11 research outputs found

    Onkogenomikai kutatás melanomában génexpresszió és a nem kódoló microRNS profil meghatározása alapján = Oncogenomic studies in melanoma through gene expression and non-coding microRNA profiling

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    Kutatásukban mikroRNS (miRNS) expressziót vizsgáltak a melanoma sejtvonalakban, szövetmintákban és melanocyta sejtekben. Céluk a melanoma komplex keletkezési és fennmaradási mechanizmusában a miRNS profil felvételével új, melanomában eddig még nem leírt, a normáltól eltérő expresszió –szabályozási hálózat keresése, amely a daganat biológiai viselkedésének hátterében állhat, tükrözve a genetikai és epigenetikai tényezők komplexitását. A talált miRNS-ek lehetőséget adhatnak a célzott génterápia bevezetésére. Eredmények: 1. A melanoma sejtvonalak miRNS összetétele eltér a normál bőr melanocitáiktól 2. Egy, sok tumorban megtalálható miRNS, a miR21 bejuttatása sejtekbe önmagában nem okoz lényeges funkcionális hatást 3. Eltérést találtak a melanoma szövetminták és a melanoma vonalak miRNS mintázatában 4. Útvonalanalízis eredményeképpen egyes molekuláris jelutak (génhálózatok) kiemelt jelentőségét ismerték fel melanomában (elsősorban a p53 út) 5. Informatikai eszközökkel célpont predikciót végeztek. Kimutatták, hogy a melanoma szövet közelében található hízósejtekben a miRNS- 132 nagyon erősen gátló hatású, amely egy új génterápia lehetőségét veti fel. | In the present research mikroRNA (miRNA) expression was analysed in melanoma cell lines, tissue and melanocytes. Our goal was to uncover the complex mechanism of the miRNA action, specific on the regulatory network of the tumor reflecting the underlying epigenetic factors. Based on these findings a novel miRNA-targeted gene therapy may provide new opportunities. Results: 1. The melanoma cell lines differ in the composition of the miRNA from that of the melanocytes of healthy skin 2. A common miRNAs, miR21 transfected alone into cells does not cause a significant functional effect 3. Differences were found in the pattern of miRNA between melanoma tissue samples and established melanoma cell lines 4. The pathway analysis of the molecular signal processes proved the utmost importance of p53 pathway. 5. Mast cells occur in close vicinity of melanoma. Based on target prediciton screening miRNA-132 was selected. This miRNA reveals a very strong inhibitory effect, which raises the possibility of a new gene therapy

    Unique patterns of CD8+ T-cell-mediated organ damage in the Act-mOVA/OT-I model of acute graft-versus-host disease.

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    T-cell receptor (TCR)-transgenic models of acute graft-versus-host disease (aGvHD) offer a straightforward and highly controlled approach to study the mechanisms and consequences of T-cell activation following allogeneic hematopoietic stem cell transplantation (aHSCT). Here, we report that aHSCT involving OT-I mice as donors, carrying an ovalbumin-specific CD8+ TCR, and Act-mOVA mice as recipients, expressing membrane-bound ovalbumin driven by the β-actin promoter, induces lethal aGvHD in a CD8+ T-cell-dependent, highly reproducible manner, within 4-7 days. Tracking of UBC-GFP/OT-I graft CD8+ T cells disclosed heavy infiltration of the gastrointestinal tract, liver, and lungs at the onset of the disease, and histology confirmed hallmark features of gastrointestinal aGVHD, hepatic aGvHD, and aGvHD-associated lymphocytic bronchitis in infiltrated organs. However, T-cell infiltration was virtually absent in the skin, a key target organ of human aGvHD, and histology confirmed the absence of cutaneous aGVHD, as well. We show that the model allows studying CD8+ T-cell responses in situ, as selective recovery of graft CD45.1/OT-I CD8+ T cells from target organs is simple and feasible by automated tissue dissociation and subsequent cell sorting. Assessment of interferon-gamma production by flow cytometry, granzyme-B release by ELISA, TREC assay, and whole-genome gene expression profiling confirmed that isolated graft CD8+ T cells remained intact, underwent clonal expansion, and exerted effector functions in all affected tissues. Taken together, these data demonstrate that the OT-I/Act-mOVA model is suitable to study the CD8+ T-cell-mediated effector mechanisms in a disease closely resembling fatal human gastrointestinal and hepatic aGVHD that may develop after aHSCT using HLA-matched unrelated donors

    Skin-homing CD8+ T cells preferentially express GPI-anchored peptidase inhibitor 16, an inhibitor of cathepsin K

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    This study sought to identify novel CD8+ T cell homing markers by studying acute graft versus host disease (aGvHD), typically involving increased T cell homing to the skin and gut. FACS-sorted skin-homing (CD8β+ /CLA+ ), gut-homing (CD8β+ /integrinβ7+ ), and reference (CD8β+ /CLA- /integrinβ7- ) T cells were compared in patients affected by cutaneous and/or gastrointestinal aGVHD. Microarray analysis, qPCR, and flow cytometry revealed increased expression of peptidase inhibitor 16 (PI16) in skin-homing CD8+ T cells. Robust association of PI16 with skin homing was confirmed in all types of aGvHD and in healthy controls, too. PI16 was not observed on CLA+ leukocytes other than T cells. Induction of PI16 expression on skin-homing T cells occurred independently of vitamin D3. Among skin-homing T cells, PI16 expression was most pronounced in memory-like CD45RO+ /CD127+ /CD25+ /CD69- /granzyme B- cells. PI16 was confined to the plasma membrane, was GPI-anchored, and was lost upon restimulation of memory CD8+ T cells. Loss of PI16 occurred by downregulation of PI16 transcription, and not by Phospholipase C (PLC)- or Angiotensin-converting enzyme (ACE)-mediated shedding, or by protein recycling. Inhibitor screening and pull-down experiments confirmed that PI16 inhibits cathepsin K, but may not bind to other skin proteases. These data link PI16 to skin-homing CD8+ T cells, and raise the possibility that PI16 may regulate cutaneous cathepsin K

    CD8+/CD103+ tissue-resident memory T cells display substantial functional plasticity and fine-tune their phenotype to meet local needs

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    n this study we sought a better understanding of t he unique phenotypic plasticity displayed by CD8+/CD103+ tissue-resident memory T cells (Trms ) in distinct tissue environments. Murine Trm cells were isolated from distinct tissues by automa ted tissue dissociation and magnetic sorting. Distinctive features of the Trm phenotype in distin ct tissues were described by microarray gene expression profiling, Q-PCR, UPLC-MS/MS, and flow c ytometry. We found that CD8+ small intestinal Trms frequently exhibit an effector-like phenotype, express various chemokines, granzymes A/B, show evi dence of degranulation, intense protein synthesis and sustained clonal expansion. In contra st, lung-resident Trms typically express lymphotoxins α/β, but not granzymes or markers of degranulation, and rarely divide due to G2/M arrest. Finally, CD8+ CD103+ T cells in the liver, a tolerogenic environment for CD8+ T cells, are mostly dormant, but often positive for CXCR4, a rec eptor of the liver-released chemokine SDF-1. We also found, however, that subtle phenotypic changes affect non-resident CD8+ T effector (Teff) cells infiltrating the same tissues, too. Although far le ss pronounced, these changes were often analogous to those observed in Trm cells. These data suggest that Trm cells display a unique phenotypic plasticity and adapt to varying environmental conditions by multiple, although not exclusively Trm-restricted mechanisms

    Mesenchymal-Stromal Cell-like Melanoma-Associated Fibroblasts Increase IL-10 Production by Macrophages in a Cyclooxygenase/Indoleamine 2,3-Dioxygenase-Dependent Manner

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    Melanoma-associated fibroblasts (MAFs) are integral parts of melanoma, providing a protective network for melanoma cells. The phenotypical and functional similarities between MAFs and mesenchymal stromal cells (MSCs) prompted us to investigate if, similarly to MSCs, MAFs are capable of modulating macrophage functions. Using immunohistochemistry, we showed that MAFs and macrophages are in intimate contact within the tumor stroma. We then demonstrated that MAFs indeed are potent inducers of IL-10 production in various macrophage types in vitro, and this process is greatly augmented by the presence of treatment-naïve and chemotherapy-treated melanoma cells. MAFs derived from thick melanomas appear to be more immunosuppressive than those cultured from thin melanomas. The IL-10 increasing effect is mediated, at least in part, by cyclooxygenase and indoleamine 2,3-dioxygenase. Our data indicate that MAF-induced IL-10 production in macrophages may contribute to melanoma aggressiveness, and targeting the cyclooxygenase and indoleamine 2,3-dioxygenase pathways may abolish MAF–macrophage interactions
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