INTRODUÇÃO: A dosagem ideal de salbutamol através de inaladores dosimetrados para o tratamento da asma aguda na infância não está bem estabelecida. Este estudo visou comparar dois regimes de dosagem de salbutamol via inalador dosimetrado com espaçador em crianças com crises moderadas ou graves de asma. As hipóteses deste estudo foram: I. Pacientes em vigência de crises de sibilância moderadas ou graves necessitam doses de salbutamol maiores que as até recentemente recomendadas pelo Global Initiative for Asthma (GINA). II. Doses maiores de salbutamol são seguras para crianças com idade igual ou superior a dois anos de idade. OBJETIVOS: I. Avaliar a eficácia (principalmente tempo de permanência na sala de emergência e taxas de internação hospitalar) de um regime de doses maiores de salbutamol em comparação com as até recentemente recomendadas pelo GINA. II. Verificar a segurança destas dosagens de salbutamol através do monitoramento de possíveis efeitos colaterais e dos níveis plasmáticos desta droga. MÉTODOS: Este foi um estudo prospectivo, randomizado, duplo-cego e controlado realizado em salas de emergência de três centros na cidade de São Paulo. Foram incluídos pacientes com 2-17 anos de idade com asma aguda moderada a grave (escore PRAM, Pediatric Respiratory Assessment Measure, >= 5). As dosagens de salbutamol via inalador dosimetrado com espaçador administradas durante a primeira hora foram: 6 (até 25 kg) ou 12 jatos ( > 25 kg) no grupo controle e 9 (até 15 kg), 12 ( > 15 a 20 kg), 15 ( > 20 a 25 kg) ou 18 jatos ( > 25 kg) no grupo estudo. Cada jato continha 100 mcg de salbutamol. Os pacientes dos dois grupos receberam corticosteroides e brometo de ipratrópio. Os desfechos principais do estudo foram o tempo de permanência na sala de emergência para os pacientes não internados e a necessidade ou não de internação hospitalar. Os desfechos secundários foram: mudança no volume expiratório forçado no primeiro segundo (VEF1) após uma hora, mudanças no escore PRAM, na oximetria de pulso e na frequência respiratória após uma hora e no tempo final (momento da alta ou internação hospitalar, máximo de quatro horas) e a necessidade de tratamentos adicionais após a primeira hora. Os desfechos de segurança incluíram mudanças nos níveis séricos de potássio, glicose, bicarbonato e pH no tempo final em relação ao tempo inicial, bem como possíveis anormalidades no eletrocardiograma, níveis plasmáticos de salbutamol, mudanças na frequência cardíaca e presença ou ausência de tremores, os dois últimos após uma hora e no tempo final. RESULTADOS: Foram incluídos 119 pacientes com condições basais semelhantes e não foram observadas diferenças significativas entre os grupos no tempo de permanência na sala de emergência (p=0.55) ou nas taxas de internação hospitalar (p=0.48). Não foram observadas diferenças significativas entre os grupos nas mudanças de VEF1 após uma hora, nas mudanças de escore PRAM, na oximetria de pulso e na frequência respiratória após uma hora e no tempo final. Não houve diferenças significativas entre os grupos na necessidade de tratamentos adicionais administrados após a primeira hora. Não foram observadas diferenças significativas nos desfechos de segurança entre os grupos. CONCLUSÕES: O uso de doses maiores de salbutamol administradas através de inalador dosimetrado com espaçador em crianças com asma aguda moderada ou grave não resultou em menor taxa de internação, menor tempo de permanência na sala de emergência ou melhora em outros desfechos de eficácia em comparação com o regime de dosagens até recentemente proposto pelo GINA. Os dois regimes de dosagens mostraram perfis de segurança semelhantes / INTRODUCTION: The ideal dosing of albuterol via metered-dose inhalers for acute childhood asthma is not well established. This study aimed to compare two dosing regimens of albuterol via metered-dose inhaler with spacer in children with moderate to severe asthma attacks. The hypothesis of this study were: I. Patients with moderate to severe asthma attacks would benefit from higher doses of albuterol than those recommended until recently by the Global Initiative for Asthma (GINA). II. Higher doses of albuterol are safe for children two years of age and older. OBJECTIVES: I. To compare the efficacy (mainly length of stay in the emergency room and admission rates) of higher doses of albuterol with those recommended until recently by the GINA. II. To assess the safety of different doses of albuterol by monitoring for possible side effects and measuring drug plasma levels. METHODS: This was a prospective, randomized, controlled, double-blind study conducted in emergency rooms of the three participating centers in the city of São Paulo. We included patients with 2-17 years old with moderate to severe acute asthma (Pediatric Respiratory Assessment Measure, PRAM, score >= 5). Dosages of albuterol via metered-dose inhaler with spacer administered during the first hour included: 6 (up to 25 kg) or 12 puffs ( > 25 kg) in the control group and 9 (up to 15 kg), 12 ( > 15 to 20 kg), 15 ( > 20 to 25 kg) or 18 puffs ( > 25 kg) in the study group. Each puff contained 100 mcg of albuterol. All patients received corticosteroids and ipratropium bromide. Primary outcomes were the length of stay in the emergency room for non-admitted patients, and rate of admission. Secondary outcomes included forced expiratory volume in one second (FEV1) changes following one hour, PRAM score, pulse oximetry and respiratory rate changes following one hour and at the final time (discharge or admission, maximum four hours) and the need for additional therapies after the first hour. Safety outcomes included changes in serum potassium, glucose, bicarbonate and pH at the final time in comparison with the initial time, as well as electrocardiogram abnormalities, plasma albuterol levels, heart rate, and tremors (the last two after one hour and at the final time). RESULTS: We included 119 patients with similar baseline conditions, and no significant differences were observed between groups in the length of stay in the emergency room (p=0.55) or admission rates (p=0.48). No significant differences were observed between groups in FEV1 changes after one hour, and PRAM score, pulse oximetry and respiratory rate changes after one hour and at the final time. There were no significant differences between groups in additional therapies administered after the first hour. No significant differences were observed in safety outcomes between groups. CONCLUSIONS: Higher dosage regimens of albuterol via metered-dose inhaler with spacer for children with moderate to severe acute asthma did not result in lower admission rate, shorter length of stay in the emergency room or improvement in other efficacy outcomes in comparison with those recommended until recently by the GINA. Both dosage regimens showed similar safety profile

INTRODUÇÃO: Indivíduos infectados pelo HIV apresentam maior risco de quadros graves de infecção por influenza sazonal e, portanto, devem receber doses anuais da vacina contra gripe. No entanto, a capacidade dos indivíduos responderem às vacinas com títulos apropriados de anticorpos depende de variáveis como tipo de antígeno vacinal, idade e grau de comprometimento imunológico no momento da imunização. OBJETIVOS: 1) Avaliar a imunogenicidade da vacina contra influenza sazonal em 37 pacientes infectados pelo HIV, em comparação com 29 indivíduos não infectados pelo HIV 2) Realizar a vigilância dos episódios de infecções respiratórias durante o período de acompanhamento após a vacinação. MÉTODOS: Ambos os grupos receberam a vacina contra o vírus da influenza sazonal recomendada para o hemisfério sul em 2008. A resposta de anticorpos contra os antígenos H1N1, H3N2 e B foi medida em amostras de sangue extraídas 1-2h antes da vacinação (T0), após 1 mês (T1) e após 6 meses (T6; apenas no Grupo HIV). A vigilância dos sintomas respiratórios foi realizada através de telefonemas semanais, durante 6 meses após a vacinação. Em indivíduos sintomáticos para infecções respiratórios foram coletadas amostras de lavado nasofaríngeo para pesquisa de vírus respiratórios por Imunofluorescência e PCR: influenza A e B, parainfluenza 1, 2 e 3, adenovírus, metapneumovírus, vírus sincicial respiratório, rinovírus e coronavírus. RESULTADOS: A idade mediana da população de estudo foi de 12 (10-18) anos. No momento T1, ambos os grupos mostraram aumento significativo nos TMGs para todos os antígenos. Contudo, o grupo controle apresentou valores mais elevados para os antígenos A/H1N1 e A/H3N2 (p = 0,002 e 0,001, respectivamente). Houve maior aumento na porcentagem de indivíduos não infectados pelo HIV com títulos protetores A/H1N1 (96,6%) em comparação aos infectados pelo HIV (67,6%). No T1 (p=0,004). A porcentagem de indivíduos do grupo controle com aumento de quatro vezes ou mais nos títulos de anticorpos para A/H1N1 e A/H3N2 foram mais elevadas que no grupo HIV (p = 0,03 e 0,01, respectivamente). Agentes virais foram detectados em 39/60 (65%) dos episódios de infecção respiratória no grupo HIV e em 17/32 (53,1%) no grupo controle. Os vírus diagnosticados no grupo HIV e grupo controle foram respectivamente: adenovirus (8,6%), metapneumovirus (1,2%), rinovirus (16,8%), coronavirus (14,0 %) e influenza B (0,1%).CONCLUSÕES: A vacina sazonal contra os vírus da influenza foram imunogenicas em ambos os grupos. Ocorreram diferença nas taxas de soroproteção entre os grupos somente para o antígeno H1, que foi mais elevadas no grupo controle. O grupo controle também mostrou valores mais altos nos TMGs para os antígenos H1 e H3 depois da imunização. Os rinovirus (27,7%) e coronavirus (22,5%) foram os agentes mais prevalentes identificados no grupo infectado pelo HIV. No grupo controle, os vírus mais freqüentes foram os rinovirus (24,2%) e adenovirus (21,2%) / INTRODUCTION: Individuals infected with HIV are at higher risk for severe cases of seasonal influenza infection and therefore should receive annual doses of influenza vaccine. However, the ability to respond to vaccines respond appropriate antibodies titres depends on variables such as vaccine antigen, age and degree of immune impairment at immunization. OBJECTIVES: 1)To evaluate the immunogenicity of a seasonal influenza vaccine in 37 HIV-infected patients (HIV Group), compared to 29 uninfected individuals (Control Group) 2) To carry out a clinical and virological surveillance of influenza in this population during a follow-up period of six months. METHODS: Both groups received the vaccine against seasonal influenza virus recommended for the southern hemisphere in 2008. The antibody response against the antigens H1N1, H3N2 and B were measured in blood samples drawn at vaccination (T0), after 30 days (T1) and after 6 months (T6; only for HIV Group). Antibody titres >1:40 were considered protective against influenza infection A surveillance of respiratory symptoms was performed weekly by telephone calls for a post-vaccination follow-up period of 6 months. Samples were collected (nasal wash) if respiratory symptoms. DFA and real time PCR was used to diagnose influenza A virus (FLU A) and B (FLU B), respiratory syncytial virus (RSV), parainfluenza virus types 1, 2 and 3 ( Paraflu 1, 2 or 3), adenovirus, coronavirus, rhinovirus, metapneumovirus and bocavirus. RESULTS: The median age of the study population was 12 (10-18) years. At T0, there were no significant differences in the antibody geometric mean titres (GMTs) against all vaccine antigens between groups. One month after vaccination (T1), both groups showed significant increases in the antibody GMTs for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively). There was a higher increase in the percentage of HIVuninfected subjects with protective A/H1N1 antibodies (96.6%) comparing to HIVinfected vaccinees (67.6%) at T1 (p = 0.004). The percentage in subjects control group with a fourfold or greater increase of A/H1N1 and A/H3N2 antibody titres was higher than that found in HIV group (p = 0.03 and p = 0.01, respectively. Viral agents were identified in 39/60 (65%) episodes of respiratory infections in HIV-infected group and in 17/32 episodes (53.1%) from the control group (P=0.273). The virus diagnosed in HIV group and control group were, respectively: Adenovirus (8;6), Metapneumovirus(1;2) Rinovirus(16;8), Coronavirus(14 ;0); Influenza B(0;1). CONCLUSIONS: The seasonal influenza vaccine was immunogenic in both groups. There were differences in seroprotection rates between groups only for AgH1, which was higher in the control group. The control group also showed a greater increase in GMTs for H1 and H3 antigens after immunization. Viral agents were identified in respiratory symptoms during the follow-up: Rhinoviruses (27.7%) and coronavirus (22.5%) were the most prevalent agents identified in HIV-infected individuals. In the control group, the viruses most frequently found were rhinoviruses (24.2%) and adenovirus (21.2%)

INTRODUÇÃO: Indivíduos infectados pelo HIV apresentam maior risco de quadros graves de infecção por influenza sazonal e, portanto, devem receber doses anuais da vacina contra gripe. No entanto, a capacidade dos indivíduos responderem às vacinas com títulos apropriados de anticorpos depende de variáveis como tipo de antígeno vacinal, idade e grau de comprometimento imunológico no momento da imunização. OBJETIVOS: 1) Avaliar a imunogenicidade da vacina contra influenza sazonal em 37 pacientes infectados pelo HIV, em comparação com 29 indivíduos não infectados pelo HIV 2) Realizar a vigilância dos episódios de infecções respiratórias durante o período de acompanhamento após a vacinação. MÉTODOS: Ambos os grupos receberam a vacina contra o vírus da influenza sazonal recomendada para o hemisfério sul em 2008. A resposta de anticorpos contra os antígenos H1N1, H3N2 e B foi medida em amostras de sangue extraídas 1-2h antes da vacinação (T0), após 1 mês (T1) e após 6 meses (T6; apenas no Grupo HIV). A vigilância dos sintomas respiratórios foi realizada através de telefonemas semanais, durante 6 meses após a vacinação. Em indivíduos sintomáticos para infecções respiratórios foram coletadas amostras de lavado nasofaríngeo para pesquisa de vírus respiratórios por Imunofluorescência e PCR: influenza A e B, parainfluenza 1, 2 e 3, adenovírus, metapneumovírus, vírus sincicial respiratório, rinovírus e coronavírus. RESULTADOS: A idade mediana da população de estudo foi de 12 (10-18) anos. No momento T1, ambos os grupos mostraram aumento significativo nos TMGs para todos os antígenos. Contudo, o grupo controle apresentou valores mais elevados para os antígenos A/H1N1 e A/H3N2 (p = 0,002 e 0,001, respectivamente). Houve maior aumento na porcentagem de indivíduos não infectados pelo HIV com títulos protetores A/H1N1 (96,6%) em comparação aos infectados pelo HIV (67,6%). No T1 (p=0,004). A porcentagem de indivíduos do grupo controle com aumento de quatro vezes ou mais nos títulos de anticorpos para A/H1N1 e A/H3N2 foram mais elevadas que no grupo HIV (p = 0,03 e 0,01, respectivamente). Agentes virais foram detectados em 39/60 (65%) dos episódios de infecção respiratória no grupo HIV e em 17/32 (53,1%) no grupo controle. Os vírus diagnosticados no grupo HIV e grupo controle foram respectivamente: adenovirus (8,6%), metapneumovirus (1,2%), rinovirus (16,8%), coronavirus (14,0 %) e influenza B (0,1%).CONCLUSÕES: A vacina sazonal contra os vírus da influenza foram imunogenicas em ambos os grupos. Ocorreram diferença nas taxas de soroproteção entre os grupos somente para o antígeno H1, que foi mais elevadas no grupo controle. O grupo controle também mostrou valores mais altos nos TMGs para os antígenos H1 e H3 depois da imunização. Os rinovirus (27,7%) e coronavirus (22,5%) foram os agentes mais prevalentes identificados no grupo infectado pelo HIV. No grupo controle, os vírus mais freqüentes foram os rinovirus (24,2%) e adenovirus (21,2%) / INTRODUCTION: Individuals infected with HIV are at higher risk for severe cases of seasonal influenza infection and therefore should receive annual doses of influenza vaccine. However, the ability to respond to vaccines respond appropriate antibodies titres depends on variables such as vaccine antigen, age and degree of immune impairment at immunization. OBJECTIVES: 1)To evaluate the immunogenicity of a seasonal influenza vaccine in 37 HIV-infected patients (HIV Group), compared to 29 uninfected individuals (Control Group) 2) To carry out a clinical and virological surveillance of influenza in this population during a follow-up period of six months. METHODS: Both groups received the vaccine against seasonal influenza virus recommended for the southern hemisphere in 2008. The antibody response against the antigens H1N1, H3N2 and B were measured in blood samples drawn at vaccination (T0), after 30 days (T1) and after 6 months (T6; only for HIV Group). Antibody titres >1:40 were considered protective against influenza infection A surveillance of respiratory symptoms was performed weekly by telephone calls for a post-vaccination follow-up period of 6 months. Samples were collected (nasal wash) if respiratory symptoms. DFA and real time PCR was used to diagnose influenza A virus (FLU A) and B (FLU B), respiratory syncytial virus (RSV), parainfluenza virus types 1, 2 and 3 ( Paraflu 1, 2 or 3), adenovirus, coronavirus, rhinovirus, metapneumovirus and bocavirus. RESULTS: The median age of the study population was 12 (10-18) years. At T0, there were no significant differences in the antibody geometric mean titres (GMTs) against all vaccine antigens between groups. One month after vaccination (T1), both groups showed significant increases in the antibody GMTs for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively). There was a higher increase in the percentage of HIVuninfected subjects with protective A/H1N1 antibodies (96.6%) comparing to HIVinfected vaccinees (67.6%) at T1 (p = 0.004). The percentage in subjects control group with a fourfold or greater increase of A/H1N1 and A/H3N2 antibody titres was higher than that found in HIV group (p = 0.03 and p = 0.01, respectively. Viral agents were identified in 39/60 (65%) episodes of respiratory infections in HIV-infected group and in 17/32 episodes (53.1%) from the control group (P=0.273). The virus diagnosed in HIV group and control group were, respectively: Adenovirus (8;6), Metapneumovirus(1;2) Rinovirus(16;8), Coronavirus(14 ;0); Influenza B(0;1). CONCLUSIONS: The seasonal influenza vaccine was immunogenic in both groups. There were differences in seroprotection rates between groups only for AgH1, which was higher in the control group. The control group also showed a greater increase in GMTs for H1 and H3 antigens after immunization. Viral agents were identified in respiratory symptoms during the follow-up: Rhinoviruses (27.7%) and coronavirus (22.5%) were the most prevalent agents identified in HIV-infected individuals. In the control group, the viruses most frequently found were rhinoviruses (24.2%) and adenovirus (21.2%)

Lung cancer is the leading cause of cancer death in the United States, but a large fraction of cases is preventable. We use a spatial smoothing algorithm to identify a geographic pattern of high lung cancer mortality, primarily in the Southeast, which we call a lung cancer belt. Disease belts are an effective mode for conveying patterns of high incidence or mortality; formally defining this lung cancer belt may encourage increased public dialogue and more focused research. Public health officials could complement existing population lung cancer data with this information to help inform resource allocation decisions.

The purpose of this dissertation was to evaluate the biological, behavioral, and psychosocial attributes of individuals diagnosed with chronic obstructive pulmonary disease (COPD). Specific aims were to: 1) explore the predictive power of spirometry measures for event-free survival in patients with heart failure and suspected COPD, focusing on the differences in survival between those with and without airflow limitation; 2) examine the psychometric properties of the Multidimensional Scale of Perceived Social Support (MSPSS) in patients with concomitant COPD and heart failure; and 3) test the efficacy of a theory-based, multidimensional, self-care educational intervention using an eHealth platform on measures of symptom severity and variability, anxiety and depressive symptoms, perceived self-care ability, perceived self-care adherence, and selfcare information needs (knowledge) in a sample of adult patients with stable COPD. Specific aim one was addressed by evaluation of the predictive power of spirometry measures (forced expiratory volume/second [FEV1], forced vital capacity [FVC], and the ratio of FEV1/FVC) for event-free time to combined hospitalization/mortality after controlling for clinical and sociodemographic variables. This analysis revealed that those patients with airflow limitation were 2.2 times more likely to experience hospitalization/mortality compared to those without airflow limitation. The second specific aim was addressed with a psychometric evaluation of the Multidimensional Scale of Perceived Social support (MSPSS) which included determination of internal consistency reliability, the factor structure and construct validity by hypothesis testing in participants with comorbid COPD and heart failure. The MSPSS was a valid and reliable instrument to measure perceived social support in patients with comorbid COPD and heart failure. The third specific aim was addressed by a trial of an eHealth educational intervention in participants with COPD (N = 20). This intervention resulted in significant change in symptom severity evaluation in patients categorized as having medium symptom severity for the following symptoms: distress due to cough, chest tightness, dyspnea with activity and fatigue; these symptoms were perceived as more severe in the intervention period. Anxiety, depressive symptoms and perceived self-care ability were unchanged; however, perceived self-care adherence scores improved, and knowledge needs were significantly reduced after the intervention.

Alterations to sphingolipid metabolism are associated with increased pulmonary inflammation, but the impact of inflammatory mediators, such as neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. NE is a protease associated CF lung disease progression, and can be found in up to micromolar concentrations in patient airways. While sphingolipids have been investigated in the context of CF, the focus has been on loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we present a novel observation: oropharyngeal aspiration of NE increases airway ceramides in mice. Using a previously characterized mouse model of NE-induced inflammation, we demonstrate that NE increases de novo ceramide production, which is likely mediated via increased SPTLC2 levels. Inhibition of de novo sphingolipid synthesis using myriocin, an SPT inhibitor, decreases airway ceramide as well as the release of pro-inflammatory signaling molecules induced by NE. Furthermore, in a retrospective study of the sphingolipid content of CF sputum—the largest of its type in this patient cohort to date, we investigated the association between NE and sphingolipids. There were linear correlations between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA) positive culture and female gender both strengthened the association of NE and sphingolipids, but higher FEV1 % predicted weakened the association, and Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. These data suggest that NE may increase sphingolipids in CF airways as it did in our in vivo model, and that this association is stronger in patients that have worse lung function, are female, and whose lungs are colonized with MRSA. Modulating sphingolipid homeostasis could provide novel pharmacological approaches for alleviating pulmonary inflammation.

Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps, 1998; El- Adhami et al., 1999). In recent studies, the OMP26, when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al., 2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focussed on the immunological and structural characterisation of the NTHi vaccine candidate, OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA, the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.

<p>IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages <i>in vitro</i>. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.</p>

Viruses replicating in the respiratory tract (RT) triggers a wide- range of cytokines and chemokines that have antiviral and pro-inflammatory features, instigating an efficient virus- specific B and T cell response that aids in virus- clearance. The majority of antibody secreting cells (ASCs) localizing in the upper RT secrete IgA that can effectively neutralize viruses. In addition, elements of B cell memory are generated that can provide protection from re-infection. Studies examining these aspects, following murine gammaherpesvirus 68 (MHV-68) infection comprise chapter 2 of the dissertation work. Our studies demonstrate that following MHV-68 infection, unlike influenza infection, resulted in a generalized deficiency of virus-specific IgA induction and deficient B cell memory establishment in the respiratory tract. The studies indicate that these aspects of B cell response are regulated by features of virus- replication in the RT. These studies lead to the speculation that these features of B cell response may represent an evolutionary adaptation of viruses that establish long-term latency and are transmitted periodically after reactivation and shedding in secretions. Following cognate interactions with CD4+ T cells, the B cells undergo proliferation, isotype-switching and differentiate towards extrafollicular (low affinity, rapid) or germinal center pathway (high affinity). It is not clear what factors regulate these pathways of B cell differentiation, especially in the context of virus infection in the RT. Studies examining these aspects following influenza infection comprise chapter 3 of the dissertation work. Our studies establish a model for the investigation of host and viral factors that modulate the quality and effectiveness of the B cell response to influenza infection. The findings indicate that the strength of the extrafollicular B cell response depends on the nature of the infecting virus. We present evidence that this pathway of rapid antiviral antibody production relates to the production of non-specifically acting factors in the lung and also dependent of the cytokine profile of virus-specific CD4+T cells. In summary, the current dissertation findings point out to an influence of virus and host associated factors in regulating features of B cell response in the RT.

IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages in vitro. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.