Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.

Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the role of the known E-selectin ligands, namely PSGL-1 and CD43, to CD44. I showed that CD44 purified from in vitro human activated T-cells or from psoriasis patients acts as a functional E-selectin ligand. Furthermore, our knock-down studies demonstrated that CD44, and not CD43, cooperates with P-selectin glycoprotein ligand-1 (PSGL-1) as a major E-selectin ligand.

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Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-12-08T11:38:49Z No. of bitstreams: 2 Dissertação - Dulce Helena Rebouças de Souza - 2012.pdf: 1582264 bytes, checksum: 81d7319b908561e96506800f7a644899 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-12-08T11:39:32Z (GMT) No. of bitstreams: 2 Dissertação - Dulce Helena Rebouças de Souza - 2012.pdf: 1582264 bytes, checksum: 81d7319b908561e96506800f7a644899 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-08T11:39:32Z (GMT). No. of bitstreams: 2 Dissertação - Dulce Helena Rebouças de Souza - 2012.pdf: 1582264 bytes, checksum: 81d7319b908561e96506800f7a644899 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-05-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lymphotropic virus human T cells 1 (HTLV-1) is a retrovirus associated with the development of diseases such as T cell leukemia in adults (ATL) and tropical spastic paraparesis (TSP) or HTLV-associated myelopathy (HAM/TSP). Transmission occurs by sexual routes, parenteral and vertical. Female sex workers (FSW) are a population vulnerable to parenteral and sexually transmitted infections since they have often risky behaviors including drug use and unprotected sex. The study aimed to investigate the seroepidemiological profile of HTLV-1 infection among a population of female sex workers in Goiânia city, using the Respondent driven Sampling methodology. A total of 402 FSWs were interviewed about demographic and risk characteristics for HTLV infection, between May 2009 and June 2010. Blood samples were collected from all females and screened by ELISA for detection of antibodies to HTLV-1/2. Positive samples were retested for confirmation by western blot and PCR, and characterized by sequencing and phylogenetic analyses. The mean age was 27.5 years (SD: 9.1 years). Most of FSWs (67.1%) were single, 47.3% had 10 a 12 years of formal education. One third of female sex workers reported illicit drug (34.1%), thought only (2.7%) used injection illicit drugs, 51.9% had more than seven sexual partners in the last week and 36.3% did not use condom with their steady sexual partners. Some women reported to recruit their clients in more than one type of venue, being nightclubs (41%), bars (27.7%) and streets (25%) predominant. Of the 402 samples screened by ELISA, three were positive and submitted to detection of DNA-HTLV for the tax, LTR and env regions. Only one was positive for HTLV-1, resulting in a prevalence of 0.2%. (CI 95%: 0.0-1.6). The virus isolate was classified as Transcontinental subgroup of the HTLV-1 Cosmopolitan subtype. These findings show a low endemicity for HTLV-1 infection in female sex workers in Goiânia-GO, however epidemiological studies of this infection are important to reinforce the need for prevention strategies based on the disclosure of the modes of transmission and status tracking serological the infected. / O vírus linfotrópico de células T humanas 1 (HTLV-1) é um retrovírus associado ao desenvolvimento de doenças como leucemia/linfoma de células T do adulto (ATL) e mielopatia associada ao HTLV-1/paraparesia espástica tropical (HAM/TSP). A transmissão ocorre por vias sexual, parenteral e vertical. As mulheres profissionais do sexo (MPS) constituem uma população vulnerável as infecções de transmissão parenteral e sexual, uma vez que apresentam comportamentos de risco, incluindo o uso de drogas e o sexo sem proteção. Este estudo teve como objetivo investigar o perfil soroepidemiologico da infecção pelo HTLV-1 em mulheres profissionais do sexo em Goiânia, GO, usando a metodologia Respondent-Driven Sampling (RDS). Um total de 402 MPS foi entrevistada sobre dados sociodemográficos e características de risco para a infecção pelo HTLV, entre maio de 2009 e junho de 2010. As amostras de sangue foram coletadas de todas as mulheres e triadas pelo ELISA para a detecção de anticorpos para HTLV-1/2. As amostras positivas foram retestadas para a confirmação por western blot e PCR, sendo caracterizadas por sequenciamento e análise filogenética. A idade média das mulheres foi 27,5 anos (Dp: 9,1 anos). A maioria (67,1%) era solteira, 47,3% tinham entre 10 e 12 anos de educação formal. Um terço das profissionais (34,1%) relatou uso de drogas ilícitas, embora apenas 2,7% usaram drogas injetáveis, 51,9% tiveram mais de sete parceiros sexuais na ultima semana e 36,3% não usaram preservativos com parceiros não pagantes. Algumas mulheres relataram recrutar seus clientes em mais de um tipo de local, boates (41%), bares (27,7%) e ruas (25%). Das 402 amostras triadas pelo ELISA, três foram positivas e submetidas à detecção do HTLV-DNA para as regiões tax, LTR e env. Apenas uma foi positiva para HTLV-1, por PCR, resultando numa prevalência de 0,2% (IC 95%: 0,0-1,6). O isolado viral foi classificado como subtipo Cosmopolita (HTLV-1a), subgrupo Transcontinental (A). Os resultados mostram uma baixa endemicidade para a infecção pelo HTLV-1 em mulheres profissionais do sexo em Goiânia-GO, entretanto estudos epidemiológicos sobre essa infecção são importantes para reforçar a necessidade de estratégias de prevenção baseadas na divulgação dos modos de transmissão e acompanhamento do status sorológico dos infectados.