In vitro studies indicate that the inflammatory response to zymosan, a fungal wall preparation, is dependent on Toll-like receptor (TLR) 2, and that this response is enhanced by the dectin-1 receptor. Complement may also play an important role in this inflammatory response. However, the relevance of these molecules within the in vivo pulmonary environment remains unknown. To examine pulmonary in vivo inflammatory responses of the lung to zymosan, zymosan was administered by intratracheal aerosolization to C57BL/6, TLR2- TLR4-, MyD88-, and complement-deficient mice. Outcomes included bronchoalveolar fluid cell counts. We next examined effects of dectin-1 inhibition on response to zymosan in alveolar macrophages in vitro and in lungs of C57BL/6, TLR2-, and complement-deficient mice. Finally, the effect of alveolar macrophage depletion on in vivo pulmonary responses was assessed. Marked zymosan-induced neutrophil responses were unaltered in TLR2-deficient mice despite a TLR2-dependent response seen with synthetic TLR2 agonists. TLR4, MyD88, and complement activation were not required for the inflammatory response to zymosan. Although dectin-1 receptor inhibition blocked the inflammatory response of alveolar macrophages to zymosan in vitro, in vivo pulmonary leukocyte recruitment was not altered even in the absence of TLR2 or complement. Depletion of alveolar macrophages did not affect the response to zymosan. Neither complement, macrophages, nor TLR2, TLR4, MyD88, and/or dectin-1 receptors were involved in the pulmonary in vivo inflammatory response to zymosan.

Pneumocystis carinii is an opportunistic fungal pathogen that causes lifethreatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis (PC) pulmonary infections. The higher incidence of PC as well as other pulmonary infections among infants is likely due to an immature immune system. The neonatal lung environment is deficient immunologically in preterm as well as term infants (1, 2). Decreased phagocytic capacity of macrophages in newborns may increase the risk of infection from inhaled pathogens (1, 2). We have previously demonstrated that there is approximately a 3-week delay in the clearance of PC organisms from pup mouse lungs compared to adults. Herein, we demonstrate that there is also a 1-week delay in the infiltration of AMs in pup compared to adult PC-infected mice. We go on to show that there is a delay in pup versus adult lung macrophage phenotypic expression and cytokine production in response to PC organisms. We demonstrated that pup AMs are competent to produce cytokine in response to LPS and that stimulation with zymosan generates cytokine production in pup AMs that is comparable to adult cytokine production. These data indicate that pup lung macrophages are specifically poorly responsive to PC organisms and likely require exogenous stimulation to mount a significant immune response and expedite clearance of the organism. We go on to show that heat-killed Escheriae coli improves cytokine response, cellular infiltration and reduces organism burden in PC-infected pup mice. The clinically relevant cytokine, GM-CSF, has been used to improve the clearance of several pulmonary infections, including PC in adult animal models. We show that monotherapy with GM-CSF is insufficient to improve PC clearance in pup mice; however, when combined with TMP/SMX it improves PC clearance and maintains a reduced PC burden following discontinuation of therapy. Furthermore, we have shown that GM-CSF improves the ability of human infant lung macrophages to phagocytose PC organsms without generating an increased inflammatory response. These data suggest that combination therapy with TMP/SMX and GM-CSF may be a viable treatment option for infants failing or intolerant to standard therapy.

Streptococcus pneumoniae (pneumococcus) is commonly found in the nasopharynx of healthy individuals, yet it can be a serious pathogen, particularly in the lower respiratory tract, where it can cause severe pneumonia. During pneumococcal pneumonia, anti-bacterial host defense requires the orchestrated expression of innate immunity mediators, initiated by alveolar macrophages and dependent on transcriptional activity driven by Nuclear Factor-𝜅B (NF-𝜅B). Although the initiation of a pulmonary inflammatory response is critical to anti-pneumococcal defense during pneumonia, how differences in pneumococcal-macrophage interactions can influence this process is unclear. To determine the functional significance of varying macrophage NF-𝜅B activation, we examined macrophage responses to pneumococcal stimulation in culture and in mice. Macrophage-pneumococcal interactions resulted in the induction of varied NF-𝜅B activation. Two main pathways were revealed regarding host response and disease outcome. Pneumococci that induced efficient macrophage NF-𝜅B activation resulted in robust anti-pneumococcal lung defense and bacterial clearance. Conversely, failure to activate effective macrophage NF-𝜅B signaling resulted in an altered macrophage response of necroptosis. Overall, we conclude that varying levels of macrophage NF-𝜅B activation by pneumococcus can directly influence the severity of infection. Furthermore, inefficient macrophage NF-𝜅B activation can also have cytotoxic effects on these critical lung resident cells during pneumonia. The induction of macrophage NF-𝜅B activation by S. pneumoniae is as diverse as the population of pneumococcal isolates in the community. A unique host-pathogen interaction exists between pneumococcus and the alveolar macrophage that plays an important role in anti-pneumococcal defense during pneumonia and in the prevention of cytotoxic consequences induced by virulent pneumococci. This interaction suggests that therapies, which modulate NF-𝜅B activation, hold promise for augmenting resistance and ameliorating deleterious effects during pneumococcal pneumonia that could lead to the development of severe disease.

No description available.

No description available.

A idade entre o terceiro e o sexto mês de vida é um período peculiar para o estabelecimento da imunidade própria dos bezerros. Nesse intervalo, há susceptibilidade e índice de letalidade à broncopneumonias maiores, provavelmente devido à imaturidade do sistema imunológico pulmonar, quer seja por uma insuficiente resposta, nos primeiros momentos, quer seja por uma resposta citotóxica exagerada, no momento subsequente. A par disso, este trabalho teve o intuito de verificar o momento em que ocorre a maturidade do sistema imunológico, como se comportam os perfis Th1 e Th2 e a existência de uma resposta citotóxica exagerada, durante esta fase de estabelecimento da imunidade ativa dos bezerros. Para tal, estudaram-se as funções de fagocitose e metabolismo oxidativo de leucócitos sanguíneos e broncoalveolares, as classes de imunoglobulinas e citocinas incriminadas nos padrões de resposta linfocitária Th1 e Th2, em 10 bezerros da raça holandesa, sadios, avaliados em sete momentos experimentais, com intervalo quinzenal, entre o terceiro e o sexto mês de vida. Os dados foram submetidos à análise estatística, pela comparação entre as médias ou medianas, confrontadas pelo teste de Anova e Tukey, quando paramétricas, e pelo teste de Kruskal Wallis e Dunn, quando não paramétricas, considerando nível de significância P≤ 0,05 e tendência P≤ 0,10. Os dados que apresentaram dinâmicas semelhantes entre si foram submetidos ao teste de correlação de Pearson. Na região broncoalveolar, observaram-se um aumento progressivo das funções dos macrófagos alveolares, equilíbrio de secreção dos isotipos IgG1 e IgG2 e predominância de citocinas compatíveis com padrão de resposta Th1, até os 150 dias de vida dos bezerros. Aos 165 dias de vida, ocorreram diminuição da função celular, aumento dos títulos de IgG2 e aumento da citocina regulatória IL-10. Aos 180 dias, retornou-se o equilíbrio entre secreção de IgG1 e IgG2, diminuiram os teores de IL-10 e ocorreu tendência a aumento de IL-12, TNF-α e metabolismo oxidativo de macrófagos alveolares, o que permitiu concluir que a resposta imune tem característica própria, nesta faixa etária, e não se torna matura até os seis meses de vida. Apesar de os fagócitos pulmonares já estarem eficientes, a partir dos 135 dias de vida, tornam-se hiperresponsivos aos 150 dias de vida, momento em que gera consequentemente uma resposta regulatória e/ou humoral aos 165 dias de vida, para que aos 180 dias de vida, o equilíbrio entre os perfis Th1 e Th2 seja atingido / The age between the third and sixth month of life is a peculiar period for the establishment of active immunity of calves. There is a greater susceptibility and lethality by bronchopneumonia, probably due to the immaturity of the pulmonary immune system, whether by an insufficient response , in the first moments , whether by an exaggerated cytotoxic response in the subsequent time. So, this work aimed to verify when the maturity of the immune system occurs, how the Th1 and Th2 profiles behave and if there is an exaggerated cytotoxic response during this active phase for immunity of the calves without maternal interference. For this purpose we studied the functions of phagocytosis and oxidative metabolism of blood and bronchoalveolar leukocytes, classes of immunoglobulins and cytokines incriminated in lymphocyte response patterns Th1 and Th2, in 10 holstein healthy calves. They were sampled every fifteen days, during the third until sixth month of life. Data were statistically analyzed by comparing the means or medians, confronted by ANOVA test and Tukey, when the data were parametric, and by Kruskal Wallis and Dunn's test, when the data were nonparametric, level of significance p ≤ 0.05 and trend p ≤ 0.10. The data that showed similar dynamics between them were subjected to Pearson correlation test. In bronchoalveolar region, until 150 days of age, the alveolar macrophages functions increased progressively, the IgG1 and IgG2 isotypes secretion showed a balance, and the cytokines profile were compatible with Th1 response. At 165 days of age, there was a decrease of cellular function, an increased of IgG2 titers and the IL-10 secretion, a regulatory cytokine, increased. At 180 days of life, we observed a balance of IgG1 and IgG2 secretion, a decreased of IL-10 levels and a tendency to increase IL-12, TNF-α and alveolar macrophage oxidative metabolism. These results indicated that the calves have an active immune response with particularities for this age group and it does not become mature until six months of life. Despite of the macrophages alveolar are already efficiency from the 135 days of age, they become more reactive at 150 days. After this moment, a regulatory and/or humoral response begins at 165 days of life, as the balance of Th1 and Th2 profiles are reached at 180 days of calves life

A idade entre o terceiro e o sexto mês de vida é um período peculiar para o estabelecimento da imunidade própria dos bezerros. Nesse intervalo, há susceptibilidade e índice de letalidade à broncopneumonias maiores, provavelmente devido à imaturidade do sistema imunológico pulmonar, quer seja por uma insuficiente resposta, nos primeiros momentos, quer seja por uma resposta citotóxica exagerada, no momento subsequente. A par disso, este trabalho teve o intuito de verificar o momento em que ocorre a maturidade do sistema imunológico, como se comportam os perfis Th1 e Th2 e a existência de uma resposta citotóxica exagerada, durante esta fase de estabelecimento da imunidade ativa dos bezerros. Para tal, estudaram-se as funções de fagocitose e metabolismo oxidativo de leucócitos sanguíneos e broncoalveolares, as classes de imunoglobulinas e citocinas incriminadas nos padrões de resposta linfocitária Th1 e Th2, em 10 bezerros da raça holandesa, sadios, avaliados em sete momentos experimentais, com intervalo quinzenal, entre o terceiro e o sexto mês de vida. Os dados foram submetidos à análise estatística, pela comparação entre as médias ou medianas, confrontadas pelo teste de Anova e Tukey, quando paramétricas, e pelo teste de Kruskal Wallis e Dunn, quando não paramétricas, considerando nível de significância P≤ 0,05 e tendência P≤ 0,10. Os dados que apresentaram dinâmicas semelhantes entre si foram submetidos ao teste de correlação de Pearson. Na região broncoalveolar, observaram-se um aumento progressivo das funções dos macrófagos alveolares, equilíbrio de secreção dos isotipos IgG1 e IgG2 e predominância de citocinas compatíveis com padrão de resposta Th1, até os 150 dias de vida dos bezerros. Aos 165 dias de vida, ocorreram diminuição da função celular, aumento dos títulos de IgG2 e aumento da citocina regulatória IL-10. Aos 180 dias, retornou-se o equilíbrio entre secreção de IgG1 e IgG2, diminuiram os teores de IL-10 e ocorreu tendência a aumento de IL-12, TNF-α e metabolismo oxidativo de macrófagos alveolares, o que permitiu concluir que a resposta imune tem característica própria, nesta faixa etária, e não se torna matura até os seis meses de vida. Apesar de os fagócitos pulmonares já estarem eficientes, a partir dos 135 dias de vida, tornam-se hiperresponsivos aos 150 dias de vida, momento em que gera consequentemente uma resposta regulatória e/ou humoral aos 165 dias de vida, para que aos 180 dias de vida, o equilíbrio entre os perfis Th1 e Th2 seja atingido / The age between the third and sixth month of life is a peculiar period for the establishment of active immunity of calves. There is a greater susceptibility and lethality by bronchopneumonia, probably due to the immaturity of the pulmonary immune system, whether by an insufficient response , in the first moments , whether by an exaggerated cytotoxic response in the subsequent time. So, this work aimed to verify when the maturity of the immune system occurs, how the Th1 and Th2 profiles behave and if there is an exaggerated cytotoxic response during this active phase for immunity of the calves without maternal interference. For this purpose we studied the functions of phagocytosis and oxidative metabolism of blood and bronchoalveolar leukocytes, classes of immunoglobulins and cytokines incriminated in lymphocyte response patterns Th1 and Th2, in 10 holstein healthy calves. They were sampled every fifteen days, during the third until sixth month of life. Data were statistically analyzed by comparing the means or medians, confronted by ANOVA test and Tukey, when the data were parametric, and by Kruskal Wallis and Dunn's test, when the data were nonparametric, level of significance p ≤ 0.05 and trend p ≤ 0.10. The data that showed similar dynamics between them were subjected to Pearson correlation test. In bronchoalveolar region, until 150 days of age, the alveolar macrophages functions increased progressively, the IgG1 and IgG2 isotypes secretion showed a balance, and the cytokines profile were compatible with Th1 response. At 165 days of age, there was a decrease of cellular function, an increased of IgG2 titers and the IL-10 secretion, a regulatory cytokine, increased. At 180 days of life, we observed a balance of IgG1 and IgG2 secretion, a decreased of IL-10 levels and a tendency to increase IL-12, TNF-α and alveolar macrophage oxidative metabolism. These results indicated that the calves have an active immune response with particularities for this age group and it does not become mature until six months of life. Despite of the macrophages alveolar are already efficiency from the 135 days of age, they become more reactive at 150 days. After this moment, a regulatory and/or humoral response begins at 165 days of life, as the balance of Th1 and Th2 profiles are reached at 180 days of calves life

This study was designed to accomplish two primary objectives: (1) to compare two test methods commonly used to evaluate toxicity of inhaled air pollutants, and (2) to observe the response as measured by each of the methods, of pulmonary alveolar macrophages exposed to 2μg hexavalent chromium. The firs t method evaluated featured use of intratracheal injections to simulate live inhalation exposures, and the second required exposure of macrophages cultured on petri plates. Pulmonary alveolar macrophages harvested from Long Evans rats were used. The two cell function parameters measured in the evaluations were chemiluminescence and oxygen consumption (which was determined for cells at rest and during phagocytosis). These two tests have been shown to be sensitive indicators of macrophage damage. Results of CL output and oxygen consumption revealed the two methods were significantly different. Evaluation of macrophages from live animals treated with CrO3 or CaCrO4showed no differences between their respective untreated controls as determined by measurement of their chemiluminescence production or of oxygen consumption rates. Alveolar macrophages that were cultured in media during treatment with the same two forms of hexavalent chromium showed statistically significant differences from untreated controls. These comparisons indicate that choices of investigative toxicological models influence interpretation of data recorded.

Indiana University-Purdue University Indianapolis (IUPUI) / Pneumocystis pneumonia (PCP) is the leading opportunistic disease in immunocompromised individuals, particularly in AIDS patients. The alveolar macrophage (AM) is the major type of cell responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PCP due to increased intracellular polyamine levels. This study examined the mechanism of this polyamine mediated apoptosis and investigated an alternative therapy for PCP by targeting this mechanism. The elevated polyamine levels were determined to be caused by increased polyamine synthesis and uptake. Increased polyamine uptake was found to be AM-specific, and recruited inflammatory cells including monocytes, B cells, and CD8+ T cells were found to be a potential source of polyamines. The expression of the antizyme inhibitor (AZI), which regulates both polyamine synthesis and uptake, was found to be greatly up-regulated in AMs during PCP. AZI overexpression was confirmed to be the cause of increased polyamine synthesis and uptake and apoptosis of AMs during PCP by gene knockdown assays. Pneumocystis organisms and zymosan were found to induce AZI overexpression in AMs, suggesting that the β-glucan of the Pneumocystis cell wall is responsible for this AZI up-regulation. In addition, levels of mRNA, protein, and activity of polyamine oxidase (PAO) were also found to be increased in AMs during PCP, and its substrates N1-acetylspermidine and N1-acetylspermine were found to induce its up-regulation. These results indicate that the H2O2 generated during PAO-mediated polyamine catabolism caused AMs to undergo apoptosis. Since increased polyamine uptake was demonstrated to be a pathogenic mechanism of PCP in this study, the potential therapeutic activity of five putative polyamine transport inhibitors against PCP was tested. Results showed that compound 44-Ant-44 significantly decreased pulmonary inflammation, organism burden, and macrophage apoptosis, and prolonged the survival of rats with PCP. In summary, this study demonstrated that Pneumocystis organisms induce AZI overexpression, leading to increased polyamine synthesis, uptake, and apoptosis rate in AMs and that targeting polyamine transport is a viable therapeutic approach against PCP.

Masters of Science / The infants of women who smoke during pregnancy have a lower birth mass than those born of women who abstain. Animal studies reveal that reduced growth due to maternal nicotine exposure during gestation is accompanied by lung hypoplasia. Biochemical analysis suggests that these lungs contain more cells which implies that lung damage occurs. In this study we examined the in vivo effects of maternal nicotine exposure (lmg/Kg/day), the equivalent of 32 cigarettes per day, on the following parameters of fetal and neonatal Wistar rat lung:(i) the content and distribution of glycogen in fetal and neonatal lung (ii) the status of connective tissue in neonatal lung (iii) the cell composition of the alveoli in neonatal lung. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Transmission electron microscope (TEM) techniques were employed to investigate the characteristics and composition of the alveolus The results show clearly that maternal nicotine exposure elevates pulmonary alveolar macrophage numbers'(PAM's) and lung glycogen levels. The quantity of elastic fibres in 1 day old neonates was significantly reduced but no changes in the quantity of reticulin and collagen fibres was observed. As a result of this change in connective tissue status, emphysema-like lesions and alveolar collapse was evident in the lungs of nicotine-exposed pups. TEM investigations revealed that changes to the composition of alveoli occurred. These included increased numbers of type II pneumocytes with high numbers of lamellar bodies with degenerative changes. Thickening of the blood-air barrier was also observed. The effect of maternal nicotine exposure has been documented in this study. However, it has not been possible to pinpoint the mechanisms involved but explanations have been proposed. Further research is required to elucidate the mechanisms by which nicotine produces these effects. Information thus obtained could help prevent the harmful effects to the fetus and neonate caused by smoking during pregnancy.