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

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

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

The impact of maternal BMI status on pregnancy outcomes with immediate short-term obstetric resource implications : a meta-analysis.

Obesity is rising in the obstetric population, yet there is an absence of services and guidance for the management of maternal obesity. This systematic review aimed to investigate relationships between obesity and impact on obstetric care. Literature was systematically searched for cohort studies of pregnant women with anthropometric measurements recorded within 16-weeks gestation, followed up for the term of the pregnancy, with at least one obese and one comparison group. Two researchers independently data-extracted and quality-assessed each included study. Outcome measures were those that directly or indirectly impacted on maternity resources. Primary outcomes included instrumental delivery, caesarean delivery, duration of hospital stay, neonatal intensive care, neonatal trauma, haemorrhage, infection and 3rd/4th degree tears. Meta-analysis shows a significant relationship between obesity and increased odds of caesarean and instrumental deliveries, haemorrhage, infection, longer duration of hospital stay and increased neonatal intensive care requirement. Maternal obesity significantly contributes to a poorer prognosis for mother and baby during delivery and in the immediate post-partum period. National clinical guidelines for management of obese pregnant women, and public health interventions to help safeguard the health of mothers and their babies are urgently required

The effect of pre-pregnancy body mass index on breastfeeding initiation, intention and duration:A systematic review and dose-response meta-analysis

Overweight and obesity not only are major risk factors for number of chronic diseases, but also a risk factor for pregnancy complications in women. The present study aims to investigate the association between pre-pregnancy BMI and the persistence and duration of BF. The electronic databases including Medline (PubMed), Scopus, Embase, Web of Science and Google Scholar were searched for papers with titles and/or abstracts including one of our keywords and published up to 15 April 2019. For dose-response relationship, the two-stage random-effects meta-analysis was performed using the �dosresmeta� function in R software. Thirty-two studies with the effect of pre-pregnancy BMI on BF initiation, intention and duration were included in the present study. Based on crude and adjusted OR models, the risk of BF cessation increased by 4 (OR = 1.04; 95 CI: 1.02�1.05) with an increase in a unit of BMI. In addition, based on crude and adjusted RR models, the risk of BF cessation increases by 2 and 1 (crude RR = 1.02; 95 CI: 1.01�1.03 and adjusted RR = 1.01; 95 CI: 0.99�1.02) with an increase in one unit of BMI. Based on the result, the health care professionals and other key stakeholders should be aware of the impact excess weight, and that women who are overweight or obese should be encouraged with continued access to guidance, counseling and support, starting from conception, to maximize BF outcomes. © 2020 The Authors Public health; Women's health; Reproductive medicine; Obstetrics and gynecology; Reproductive system; Clinical research; Pre-pregnancy, Body mass index, Breastfeeding, Initiation, Duration. © 2020 The Author