Hisztamin hatása a sejtdifferenciációra, összehasonlító vizsgálatok tumor - és embrionális őssejteken = Effect of histamine on cell differentiation, comparative study of tumor-and embronic stem cells

Célkitűzésünk a genetikailag hisztamin hiányos és WT dermatofibrosarcoma tumorsejtek és embrionális őssejtek differenciációs markereinek összehasonlítása volt. Az indukált tumorokból alapított sejttenyészetekből tumor spheroid formájában fenntartható klónokat hoztunk létre, melyek jellemzőikben hasonlítottak az embrionális testet (EB) képző őssejtekre. A továbbiakban microarray analízissel a letapadó, illetve spheroidként/EB-ként növő sejtek génexpressziós mintázatát vizsgáltuk, különös tekintettel az egyedfejlődésben szerepet játszó, és a sejt önmegújulásért felelős transzkripciós faktorokra. Mivel az array adatok igen nagyszámú gén aktivitásában mutattak különbséget, ezért az Ingenuity pathway analízis nyomán csak a Wnt jelátviteli útra koncentráltunk a továbbiakban. Az így kiemelt Id2, Sox2, Nestin, Col2A1 génhálózat expressziós tendenciáit real-time PCR-rel és Western blottal próbáltuk validálni, azonban a 3 módszerrel detektált változások tendenciái nem vagy csak részben mutattak egyezést. Mivel a spheroid- és EB-képzésben a sejt-mátrix kapcsolatoknak is szerepe van (erre utalhat a Col2A1) ezért ebbe az irányba terjesztettük ki kutatásainkat, az adherens és spheroid/EB sejtek adhéziós viselkedését vizsgálva, különös tekintettel egy másik (a hisztaminnal összefüggésben már igazolt szerepű) mátrix komponensre, a fibulin5-re. A HDC KO sejtek fokozottabb adhézióval és fibulin5 expresszióval rendelkeznek, ez összhangban áll a korábban melanoma sejteken tett megfigyelésünkkel. | Our aim was to compare the differentiation markers of BalB/c WT and HDC KO dermatofibrosarcoma tumor cells and embryonic stem cells with the same genetic background. From the induced tumors cell cultures and non-adhesive spheroid forming clones were established, which were similar embryoid bodies (EBs). Subsequently microarray analyses on Agilent platform were carried out to determine the gene expression patterns of the adherent and spheroid or EB forming cells. We focused our interest to those transcription factors involved in cellular self-renewal and in early embryogenesis. As the array data showed differences in hundreds of genes, we concentrated our efforts - after using Ingenuity pathway analysis - the Wnt pathway, important in self-renewal and tumorigenesis. Id2, Sox2, Nestin and Col2A1 elements of this network were used for validation via real-time PCR and Western blotting. Unfortunately the tendencies observed here did not fit well the data derived from microarray analysis. Since cell-matrix connections are important in both spheroid and EB formation (reflected by the Col2A1 expression, too) we expand our efforts to study the adhesive behaviour of adherent and spheroid/body forming cells in connection with an other matrix component fibulin5, its role related to histamine has been proven earlier by us. The HDC KO cells had better adhesion characteristics and increased FBLN5 expression, which is in good correlation our earlier observations made on melanoma cells

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

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

TP53inp1 Gene Is Implicated in Early Radiation Response in Human Fibroblast Cells

Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. gamma-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-beta-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts

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

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

Histidine Decarboxylase Expression in Human Melanoma

SummaryHistamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma

Low dose cranial irradiation-induced cerebrovascular damage is reversible in mice

BACKGROUND: High-dose radiation-induced blood-brain barrier breakdown contributes to acute radiation toxicity syndrome and delayed brain injury, but there are few data on the effects of low dose cranial irradiation. Our goal was to measure blood-brain barrier changes after low (0.1 Gy), moderate (2 Gy) and high (10 Gy) dose irradiation under in vivo and in vitro conditions. METHODOLOGY: Cranial irradiation was performed on 10-day-old and 10-week-old mice. Blood-brain barrier permeability for Evans blue, body weight and number of peripheral mononuclear and circulating endothelial progenitor cells were evaluated 1, 4 and 26 weeks postirradiation. Barrier properties of primary mouse brain endothelial cells co-cultured with glial cells were determined by measurement of resistance and permeability for marker molecules and staining for interendothelial junctions. Endothelial senescence was determined by senescence associated β-galactosidase staining. PRINCIPLE FINDINGS: Extravasation of Evans blue increased in cerebrum and cerebellum in adult mice 1 week and in infant mice 4 weeks postirradiation at all treatment doses. Head irradiation with 10 Gy decreased body weight. The number of circulating endothelial progenitor cells in blood was decreased 1 day after irradiation with 0.1 and 2 Gy. Increase in the permeability of cultured brain endothelial monolayers for fluorescein and albumin was time- and radiation dose dependent and accompanied by changes in junctional immunostaining for claudin-5, ZO-1 and β-catenin. The number of cultured brain endothelial and glial cells decreased from third day of postirradiation and senescence in endothelial cells increased at 2 and 10 Gy. CONCLUSION: Not only high but low and moderate doses of cranial irradiation increase permeability of cerebral vessels in mice, but this effect is reversible by 6 months. In-vitro experiments suggest that irradiation changes junctional morphology, decreases cell number and causes senescence in brain endothelial cells

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly