<p>A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. </p><p>An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. </p><p>The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/10⁶ cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8).</p><p>In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/10⁶ cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. </p><p>Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed <i>in vivo</i> after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a. </p>

<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>

Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release in vivo and ex vivo during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure. In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO. The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM. ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.

A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/106 cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8). In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/106 cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed in vivo after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a.

Orientador: Gilberto De Nucci / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T16:13:57Z (GMT). No. of bitstreams: 1 Maziero_AlineMendes_D.pdf: 2175096 bytes, checksum: b3e712ed8baf6a7f7d4ef12131b9e85a (MD5) Previous issue date: 2014 / Resumo: Os eosinófilos participam de processos inflamatórios e alérgicos. Estando relacionados com o sistema de imunidade inata do organismo, eles representam uma linha fundamental de defesa contra invasão microbiana e, quando ativados, produzem uma série de mediadores solúveis que atuam nas respostas inflamatórias e alérgicas. A relação entre a atividade dos eosinófilos e plaquetas foi observada nas últimas décadas por muitos cientistas. Estas observações incluem o aumento do número de eosinófilos associados a desordens plaquetárias, incluindo alterações na cascata de coagulação e agregação plaquetária. Com base nessas observações, a interação entre os eosinófilos e plaquetas foram analisadas na agregação plaquetária. Plaquetas humanas foram incubadas com a fração citosólica de eosinófilos, linhagem celular promielocítica humana HL-60 clone 15 e proteína catiônica do eosinófilo (ECP). A agregação em plasma rico em plaquetas (PRP) foi induzida por difosfato de adenosina, fator de ativação plaquetária, ácido araquidônico e colágeno, e as plaquetas lavadas (PL) foram ativadas por trombina. A agregação induzida por todos os agonistas foi inibida de maneira concentração de células dependente pela fração citosólica de eosinófilos. Esta inibição foi apenas parcialmente revertida pela prévia incubação dos eosinófilos com L-Nitro-Arginina-metil-éster (L-NAME). A prévia incubação com indometacina não impediu a inibição induzida pela fração citosólica. A separação da fração citosólica de eosinófilos por gel filtração em Sephadex G-75 mostrou que a atividade inibitória foi concentrada na fração de peso molecular mais baixo. As células HL -60 clone 15 diferenciadas em eosinófilos por 5 e 7 dias foram capazes de inibir a agregação plaquetária. A proteína de ECP inibiu a agregação plaquetária em PRP e PL. Esta inibição foi mais evidente em PL, e o ensaio de citotoxicidade com MTT demonstrou a viabilidade de plaquetas testadas, indicando que a inibição observada pela proteína ECP não ocorre simplesmente pela morte celular. A proteína EDN, clonada e expressa em sistema eucarioto, também apresentou efeito de inibição sobre a agregação plaquetária em PRP, enquanto que a proteína MBP não apresentou efeito de inibição da agregação plaquetária significativo. Os nossos resultados indicam que os eosinófilos desempenham um papel fundamental na inibição da agregação plaquetária / Abstract: Eosinophils participate in allergic and inflammatory processes, being related to innate immunity system of the body, they represent a fundamental line of defense against microbial invasion and when activated produce a number of soluble mediators that act in inflammatory and allergic responses. The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with L-Nitro-Arginine-Methyl-Ester (L-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. The EDN protein, cloned and expressed in eukaryotic system, also showed inhibitory effect on platelet aggregation in PRP, whereas the protein MBP had no effect significative inhibiting platelet aggregation. Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition / Doutorado / Farmacologia / Doutora em Farmacologia