A hepatitis C virus (HCV) fertőzés pathogenesise: a genetikai és az immunológiai tényezők, valamint az oxidativ stress szerepe és a kórokozó virus sajátosságai, különös tekintettel a HCV-okozta betegségekben és a tünetmentes virushordozó állapotban = Pathogenesis of hepatitis C virus (HCV) infection: the role of the genetic and immunological factors, the oxidative stress and the viral features in the HCV-related diseases and in the symptomfree virus carriers

Genetikai, immunológiai és környezeti tényezők potenciális szerepe krónikus hepatitis C virus (HCV) infekcióban: összehasonlitó vizsgálatok aktiv HCV hepatitises betegekben és tünetmentes "egészséges" virushordozókban. 1. Sem a haemochromatosis gen mutációk, sem a CTLA4 polymorfizmusok nem befolyásolják a HCV-okozta májbetegség aktivitását, az angiotensin convertalo enzym (ACE) gen deletio azoban kedvező hatásu az anti-HCV terapia kimenetelére. 2. Az NK és B sejek felszinén levő CD81 molekula overexpressioja, valamint a regulatoros T sejtek termelte transformáló növekedési faktor (TGFbeta1), és az utóbbi által a cytotoxikus sejteken downregulált NKG2D aktiváló receptor - meghatározó szerepet játszanak a HCV infecióban kulcsfontosságu károsodott cellularis immunválaszban 3. A plasma TGFbeta1, a hyaluronsav és a procollagen-III-peptid szintek - mint a fibrogenesis markerei - kórosan emelkedettek krónikus aktiv HCV hepatitisben, de nem a tünetmentes virus-hordozókban. Az interferon + ribavirin antiviralis terapia gátolhatja a fibrogenesist, függetlenül a virológiai választól. 4. Mint additiv környezeti tényező, a SEN virus ko-infekció, gyakran előfordul HCV fertőzésben, és jelenléte negativan befolyásolja az anti-HCV terapia hatását krónikus C hepatitisben. | Genetic, immunologic and environmental factors and their potential role in the pathogenesis of chronic hepatitis C virus (HCV) infection: a comparison between active HCV hepatitis and symptomfree 'healthy' virus carriers. 1. Neither haemochromatosis gene mutations, nor CTLA4 polymorphisms influence the activity of HCV-related liver disease, however, ACE gene deletion has a favourable effect on the outcome of anti-HCV treatment in chronic hepatitis C. 2. Overexpression of CD81 molecule on NK and B cells, as well as transforming growth factor beta1 (TGFbeta1) secreted by regulatory T cells, and downregulated NKG2D activating receptor on cytotoxic effector cells, may play a privotal role in the impaired cell mediated immunity in active HCV infection 3. Plasma TGFbeta1, hyalurinic acid and procollagene-III-peptide levels - as markers of fibrogenesis - are elevated in active hepatitis C patients, but not in symptomfree carriers. Interferon + ribavirin therapy may inhibit fibrogenesis independently of virological response in chronic HCV hepatitis. 4. SEN virus co-infection, - as an additive environmetal factor - frequently occuring in HCV patients, decreases the effect of anti-HCV treatment

Effects of Photosensitization and Low-Power Helium-Neon Laser Irradiation on Liposomes and Cell Membranes

Low-power He-Ne laser irradiation causes a well-defined and energy dependent cell destruction of in vitro cultured cell lines sensitized by hematoporphyrin derivative (HPD). The mechanism of this photosensitization was studied by measuring with polarization microscopic, scanning electron microscopic, and electron-spin-resonance (ESR) spectroscopic parameters. The cell damage caused by photosensitization and laser irradiation seems to be a complex process, however the biological membranes seem to be one of the primary targets. The energy of laser light causes rotation and resonance changes of macromolecules and the water molecules, resulting in an increased structural order of the submembraneous components in the living cells, detectable microscopically. During the photosensitization process, the red (630 nm) He-Ne laser light, during a one-photon energy activation, causes excitation of hematoporphyrin molecules to their triplet state. The excitation of HPD molecules results in a multi-step, free-radical generating effect, measured by ESR spectroscopy and studied by the ultrastructural changes of membrane organization and cell shape. Similar effects could be observed on in vitro lipid-water liposome membranes

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

STUDIES ON THE RECONSTITUTION OF T CELL PRODUCTION IN ZAP-70 DEFICIENT MICE

The ZAP-70 (70 kDa Zeta-Chain Associated Protein) kinase plays a crucial role in the signal transduction by the antigen receptor during T cell activation. It is essential in T cell differentiation as well, in its absence, T cell maturation is blocked in the double positive (CD4+CD8+) stage in the thymus, leading to the complete lack of mature αβ T cells in the periphery which results in severe immunodeficiency in both human and mice. T cell maturation could be stably restored in ZAP-70 deficient mice by simple intraperitoneal (ip.) injection of thymocytes isolated from their wild type (ZAP-70 expressing) siblings. T cells appeared in the blood and lymphoid organs of the transferred mice and, importantly, the survival of the animals increased significantly, which clearly demonstrated the correction of the severe immunodeficiency. In our present work investigated the characteristics and kinetics of the T cell reconstitution by monitoring the changes in the morphology and cellular composition of the thymus and peripheral lymphoid organs. Following the thymocyte transfer of ZAP-70-/- mice the histology of the thymus and the appearance of T cells in the periphery were analyzed regularly. Flow cytometry revealed that 3 weeks after the ip. thymocyte transfer αβ T cells appeared in significant numbers in the blood, spleen and lymph nodes of the animals. This was preceded by the appearance of mature CD4+ or CD8+ cells in the thymus 2 weeks after the transfer. Using quantitative immunohistology we have found that the area of the medullary region increased after the transfer, which also indicated the normalisation of T cell maturation. In future experiments, we plan to test the in vivo effects of ZAP-70 point mutations using ZAP-70 deficient mice. We will reconstitute the ZAP-70 molecule under the control of T cell specific promoter(s) into T cell precursors of ZAP-70 deficient mice. Using various mutant forms of ZAP-70 or the normal molecule we can investigate the effects on T cell development and function. As an important preliminary step, we have cloned the T cell specific proximal- and distal Lck promoters and a CD4 minimal promoter construct. The T cell-specific activity of these promoter constructs was verified on cell lines using a lentiviral expression system. We have managed to correct the severe T cell deficiency using adoptive thymocyte transfer. According to our results the transferred T cell progenitors with normal ZAP-70 expression level can induce long-term normalisation of T cell maturation. Combined with the T cell-specific expression of ZAP-70 variants we will possess an excellent tool to study T cell differentiation and function. This work was funded by OTKA, K101493. and Bolyai Janos Reseach Scholarship to FB

A T sejt képzés helyreállításának kinetikai vizsgálata ZAP-70 deficiens egerekben.

A ZAP-70 kináz (70kDa zéta lánc aszociált kináz) központi szerepet tölt be a T sejtek antigén receptoron keresztüli aktivációjának jeltovábbításában. A ZAP-70 kináz a T sejt differenciáció során is kritikus szerepet tölt be, hiányában a T sejtek fejlődése leáll a thymusban a kettős pozitív (CD4+CD8+) stádiumban, aminek következtében a perifériás nyirokszervekben nincsenek érett αβ T sejtek, így súlyos immundeficiencia alakul ki. Előzetesen már kimutattuk, hogy ZAP-70 deficiens egerekben, vad típusú (azaz ZAP-70-et expresszáló) tetvéreikből izolált csontvelő vagy thymus sejtek adoptív transzferével stabilan helyreállítható a T sejt fejlődés. A transzferált állatok vérében ill. nyirokszerveiben T sejtek jelentek meg az egészségesekhez hasonló módon, továbbá az immundeficiencia megszűnésére utalt a szignifikánsan meghosszabodott élettartam is. Jelen vizsgálataink célja a T sejt képzés helyreállítás kinetikájának ill. korai eseményeinek pontosabb megismerése volt. Ehhez 15-20 ZAP-70-/- egér egyidejű transzferét követően rendszeresen ellenőriztük a thymus összetételét ill. a T sejtek megjelenését a periférián. Áramlási citometriás eredményeink szerint, intraperitonealis thymocyta transzfert követően 3 héttel jelentek meg szignifikáns mennyiségben az αβ T sejtek a vérben, a nyirokcsomókban illetve a lépben. Ezt valamivel megelőzve, már a transzfert követő második héten a thymusban is megjelentek CD4+ ill CD8+ érett sejtek. Kvantitatív immunhisztológia segítségével igazoltuk, hogy a thymusban megnövekedett a medulláris állomány, amely szintén a T sejt érés fokozódására utalt. Intrahepatikus csontvelő sejt transzfer után hasonlóképpen megnövekedett medulláris állomány volt detektálható. Transzfer kísérleteink segítségével sikerült a ZAP-70 deficiencia által okozott T sejt hiányos immundeficienciát korrigálnunk. Eredményeink szerint a bejuttatott normális ZAP-70 expressziójú T sejt progenitorok a recipiensben megtapadva képesek stabilan fokozni a T sejt képzést. Munkánkat az OTKA-K101493. sz pályázata támogatta. Boldizsár Ferenc MTA, Bolyai János Kutatói Ösztöndíjban részesül

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

Imported Infections Versus Herd Immunity Gaps; A Didactic Demonstration of Compartment Models Through the Example of a Minor Measles Outbreak in Hungary

Introduction: In Hungary, where MMR vaccine coverage is 99%, in 2017, a minor measles epidemic started from imported cases due to two major factors – latent susceptible cohorts among the domestic population and the vicinity of measles-endemic countries. Suspended immunization activities due to the COVID-19 surge are an ominous precursor to a measles resurgence. This epidemiological demonstration is aimed at promoting a better public understanding of epidemiological data. Materials and Methods: Our previous MMR sero-epidemiological measurements (N of total measles cases = 3919, N of mumps cases = 2132, and N of rubella cases = 2132) were analyzed using open-source epidemiological data (ANTSZ) of a small-scale measles epidemic outbreak (2017, Hungary). A simplified SEIR model was applied in the analysis. Results: In case of measles, due to a cluster-specific inadequacy of IgG levels, the cumulative seropositivity ratios (measles = 89.97%) failed to reach the herd immunity threshold (HIT Measles = 92–95%). Despite the fact that 90% of overall vaccination coverage is just slightly below the HIT, unprotected individuals may pose an elevated epidemiological risk. According to the SEIR model, ≥74% of susceptible individuals are expected to get infected. Estimations based on the input data of a local epidemic may suggest an even lower effective coverage rate (80%) in certain clusters of the population. Conclusion: Serological survey-based, historical and model-computed results are in agreement. A practical demonstration of epidemiological events of the past and present may promote a higher awareness of infectious diseases. Because of the high R0 value of measles, continuous large-scale monitoring of humoral immunity levels is important

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

THE ROLE OF ZAP-70 KINASE IN THE FINE-TUNING OF TCR SIGNALLING : IMPLICATIONS FOR IMMUNOPATHOLOGY AND –THERAPY

ZAP-70 (zeta-chain associated 70 kDa) kinase is a key regulator of T cell receptor signaling. After ligand binding of the T cell receptor (TcR), Lck kinase phosphorylates tyrosine (Y) residues of the CD3 ζ chains and ZAP-70, which, in turn, phosphorylates a number of downstream target proteins (eg. LAT, SLP-76, PLCγ, Cbl). ZAP-70 itself contains a number of Y residues, which can be phosphorylated. Using an array of mutant cell lines where targeted Y-Phenylalanine (F) mutations were introduced into ZAP-70, we were able to characterize the fine details of TcR signaling. Our data confirmed the function of earlier described activator (Y315, Y493) and inhibitory (Y292, Y492) residues; moreover, we described the regulatory role of previously less-known (Y069, Y126, Y178) positions. Glucocorticoid treatment is widely used for suppressing the immune response, primarily through the inhibition of T cell functions. Our earlier work demonstrated, that ZAP-70 is also involved in non-genomic (rapid) GC signaling mechanisms. Using our Y-F mutant ZAP-70 expressing cell line array, we identified that Y315 and Y492 were phosphorylated upon short-term high dose GC analogue treatment. These results confirmed that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Moreover, potential role of ZAP-70 kinase was implicated in chronic lymphoid leukemia (CLL) and autoimmune arthritis. It has been shown in a subgroup of patients with CLL that the malignant B-lymphocytes express ZAP-70 kinase, which was associated with inferior clinical outcome and prognosis. Using two ZAP-70 specific antibodies recognizing different epitopes in the kinase, we performed intracellular staining of malignant B cells from CLL patients. Based on our preliminary experiments, it seems possible that the ZAP-70 molecule expressed in the tumorous B-cells is structurally different from that found in normal T-cells, as some patients showed positivity with either one or the other antibody, while the normal T-cells were positive with both antibodies, just as expected. A spontaneous single point mutation at 163 from Triptophane (W) to Cysteine (C) in the SH2 domain of ZAP-70 caused altered thymic selection and leads to the development of autoimmune arthritis in SKG mice. Another study has shown that targeted simultaneous mutation at positions Y315 and Y319 to Alanine led to similar defects in T cell development than in SKG mice, interestingly, however, these mice did not develop autoimmune arthritis despite the presence of rheuma factor in the sera, increased IL-17 production and impaired Treg development. These data clearly show, how our understanding about ZAP-70 kinase has emerged from being exclusively a T cell specific signaling molecule to an important therapeutic target and potential regulator of pathologies like CLL or autoimmune arthritis