Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2015-07-17T18:21:40Z No. of bitstreams: 1 CAMPOS, Maurício de Souza.pdf: 2732552 bytes, checksum: 0aae28f6faaa3ad7fd787acbca547eea (MD5) / Made available in DSpace on 2015-07-17T18:21:40Z (GMT). No. of bitstreams: 1 CAMPOS, Maurício de Souza.pdf: 2732552 bytes, checksum: 0aae28f6faaa3ad7fd787acbca547eea (MD5) / PRONEX e CAPES / Introdução: A infecção pelo vírus da hepatite B (HBV) compreende um amplo espectro de quadros clínicos que vai desde a manifestação aguda e autolimitada até a forma crônica, com possibilidade de desenvolvimento de cirrose hepática e carcinoma hepatocelular. A proteína 10 induzida por INF-γ (IP-10) é uma quimiocina CXC que pode ser secretada pelos hepatócitos e endotélio sinusoidal no fígado de pacientes com hepatite viral. Por ligação ao receptor CXCR3, IP-10 exerce efeito quimiotático em células NK, células T ativadas e células dendríticas, modulando, dessa forma, a resposta imunológica. Sabe-se que o IFN-γ estimula a secreção de IP-10 por células infectadas pelo vírus da hepatite C, mas no contexto da infecção pelo HBV, essa relação ainda é pouco conhecida. Objetivo: descrever os níveis das citocinas e quimiocinas séricas em pacientes com hepatite B crônica naive e sob tratamento antiviral. Material e métodos: foi realizada a dosagem de citocinas séricas de 24 voluntários com hepatite B crônica em tratamento e 33 voluntários com hepatite B crônica naive, utilizando o kit de CBA (Citokine Beads Array) da eBioscience e análise em FACScalibur BD. A citocina IP-10 foi quantificada utilizando o kit CBA da empresa BD conforme padronização prévia e recomendação do fabricante, e também analisada em FACScalibur BD. Os softwares SPSS versão 18 e GraphPad versão 6 foram utilizados para análise estatística. Resultados: foram encontradas diferenças estatísticas na comparação dos níveis de IP-10, IL-5 e TGF – β entre indivíduos com hepatite B crônica tratados e naive. Foram encontrados valores mais elevados de citocinas Th1 no soro de pacientes naive, quando comparados aos pacientes tratados. Os níveis séricos de IP-10 nos pacientes tratados e não tratados são mais elevados que os de INF-γ. Conclusão: os níveis séricos de citocinas Th1 entre pacientes não tratados estavam mais elevados do que em pacientes tratados. A amplitude dos níveis de INFγ foi maior nas amostras dos indivíduos com hepatite B sem tratamento. A correlação entre os níveis de INFγ e IL- 10 foi inversa em amostras de indivíduos não tratados. O tratamento pareceu modular a produção de INFγ sem interferir nos níveis de IP-10 e IL-10. A correlação entre os níveis de IP-10 e IL-10 também foi inversa em amostras de indivíduos não tratados. A IP-10 pode ser um marcador de maior sensibilidade que o INF-gama anteriormente descrito como a citocina chave no processo inflamatório na HBV / Background: infection with the hepatitis B virus(HBV) comprises a broad spectrum of clinical presentations ranging from acute and self-limited to the chronic form, with the possibility of development of liver cirrhosis and hepatocellular carcinoma. The protein10 induced by INF-γ(IP-10) is a CXC chemokine that can be secreted by hepatocytes and sinusoidal endothelium in the liver of patients with viral hepatitis. By binding to CXCR3 receptor, IP-10 exerts a chemotactic effect on NK cells, activated T cells and dendritic cells, potentializing, thus, the immune response. It is known that IFN-γ stimulates IP-10 secretion by cells infected with hepatitis C, but in the context of HBV infection, this relationship is still poorly known. Aim: to describe the levels of cytokines and chemokines in serum patients with chronic hepatitis B naïve and under antiviral treatment. Methods: the dosage of serum cytokines of 24 volunteers with chronic hepatitis B treatment and 33 volunteers with chronic hepatitis B naïve was performed using the CBA kit (Citokine Beads Array) from eBioscience and analyzed using FACScalibur BD. IP-10 cytokine was quantified using the CBA kit from BD company as previous standardization and recommendation of the manufacturer and also analyzed using FACScalibur BD. The SPSS software version 18 and GraphPad version 6 were used for statistical analysis. Results: statistical differences were found when comparing the IP-10 levels, IL-5 and TGF-β among individuals with chronic hepatitis B treated and naive. Higher values of Th1cytokines were found in the serum of treated patients when compared to naïve patients. The IP-10 serum levels in treated and untreated patients are higher than those of INF-γ. Conclusion: the serum levels of Th1cytokines from untreated patients were higher than in patients treated. The breadth of the INFγ levels were higher in samples of individuals without hepatitis B treatment. The correlation between the levels of INFγ and IL-10 was reverse on samples from untreated individuals. The treatment appeared to modulate INFγ production without interfering on IP-10and IL-10 production levels. The correlation between the IP-10 and IL-10 levels was also reverse in samples of untreated individuals. IP-10 may be a higher sensibility marker than the INFγ priorly described as a key cytokine in the inflammatory process in hepatitis B.

Die Diagnostik eines Glioblastoms (GBM) stützt sich z.Z. neben klinischer Symptomatik auf bildgebende Diagnostik sowie die histologische Untersuchung. In letzter Zeit werden zuneh-mend Serumproteine beschrieben und untersucht, die mit einer GBM-Erkrankung assoziiert sind. Elstner, Stockhammer und Kollegen haben 2011 ein Serumproteinprofil identifiziert, das aus CXCL10/IP-10, BMP-2 und HSP70 besteht und in einem Kollektiv von 23 GBM-Erkrankten und 9 Gesunden eine Sensitivität von 89% und eine Spezifität von 96% besaß. Dieses Profil wurde nun in einer unizentrischen klinischen Studie an 35 GBM-Erkrankten und 37 Patienten mit differenzialdiagnostisch relevanten Erkrankungen (v. a. Hirnmetastasen und primären ZNS-Lymphomen) überprüft. Dabei wurden die präoperativ abgenommenen Blut-proben mittels des ELISA-Nachweisverfahrens untersucht und die jeweiligen Konzentratio-nen in die von Elstner et al. (2011) entwickelte Regel eingesetzt. In diesem Kollektiv konnte das Profil nicht zwischen einem GBM und seinen Differenzialdiagnosen unterscheiden (Sensitivität 31%, Spezifität 54%). Es ist nicht als Hilfsmittel zur Diagnostik von Glioblastomen geeignet.

Background: The p53-up-regulated modulator of apoptosis (PUMA) protein is a pro-apoptotic, BH3-only member of the BCL2 family of effector proteins responsible for promoting organized cell death. PUMA is required for resolution of pneumococcal pneumonia in mice, as mice deficient of PUMA exhibit greater numbers of S. pneumoniae CFU within tissues and higher mortality rates than observed in Puma+/+ mice. Methods: Puma+/+ and Puma-/- mice were intranasally challenged with TIGR4 pneumococcus and sacrificed 24 h post-infection. Differences in cytokine levels from blood and whole lung tissue were detected by MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. Lung transcriptomes from Puma+/+ and Puma-/- mice were prepared from total lung RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina. Libraries were read by Illumina NovaSeq and transcript reads were referenced to Mus musculus. Results: Puma-/- mice exhibited significant differences in G-CSF, GM-CSF, IFN-gamma, IL-1-alpha and -beta, -6, -9, -10, -12 (p40 and p70), -13, and -17, IP-10, KC, MCP-1, MIP- iv 1alpha and -beta, MIP-2, RANTES, and TNF-alpha compared to Puma+/+ mice. Puma-/- lungs exhibited higher levels of IL-12, IFN-gamma, and IP-10. Loss of PUMA also resulted in expression of the pro-angiogenic genes Adam19 and Neurexin2. Additionally, Puma+/+ and Puma-/- mice displayed similar levels of colonization, but Puma-/- mice were more susceptible to subsequent dissemination to the lungs and blood. Conclusion: Polymorphonuclear cells (PMNs) were previously demonstrated to be one of the innate cell types responsible for Puma-dependent resolution of pneumococcal pneumonia in mice. Observations reported here suggest that this resolution is propelled by suppressing the inflammatory response via the inhibition of IL-12/IFN-gamma/IP-10 pro-inflammatory axis. Pulmonary tissue transcriptomic analysis also suggests PUMA-dependent positive regulation of homeostatic control of pulmonary vasculature, smooth muscle innervation, and maintenance of the interstitium. Gene ontological analysis further demonstrated Puma's modulatory role in Type I and II IFN signaling. For the first time, we report Puma's regulatory effects on pro-inflammatory cytokine signaling and gene expression during pneumococcal pneumonia.

No description available.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

B virus (Macacine herpesvirus 1, Cercopithecine herpesvirus 1, herpes B virus) is an Old World monkey simplex virus endemic in macaques. B virus infection in its natural host, macaque, is very similar to HSV-­‐1 infection in humans causing mild or asymptomatic infection. On the other hand, zoonotic infection in humans results in death in the absence of early initiation of antiviral drugs. Viruses evade host immune responses in order to survive and propagate. Most herpes viruses including HSV-­‐1 down-­‐regulate major histocompatibility complex class I (MHC class I) surface expression on infected cells in order to prevent CD8+ T-­‐cell recognition and subsequent cell lysis. MHC class I molecules bind to the inhibitory receptors of NK cells and prevent NK cell activity. Thus, this mechanism protects HSV-­‐1 infected cells from CD8+ T-­‐cell lysis, making them sensitive to natural killer (NK) cell cytotoxicity. To investigate if B virus pathogenicity is a result of novel immune evasion mechanisms employed by B virus, we determined NK cell regulation during B virus infection. To this end, our experiments demonstrate that B virus does not down-­‐ regulate MHC I expression as effectively as HSV-­‐1, leading us to hypothesize that B virus in-­‐ fected cells are resistant to NK cell activity. We examined the expression of MHC I chain related genes (MICA/ MICB), which are activation ligands to NKG2D receptors on NK cells. Our results show that there is no significant difference in MICA and MICB expression between HSV-­‐1 and B virus infected cells. Furthermore, we tested for the up-­‐regulation of cytokines and chemokines responsible for NK cell activation and migration. Our results indicate a significant up-­‐regulation of IFN-­‐α from PBMCs co-­‐cultured with HSV-­‐1 infected cells, which plays an important role in activating NK cells. NK cells within these PBMCs up-­‐regulate perforin release indicative of NK cell activity. PBMCs co-­‐cultured with B virus infected cells do not up-­‐regulate any cytokines or chemokines responsible for NK cell activity. As a result the NK cells within these PBMCs do not significantly up-­‐regulate perforin release. These results demonstrate that B virus employs a novel immune evasion mechanism to subvert NK cell activity.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

No description available.

No description available.

<p> Virus-induced modulation of dendritic cell function is thought to be an effective mechanism for viral-immune evasion. The severe-acute respiratory syndrome coronavirus (SARS-CoV) has been shown to infected human myeloid dendritic cells (MDCs) and directly modulate the cellular cytokine production. The ability of other human coronaviruses to infect MDCs and impair cell immune function has not been assessed. </p> <p> This thesis describes the infection of human MDCs with coronavirus 229E, NL63, and OC43. 229E showed productive, but limited genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. 229E infection stimulated IFN-α, IL-6 and MCP-1 production in MDCs, but little to no IL-12, TNF-α, IL-8, IP-10, or RANTES . 229E-infected MDCs showed poor CD80 expression, down-regulated CD86 and HLA-DR expression and were poor stimulators of CD4+ T cell proliferation. In contrast to 229E, OC43 showed persistent and productive genomic replication, nucleocapsid protein synthesis and infectious progeny release in MDCs. OC43 infection stimulated IFN-α, IL-12, IP-10 and MCP-1 production in MDCs, but little to no TNF-α, IL-6, IL-8 or RANTES . The up-regulation of maturation molecules and CD4+ T cell stimulatory capacity in OC43-infected MDCs was donor cell-dependent. In contrast to 229E and OC43, NL63 infection of MDCs was non-productive, showing no viral genomic replication, protein production or infectious progeny release. NL63 infection stimulated strong cytokine (IFN-α, IL-12, TNF-β and IL-6) and chemokine (IL-8, IP-10, RANTES and MCP-1) responses in MDCs. NL63-infected MDCs showed up-regulated CD80, CD83, CD86 and HLA-DR expression and were efficient stimulators of CD4+ T cell proliferation. </p> <p> This study provides the first evidence that human coronaviruses other than SARSCo V can abrogate MDC immune effector function. It also provides the first side-by-side comparison of 229E, NL63 and OC43 and identifies the potential of 229E and OC43 to impair MDC cytokine production and T cell stimulation as a mechanism of immune response evasion. <p> / Thesis / Doctor of Philosophy (PhD)