Vilmos Tubak

Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1.

Galectin-1 (Gal-1) has been implicated in tumor progression partly via the induction of T-cell apoptosis. However the mechanism of Gal-1 induced T-cell death was mostly studied using recombinant, soluble Gal-1 producing controversial results. To explore the true mechanism of Gal-1 and hence tumor cell-induced T-cell death, we applied co-cultures of tumor cells and T-cells thus avoiding artificial circumstances generated using recombinant protein. T-cells died when co-cultured with Gal-1-expressing but survived with Gal-1 non-expressing tumor cells. Removing tumor cell surface Gal-1 or knocking down Gal-1 expression resulted in diminution of T-cell apoptosis. Gal-1 transgenic or soluble Gal-1 treated HeLa cells became cytotoxic. Stimulation of apoptosis required interaction between the tumor and T-cells, presence of p56lck and ZAP70, decrease of mitochondrial membrane potential and caspase activation. Hence tumor cell-derived Gal-1 might efficiently contribute to tumor self-defense. Moreover this system resolves the discrepancies obtained using recombinant Gal-1 in T-cell apoptosis studies.

Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment.

Novel method to load multiple genes onto a mammalian artificial chromosome.

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

Inhibition of fatty acid amide hydrolase exerts cutaneous anti-inflammatory effects both in vitro and in vivo

Inhibition of fatty acid amide hydrolase exerts cutaneous anti-inflammatory effects both in vitro and in vivo Attila Oláh, Lı́dia Ambrus, Simon Nicolussi, J€ urg Gertsch, Vilmos Tubak, Lajos Kemény, Michael Soeberdt, Christoph Abels and Tamás Bı́ró DE-MTA ‘Lend€ ulet’ Cellular Physiology Research Group, Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; Institute of Biochemistry and Molecular Medicine, NCCR TransCure, University of Bern, Bern, Switzerland; Creative Laboratory Ltd., Szeged, Hungary; MTA-SZTE Dermatological Research Group, University of Szeged, Szeged, Hungary; Dr. August Wolff GmbH & Co. KG Arzneimittel, Bielefeld, Germany; Department of Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary Correspondence: Tamás Bı́ró, MD, PhD, DSc, 4032 Debrecen, Nagyerdei krt. 98., Hungary, Tel.: +36-52-255-575, Fax: +36-52-255-116, e-mail: biro.tamas@med.unideb.hu

Genome Wide Transcriptome Analysis of Dendritic Cells Identifies Genes with Altered Expression in Psoriasis

Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS) or peptidoglycan (PGN) induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs) upon PGN induced tolerance. Using SAGE-Seq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (Kegg) analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified numerous genes with altered expression to date not associated with role in chronic inflammation, underlying the relevance of our in vitro model for further characterization of IFN-primed iDCs.

The expression of TAM receptors and their ligand Gas6 is downregulated in psoriasis

Psoriasis, a common chronic skin disease is, among others, characterized by epidermal hyperplasia and altered cytokine milieu. The most widely held model of psoriasis pathogenesis proposes that keratinocyte hyperproliferation is triggered by cutaneous lymphocyte infiltration, activation and differentiation of inflammatory cells and that these events generate a localized cytokine environment [1]. Despite the heterogeneity of cytokine networks [2] these effector molecules both sustain and reinforce the pathogenic cascade in psoriasis. Hence, psoriasis plaques formation involves the interaction between inflammatory and resident tissue cells, primarily mediated by various cytokines

Galectin-1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr-Xxx-Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. 15N,1H HSQC titrations with 15N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.

Bacterial Sepsis Increases Survival in Metastatic Melanoma: Chlamydophila Pneumoniae Induces Macrophage Polarization and Tumor Regression

Complete List of Authors: Buzas, Krisztina; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry; University of Szeged, Faculty of Dentistry Marton, Annamaria; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry Vizler, Csaba; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry Gyukity-Sebestyen, Edina; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry Harmati, Maria; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry Nagy, Katalin; University of Szeged, Faculty of Dentistry Zvara, Agnes; Hungarian Academy of Sciences, Biological Research Centre, Laboratory of Functional Genomics Katona, Róbert; Biological Research Centre, Institute of Genetics Tubak, Vilmos; Creative Laboratory Ltd., Creative Laboratory Ltd. Endresz, Valeria; University of Szeged, Department of Medical Microbiology and Immunbiology Németh, István; University of Szeged, Department of Dermatology and Allergology Olah, Judit; University of Szeged, Department of Dermatology and Allergology Szeged, Hungary Vigh, Laszlo; Hungarian Academy of Sciences, Biological Research Centre, Institute of Biochemistry Biro, Tamas; University of Debrecen, Medical and Health Science Center, Department of Physiology Kemeny, Lajos; University of Szeged, MTA-SZTE Dermatological Research Group