<p>Eosinophil granulocytes are tissue dwelling leukocytes that are implicated in host defence, particularly against helminthic parasites; they also participate in most inflammatory disorders. Although eosinophils have important roles in host defence mechanisms, their actions can also be harmful to the host as in the allergic inflammation where lung epithelium is destructed due to the release of toxic granule proteins. </p><p>The focus of the present thesis work has been to characterize the molecular and functional heterogeneity of the granule protein Eosinophil Cationic Protein (ECP). We investigated a coding ECP gene polymorphism (arg97thr) in an African population endemically exposed to the <i>Schistosoma mansoni</i> parasite and found a correlation between ECP genotype and disease manifestations; ECP^97arg was more effective in terms of host defence against the parasite, but was also correlated to development of liver fibrosis in infected subjects. </p><p>We purified ECP^97arg and ECP^97thr from healthy blood donors and showed that they differ in their cytotoxic activities; ECP^97arg was cytotoxic whereas ECP^97thr was non-cytotoxic. They did not differ in terms of RNase activity or in their ability to stimulate fibroblast-mediated collagen gel contraction. </p><p>We developed a new SELDI-TOF MS assay to enable the study of the structure of ECP in more detail and showed that ECP is produced in several glycosylated forms, and that the degree of glycosylation determines the cytotoxic activity. Enzymatic deglycosylation significantly enhanced the cytotoxic activity of highly glycosylated ECP-variants.</p><p>To summarize, in this thesis we demonstrated that the cytotoxic activity of ECP is dependent on both a gene polymorphism and post-translational modifications, and that the cytotoxic activity is distinct from other functions of ECP. We speculate that ECP is synthesised in heavily glycosylated variants as a means to protect the host from its harmful effects and that ECP is activated by deglycosylation when required at the site of inflammation.</p>

Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified. The thrombin inhibitor melagatran completely blocked the IBMIR in an in vitro tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.

Eosinophil granulocytes are tissue dwelling leukocytes that are implicated in host defence, particularly against helminthic parasites; they also participate in most inflammatory disorders. Although eosinophils have important roles in host defence mechanisms, their actions can also be harmful to the host as in the allergic inflammation where lung epithelium is destructed due to the release of toxic granule proteins. The focus of the present thesis work has been to characterize the molecular and functional heterogeneity of the granule protein Eosinophil Cationic Protein (ECP). We investigated a coding ECP gene polymorphism (arg97thr) in an African population endemically exposed to the Schistosoma mansoni parasite and found a correlation between ECP genotype and disease manifestations; ECP97arg was more effective in terms of host defence against the parasite, but was also correlated to development of liver fibrosis in infected subjects. We purified ECP97arg and ECP97thr from healthy blood donors and showed that they differ in their cytotoxic activities; ECP97arg was cytotoxic whereas ECP97thr was non-cytotoxic. They did not differ in terms of RNase activity or in their ability to stimulate fibroblast-mediated collagen gel contraction. We developed a new SELDI-TOF MS assay to enable the study of the structure of ECP in more detail and showed that ECP is produced in several glycosylated forms, and that the degree of glycosylation determines the cytotoxic activity. Enzymatic deglycosylation significantly enhanced the cytotoxic activity of highly glycosylated ECP-variants. To summarize, in this thesis we demonstrated that the cytotoxic activity of ECP is dependent on both a gene polymorphism and post-translational modifications, and that the cytotoxic activity is distinct from other functions of ECP. We speculate that ECP is synthesised in heavily glycosylated variants as a means to protect the host from its harmful effects and that ECP is activated by deglycosylation when required at the site of inflammation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Disertaciniame darbe analizuojama ankstyvo ir metastazavusio krūties vėžio prognozė priklausomai nuo skirtos chemoterapijos dozės ir molekulinių žymenų. Tyrimas susidėjo iš trijų etapų. I tyrimo etape buvo atlikta molekulinių žymenų (p53 baltymo, HER2, estrogeno ir progesterono receptorių) prognozinės vertės analizė retrospektyviojo ankstyvo krūties vėžio tyrimo metu, kartu įvertinta sumažintos chemoterapijos dozės įtaka ligos išeitims. II tyrimo etape ištirtas naujo rGKSF filgrastimo saugumas ir veiksmingumas gydant chemoterapija metastazavusį krūties vėžį. III tyrimo etape atlikta molekulinių žymenų prognozinės vertės analizė, eliminavus chemoterapijos dozės įtaką klinikinėms ligos išeitims, gydymą optimalia cheminio preparato doze užtikrinus prospekyviajame daugiacentriame naujo rGKSF saugumo ir veiksmingumo tyrime. Darbo uždaviniai 1. Nustatyti antraciklinų dozės įtaką sergančiųjų ankstyvu krūties vė¬žiu išgyvenamumui. 2. Ištirti p53 baltymo raiškos, kitų molekulinių žymenų ir gydymo ypatumų įtaką ankstyvo krūties vėžio prognozei. 3. Įvertinti febrilios neutropenijos profilaktikai skiriamo naujo rGKSF saugumą, gydant metastazavusį krūties vėžį. 4. Įvertinti febrilios neutropenijos profilaktikai skiriamo naujo rGKSF veiksmingumą, gydant metastazavusį krūties vėžį. 5. Nustatyti metastazavusiu krūties vėžiu sergančių pacienčių išgyvenamumo priklausomybę nuo atsako į skiriamą gydymą. 6. Įvertinti krūties vėžio prognozę įtakojančių molekulinių žymenų tarpusavio sąsajas. / This study investigated the clinical outcomes in early and metastatic breast cancer patients depending on chemotherapy and molecular markers. The study comprised of 3 parts: Part I - retrospective investigation of molecular markers (p53 protein, HER2 receptors, estrogene and progesterone receptors) and chemotherapy dose reduction in early stage breast cancer; Part II - prospective open-label multicenter phase IV clinical trial on safety and effectiveness of GCSF used in chemotherapy of metastatic breast cancer; Part III - investigation of prognostic value of p53 protein expression and other molecular markers in metastatic breast cancer when optimal chemotherapy dose was adjusted in prospective multicenter trial of new rGCSF on safety and effectiveness used in chemotherapy of metastatic breast cancer. Objectives: 1. To establish the influence of anthracycline dose on survival of early stage breast cancer patients. 2. To evaluate the influence of p53 protein expression and other molecular markers as well as treatment on prognosis of early stage breast cancer. 3. To establish the safety of new rGCSF prescribed for prevention of febrile neutropenia in chemotherapy of metastatic breast cancer. 4. To analyze the effectiveness of new rGCSF prescribed for prevention of febrile neutropenia in chemotherapy of metastatic breast cancer. 5. To establish the relationship between response to treatment and survival in metastatic breast cancer patients. 6. To evaluate the associations among... [to full text]

Typescript (Photocopy) / Bibliography: leaves 170-198. / xx, 198 leaves, 1 leaf of col. plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1991

Bibliography: leaves 175-203. / xxix, 203 leaves, [14] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates whether cytokines influence the development of the embryo prior to implantation. / Thesis (Ph.D.)--University of Adelaide, Depts. of Obstetrics and Gynaecology and Microbiology and Immunology, 1993

Thesis (Ph. D.)--University of Missouri--Columbia, 2005. / "May 2005." Typescript. Vita. Includes bibliographical references (leaves 139-157). Also issued on the Internet.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-03-22T14:03:31Z No. of bitstreams: 1 Tulio_Queto.pdf: 43727261 bytes, checksum: 24d32a6a536f42194f28e8e2b42a6fe1 (MD5) / Made available in DSpace on 2013-03-22T14:03:31Z (GMT). No. of bitstreams: 1 Tulio_Queto.pdf: 43727261 bytes, checksum: 24d32a6a536f42194f28e8e2b42a6fe1 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A provocação por via respiratória com ovalbumina (OVA) em camundongos sensibilizados promove, na medula óssea (MO), eosinopoiese, resposta exacerbada à Interleucina(IL)-5, e mudanças no padrão de resposta em cultura à eotaxina, à IL- 13 e aos antiinflamatórios não esteroidais (NSAIDs). Em cultura de MO, a Prostaglandina (PG) E2 induz apoptose de eosinófilos, por via dependente de NO sintase indutível (iNOS) e ligante de CD95 (CD95L), enquanto a dexametasona (DEXA) promove eosinopoiese, gerando eosinófilos agregados e morfológicamente imaturos e protege contra da apoptose induzida por PGE2, provavelmente por mecanismos que regulem a expressão ou ativação de integrinas. No presente trabalho avaliamos se: a) in vitro, os efeitos dos NSAIDs, da IL-13 e da eotaxina dependem da produção de cisteinil-leucotrienos (CisLT) e da sinalização via receptor 1 de CisLT (CysLT1R); b) in vitro, a DEXA regula expressão de VLA-4, que promoveria a agregação e imaturidade dos eosinófilos, e se PGE2 contrapõe a ação da DEXA; c) in vivo, G-CSF e dietilcarbamazina (DEC) promoveriam eosinopenia medular e sistemica e se inibiriam a inflamação pulmonar alérgica. Resultados: a) NSAIDs, eotaxina e IL-13 potencializam a eosinopoiese via produção de CisLT e sinalização via CysLT1R. Os NSAIDs ainda protegem os eosinófilos da apoptose induzida por PGE2 exógena. b) A interação farmacológica entre PGE2 e DEXA modificam a ação de ambas, de forma estreitamente relacionada sobre a expressão de VLA-4 e, em condições específicas, esses agentes sinergizam gerando quantidades aumentadas de eosinófilos maduros. c) A DEC, inibidor da síntese de LTs, que na filariose experimental possivelmente atua via iNOS, inibe a eosinopoiese e os efeitos da provocação sobre o pulmão e a MO, através da via iNOS-CD95L. O Fator Estimulante de Colônias de Granulócitos (G-CSF), estimulante da neutropoiese, igualmente inibiu a inflamação pulmonar alérgica através da inibição da eosinopoiese. Este trabalho é parte do projeto intitulado "Eosinofilia na Asma Experimental", licenciado pela Comissão de Ética no Uso de Animais (CEUA) da Fiocruz, sob nos L010/04 e L002/09. / Our laboratory has previously shown that airway allergen challenge in ovalbumin-sensitized mice rapidly induces an increase in bone-marrow (BM) eosinophil production (eosinopoiesis), along with an increased response to Interleukin(IL)-5 in BM culture, changes in the ex vivo responses to cytokines and immunomodulators, including nonsteroidal anti-inflammatory drugs (NSAIDs) and cysteinyl-leukotrienes (CysLT), and colonization of the lungs by eosinophil progenitors. Early in the course of IL-5-induced eosinophil differentiation in BM culture, Prostaglandin E2 (PGE2) induces apoptosis, through a pathway dependent on the inducible isoform of NO synthase (iNOS) and the ligand for CD95 (CD95L). NSAIDs enhance eosinopoiesis and protect eosinophils in this critical period from exogenous PGE2, through a novel mechanism of action at the celular level. In this study, we show that indomethacin and aspirin act through endogenously synthesized CysLT, by establishing the essential roles of 5-lipoxygenase, LTC4 synthase and CysLT1R receptors, as well as the cytoprotective effect of CysLT against exogenous PGE2. The similarity between the effects of NSAIDs and those of eotaxin and IL-13 prompted us to reevaluate the contribution of endogenous CysLT to the effects of these cytokines. We confirmed that eotaxin and IL-13 act through this mechanism, and expand therefore the list of agents that, through CysLT, enhance eosinopoiesis, protecting immature eosinophils from apoptosis induced through the iNOS-CD95L pathway. Dexamethasone promotes BM eosinopoiesis, generating aggregated, cytologically immature eosinophils, which are nevertheless protected from PGE2- induced apoptosis. We examined therefore the possibility that dexamethasone upregulates integrin expression/activation, thereby maintaining an immature celular phenotype in cultured eosinophils, while PGE2 would have opposite effects on both integrin function and cytological maturation. We show that the proapoptotic effects of PGE2 are profoundly modified by its pharmacological interaction with dexamethasone, paralleling the effects of both drugs on integrins, and leading to a synergic generation of increased numbers of mature eosinophils, in very specific experimental conditions. Diethylcarbamazine (DEC) is an antihelminthic drug that blocks leukotriene synthesis, and possibly acts in experimental filarial infection through iNOS. We have examined, for the first time, the effects of DEC in a model of allergic pulmonary inflammation, showing that DEC is very effective in preventing the impact of airway allergen challenge on BM and lungs, through the in vivo operation of the iNOS-CD95L pathway. Granulocyte Colony-stimulating Factor (G-CSF), which stimulates neutropoiesis, mobilizes CD34+ hemopoietic progenitors from BM, and exerts complex immunoregulatory effects, was shown in our study to have a strong impact in a murine model of allergic pulmonary inflammation. Like DEC, G-CSF suppressed BM eosinopoiesis, although through a different mechanism, since DEC suppressed neutrophilia in the lungs with no effect on BM neutrophilia/neutropoiesis, while G-CSF promoted neutropoiesis and induced blood neutrophilia, even though it suppressed eosinopoiesis. This work was part of the Research Project “Eosinophilia in Experimental Asthma”, licensed by the Committee on the Ethical Use of Laboratory Animals (CEUA) at FIOCRUZ, under numbers L010/04 and L002/09.

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.