Comparative study of physiological and pathological autoimmune models

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Proteoglycan Aggrecan Conducting T Cell Activation and Apoptosis in a Murine Model of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease and its targeting of the joints indicates the presence of a candidate autoantigen(s) in synovial joints. Patients with RA show immune responses in their peripheral blood to proteoglycan (PG) aggrecan. One of the most relevant animal models of RA appears to be proteoglycan-induced arthritis (PGIA), and CD4+ T cells seem to play a crucial role in the initiation of the disease. In this review, the role of various T cell epitopes of aggrecan in the induction of autoreactive T cell activation and arthritis is discussed. We pay special attention to two critically important arthritogenic epitopes, 5/4E8 and P135H, found in the G1 and G3 domains of PG aggrecan, respectively, in the induction of autoimmune arthritis. Finally, results obtained with the recently developed PG-specific TCR transgenic mice system showed that altered T cell apoptosis, the balance of activation, and apoptosis of autoreactive T cells are critical factors in the development of autoimmunity

T sejt rekonstitúciós vizsgálatok ZAP-70 deficiens egerekben

A ZAP-70 kináz (70kDa-os zéta lánc aszociált kináz) központi szerepet tölt be a T sejtek antigén receptoron keresztüli aktivációjának jeltovábbításában. A molekula fontosságát jól jelzi, hogy hiányában számos jelátviteli folyamat gátolt, illetve egérben és emberben is súlyos T sejtes immundeficiencia alakul ki. Ez utóbbi oka, hogy a ZAP-70 kináz a T sejt differenciáció során is kritikus szerepet tölt be, hiányában a T sejtek fejlődése blokkolódik a thymusban a kettős pozitív (CD4+CD8+) stádiumban, aminek következtében a perifériás nyirokszervekben nem találhatunk érett T sejteket. Munkánk során ZAP-70 deficiens egerekben tanulmányoztuk a T sejt hiány helyreállítás lehetőségeit. Ehhez adoptív transzfer vizsgálatokat hajtottunk végre, melyek során a ZAP-70-/- egereket vad típusú (azaz ZAP-70-et expresszáló) tetvéreik csontvelő illetve thymus sejtjeivel rekonstituáltuk intrahepatikus ill. intraperitonealis utakon. Eredményeink szerint mindkét transzfer technika hatékonynak bizonyult a T sejtek helyreállítátban. A sejtek transzferét követően 2 hetenként vérvétellel igazoltuk a T sejtek megjelenését a vérben. Azon állatok túlélése, melyekben T sejtek jelentek meg szignifikánsan meghaladta, az immundeficiens egerekét. A kísérletek végén (a transzfert követő végzett áramlási citometriás vizsgálatok illetve immunhisztokémia is azt igazolta, hogy T sejtek jelentek meg a transzferált egerek lépében, nyirokcsomóiban illetve a bélhez aszociált nyirokszövetekben. Továbbá a thymus sejt összetételének vizsgálata során megállapítottuk, hogy szignifikánsan emelkedett az érett CD4- vagy CD8 egyszeresen pozitív sejtek aránya, amely a T sejt érés normalizálódását jelezte. Sikerült kétféle transzfer módszerrel stabil kimérizmust létrehoznunk ZAP-70 hiányos egerekben. Bizonyítottuk, hogy mind a thymus mind a csontvelői eredetű sejtek képesek voltak a T sejt fejlődés helyreállítására

T CELL RECONSTITUTION STUDIES IN ZAP-70 DEFICIENT MICE

The ZAP-70 kinase (70kDa Zeta-Chain Associated Protein) plays a central role in signal transduction through the antigen receptor during T cell activation. The importance of the molecule is clearly demostrated when it is absent: several signaling pathways are inhibited, and severe T-cell immunodeficiency appears both in humans and mice. The reason of the latter is that ZAP-70 is indispensable in T cell differentiation: in its absence the maturation of T cells in the thymus is blocked in the double positive (CD4+CD8+) stage, and, as a consequence no mature T-cells can be found in the peripheral lymphoid organs. In our work we studied the possibilities of T cell reconstitution in ZAP-70 deficient mice. We performed adoptive transfer experiments, where ZAP-70-/- mice were reconstituted with bone marrow or thymus cells from their wild type (ZAP-70 expressing) siblings intrahepatically or intraperitoneally. According to our results both transfer techniques were effective in restoring T cells. After the cell transfers, blood was taken every 2 weeks to detect the presence of T cells in the blood. The survival of those animals which had T cells reconstituted exceeded significantly those which were immunodeficient. Both flow cytometric measurements and immunohistochemistrical staining performed after the experiments (following the transfers) proved that T cells appeared in the spleen, lymph nodes and gut associated lymphoid tissues of the animals. Furthermore, during the investigation of cell constitution of the thymus, we have found that the ratio of CD4+ or CD8+ single positive cells increased significantly, which indicated the normalisation of T-cell maturation. Thus, we managed to establish stable chimerism in ZAP-70 deficient mice with two methods. We proved that both thymus and bone marrow originated cells were able to restore the development of T-cells. This work was supported by the OTKA-K101493 research grant to Ferenc Boldizsár. Ferenc Boldizsár recieves Bolyai János Research Scholarship from the Hungarian Academy of Sciences

THE EFFECT OF ZAP-70 DEFICIENCY UPON THE HISTOLOGICAL COMPOSITION OF PRIMARY- AND SECONDARY LYMPHOID ORGANS IN MICE

The ZAP-70 kinase (70kDa Zeta-Chain Associated Protein) plays a critical role in both T cell signalling and maturation. In its absence, the development of thymocytes is blocked in the double positive (CD4+CD8+) stage, thus no mature T cells can be found in the peripheral lymphoid organs, which results in severe combined immunodeficiency (SCID) in both humans and mice. In our work we wanted to investigate the effect of ZAP-70 deficiency upon the lymphoid structures of the thymi, spleens and lymph nodes of wild type, heterozygous and homozygous ZAP-70 knockout mice using immunohistochemistry and flow cytometry. During the investigation of thymic structure we have observed that no single positive (CD4+ or CD8+) thymocytes are present in samples from homozygous knockout animals. This change was accompanied with the disappearance of mature medullary epithelial cells. According to our results heterozygous knockout animals do have single positive thymocytes and medullary regions, but in diminished number and size, respectively. In spleens and lymph nodes of homozygous knockout animals the T cell zones completely disappeared, which was coupled with an increased size of B cell zones. In heterozygous knockout animals similar, but not so fundamental changes were observable. Some scattered CD3 positivity was detectable in ZAP-70-/- animals’ lymph nodes, which were identified as γδ T cells. All of our immunohistochemical results were supported by quantitative, flow cytometric measurements. Our results support the theory that there is a mutual cross-talk between the stromal- and lymphoid cells in the thymus during T cell development. ZAP-70 deficiency inhibits the formation of T cell zones in the peripheral lymphoid organs, however it has less effect on the development of γδ T cells. This work was supported by the OTKA-K101493 research grant to Ferenc Boldizsár. Ferenc Boldizsár recieves Bolyai János Research Scholarship from the Hungarian Academy of Sciences

Analysis of partial ZAP-70 deficiency in a murine model of rheumatoid arthritis

Recombinant human G1 (rhG1) induced arthritis (GIA) model resembles human rheumatoid arthritis (RA) both in immunological characteristics and clinical parameters. Immunization of BALB/c mice with rhG1 domain of human proteoglycan aggrecan induces arthritis. T cells are involved in the pathogenesis of arthritis; their activation is regulated by ZAP-70, a key molecule in T cell receptor signaling. The aim of our study was to assess the effect of partial ZAP-70 deficiency on autoimmune arthritis in the GIA model. Wild-type BALB/c (WT) and ZAP-70 heterozygous knockout (ZAP-70+/-) mice were immunized with rhG1 side-by-side. Surprisingly, partial ZAP-70 deficiency did not inhibit the development of GIA; moreover, the arthritis was more severe in ZAP-70+/- mice than in WT as assessed by the physical scoring system. Luminescence imaging confirmed the increased inflammatory activity in affected limbs of ZAP-70+/- mice compared to WT animals. There was a clear correlation between the results of the functional test (hanging time measurements) and the clinical scores. Alterations in the physical performance preceded the clinical onset of arthritis. Investigation of the T cell mediated immune response indicated decreased T cell proliferation and IL-4,-6 production accompanied by significant IL-17, IFNγ and TNFα production measured from in vitro splenocyte cultures. Contrary to our expectations partial deficiency of ZAP-70 did not ameliorate the severity of arthritis in GIA model, which may be due to alterations in T cell activation and apoptosis. This work was funded by OTKA, K101493. and Bolyai Janos Reseach Scholarship of the Hungarian Academy of Sciences to FB

A ZAP-70 kináz részleges hiányának vizsgálata rheumatoid arthritis egér modellben

A rekombináns humán G1 (rhG1) domén indukált arthritis (GIA) egér modell sok tekintetben hasonlít a humán rheumatoid arthritishez (RA) főbb klinikai tüneteit és immunológiai paramétereit tekintve. Az autoimmun arthritis BALB/c egerekben humán proteoglikán aggrekán rekombináns G1 doménjével történő immunizálással váltható ki. Ismert, hogy a T-sejtek fontos szerepet töltenek be az arthritis indukciójában; aktivációjuk szabályozásában a T-sejt receptor jelátvitel egyik különösen fontos molekulája a ZAP-70 tirozin kináz is szerepet játszik. Munkánk során azt vizsgáltuk, hogy a ZAP-70 részleges hiánya milyen hatással van az autoimmun arthritis kialakulására a GIA modellben. Vad típusú BALB/c (WT) és ZAP-70 heterozigóta knockout (ZAP-70+/-) egereket párhuzamosan háromhetente immunizáltunk rhG1-el, összesen három alkalommal. A betegség klinikai tüneteit egy 4-es pontrendszer segítségével értékeltük, a gyulladást in vivo lumineszcens képalkotó vizsgálattal jelemeztük, a végtagfunkció mérésére pedig kapaszkodási tesztet végeztünk. A T sejt közvetített immunválasz jellemzésére in vitro lépsejt kultúrákban proliferációs tesztet végeztünk és citokin termelést mértünk. A részleges ZAP-70 hiány érdekes módon nem gátolta a GIA kialakulását, sőt várakozásainkkal ellentétben a pontrendszer és a kapaszkodási idők alapján ZAP-70+/- egerekben WT egerekhez képest kissé súlyosabb arthritis alakult ki. Egyértelmű összefüggést találtunk a pontszám és a funkcionális teszt eredményei között, valamint megfigyeltük, hogy a fizikai teljesítményben jelentkező változások (pl. csökkent kapaszkodási idő) megelőzték az izületi gyulladás megjelenését. A lumineszcens képalkotó vizsgálatok szintén megnövekedett gyulladásos aktivitást mutattak ki a ZAP-70+/- egerek érintett végtagjaiban a WT állatokéhoz képest. A ZAP-70+/- egerek in vitro tesztekben csökkent T sejt proliferációt és IL-4,-6 termelést mutattak, szignifikáns IL-17, IFNγ és TNFα termeléssel. Előzetes várakozásainkkal ellentétben a T-sejt jelátvitelben kiemelkedő fontosságú ZAP-70 molekula részleges hiánya nem csökkentette az autoimmun arthritis súlyosságát GIA egérmodellben. Ebben feltételezhetően a megváltozott T sejt aktiváció, differenciáció és apoptózis játszhat szerepet. Munkánkat az OTKA-K101493. sz pályázata támogatta. Boldizsár Ferenc MTA, Bolyai János Kutatói Ösztöndíjban részesül

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1β, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation

Investigation of the RF Sputtering Process and the Properties of Deposited Silicon Oxynitride Layers Under Varying Reactive Gas Conditions

In a single process run, an amorphous silicon oxynitride layer was grown, which includes the entire transition from oxide to nitride. The variation of the optical properties and the thickness of the layer was characterized by Spectroscopic Ellipsometry (SE) measurements, while the elemental composition was investigated by Energy Dispersive Spectroscopy (EDS). It was revealed that the refractive index of the layer at 632.8 nm is tunable in the 1.48–1.89 range by varying the oxygen partial pressure in the chamber. From the data of the composition of the layer, the typical physical parameters of the process were determined by applying the Berg model valid for reactive sputtering. In our modelling, a new approach was introduced, where the metallic Si target sputtered with a uniform nitrogen and variable oxygen gas flow was considered as an oxygen gas-sputtered SiN target. The layer growth method used in the present work and the revealed correlations between sputtering parameters, layer composition and refractive index, enable both the achievement of the desired optical properties of silicon oxynitride layers and the production of thin films with gradient refractive index for technology applications