Abstract The cytochrome P450 (CYP) enzyme system in human lung is an essential component in the pulmonary carcinogenicity of several inhaled xenobiotic compounds. The aim of this study was to elucidate the expression and regulation of xenobiotic-metabolizing CYP enzymes in human lung. To evaluate which of the two is a better surrogate cell model for lung tissue, the expression patterns of CYP enzymes in alveolar macrophages and peripheral blood lymphocytes were clarified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared to the expression in lung tissue. The pattern of CYP expression in alveolar macrophages was found to closely resemble the expression pattern in human lung tissue, while the pattern in lymphocytes was markedly different. The expression of CYP2B6, CYP2C, CYP3A5, and CYP4B1 mRNAs in alveolar macrophages was demonstrated for the first time. To facilitate mechanistic studies on human pulmonary CYP induction, the A549 lung adenocarcinoma cell line was characterized by RT-PCR, and the CYP expression pattern was found to compare reasonably well to human lung epithelial cells. The induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) behaved as predicted, and CYP1B1 and CYP3A5 were also inducible by TCDD and dexamethasone, respectively. TCDD elevated the level of CYP1A1 mRNA (56-fold), while the induction of CYP1B1 mRNA was more modest (2.5-fold). The tyrosine kinase inhibitor genistein and the protein kinase C inhibitor staurosporine blocked CYP1A1 induction by TCDD, but did not affect CYP1B1 induction. The serine/threonine protein phosphatase inhibitor calyculin A and okadaic acid enhanced CYP1B1 induction slightly, but did not alter CYP1A1 induction. The expression of CYP3A forms in human pulmonary tissues was studied with RT-PCR and immunohistochemistry, and both methods established CYP3A5 as the main CYP3A form. CYP3A4 was expressed in only about 20% of the cases. In A549 cells, CYP3A5 was induced about 4-fold by the glucocorticoids budesonide, beclomethasone dipropionate, and dexamethasone. Maximal induction was achieved by concentrations as low as ~100 nM, suggesting that CYP3A5 could be induced in vivo in patients using inhaled glucocorticoids. However, there were no differences in CYP3A5 expression in alveolar macrophages in current glucocorticoid users, ex-users, and non-users. Cigarette smoking had a marked decreasing effect on CYP3A5 levels in alveolar macrophages. The presence and possible induction of CYP3A5 by glucocorticoids in human lung could have consequences for the maintenance of physiological steroid hormone balance in lung and the individual susceptibility to lung cancer of patients using glucocorticoids.

L’asthme équin est une maladie obstructive récurrente des voies respiratoires. De nombreux facteurs étiologiques ontété déterminés comme pouvant initier ou maintenir l’asthme équin sévère (AES), cependant l’exposition au foin restele facteur de risque principal. L’utilisation de traitements à base de corticoïdes sont efficaces en cas de crise maisinefficaces en l’absence de mesures sanitaires. Ainsi, depuis quelques années un dispositif permettant de « purifier »le foin à la vapeur a été développé, afin de diminuer la teneur en poussières et les antigènes microbiologiques présentsdans le foin. La physiopathologie de l’AES est complexe. Parmi les principaux acteurs de cette réponse immunitaire,les macrophages alvéolaires ont un rôle prédominant dans l’initiation et l’orientation de la réponse immunitaire. Ainsiétudier le rôle des macrophages des chevaux AES permettrait de mieux comprendre l’initiation de la réponseimmunitaire dans l’AES. Dans ce contexte, nous nous sommes interrogés sur l’influence du foin « à la vapeur » surla réponse immunitaire des chevaux AES lors d’un challenge in vivo et lors de la stimulation in vitro des macrophagesalvéolaires. Ainsi nous avons d’abord étudié la réponse systémique et locale des chevaux soumis à un challenged’exposition in vivo à du foin sec ou « à la vapeur ». La première partie de ce travail nous a permis d’observer uneffet bénéfique du foin « à la vapeur » sur le score de mucus trachéal des chevaux CTL et AES cependant nousn’avons pas observé un effet bénéfique significatif du foin « à la vapeur » sur la réponse cytologique et immunitaire.La deuxième partie du travail a permis d’étudier la réponse au foin « à la vapeur » à l’échelle des macrophagesalvéolaires. La microscopie en temps réel a mis en évidence des différences dans le comportement des macrophagesen réponse (1) à différents stimuli, (2) entre les chevaux AES et CTL, (3) entre les HDS provenant de foin sec ou defoin « à la vapeur ». La concentration en IL-1β était significativement plus élevée et la concentration en IL-10significativement plus faible chez les MA des chevaux AES comparés aux chevaux CTL. Une concentration en TNF-α plus élevée après stimulation in vitro a été observée chez les MA des chevaux AES et CTL. Cet état des lieux surla réponse des macrophages alvéolaires stimulés in vivo et in vitro pourra servir de base aux études futures nécessairespour conclure sur le rôle des MA dans le cas de l’AES. Cette triple approche à l’échelle du cheval, du poumon et dela cellule permet une vision globale de la réponse à un challenge d’exposition à du foin sec ou « à la vapeur » etpermet tout de même d’avoir un regard optimiste sur l’utilisation du foin « à la vapeur » pour les chevaux atteintsd’AES. / Equine asthma is a recurrent obstructive disease of respiratory tract. Several aetiologic factors are known to induceor maintain the severe equine asthma (sEA), however the hay exposure is the main risk factor. The use of treatmentswith corticoids are effective in case of crisis, but ineffective in the absence of sanitary measures. So, since severalyears a device allowing to purify the hay with steam was developed, in order to decrease the dust content and themicrobiological antigens within the hay. The pathophysiology of sEA is complex. Among the main actors of thisimmune response, the alveolar macrophages have an important role in the intiatiation and orientation of the immuneresponse. So, investigating the role of the equine alveolar macrophages of sEA horses would allow to betterunderstand the initiation of the immune response in the sEA. In this context, we focused on the influence of steamedhay on the immune response of sEA horses during an in vivo challenge and an in vitro stimulation of the alveolarmacrophages. First, we studied the systematic and local responses of horses submitted to an in vivo challengeexposure to dry and steamed hay. The first part of this work allowed us to show a beneficial effect of the steamedhay on the mucus tracheal score of sEA horses, however we did not observe any beneficial effect of the steamed hayon the cytological and immune response. The second part of the study allowed us to investigate the response of thealveolar macrophages to steamed hay. The real time microscopy showed differences in the behavior of macrophagesin response (1) to various stimuli, (2) between sEA and CTL horses (3) between HDS resulting from dry hay or fromsteamed hay. Moreover, the protein quantification of IL-1β was signifantly higher and the concentration of IL-10significantly lower in AM supernatant of sEA horses compared to CTL. The TNF-α concentration was higher on AMafter in vitro stimulation in sEA and CTL horses. These results on the alveolar macrophages reponse after both invivo challenge and in vitro stimulation, can be used as a basis for future studies in order to further characterize therole of AM in case of sEA. This triple approach on the horse, the lung and the cell scale allows a global vision of theresponse to an exposure challenge to dry or steamed hay and allows having an optimistic preliminary look on the useof the steamed hay for sEA horses.

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A2α (cPLA2α) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA2α in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the β-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1-/- alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA2α on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-α production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA 2α in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

Pneumonia is considered to be one of the leading causes of death worldwide. The outcome depends on both, proper antibiotic treatment and the effectivity of the immune response of the host. However, due to the complexity of the immunologic cascade initiated during infection, the latter cannot be predicted easily. We construct a biomathematical model of the murine immune response during infection with pneumococcus aiming at predicting the outcome of antibiotic treatment. The model consists of a number of non-linear ordinary differential equations describing dynamics of pneumococcal population, the inflammatory cytokine IL-6, neutrophils and macrophages fighting the infection and destruction of alveolar tissue due to pneumococcus. Equations were derived by translating known biological mechanisms and assuming certain response kinetics. Antibiotic therapy is modelled by a transient depletion of bacteria. Unknown model parameters were determined by fitting the predictions of the model to data sets derived from mice experiments of pneumococcal lung infection with and without antibiotic treatment. Time series of pneumococcal population, debris, neutrophils, activated epithelial cells, macrophages, monocytes and IL-6 serum concentrations were available for this purpose. The antibiotics Ampicillin and Moxifloxacin were considered. Parameter fittings resulted in a good agreement of model and data for all experimental scenarios. Identifiability of parameters is also estimated. The model can be used to predict the performance of alternative schedules of antibiotic treatment. We conclude that we established a biomathematical model of pneumococcal lung infection in mice allowing predictions regarding the outcome of different schedules of antibiotic treatment. We aim at translating the model to the human situation in the near future.

No description available.

In the last decade, the potential of β-glucan, a fungal cell wall component, to induce epigenetic and functional modification of innate immune cells, signified as trained innate immunity (TII) has been demonstrated in several pre-clinical and clinical studies. Parenteral administration of β-glucan has resulted in centrally induced TII in the bone marrow/circulating monocytes. Such trained innate immune cells play a critical role in protection against secondary infections. However, there are now indications that inducing local long-lasting immunity at mucosal barrier tissues such as the lung is warranted for protective immunity against respiratory pathogens. Currently, it remains unclear whether respiratory mucosal administration of β-glucan will induce long-lasting resident-memory macrophages and TII and if so, what are the underlying mechanisms of development and maintenance of memory macrophages at respiratory mucosa. To address this, and kinetics of immune responses in the lung were studied. Profound changes in airway macrophage (AM) pools were observed starting from 3 days post-exposure, which was associated with monocyte recruitment, and this was followed by a series of phenotypic shifts in AMs. The altered AM phenotype profile persisted for up to 8 weeks post-exposure. Importantly, β-glucan-trained AMs demonstrated heightened MHC II expression, enhanced responses to secondary stimulation and improved capacity to perform bacterial phagocytosis. Furthermore, mice with, β-glucan-trained AMs displayed higher rates of survival and improved bacterial control, in the lung and periphery, following a lethal S. pneumoniae infection. Our findings together indicate that a single intranasal delivery of β-glucan is able to train AMs. Further work into epigenetics, metabolism, and the contribution of AMs in protection is needed. / Thesis / Master of Health Sciences (MSc) / The immune system has been classically divided into two major compartments known as the innate and adaptive immune system. For decades, the predominant consensus amongst the field was that only the adaptive immune system can form memory against any pathogens encountered. It has been well established that plants and invertebrates only possess an innate immune system and still show boosted responses and enhanced protection against previously encountered as well as new pathogens. Recently, such capacity for innate immune memory has also been demonstrated in humans and pre-clinical animal models. Innate immune memory provides non-specific, broad- spectrum protection whereas adaptive memory is specific to a singular pathogen. Inducing broad-spectrum protection can be crucial for the future of human medicine. Activation of both adaptive and innate immune arms could prove to be extremely beneficial in vaccination strategies. Through the use of a pre-clinical model, we have found that administering β-glucan, a component of fungal cell wall, directly into the lung significantly alters the phenotype and functionality of lung immune cells, and also provides enhanced protection against a heterologous infection.

Die Ergebnisse der Arbeit weisen mehrfach auf eine defizitäre IL-10 Produktion in Alveolarmakrophagen von Asthmatikern hin. Die reduzierte IL-10 Expression auf Protein- und Genebene korrelierte mit einer erhöhten Produktion proinflammatorischer Zytokine (TNF-?, MIP1-?, GM-CSF). Diese Beobachtung impliziert einen Defekt in der IL-10 Synthese, der in einer verstärkten und prolongierten pulmonalen Entzündungsantwort resultiert. Daraus läßt sich schlußfolgern, dass beim Asthma bronchiale eine Dysbalance zwischen pro- und antiinflammatorischen Zytokinen pathogenetisch von Bedeutung ist. Die verringerte Sensitivität von Alveolarmakrophagen auf die inhibitorischen Effekte von exogenem IL-10 im Vergleich zu Blutmonozyten ist durch Unterschiede in den Mechanismen der Signaltransduktion bedingt (37, 54). Der Nachweis der Expression von proinflammatorischen Zytokinen in Bronchialmyozyten (RANTES, IL-8) führte zu einer Neubewertung dieser Zellen als Immuneffektorzellen in der Pathogenese des Asthma bronchiale. Neben der Kontraktilität sind Myozyten auch aktiv an der Aufrechterhaltung der Atemwegsentzündung beteiligt. Die inhibitorischen Effekte von IL-10 und IL-13 auf die Synthese proinflammatorischer Chemokine (RANTES, IL-8, MIP-1() in migrierten Entzündungszellen und residenten Bronchialmyozyten konnten in verschiedenen Arbeiten gut dokumentiert werden. Die Vielzahl antiinflammatorischer Effekte von IL-10, die sich auf unterschiedliche Zellsysteme wie Monozyten, Makrophagen und Bronchialmyozyten erstrecken, unterstreicht die pathogenetische Bedeutung dieses Zytokins. Der molekulare Mechanismus, welcher die IL-10 Wirkung vermittelt, ist derzeit noch nicht vollständig aufgeklärt. Angenommen wird eine rezeptorvermittelte Inhibition von Transkriptionsfaktoren des Stat Systems und NF-(B (76). Zukünftige molekularbiologische und klinische Studien sind jedoch notwendig, um den Kenntnisstand der Effekte antiinflammatorischer Zytokine zu vertiefen, und die Gabe von rekombinantem IL-10 als möglichen Ansatz zur Therapie chronisch entzündlicher Lungenerkrankungen zu evaluieren (81). / The results of this present thesis show a deficiency of IL-10 production in alveolar macrophages in asthma. The reduced IL-10 expression on protein and m-RNA level correlated with an increased production of pro-inflammatory cytokines such as TNF-(, MIP1- ( and GM-CSF. These observations implicate an impaired IL-10 synthesis in asthma with a subsequent prolongation of the inflammatory response. This leads to the conclusion that a dysbalance between pro- and anti-inflammatory cytokines is present in asthma and may be therefore of pathogenetic importance. The reduced sensitivity of alveolar macrophages to the inhibitory effects of exogenous IL-10 compared to peripheral blood monocytes may be caused by different signal transduction mechanisms. The expression of the proinflammatory cytokines RANTES and IL-8 in cultured human airway smooth muscle cells led to the conclusion that airway smooth muscle cells may act beside their contractile function as immunomodulatory cells in the pathogenesis of asthma. The inhibitory effects of IL-10 and IL-13 on the synthesis of proinflammatory cytokines (RANTES, IL-8, MIP1-() in immigrated inflammatory cells and resident cells such as airway smooth muscle cells have been shown in several publications that are part of the present thesis. The numerous antiinflammatory effects of IL-10 on different inflammatory cell systems such as monocytes/macrophages and smooth muscle cells underline the pathogenetic importance of this cytokine. The molecular mechanisms that mediate the IL-10 effects involve the transcription factors NF-(B and the Stat-System. Future studies are needed to determine the molecular mechanisms of the anti-inflammatory effects of IL-10 and IL-13 more deeply and to evaluate their application for the therapy of chronic inflammatory pulmonary diseases.

Abstract Aptosis denotes a biochemically and morphologically distinct chain of events leading to self-destruction of cell. It is pivotal in the maintenance of tissue homeostasis and also plays a role in neoplasm. In this work, the extent of apoptosis and apoptosis regulating proteins and factors was studied in a total of 94 patients operated for lung carcinoma, including 56 small cell lung carcinomas (SCLC) and 38 large cell lung carcinomas (LCLC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using 3'- end labelling of the apoptotic DNA. The extent of apoptosis in SCLC was compared with the cell proliferation activity as determined by Ki-67 immunohistochemistry, with the volume density of necrosis and with the occurrence of immunohistochemically detectable p53 and bcl-2 proteins. In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and with low differentiation degree in LCLC and that it is regulated by bcl-2 family proteins, the extent of apoptosis and tumour necrosis was analysed in relation to the expression of bcl-2 family proteins bcl-2, mcl-1, bax and bak. Apoptosis, tumour infiltrating lymphocytes (TILs), and angiogenesis are important factors that contribute to tumour growth. In the present study immunohistochemical methods were used to investigate the relationships of these factors and their role in the prognosis of the patients with LCLC and SCLC. A remarkably high apoptotic activity was detected in both SCLC and LCLC. The mean apoptotic index in SCLC was 2.70 % and in LCLC 2.49 %. Exceptionally high proliferation activity and high percentage of tumour necrosis was seen in SCLC. 58 % of SCLC showed more than 40 % of Ki-67 positive nuclei, and tumour necrosis was seen in 83 % of the cases. P53 protein accumulation was detected in 38 % and bcl-2 expression in 50 % of SCLC. The extent of apoptosis in SCLC was inversely related to tumour necrosis and p53 protein accumulation. In LCLC, bcl-2 expression was detected in 40 % of the cases. It was associated with neuroendocrine differentiation and predicted favourable prognosis of the patients. A high number of T cells and macrophages with a small number of B cells was detected in both SCLC and LCLC. The occurrence of intratumoural cytotoxic CD8 cells was associated with the occurrence of apoptotic bodies in SCLC. The increased number of intratumoural T cells, CD8-positive cells and macrophages predicted favourable prognosis of the patients with SCLC. In LCLC, an increased number of B cells and macrophages, but not T cells, was associated with better survival. Iaddition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test whether such cells could be found in sputum smears and if their presence could be utilised as a marker of malignancy in tumour diagnosis, the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) was analysed in 84 sputum samples and 13 broncho-alveolar lavage (BAL) specimens from patients with and without lung carcinoma. AMWABs could be found in cytological samples of the patients with lung carcinoma. In sputum and BAL specimens, enhanced apoptosis, as measured by an increased number of AMWABs reflected and was indicative of malignancy. This was also true for cytological specimens of the patients even when the actual malignant cells were not found. Therefore the AMWABs served as a marker of pulmonary malignancy.

African Swine Fever Virus (ASFV) is an Asfivirus and is the only member of the family Asfarviridae. It manifests as a disease that varies from acute to sub-acute or chronic forms. A true carrier state in domestic pigs is unknown but chronically affected individuals may carry and spread the virus for extended periods. African Swine Fever (ASF) is a socio-economically important disease characterized by high morbidity and mortality affecting the livelihood of many small to big scale farmers and seriously compromising international trade. Strategic measures to control this disease are by physical containment and culling in outbreak situations. There is no vaccine available. Nevertheless, every pork producer should ideally be actively involved in having biosecurity measures in place to avoid contamination and contacting their veterinary services in case of suspicion of ASF to have appropriate samples analysed. Official veterinary services must be equipped with proper diagnostic tools in order to provide a quick response. The sensitivity of currently available diagnostic tests at the Transboundary Animal Diseases Programme, Onderstepoort Veterinary Institute was analysed in order to report the best technique available. Sensitivity to ASF virus infection and therefore diagnostic potential of cell primary cultures as bone marrow macrophages, blood macrophages and alveolar macrophages was done via comparison of titre results from inoculations of ASFV SPEC 257 as control, and ASFV MOZ 1/98. In addition, molecular detection of specific DNA fragments within the viral genome were compared using five different PCRs. Bone marrow macrophage cultures and blood macrophage cultures were the most reliable cells whereas alveolar macrophages more often showed contamination. Results show that PPA PCR and real time PCR detected the highest diluted samples, thus the lowest concentration of virus, in both trials done with ASFV MOZ 1/98 and ASFV SPEC 257. In addition, animal trials were performed by inoculating domestic pigs with four different ASFV isolates of varying pathogenicity. These viruses were all from distinct geographic origins. Non-virulent ASFV OURT 3/88 and high virulent ASFV BENIN 1/97 were previously described and used as reference viruses. ASFV MOZ 1/98, suspected of having high virulence and ASFV MKUZE, which was thought to be of low virulence were included in this study to provide further information on the pathological and clinical outcome of the disease as well as measuring viral replication in various organs and blood. The study showed that ASFV MKUZE was of intermediate virulence, whilst ASFV MOZ 1/98 was highly virulent with a high mortality rate. Results confirmed the inadequacy of ASFV MKUZE to act as vaccine opposed to ASFV OURT 3/88. Following this, a potential vaccine by use of attenuated Portuguese ASFV OURT 3/88 tested against virulent heterologous challenge with a strain now known with certainty to cause acute ASF, the isolate ASFV MOZ 1/98 collected from a diseased pig in Mozambique. Domestic commercial pigs where submitted to either one or two vaccinations before challenge. Viral load in blood and tissue samples was higher in unvaccinated animals and higher in single vaccinated than in pigs vaccinated twice. However, acute ASF afflicted all groups with severe clinical signs and post-mortem lesions. Although it did not confer total immunity it was determined that pigs vaccinated with European attenuated ASFV OURT 3/88 acquired partial protection against challenge with virulent southern Africa ASFV MOZ 1/98. / Dissertation (MSc)--University of Pretoria, 2012. / gm2014 / ab2015 / Veterinary Tropical Diseases / unrestricted

Le Virus Respiratoire Syncytial (VRS) est responsable de 70 % des cas de bronchiolite chez les enfants de moins de cinq ans. La survenue de bronchiolites sévères chez le nourrisson est un facteur de risque de développement d’asthme en grandissant. Aucun vaccin contre le VRS n’est disponible chez l’Homme. Le système immunitaire inné est la première ligne de défense de l’organisme contre les infections. De plus, en interaction avec la flore bactérienne commensale des poumons, l’immunité innée participe à la maturation de la réponse immunitaire adaptative qui confère à l’individu une protection sur le long terme vis-à-vis des pathogènes. Afin d’expliquer la susceptibilité néonatale au VRS, nous avons caractérisé un nouveau mécanisme de contrôle de la réponse innée antivirale lors de l’infection de souriceaux. Nous avons également testé une nouvelle approche de modulation de la réponse immunitaire au VRS par le microbiote pulmonaire. Ainsi, mieux comprendre les mécanismes immunologiques et virologiques responsables de bronchiolites sévères en période néonatale permettra de développer des moyens de lutte sûrs et efficaces contre l’infection par le VRS. / Respiratory Syncytial Virus (RSV) is responsible for 70 % of bronchiolitis in children under five years old. Severe bronchiolitis in infants is a risk factor for asthma development. No vaccine against RSV is available in humans. The innate immune system is the first line of defense against infections. Moreover, in interaction with lung microbiota, innate immunity shapes adaptive immune response responsible for long-term protection against pathogens. To explain the susceptibility of young children to RSV, we characterized a novel regulatory mechanism of the innate antiviral response during neonatal RSV infection in the murine model. We also tested a new approach for modulating immune responses to RSV by the pulmonary microbiota. Thus, a better understanding of immunological and virological mechanisms responsible for severe bronchiolitis during the neonatal period will allow the development of safe and effective therapeutic strategies against RSV infection.