The objective of this study was to purify IgG from commercially available hyperimmune Rhodococcus equi plasma and to assess the delivery of IgG as an aerosol to the equine lung. IgG was purified from plasma, and the IgG concentration of both the plasma and the purified IgG was determined by ELISA. The purified IgG was aerosolized using a vibrating mesh nebulizer and aerosol characterization was performed using cascade impaction. The purified IgG was nebulized to six healthy adult horses in order to assess the efficacy of pulmonary delivery and safety of administration. Bronchoalveolar fluid was retrieved endoscopically using a low volume technique prior to aerosolization (time 0) and at 0.5, 4 and 24 hours post aerosolization. The BAL fluid IgG concentration was determined and cytologic analysis was performed. The IgG concentrations of the plasma and purified IgG were 2,175 mg/dL and 1,145 mg/dL, respectively. The MMAD of the purified IgG aerosol was 4.7 microns. The mean BAL fluid IgG concentration increased 61% from 19.33 µg/dL at time 0 to 31.5 µg/dL at 0.5 hours, but this increase was not significant (P=0.603). No significant change was observed in inflammatory cell numbers over time or at any time point during the study. This study demonstrated that IgG antibodies were purified at a concentration acceptable for nebulization, and that the nebulization unit generated aerosol particles from the IgG solution of appropriate size for pulmonary delivery. Nebulization of purified IgG to adult horses was well tolerated and caused no local or systemic adverse effects. / Master of Science

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vii, 43 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 31-41).

A Mannheimia haemolytica é uma importante bactéria relacionada ao Complexo Doença Respiratória dos Bovinos e a essa atribui-se uma evolução para uma forma grave de pneumonia fibrinonecrótica. É considerada um habitante comensal da nasofaringe que em situações de comprometimento da resposta imune adquire um perfil oportunista. O presente estudo buscou observar por meio de modelo de infecção experimental, as possíveis alterações locais e sistêmicas causadas pela M. haemolytica em bezerros experimentalmente inoculados. Dessa forma seria possível de maneira longitudinal, acompanhar a dinâmica dos principais aspectos de defesa das vias aéreas posteriores durante a infecção e após o tratamento com o antimicrobiano norfloxacina associado ou não à flunexina meglumina. Avaliou-se por exame físico acrescido de broncoscopia, alterações funcionais das células de defesa e mediadores inflamatórios, tanto séricos quanto locais e a atividade in vitro da norfloxacina sobre a função dos fagócitos sanguíneos e do lavado broncoalveolar (LBA). Para tal foram utilizados 12 bezerros sadios que foram experimentalmente infectados por M. haemolytica dos quais foram avaliadas as alterações clínicas e, quantitativamente e funcionalmente, as populações leucocitárias no sangue e no LBA, assim como o efeito da norfloxacina sobre a atividade funcional dos fagócitos no sangue e no LBA. Os resultados do presente estudo demonstraram o sucesso da infecção experimental por M. haemolytica pelos achados clínicos, broncoscópicos e citológicos. Ademais, a infecção experimental por M. haemolytica foi associada a alterações nas subpopulações de linfócitos T CD8+ e уδ, na produção intracelular de espécies reativas de oxigênio (ERO), fagocitose e viabilidade pelas células CD14+ sanguíneas e do LBA e granulócitos do sangue e, expressão de L-selectina pelos leucócitos polimorfonucleares do sangue. Nenhuma alteração evidente foi observada na expressão de citocinas IL-1β, IL-8 e TNF-α nas células sanguíneas e do LBA. O tratamento com antimicrobiano associado ou não ao anti-inflamatório foi capaz de curar a infecção e reestabelecer os parâmetros avaliados à sua condição basal. Portanto, não se observou benefícios com a utilização adicional do anti-inflamatório no reestabelecimento do quadro clínico e da resposta imune neste experimento. Contudo, apesar de algumas alterações na resposta sistêmica durante o quadro infeccioso, as alterações locais foram mais perceptíveis. Outro aspecto importante encontrado foi o efeito in vitro da norfloxacina na produção intracelular de ERO, fagocitose bacteriana pelas células CD14+ sanguíneas e do LBA e em leucócitos polimorfonucleares no sangue. Conclui-se que as alterações funcionais dos fagócitos apresentaram papel importante na patogenia da mannheimiose, que foram condizentes com os achados clínicos da mannheimiose e da evolução do tratamento quando realizado no início do processo nosológico. / Mannheimia haemolytica is an important bacterial pathogen associated with Bovine Respiratory Disease Complex (BRDC) and it is believed to be the predominant cause of the disease’s evolution into a fibrinonecrotic pneumonia. A commensal inhabitant of the nasopharynx, M. haemolytica acts as an opportunist when host defenses are compromised. This study used an experimental infection model to investigate the possible local and systemic changes caused by M. haemolytica in inoculated calves. It sought to linearly follow the dynamics of the lower respiratory tract defense mechanisms, during the course of infection and after treatment with the antibacterial norfloxacin, which was administrated both with and without the anti-inflammatory flunixin meglumine. With clinical examination followed by bronchoscopy, this study evaluated the physiological modifications in defense cells and mediators of inflammation, and the in vitro influence of norfloxacin on phagocytes from the peripheral blood and Bronchoalveolar Lavage Fluid (BLF). Twelve (12) healthy calves were infected with M. haemolytica and posteriorly physically examined, and had samples of white cells from the peripheral blood and BLF analyzed for changes in count and physiology, further, the norfloxacin effect on phagocytes from the peripheral blood and BLF was also studied. The experimental infection proved itself to be successful based on clinical, bronchoscopic and cytological findings. Furthermore, the M. haemolytica experimental infection was associated with modifications in the subpopulations of lymphocytes CD8+ and уδ T cells, in intracellular production of Reactive Oxygen Species (ROS), viability and phagocytosis activity of CD14+ cells from the peripheral blood and BLF and granulocytes from the peripheral blood. No obvious change was observed in the expression of cytokines IL-1β, IL-8 e TNF-α by cells from the peripheral blood or BLF. The treatment with the antibacterial agent, with or without the anti-inflammatory, was proved to be successful in curing the disease, thus, the addition of an anti-inflammatory was considered unnecessary to revert the clinical infection and in the immune response. Although there was a systemic response during the course of infection, the local response was more noticeable. Another key finding of the present study was the in vitro effect of norfloxacin on the intracellular production of ROS and on phagocytosis activity of CD14+ cells from the peripheral blood and BLF and granulocytes from the peripheral blood. In conclusion, the functional changes in phagocytes play an important role in the pathogenesis of pulmonary infection caused by M. haemolytica, as they were consistent with the clinical findings of mannheimiosis and with the treatment when it was administrated in the beginning of the infection.

Objetivou-se caracterizar clinicamente a Doença Inflamatória de Vias Aéreas (DIVA) assim como verificar o comportamento da atividade dos macrófagos alveolares destes animais. Utilizaram-se 17 eqüinos adultos machos e fêmeas, com idade variando entre 11 e 24 anos, sendo oito do Regimento Escola de Cavalaria Andrade Neves (grupo Controle) e nove da Polícia Militar do Rio de Janeiro (grupo Doente, com DIVA). Estes animais não apresentavam história pregressa recente (dois meses) compatível com doença respiratória, nesse período não foram submetidos a nenhum tipo de tratamento e apresentavam resultados de leucogramas e determinações de fibrinogênio plasmático dentro da normalidade. Realizaram-se exames físicos, endoscópicos, mensurações da diferença de pressão intrapleural máxima (Ventigrafia), e citologia broncoalveolar. Para o grupo Controle os animais foram selecionados levando-se em conta os resultados de normalidade nos exames executados. O grupo Doente foi selecionado considerando-se achados de anormalidade compatíveis com alterações inflamatórias pulmonares no mínimo em dois exames, sendo obrigatórios os exames endoscópicos e de citologia broncoalveolar. Para a avaliação da atividade macrofágica, macrófagos alveolares obtidos através de lavagem broncoalveolar foram cultivados a 37ºC em atmosfera de 7% CO2, aderidos em lamínulas de vidro estéril, de 13 mm de diâmetro em placas de 24 poços contendo 300 µL de RPMI 1640 enriquecido com 10% de Soro Fetal Bovino inativado pelo calor. Realizaram-se testes de fagocitose de Zymosan (por uma hora) e de interação com Promastigotas de Leishmania braziliensis cepa 3456 (por uma até 48 horas pós infecção). A análise das funções vitais e das mensurações da diferença de pressão intrapleural máxima revelou, ainda que dentro de valores fisiológicos, diferenças significativas apenas para a temperatura corpórea e freqüência cardíaca. Para os achados do exame do exame físico, não foi detectada diferença estatística entre os grupos. Com relação ao lavado broncoalveolar, o grupo Doente apresentou aumento na contagem celular total, moderado aumento na porcentagem de neutrófilos, discreta redução na porcentagem de macrófagos, porém com aumento no número de macrófagos espumosos e de células epiteliais além de discreto aumento na porcentagem de eosinófilos. Não observou-se diferença, entre os grupos, na capacidade fagocítica com relação ao Zymosan. Porém, uma diferença significativa na capacidade fagocítica após uma hora de interação e redução do índice de sobrevivência de L. braziliensis após 48 horas de cultivo no grupo Doente foi observada. Os resultados demonstraram que a DIVA nos animais de policiamento apresentou caráter assintomático e que os macrófagos alveolares provenientes destes animais, quando comparados aos do grupo Controle, não apresentaram alteração em sua capacidade fagocítica para Zymosan, mas sim para L. braziliensis e um maior estado de ativação, caracterizado pela maior redução no índice de sobrevivência deste parasita após 48 horas de cultivo. / The objectives were to characterize clinically the inflammatory airway disease (IAD), as well as to verify the behavior of the alveolar macrophages activity in the horses. Seventeen adult horses, from both sexes, from 11 to 24 years old, were used, eight from the Regimento Escola de Cavalaria Andrade Neves (Control group) and nine from the Polícia Militar do Rio de Janeiro (Diseased group, with IAD). These animals did not present recente previous history (two months) compatible with respiratory disease. In this period the horses did not undergo any kind of treatment and presented leucogram and plasmatic fibrinogen determination results under normality. Physical and endoscopic examination, measurement of the maximal intrapleural pressure (Ventigraphy) and bronchoalveolar cytology were done. For the Control group, the animals were selected considering the normal results of the exams. The Diseased group was selected considering abnormal findings compatible with inflammatory pulmonary alterations at least in two exams, obligatory the endoscopic examination and bronchoalveolar cytology. To evaluate the macrophagic activity, alveolar macrophages obtained through bronchoalveolar lavage were cultured under 37oC in a 7% CO2 atmosphere, adhered to 13 mm diameter sterile glass cover slips , in 24 wells polystyrene plates containing 300µL of RPMI 1640 enriched with 10% Foetal Calf Serum inactivated by heat. Assays for phagocytosis of Zymosan (for 1h) and for promastigotes of Leishmania braziliensis strain 3456 binding (for one until 48 hours post-infection) were done. The vital functions and the measurement of the maximal intrapleural pressure measurement analyses showed, even under physiological values, significant differences for body temperature and heart rate. There was not statistical difference between the groups for the physical examination findings. In relation to the bronchoalveolar lavage, the Diseased group presented an increase in total cell count, a moderate increase in neutrophil percentage, a discrete reduction in macrophage percentage, but with a higher number of foamy macrophages and epithelial cells, and also a discrete increase of eosinophil percentage. It was not observed any difference between groups in the phagocytic capability in relation to Zymosan. However, a significant difference in the phagocytic capability after one hour interaction and a reduction of the survival index of L. braziliensis after 48 hours of culture in the Diseased group was observed. The results demonstrated that IAD in the police horses presented an asymptomatic characterization and that the alveolar macrophages from these horses, when compared to the Control group, did not present alteration in their phagocytic capacity for Zymosan, but did for L. braziliensis, and a greater activation status, characterized by a greater reduction of the survival index of L. braziliensis after 48 hours of culture.

As influências etárias do sistema imune de bezerros são descritas na primeira fase de vida desses animais e a hipótese de também ocorrerem ariações nos principais mecanismos de resposta inata do pulmão pode identificar períodos de maior suscetibilidade às principais doenças respiratórias que acometem os bezerros nesse período. Com a finalidade de minimizar os prejuízos econômicos associados às doenças respiratórias em bezerros, este estudo objetivou avaliar a dinâmica imunológica inata do sistema respiratório de bezerros sadios nos três primeiros meses de vida, no qual nove bezerros sadios foram acompanhados por três meses e submetidos a oito avaliações imunológicas. O material recuperado do lavado broncoalveolar colhido por broncoscopia foi submetido à avaliação funcional dos macrófagos alveolares utilizando as provas de fagocitose (SaPI e E.coli), burst oxidativo, quantificação de imunoglobulinas e expressão de CD14. Os dados foram avaliados pelo teste ANOVA oneway (unstacked) (paramétricos) e pelo teste Mann-Whitney (não paramétricos). Verificaramse alterações funcionais de fagócitos CD14+, que apesar de se manterem constantes em seus valores relativos durante todo o período, apresentou intensidade de fagocitose elevada pontual na terceira semana de vida e um aumento da fagocitose por mononucleares CD14+ aos 45 dias de idade com diminuição da intensidade da fagocitose por essas mesmas células a partir dessa idade. Conclui-se que a partir de 45 dias de vida os animais começam a montar uma resposta imune própria, porém pontual e que até os 90 dias não atingem a estabilidade necessária para atestar a conclusão do processo de maturação da resposta inata local. / The influences of age in calves\' immune system are described in their first phase of life. The hypothesis that variations occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. With the purpose of minimizing the economic losses associated with respiratory disease in calves, this study aimed to evaluate the innate immune dynamics of the respiratory system of healthy calves in the first three months of life. Nine healthy calves were monitored for three months and eight immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. Then, the alveolar macrophages in samples were identified by protein expression of CD14 and undergone functional evaluation of phagocytosis (SAPI and E.coli) and oxidative burst. Immunoglobulin were also quantified in samples. Data was assessed by one-way ANOVA (unstacked) (parametric) and the Mann-Whitney test nonparametric). Functional alterations in phagocytes CD14 + were observed, and although their relative values were kept throughout the period, higher intensity of phagocytosis in the third week and increased phagocytosis by macrophages CD14 + at 45 days of life was observed. Decreased intensity of phagocytosis was observed after this age. It is concluded that from 45 days of life on, calves began to maintain their immune response, but until 90 days of life they did not achieve the stability to conclude the maturation of local innate response.

Bronchoalveolar lavage (BAL) is an established method providing diagnostic support and evaluation of disease activity in interstitial lung disease (ILD). The aims of the present investigation were 1) to study the inflammatory response in pneumonitis evoked by irradiation. 2) to evaluate how well lung tissue inflammation is reflected in BAL findings. 3) to study the effect of smoking on radiation-induced pneumonitis. BAL was performed in 21 patients (11 smokers, 10 non-smokers) who were treated for breast cancer, stage 1 (TjMaNq) by post-surgery irradiation to an accumulated target dose of 56 Gy. It was founa that irradiation induced an alveolitis in the non-smoking patient group while the smoking patients did not differ from their smoking controls. The alveolitis in non-smokers was characterized by an increase in lymphocytes, mast cells and elevated concentrations of hyaluronan (HA), and fibronectin (FN). Three of the non-smoking patients had chest X-ray infiltrates indicating the presence of pneumonitis. An animal experimental model for radiation-induced pneumonitis and fibrosis was established in rats, allowing comparative analysis of BAL fluid and morphology. In the rat model a divergence was noted between the differential cell counts in BAL and cells observed in the interstitial tissue, which was most notable for neutrophils (PMN) and mast cells whereas there was a good correlation between HA content in BAL and HA deposition in the lung tissue. A marked infiltration of intraseptally-located mast cells occurred during the pneumonitis-phase, and this increase was paralleled by a deposition of HA in the interstitial tissue. Histochemical fixation and staining properties of the mast cells revealed that the majority of these cells were of connective tissue mast cell type (CTMC). Compound 48/80, a mast cell secretagogue, significantly altered the HA content both in BAL and in lung tissue in the irradiated animals. Regular treatment throughout the whole experimental period induced depletion of mast cell granules and a decrease in HA deposition whereas 48/80 treatment during the pneumonitis phase enhanced HA deposition. A rat model with smoke exposure was developed, and the effect of cigarette smoke on radiation-induced inflammation was studied. Rats that smoked 3 weeks prior to irradiation and continued to smoke throughout the observation period (7 weeks) had a significantly reduced inflammatory response compared to irradiated non-smoking rats. The most prominent BAL findings in the smoke-exposed rats were a decrease in PMN, mast cells and a decrease in HA. In conclusion, irradiation induces an alveolitis characterized mainly by mononuclear cells. Mast cells seem to be of importance in the remodelling of the connective tissue in the radiation-induced inflammatory response. Hyaluronan is an important component in the early connective tissue response preceding later collagen deposition, and its interstitial deposition is very well reflected in BAL. Moreover, tobacco-smoke suppresses the radiation-induced inflammation with a decreased recruitment of effector cells including mast cells. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1992, härtill 5 uppsatser.</p> / digitalisering@umu

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Objetivou-se caracterizar clinicamente a Doença Inflamatória de Vias Aéreas (DIVA) assim como verificar o comportamento da atividade dos macrófagos alveolares destes animais. Utilizaram-se 17 eqüinos adultos machos e fêmeas, com idade variando entre 11 e 24 anos, sendo oito do Regimento Escola de Cavalaria Andrade Neves (grupo Controle) e nove da Polícia Militar do Rio de Janeiro (grupo Doente, com DIVA). Estes animais não apresentavam história pregressa recente (dois meses) compatível com doença respiratória, nesse período não foram submetidos a nenhum tipo de tratamento e apresentavam resultados de leucogramas e determinações de fibrinogênio plasmático dentro da normalidade. Realizaram-se exames físicos, endoscópicos, mensurações da diferença de pressão intrapleural máxima (Ventigrafia), e citologia broncoalveolar. Para o grupo Controle os animais foram selecionados levando-se em conta os resultados de normalidade nos exames executados. O grupo Doente foi selecionado considerando-se achados de anormalidade compatíveis com alterações inflamatórias pulmonares no mínimo em dois exames, sendo obrigatórios os exames endoscópicos e de citologia broncoalveolar. Para a avaliação da atividade macrofágica, macrófagos alveolares obtidos através de lavagem broncoalveolar foram cultivados a 37ºC em atmosfera de 7% CO2, aderidos em lamínulas de vidro estéril, de 13 mm de diâmetro em placas de 24 poços contendo 300 µL de RPMI 1640 enriquecido com 10% de Soro Fetal Bovino inativado pelo calor. Realizaram-se testes de fagocitose de Zymosan (por uma hora) e de interação com Promastigotas de Leishmania braziliensis cepa 3456 (por uma até 48 horas pós infecção). A análise das funções vitais e das mensurações da diferença de pressão intrapleural máxima revelou, ainda que dentro de valores fisiológicos, diferenças significativas apenas para a temperatura corpórea e freqüência cardíaca. Para os achados do exame do exame físico, não foi detectada diferença estatística entre os grupos. Com relação ao lavado broncoalveolar, o grupo Doente apresentou aumento na contagem celular total, moderado aumento na porcentagem de neutrófilos, discreta redução na porcentagem de macrófagos, porém com aumento no número de macrófagos espumosos e de células epiteliais além de discreto aumento na porcentagem de eosinófilos. Não observou-se diferença, entre os grupos, na capacidade fagocítica com relação ao Zymosan. Porém, uma diferença significativa na capacidade fagocítica após uma hora de interação e redução do índice de sobrevivência de L. braziliensis após 48 horas de cultivo no grupo Doente foi observada. Os resultados demonstraram que a DIVA nos animais de policiamento apresentou caráter assintomático e que os macrófagos alveolares provenientes destes animais, quando comparados aos do grupo Controle, não apresentaram alteração em sua capacidade fagocítica para Zymosan, mas sim para L. braziliensis e um maior estado de ativação, caracterizado pela maior redução no índice de sobrevivência deste parasita após 48 horas de cultivo. / The objectives were to characterize clinically the inflammatory airway disease (IAD), as well as to verify the behavior of the alveolar macrophages activity in the horses. Seventeen adult horses, from both sexes, from 11 to 24 years old, were used, eight from the Regimento Escola de Cavalaria Andrade Neves (Control group) and nine from the Polícia Militar do Rio de Janeiro (Diseased group, with IAD). These animals did not present recente previous history (two months) compatible with respiratory disease. In this period the horses did not undergo any kind of treatment and presented leucogram and plasmatic fibrinogen determination results under normality. Physical and endoscopic examination, measurement of the maximal intrapleural pressure (Ventigraphy) and bronchoalveolar cytology were done. For the Control group, the animals were selected considering the normal results of the exams. The Diseased group was selected considering abnormal findings compatible with inflammatory pulmonary alterations at least in two exams, obligatory the endoscopic examination and bronchoalveolar cytology. To evaluate the macrophagic activity, alveolar macrophages obtained through bronchoalveolar lavage were cultured under 37oC in a 7% CO2 atmosphere, adhered to 13 mm diameter sterile glass cover slips , in 24 wells polystyrene plates containing 300µL of RPMI 1640 enriched with 10% Foetal Calf Serum inactivated by heat. Assays for phagocytosis of Zymosan (for 1h) and for promastigotes of Leishmania braziliensis strain 3456 binding (for one until 48 hours post-infection) were done. The vital functions and the measurement of the maximal intrapleural pressure measurement analyses showed, even under physiological values, significant differences for body temperature and heart rate. There was not statistical difference between the groups for the physical examination findings. In relation to the bronchoalveolar lavage, the Diseased group presented an increase in total cell count, a moderate increase in neutrophil percentage, a discrete reduction in macrophage percentage, but with a higher number of foamy macrophages and epithelial cells, and also a discrete increase of eosinophil percentage. It was not observed any difference between groups in the phagocytic capability in relation to Zymosan. However, a significant difference in the phagocytic capability after one hour interaction and a reduction of the survival index of L. braziliensis after 48 hours of culture in the Diseased group was observed. The results demonstrated that IAD in the police horses presented an asymptomatic characterization and that the alveolar macrophages from these horses, when compared to the Control group, did not present alteration in their phagocytic capacity for Zymosan, but did for L. braziliensis, and a greater activation status, characterized by a greater reduction of the survival index of L. braziliensis after 48 hours of culture.