Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions. / Master of Science

Objective - evaluate horses with acute airway obstruction using three treatment regimens: tapering doses of dexamethasone (DEX), environmental modification (ENV), and a combination of both treatments (DEX + ENV) by analyzing clinical parameters, pulmonary function testing, bronchoalveolar lavage fluid (BALF) cytology and BALF cell expression of the cytokines IFN-? and IL-4 Animals - 6 horses with recurrent airway obstruction (RAO) Procedures - Clinical examination, pulmonary function test, and collection of BALF prior to treatment and during 22 day treatment period Hypothesis - Alterations in clinical parameters, pulmonary function and airway inflammation in acute equine RAO will return to remission values by treating with DEX, ENV or DEX + ENV Results - All horses demonstrated clinical disease, reduced pulmonary dynamic compliance (Cdyn) and an increased maximum change in pleural pressures (?Pplmax) when in a challenge environment. All treatments improved clinical parameters, ?Pplmax and Cdyn. BALF cytology during an RAO crisis demonstrated neutrophilic inflammation. ENV or DEX + ENV resulted in a significant decrease in airway neutrophilia that was maintained throughout the treatment period. In contrast, treatment with DEX caused a reduction in airway neutrophilia initially followed by a rebound neutrophilia as the period between administrations of dexamethasone (0.05mg/kg) was increased to 72 hours. The rebound neutrophilia was not accompanied by equivalent deterioration in clinical parameters or pulmonary function. Conclusions - Environmental modification is important in the management of RAO horses. Treatment of clinical RAO with a decreasing dosage protocol of corticosteroids in the absence of environmental modification results in the persistence of airway inflammation without recrudescence of clinical disease. / Master of Science

Introduction: The pathogenesis and treatment of allergic bronchopulmonary aspergillosis (ABPA), severe asthma-non fungal sensitised (SANFS) and severe asthma with fungal sensitization (SAFS) is poorly understood. IL-17A, IgE and microbiome may be associated with pathogenesis of asthma, but their role in fungal-associated asthma is uncertain. Further, the efficacy of voriconazole, posaconazole and nebulised amphotericin B (NAB) in ABPA and SAFS has not been fully studied. Aims and objectives: The aim of this PhD thesis was to evaluate the role of IL-17A, IgE and lung microbiome in patients with SANFS, SAFS and ABPA. We also studied the efficacy and safety of NAB, voriconazole and posaconazole. Methods: Airway lymphocytes and peripheral blood mononuclear cells (PBMC) from patients with ABPA (n=16), SAFS (n=15), SANFS (n=11), mild asthma (MA) (n=6) and NH (n=11) were characterized by flow cytometric analysis (FACS) to determine the % of CD (+) IL-17A expressing cells. We also evaluated microbiome population using culture and PCR plus sequencing from BAL of these patients. In chapter 3, we analysed total and specific IgE in blood from adult cohorts of SAFS (n=34) and ABPA (n=48) using ImmunoCAP 100. In chapter 5 we studied the efficacy of voriconazole and posaconazole and in chapter 6; we studied the efficacy of NAB.Results: %CD4+IL-17A expressing cells were significantly higher in patients with severe asthma and correlated positively with serum neutrophil and presence of fungi in the airways. ABPA, SAFS and SANFS were similar but all were significantly higher than MA and NH. There were no differences in IL-17A expression between blood and the lung. Fungi were more frequently associated with severe asthma and low FEV1. Steroid treatment significantly increased airway fungal load. IgE against staphylococcal aureus (SE-IgE) correlated positively with FEV1 and OCS dose. Voriconazole and posaconazole improved asthma severity and radiological abnormalities. NAB was associated bronchospasm, but was extrely effective in the few patients (n=3) that took treatment for >12 months. These responders had unique characteristics. Conclusions: IL-17A, SE-IgE, and lung microbiome are associated with asthma severity. Steroid use in these patients may increase airway fungal load. Whereas voriconazole and posaconazole are efficacious, the use of NAB is associated with significant bronchospasm. SE-IgE -high asthma patients may be a distinct asthma phenotype. Larger studies are needed.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-17T14:40:56Z No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T14:35:33Z (GMT) No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) / Made available in DSpace on 2016-06-28T14:35:33Z (GMT). No. of bitstreams: 1 anaclaudiacarvalhogouveia.pdf: 2060044 bytes, checksum: a2757483182ff953fdffbf86f346380b (MD5) Previous issue date: 2012-08-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A asma alérgica é uma doença inflamatória crônica das vias aéreas, caracterizada por uma resposta de hipersensibilidade imediata, obstrução brônquica, inflamação pulmonar e níveis elevados de IgE. A doença é mediada principalmente por uma resposta imunológica alérgeno-específica tipo Th2. Nas últimas décadas, a prevalência da asma alérgica tem aumentado significativamente, sobretudo nos países desenvolvidos. A Hipótese da Higiene atribui este aumento a uma menor exposição a determinados microrganismos durante a infância, quando o amadurecimento adequado do sistema imunológico requer estímulos que induzam respostas imunológicas de perfil Th1, fundamentais para o equilíbrio de respostas Th2 exacerbadas. Diversos trabalhos epidemiológicos parecem comprovar esta hipótese, evidenciando a existência de uma relação inversa entre o contato com microrganismos indutores de uma resposta Th1 e o desenvolvimento de asma alérgica. Paralelamente, estudos em modelos murinos constataram que o tratamento com Mycobacterium bovis BCG (BCG) reduz respostas Th2 alérgenoespecíficas. No entanto, os mecanismos pelos quais a micobactéria inibe o desenvolvimento da resposta alérgica são ainda pouco conhecidos. Este estudo avaliou o efeito da administração do BCG sobre a resposta imunológica ocorrida na alergia pulmonar em camundongos BALB/c previamente sensibilizados e desafiados com OVA. Vinte e quatro horas após o último desafio, o sangue e o lavado broncoalveolar foram coletados para análises de imunoglobulinas e contagem de células, respectivamente. Adicionalmente, os pulmões foram submetidos à análise histológica, avaliação da atividade de EPO e dosagens de citocinas e quimiocinas, assim como avaliação da expressão de CTLA-4, Foxp3 e IL-10 por citometria de fluxo. Os resultados obtidos indicam que o tratamento com BCG melhorou o processo alérgico através da redução dos principais parâmetros relacionados à resposta Th2, como o infiltrado eosinofílico pulmonar, a atividade de EPO, IL-4, IL-13, CCL11, além de IgE e IgG1 específicas anti-OVA. Por outro lado, a administração da micobactéria aumentou os níveis de IFN-γ, IL-10 e TGF-β, além das expressões de Foxp3 e CTLA-4 pelos linfócitos T CD4+. Paralelamente, houve um aumento na produção de IL-10 pelos linfócitos T CD8+. Esses dados sugerem que, além da indução de uma resposta imune Th1, a ação imunomoduladora do BCG está relacionada também à indução de mecanismos reguladores. / Atopic asthma is a chronic respiratory disease characterized by airway hyperresponsiveness, reversible airway obstruction, lung inflammation, and high levels of allergen-specific IgE, driven by allergen-specific Th2 cells. The increasing prevalence of allergic diseases, particularly in industrialized countries, has led to the hygiene hypothesis, which states that the newborn infant’s immune system is skewed toward Th2 responses and needs timely and appropriate environmental stimulus to create a balanced immune response. Supporting this hypothesis, epidemiological and experimental evidence has shown an inverse correlation between Th1-induced microbial infections and atopic asthma. Similarly, some animal studies have demonstrated that exposure to Mycobacterium tuberculosis or to environmental mycobacteria is able to protect against the development of allergic responses. However the exact mechanism underlying this inhibition still remains poorly understood. This study aimed to evaluate the ability of BCG to suppress an established allergic response in a mouse model of OVA-induced airway inflammation. To achieve this, OVA sensitized and challenged BALB/c mice were twice treated with BCG via nasal and 21 days after the first treatment, mice were rechallenged with OVA. Twenty-four hours after the last challenge, blood samples were collected to detect anti-OVA immunoglobulin isotypes, and bronchoalveolar lavage (BAL) was harvested for cell count. Additionally, lungs were collected for histological analysis, detection of EPO activity and measurement of cytokines and chemokines. The expression of CTLA-4, Foxp3 and IL-10 was also determined in lung tissue by flow cytometry. The data indicated that BCG treatment was able to inhibit an established allergic Th2-response by decreasing the allergen-induced eosinophilic inflammation, EPO activity, levels of IL-4, IL-13, CCL11 and serum levels of IgE and IgG1. Mycobacteria treatment increased lung levels of IFN-γ, IL-10 and TGF-β, and expressions of Foxp3 and CTLA-4 in CD4+T cells. Additionally, an increased production of IL-10 by CD8+ T cells was observed, even though no detectable changes in CD4+IL-10+ was noticed. Altogether, these results suggest that the mechanism underlying the down-regulatory effects of BCG on OVA-induced airway inflammation appear to be associated with the induction of both Th1 and T regulatory immune responses.

Les immunoglobulines intraveineuses (IVIg) constituent une préparation polyclonale d’IgG isolée et regroupée à partir du plasma sanguin de multiples donneurs. Initialement utilisé comme traitement de remplacement chez les patients souffrant d’immunodéficience primaire ou secondaire, les IVIg sont maintenant largement utilisées dans le traitement de plusieurs conditions auto-immunes, allergiques ou inflammatoires à une dose élevée, dite immunomodulatrice. Différents mécanismes d’action ont été postulés au fil des années pour expliquer l’effet thérapeutique des IVIg dans les maladies auto-immunes et inflammatoires. Entre autre, un nombre grandissant de données issues de modèles expérimentaux chez l’animal et l’humain suggère que les IVIg induisent l’expansion et augmentent l’action suppressive des cellules T régulatrices (Tregs), par un mécanisme qui demeure encore inconnu. Également, les patients atteints de maladies auto-immunes ou inflammatoires présentent souvent un nombre abaissé de Tregs par rapport aux individus sains. Ainsi, une meilleure compréhension des mécanismes par lesquels les IVIg modulent les cellules T régulatrices est requise afin de permettre un usage plus rationnel de ce produit sanguin en tant qu’alternative thérapeutique dans le traitement des maladies auto-immunes et inflammatoires. Par le biais d’un modèle expérimental d’allergie respiratoire induite par un allergène, nous avons démontré que les IVIg diminuaient significativement l’inflammation au niveau des voies aériennes ce, en association avec une différenciation des Tregs à partir des cellules T non régulatrices du tissu pulmonaire. Nous avons également démontré qu’au sein de notre modèle expérimental, l’effet anti-inflammatoire des IVIg était dépendant des cellules dendritiques CD11c+ (CDs) pulmonaires, puisque cet effet pouvait être complètement reproduit par le transfert adoptif de CDs provenant de souris préalablement traitées par les IVIg. À cet effet, il est déjà établi que les IVIg peuvent moduler l’activation et les propriétés des CDs pour favoriser la tolérance immunitaire et que ces cellules seraient cruciales pour l’induction périphérique des Tregs. C’est pourquoi, nous avons cherché à mieux comprendre comment les IVIg exercent leur effet sur ces cellules. Pour la première fois, nous avons démontré que la fraction d’IgG riche en acide sialique (SA-IVIg) (constituant 2-5% de l’ensemble des IgG des donneurs) interagit avec un récepteur dendritique inhibiteur de type lectine C (DCIR) et active une cascade de signalement intracellulaire initiée par la phosphorylation du motif ITIM qui est responsable des changements observés en faveur de la tolérance immunitaire auprès des cellules dendritiques et des Tregs. L’activité anti-inflammatoire de la composante SA-IVIg a déjà été décrite dans des études antérieures, mais encore une fois le mécanisme par lequel ce traitement modifie la fonction des CDs n’a pas été établi. Nous avons finalement démontré que le récepteur DCIR facilite l’internalisation des molécules d’IgG liées au récepteur et que cette étape est cruciale pour permettre l’induction périphérique des Tregs. En tant que produit sanguin, les IVIg constitue un traitement précieux qui existe en quantité limitée. La caractérisation des mécanismes d’action des IVIg permettra une meilleure utilisation de ce traitement dans un vaste éventail de pathologies auto-immunes et inflammatoires. / Intravenous immunoglobulin (IVIg) is a therapeutic preparation of normal human polyclonal IgG derived from pooled plasma from a large number of healthy donors. Initially used as replacement therapy for patients with primary and secondary immune deficiencies, IVIg is now also widely used for the treatment of a variety of autoimmune, allergic and systemic inflammatory disorders, at high immunomodulatory doses. The beneficial effect of IVIg in autoimmune and inflammatory diseases has been attributed to different mechanisms. Increasing evidence shows that IVIg induces expansion and enhances the suppressive function of regulatory T cells (Tregs) in different experimental animal models and human subjects, through an unknown mechanism. Human inflammatory and autoimmune diseases are known to be associated with Treg deficiency. Therefore, a more precise understanding of the mechanisms by which IVIg modulate Treg populations seems to be needed for more rational use of this compound as an alternative therapy in context of various inflammatory and autoimmune disorders. Using a robust antigen-driven model of allergic airway disease, we have demonstrated that IVIg markedly attenuates airway inflammation and this effect is associated with the induction of Tregs from non-regulatory T cells in pulmonary tissues. We have also demonstrated that the antiinflammatory actions of IVIg, in our model are dependent on a population of pulmonary CD11c+ dendritic cells (DCs), as the action of IVIg could be completely replicated by adoptive transfer of CD11c+ DCs from IVIg-treated mice. we have shown that tolerogenic DCs involve in the peripheral induction of Tregs. Given the requirement of DCs in the induction of Tregs, we explored the mechanism by which IVIg interacts and modulate these cells and for the first time demonstrated that the purified sialylated fraction of human IgG (SA-IVIg) (that consists 2-5% of whole IgG) interacts with an inhibitory C-type lectin receptor on dendritic (DCIR) and this interaction triggers an ITIM intracellular signaling cascade. This subsequently results in rendering tolerogenic activities to DCs and peripheral induction of Tregs. The anti-inflammatory activity of SA-IVIg has been shown in previous studies, but the mechanism by which it modulates DCs functions is not well understood. We also demonstrated that DCIR facilitates the internalization of IgG molecules into DC and this internalization appears to be a crucial step for induction of Tregs. IVIg is a costly therapeutic compound. Characterization of the mechanism of action of IVIg can lead to a better application of this plasma based therapy in a wide range of autoimmune and inflammatory diseases.

Les immunoglobulines intraveineuses (IVIg) constituent une préparation polyclonale d’IgG isolée et regroupée à partir du plasma sanguin de multiples donneurs. Initialement utilisé comme traitement de remplacement chez les patients souffrant d’immunodéficience primaire ou secondaire, les IVIg sont maintenant largement utilisées dans le traitement de plusieurs conditions auto-immunes, allergiques ou inflammatoires à une dose élevée, dite immunomodulatrice. Différents mécanismes d’action ont été postulés au fil des années pour expliquer l’effet thérapeutique des IVIg dans les maladies auto-immunes et inflammatoires. Entre autre, un nombre grandissant de données issues de modèles expérimentaux chez l’animal et l’humain suggère que les IVIg induisent l’expansion et augmentent l’action suppressive des cellules T régulatrices (Tregs), par un mécanisme qui demeure encore inconnu. Également, les patients atteints de maladies auto-immunes ou inflammatoires présentent souvent un nombre abaissé de Tregs par rapport aux individus sains. Ainsi, une meilleure compréhension des mécanismes par lesquels les IVIg modulent les cellules T régulatrices est requise afin de permettre un usage plus rationnel de ce produit sanguin en tant qu’alternative thérapeutique dans le traitement des maladies auto-immunes et inflammatoires. Par le biais d’un modèle expérimental d’allergie respiratoire induite par un allergène, nous avons démontré que les IVIg diminuaient significativement l’inflammation au niveau des voies aériennes ce, en association avec une différenciation des Tregs à partir des cellules T non régulatrices du tissu pulmonaire. Nous avons également démontré qu’au sein de notre modèle expérimental, l’effet anti-inflammatoire des IVIg était dépendant des cellules dendritiques CD11c+ (CDs) pulmonaires, puisque cet effet pouvait être complètement reproduit par le transfert adoptif de CDs provenant de souris préalablement traitées par les IVIg. À cet effet, il est déjà établi que les IVIg peuvent moduler l’activation et les propriétés des CDs pour favoriser la tolérance immunitaire et que ces cellules seraient cruciales pour l’induction périphérique des Tregs. C’est pourquoi, nous avons cherché à mieux comprendre comment les IVIg exercent leur effet sur ces cellules. Pour la première fois, nous avons démontré que la fraction d’IgG riche en acide sialique (SA-IVIg) (constituant 2-5% de l’ensemble des IgG des donneurs) interagit avec un récepteur dendritique inhibiteur de type lectine C (DCIR) et active une cascade de signalement intracellulaire initiée par la phosphorylation du motif ITIM qui est responsable des changements observés en faveur de la tolérance immunitaire auprès des cellules dendritiques et des Tregs. L’activité anti-inflammatoire de la composante SA-IVIg a déjà été décrite dans des études antérieures, mais encore une fois le mécanisme par lequel ce traitement modifie la fonction des CDs n’a pas été établi. Nous avons finalement démontré que le récepteur DCIR facilite l’internalisation des molécules d’IgG liées au récepteur et que cette étape est cruciale pour permettre l’induction périphérique des Tregs. En tant que produit sanguin, les IVIg constitue un traitement précieux qui existe en quantité limitée. La caractérisation des mécanismes d’action des IVIg permettra une meilleure utilisation de ce traitement dans un vaste éventail de pathologies auto-immunes et inflammatoires. / Intravenous immunoglobulin (IVIg) is a therapeutic preparation of normal human polyclonal IgG derived from pooled plasma from a large number of healthy donors. Initially used as replacement therapy for patients with primary and secondary immune deficiencies, IVIg is now also widely used for the treatment of a variety of autoimmune, allergic and systemic inflammatory disorders, at high immunomodulatory doses. The beneficial effect of IVIg in autoimmune and inflammatory diseases has been attributed to different mechanisms. Increasing evidence shows that IVIg induces expansion and enhances the suppressive function of regulatory T cells (Tregs) in different experimental animal models and human subjects, through an unknown mechanism. Human inflammatory and autoimmune diseases are known to be associated with Treg deficiency. Therefore, a more precise understanding of the mechanisms by which IVIg modulate Treg populations seems to be needed for more rational use of this compound as an alternative therapy in context of various inflammatory and autoimmune disorders. Using a robust antigen-driven model of allergic airway disease, we have demonstrated that IVIg markedly attenuates airway inflammation and this effect is associated with the induction of Tregs from non-regulatory T cells in pulmonary tissues. We have also demonstrated that the antiinflammatory actions of IVIg, in our model are dependent on a population of pulmonary CD11c+ dendritic cells (DCs), as the action of IVIg could be completely replicated by adoptive transfer of CD11c+ DCs from IVIg-treated mice. we have shown that tolerogenic DCs involve in the peripheral induction of Tregs. Given the requirement of DCs in the induction of Tregs, we explored the mechanism by which IVIg interacts and modulate these cells and for the first time demonstrated that the purified sialylated fraction of human IgG (SA-IVIg) (that consists 2-5% of whole IgG) interacts with an inhibitory C-type lectin receptor on dendritic (DCIR) and this interaction triggers an ITIM intracellular signaling cascade. This subsequently results in rendering tolerogenic activities to DCs and peripheral induction of Tregs. The anti-inflammatory activity of SA-IVIg has been shown in previous studies, but the mechanism by which it modulates DCs functions is not well understood. We also demonstrated that DCIR facilitates the internalization of IgG molecules into DC and this internalization appears to be a crucial step for induction of Tregs. IVIg is a costly therapeutic compound. Characterization of the mechanism of action of IVIg can lead to a better application of this plasma based therapy in a wide range of autoimmune and inflammatory diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.