with nonatopic phenotypes. Among asthmatic children carrying variant MBL alleles there were significantly more patients positive for C pneumoniae (CP)-specific IgG, than among control children with variant MBL genotypes. Significantly more allergic asthmatic patients were positive for (CP)-specific IgA and IgA+IgG than nonallergic asthmatic patients. Among asthmatic children carrying the TNF? -308A allele, there were significantly more patients positive for (CP)-specific IgG, than among control children carrying the same allele. In the asthmatic group significantly more children were positive for Mycoplasma pneumoniae (MP) -specific antibodies, than in the control group (31.1% vs. 18.1%). The allelic frequency of the CCR5?32 was significantly higher among MP-seropositive and than among MP-seronegative children. Significantly less MP-seropositive asthmatic than MP-seropositive control carries the CCR5?32 allele. We collected genomic DNA and clinical data from 425 children with asthma, 304 allergic, but without asthma and more than 500 healthy controls. We started the partial genome screening of asthma with genotyping 67 SNPs in chromosome 11q13

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4 degrees C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4 degrees C, or UC performed at 37 degrees C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield

Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100nm and 100-1000nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, render EVs ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3x5-5min global ischemia and reperfusion (IPC) or 30min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30min global ischemia and 120min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9+/-1,6% vs. 25.0+/-2.7%), similarly to cardioprotection afforded by IPC (7.3+/-2.7% vs. 22.1+/-2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0+/-2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection

Extracelluláris vezikulák és hematológiai malignitásokban játszott szerepük

Absztrakt Extracelluláris vesiculák minden szervezetben képződnek. Három legintenzívebben vizsgált csoportjuk az apoptotikus testek, a microvesiculák és az exosomák. A sejtek közötti kommunikációban, immunreakciókban, angiogenezisben betöltött szerepük csak néhány az eddig megismertek közül. A fiziológiás folyamatok mellett sokféle betegségben leírták változásaikat; a patomechanizmusban betöltött szerepük mellett felvetődik potenciális használatuk biomarkerekként. A szerzők betekintést kívánnak nyújtani az extracelluláris vesiculák kutatásába, kiemelve azt a néhány tanulmányt, amely a hematológiai malignitásokra fókuszált. A microvesiculák és exosomák vérplazmában mért mennyisége, a terápia során megfigyelt minőségi változása miatt felmerült, hogy a diagnosztikában, prognosztikában, illetve a minimális residualis betegség monitorozásában is használhatók lehetnek. Akut myeloid leukaemiában a természetes ölősejtek aktivitásának szupresszálásában bizonyított a blasteredetű exosomák szerepe. Krónikus lymphoid leukaemiában a microvesiculák közreműködése valószínű a gyógyszer-rezisztencia kialakulásában is. Orv. Hetil., 2016, 157(35), 1379–1384. | Abstract Extracellular vesicles are produced in all organisms. The most intensively investigated categories of extracellular vesicles include apoptotic bodies, microvesicles and exosomes. Among a very wide range of areas, their role has been confirmed in intercellular communication, immune response and angiogenesis (in both physiological and pathological conditions). Their alterations suggest the potential use of them as biomarkers. In this paper the authors give an insight into the research of extracellular vesicles in general, and then focus on published findings in hematological malignancies. Quantitative and qualitative changes of microvesicles and exosomes may have value in diagnostics, prognostics and minimal residual disease monitoring of hematological malignancies. The function of extracellular vesicles in downregulation of natural killer cells’ activity has been demonstrated in acute myeloid leukemia. In chronic lymphocytic leukemia, microvesicles seem to play a role in drug resistance. Orv. Hetil., 2016, 157(35), 1379–1384

Allergia genomika: ismert és teljes genomszűréssel kiemelt asthmára hajlamosító gének vizsgálata emberben és transzgenikus állatmodellen valamint európai összevetésre alkalmas hazai asthma biobank létrehozása = Allergy genomics: Studies on known and other asthma susceptibility genes found by whole genome search in human and transgenic animal models; generation of Hungarian asthma biobank for European comparative studies

A munka során egérmodellen és emberi mintákon allergiagenomikai (ezen belül asztmagenomikai) kutatást végeztek. Ennek során: 1. az allergiás immunválaszban szerepelő egyes molekulák génjeiben polimorfizmusokat (SNP) találtak, melyek szorosan kapcsoltak a betegséggel; 2. microarray (chip) génexpressziós analízissel az allergiás folyamatban kulcsszerepet játszó hízósejtekben eddig nem ismert géneket azonosítottak. Figyelmük elsősorban a hízósejt proteázaira irányították, ezek funkciója ugyanis kitekintést ad a hízósejt-tumor kölcsönhatásra is; 4. Létrehozták a hazai asztma biobankot és bekapcsolódtak a nemzetközi biobank hálózatba; 5. Kialakítottak egy egér asztmamodellt, amelyen részben igazolták a hisztamin szerepét az asztmában, másrészt új géneket azonosítottak az asztma patomechanizmusában; 6. Az állatkisérletek alapján az emberi genomban funkcionálisan jellemezték homológ géneket; 7. új RNS interferenciára (siRNS) alapuló asztma génterápia beállítását kezdték el. | In our work we carried out allergy -genomics (asthma-genomics) studies on murine model and human samples. The results include: 1. Finding genetic polymorphisms (SNP) related to the disease in the genes of molecules involved in allergic immune response; 2. Applying microarray (chip) gene expression analysis new (formerly unknown) important genes were identified. The attention has been focused on mast cell proteases, related to mast cell tumour interactions; 4. A Hungarian asthma biobank has been established and connected into the international biobanking network; 5. A murine asthma model has been generated, serving for examining the role of histamine in asthma and identification of novel genes in pathomechanism of asthma; 6. Based on animal models, homologous genes were characterized in human genome; a new gene therapy based on siRNS technology has been started

Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles

The past decade has witnessed an exponential development in the field of extracellular vesicles. Sporadic observations have reached a critical level and the scientific community increasingly recognizes the potential biomedical significance of these subcellular structures present in all body fluids as significant components of the cellular secretome. The Educational Committee of the International Society for Extracellular Vesicles prepared two posters ("Basic aspects of extracellular vesicles" and "Clinical aspects of extracellular vesicles") to provide essential pieces of information on extracellular vesicles at glance for anyone not familiar with the field

Extracellular Vesicle-Based Communication May Contribute to the Co-Evolution of Cancer Stem Cells and Cancer-Associated Fibroblasts in Anti-Cancer Therapy

Analogously to the natural selective forces in ecosystems, therapies impose selective pressure on cancer cells within tumors. Some tumor cells can adapt to this stress and are able to form resistant subpopulations, parallel with enrichment of cancer stem cell properties in the residual tumor masses. However, these therapy-resistant cells are unlikely to be sufficient for the fast tumor repopulation and regrowth by themselves. The dynamic and coordinated plasticity of residual tumor cells is essential both for the conversion of their regulatory network and for the stromal microenvironment to produce cancer supporting signals. In this nursing tissue “niche”, cancer-associated fibroblasts are known to play crucial roles in developing therapy resistance and survival of residual stem-like cells. As paracrine messengers, extracellular vesicles carrying a wide range of signaling molecules with oncogenic potential, can support the escape of some tumor cells from their deadly fate. Here, we briefly overview how extracellular vesicle signaling between fibroblasts and cancer cells including cancer progenitor/stem cells may contribute to the progression, therapy resistance and recurrence of malignant tumors