Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Wir untersuchten Liquor- und Serumproben von 40 an einem shuntversorgten Hydrocephalus erkrankten Kindern auf die Konzentration der Zytokine bFGF, TGF-β1, VEGF, IL-6, IGF-1 und Leptin sowie deren Korrelation mit dem Risiko von Shuntinsuffizienzen. Dabei konnten wir die Hypothese bestätigen, dass erhöhte Konzentration der fibrogenen Zytokine bFGF und TGF-β1 im Serum bzw. Liquor mit einem erhöhten Risiko für operationspflichtige Shuntinsuffizienzen durch Obstruktion des Schlauchsystems einhergehen, und dass diese Komplikationen mit steigenden Zytokinkonzentrationen umso eher eintreten. Außerdem war bFGF im Liquor von Kindern, die zum Abnahmezeitpunkt an einer Shuntdysfunktion durch Obstruktion oder Einwachsen des Shunts litten, signifikant höher als bei Kindern, die zum Zeitpunkt der Abnahme keine Shuntdysfunktion aus eben genannten Gründen hatten. Des Weiteren fanden wir Konzentrationsunterschiede für IL-6 im Liquor zwischen den einzelnen Ursachen der Erkrankung, wobei das Zytokin am höchsten bei Tumorpatienten war, gefolgt von posthämorrhagischem und postmeningitischem Hydrocephalus, und am niedrigsten bei Kindern mit kongenitaler ZNS-Fehlbildung.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

Les cytokines de la famille γc sont essentielles au développement, à la différenciation thymique et à la survie périphérique des lymphocytes T naïfs. Transmettant leurs signaux par des récepteurs qui ont en commun la chaîne γc, les interleukines -2, -7, -15 et -21 sont des facteurs solubles pléiotropes. De par leur redondance lors d'une réponse immunitaire, le rôle individuel des cytokines γc dans l'homéostasie des lymphocytes T CD8 et dans la réponse anti-virale n'a été que partiellement élucidé. De plus, l'état actuel des connaissances ne permet pas de savoir avec précision à quel moment de la différenciation et selon quels mécanismes ces cytokines interviennent. Afin d'évaluer le rôle des cytokines γc dans l'homéostasie des lymphocytes T CD8 naïfs, nous avons comparé des cellules monoclonales CD8 issues de souris TCR transgéniques P14 γc-compétentes ou γc-déficientes. Nous avons montré que les cellules T CD8 naïves γc -/- ne s'accumulent pas dans les organes lymphoïdes secondaires et que les quelques cellules résiduelles se caractérisent par une petite taille, une diminution de l'expression du CMH de classe I et une augmentation de l'apoptose. Nous avons ensuite corrigé le défaut intrinsèque de survie des cellules T CD8 γc -/-naïves, en surexprimant la molécule humaine Bcl-2, un facteur anti-apoptotique. Cette approche nous a permis de restaurer le nombre de lymphocytes T CD8 naïfs en périphérie, malgré l'absence de chaîne γc. Par contre, tout comme ce qui avait été démontré pour les cellules T CD4, l'expression de Bcl-2 ne permet pas de corriger le défaut de taille et de synthèse protéique des cellules γc-déficientes. Nous concluons donc que les cytokines γc génèrent des signaux Bcl-2-dépendants et Bcl-2-indépendants pour maintenir le phénotype et l'homéostasie des lymphocytes T CD8 naïfs. Afin de définir l'implication précise des cytokines γc au cours de la différenciation des cellules T CD8, nous avons évalué la réponse des cellules T CD8 Bcl-2+ γc +/+ ou γc -/- après infection par le virus de la chorioméningite lymphocytaire. De façon tout à fait étonnante, nous avons démontré que de nombreuses étapes de la réponse anti-virale primaire se déroulent normalement en l'absence de chaîne γc. En effet, l'expansion clonale, les changements phénotypiques associés à une activation et l'acquisition de fonctions effectrices par les lymphocytes T CD8 γc-déficients sont préservés. Par contre, les signaux dépendants de la chaîne γc s'avèrent essentiels à la différenciation et la prolifération des effecteurs tardifs ainsi qu'à la génération et le maintien des lymphocytes T CD8 mémoires. Nous proposons donc que les cytokines γc-dépendantes ne sont pas indispensables à l'acquisition de fonctions cytotoxiques et à la réponse anti-virale, mais génèrent des signaux Bcl-2-indépendants essentiels à la survie et à la prolifération des cellules T CD8 mémoires.

Respiratory virus infections are associated with asthma pathogenesis and exacerbations in children and adults. Unfortunately, it remains largely unknown whether innate and adaptive T cell anti-viral immunity differs in allergic disease versus health. Here, we established a short-term primary cell culture system using human peripheral blood mononuclear cells (PBMC) optimized for measuring immune responses to reovirus, respiratory syncytial virus (RSV) and metapneumovirus (MPV) based on virus-specific cytokine and chemokine production. The prevalence and intensity of innate and adaptive responses in children and adult populations was addressed. Using this in vitro model of human anti-viral immunity, we tested our global hypothesis that asthmatics mount anti-viral cytokine responses to respiratory viruses that differ from those of healthy individuals. MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Reovirus induced exceptionally strong IFN recall responses concomitant with intense IL-10 production, which were independent of viral replication in PBMC. Surprisingly, despite Type 1 cytokine production dominated adaptive immune responses in both asthmatic and non-asthmatic individuals, asthmatics exhibited significantly stronger pro-inflammatory IFNγ and IL-10 production towards virus stimulation than non-asthmatic children and adults. Moreover, children with current AHR, regardless of asthmatic status, exhibit a greater frequency and intensity of IFNγ responses towards pneumoviruses than do non-AHR counterparts. Conversely, expression of chemokine CCL5 was substantially weaker in asthmatics, and was further decreased in children with AHR and familial history of asthma. This pattern of enhanced pro-inflammatory and deficient anti-viral CCL5 responses towards pneumoviruses in children with markers of symptomatic asthma or AHR may underlie the enhanced sensitivity of these children to experience breathing difficulties following infection with respiratory viruses. Furthermore, we have clearly demonstrated a gene by environment interaction, whereby ETS exposure in children with familial asthma results in suppressed anti-viral IFNγ and IL-10 production. Therefore, we have attempted to determine whether genetic variation affects the intermediate phenotype of anti-viral immunity, in the population and dependent on clinical status. In summary, we have demonstrated that asthma, childhood exposures and genetics shape anti-viral cytokine responses in human. These findings have a substantial impact for physicians deciding the contextually appropriate treatment for asthma symptoms in their patients and could have implications for experimentation relating to mechanisms of disease, clinical practice and development of appropriate therapeutics. / October 2007

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Lyme borreliosis was first described some 30 years ago in the USA. Today, it is the most common vector borne disease in Europe and the USA. The disease can have multiple stages and symptoms can manifest from various parts of the body; joints, skin heart and nervous system. In Europe, neuroborreliosis is the most frequent late stage diagnosis. Although Lyme borreliosis is treatable with antibiotics and the causative spirochete has not been shown to be resistant to drugs, some patients do not recover completely. They have persistent symptoms and are diagnosed with chronic or persistent Lyme borreliosis. The mechanism behind the lingering symptoms is unclear but might be due to tissue damage caused by the immune system. The aim of this thesis was to study the immunological differences between patients with different outcome of Lyme borreliosis, i.e. chronic, subacute and asymptomatic, and various factors that might influence the course of the disease. The Borrelia-specific IFN-γ and IL-4 secretion was detected in blood and cerebrospinal fluid from patients with chronic and subacute neuroborreliosis during the course of the disease. Blood samples were also obtained from patients with erythema migrans (EM) and acrodermatitis chronicum atrophicans. An early increase of IFN-γ with a later switch to an IL-4 response was observed in patients with a subacute disease course whereas the IFN-γ secretion continued to be elevated in chronic patients. The Borrelia-specific Th1-response was further investigated in chronic, subacute and asymptomatic individuals by studying the expression of the Th1-marker IL-12Rβ2, on a protein and mRNA level. The cytokine secretion and Foxp3, a marker for regulatory T-cells, were also analyzed. Chronic patients had a lower IL-12Rβ2 expression on CD8+ T-cells and a lower number of Borrelia-specific IFN-γ secreting cells compared to asymptomatic individuals. Chronic patients also displayed a higher expression of Borrelia-specific Foxp3 than healthy controls. The conclusions for these tow studies were that a strong Th1-response early in the infection with a later switch to a Th2-response is beneficiary whereas a slow or weak Th1-response corresponds to a prolonged disease course. The influence of a previous infection with another pathogen, seen to suppress the immune response in animals, and the possible gender difference in immune response was also investigated. Patients with EM were screened for antibodies to Anaplasma phagocytophilum (Ap) as a sign of a previous exposure to these tick-borne bacteria. Blood lymphocytes from Ap seronegative, Ap seropositive and healthy controls were stimulated with Borrelia antigen and the secretion of IL-4, IL-5, IL-12, IL-13 and IFN-γ was detected by ELISPOT. Ap seropositive patients had a lower number of cells responding with IL-12 secretion compared to the other groups which might indicate an inhibited Th1-response. Reinfections with Lyme borreliosis was in a previous study, done by Bennet et al, found to be more frequent in postmenopausal women than in men. To investigate if there was an immunological explanation to the gender discrepancy, blood lymphocytes from individuals reinfected with Lyme borreliosis and individuals infected only once were stimulated with various antigens. The cytokine secretion was detected by ELISPOT, ELISA and Immulite. There were no differences between reinfected and single infected individuals. However, women, regardless of times infected, displayed a Th2-derived and anti-inflammatory spontaneous immune response compared to men. A previous infection with the bacteria Ap might possibly have a long term effect on the immune system and might be of disadvantage when mounting a Th1-response to a Borrelia infection. Also, the Th2-derived response displayed by postmenopausal women could indicate why more women than men get reinfected with Borrelia burgdorferi. / On the day of the public defence date of the doctoral thesis the status of article III was Accepted; the status of article IV was Submitted and the title was "Importance of induction and secretion of interferon-gamma for optimal resolution of human Lyme borreliosis: differencesbetween different outcomes of the infection".

L'objet de cette thèse est l'évaluation de l'effet du traitement chiropratique sur des variables physiologiques. Il y a peu d'information sur l'effet du traitement chiropratique sur la température cutanée paraspinale. De plus, il y a peu d'information sur la technologie de thermométrie utilisée pour faire ces évaluations. Plusieurs recherches ont été effectuées pour mesurer l'effet des traitements chiropratiques cervicaux ct dorsaux sur la variabilité du rythme cardiaque, mais il n'y a aucune étude qui mesure les effets des traitements chiropratiques lombaires. La littérature fournit peu de documentation sur l'effet des traitements chiropratiques sur les hormones pro-inflammatoires. Il n'y a aucune information sur l'effet des traitements chiropratiques sur l'interleukine-6 et la protéine réactive C. Les hypothèses visaient à mesurer s'il y a des effets produits par des traitements chiropratiques sur les variables énumérées ci-haut : la température cutanée paraspinale; la variabilité du rythme cardiaque et les hormones pro-inflammatoires. La méthodologie était progressive. Le premier projet évaluait l'effet du traitement chiropratique sur des sujets sans douleurs au cours d'un seul traitement. Le traitement chiropratique a été effectué avec un instrument (Activator IV). Deux périodes d'adaptation furent utilisées pour mesurer la réaction de la température cutanée paraspinale selon la période d'adaptation. Le deuxième projet évaluait l'effet du traitement chiropratique de sujets en douleurs lors d'un seul traitement traditionnel et il n'y a eu qu'une seule période d'adaptation. La période d'adaptation a été choisie à partir du projet 1 et elle représentait la période d'adaptation qui semblait la mieux adaptée cliniquement. Le traitement a été effectué avec la main, selon la méthode Diversified. Ce projet nous a permis de mesurer les effets de l'ajustement chiropratique sur la TC de sujets en douleurs et de comparer ces résultats à ceux des sujets sans douleur du projet 1. De plus, il a été possible de mesurer le transfert de chaleur de la main du clinicien et de comparer ces mesures à l'effet de l'instrument utilisé dans le projet 1. Pour le troisième projet, nous avons recruté des sujets avec douleurs et des sujets sans douleurs. Ce projet évaluait l'effet du traitement chiropratique sur la variabilité du rythme cardiaque. Les deux techniques chiropratiques des projets 1 et 2 ont été utilisées. Il y avait une période d'acclimatation de trois minutes et les mesures de la VRC ont été enregistrées pendant les cinq minutes qui suivaient ces trois minutes initiales. Le traitement chiropratique a été effectué et il y avait une autre période d'enregistrement de cinq minutes après le traitement chiropratique. Les mesures temporelles et spectrales ont été analysées. Le quatrième projet évaluait l'effet du traitement chiropratique sur des sujets en douleurs chroniques pour une période de neuf traitements échelonnée sur deux semaines. Il y avait un groupe-témoin dont les sujets étaient sans douleurs. Nous avons mesuré pour chaque groupe l'indice de fonctionnalité de Oswestry (questionnaire sur la douleur et la fonctionnalité); la température cutanée paraspinale; la variabilité du rythme cardiaque et les hormones pro-inflammatoires, soit l'interleukine-6 et la protéine réactive C. Les résultats du projet 1 ont démontré que l'ajustement chiropratique avec instrumentation avait un effet sur la température cutanée paraspinale. Toutefois, pour le projet 2, il y a une différence lors de l'évaluation de la mesure obtenue immédiatement après le traitement. Dans le projet 1, il y avait un refroidissement alors que dans le projet 2, il y avait un réchauffement. Le reste des enregistrements étaient très similaires. Les résultats du projet 3 ont été mitigés et ont révélé que la variabilité du rythme cardiaque avait subi une certaine influence provenant d'une manipulation lombaire. Les résultats du projet 4 étaient multiples. L'indice d'Oswestry a démontré que les valeurs du groupe-traitement avaient tendance à se rapprocher des valeurs du groupe-témoin. Pour ce qui est de la température cutanée paraspinale, celle du groupe-traitement était plus froide que celle du groupe-témoin avant le traitement. Après neuf traitements, les valeurs des variables du groupe-traitement avaient tendance à se rapprocher des valeurs des variables du groupe-témoin, les valeurs pré et post des variables du groupe témoin n'ayant pas changé. Les données sur la variabilité du rythme cardiaque ont démontré que les valeurs du groupe-traitement avaient tendance à se rapprocher des valeurs du groupe-témoin. De même, les valeurs des données de l’IL-6 et de la PRC ont démontré la même tendance, à savoir se rapprocher des valeurs du groupe-témoin. En conclusion, même s'il y avait des effets sur la température cutanée paraspinale, il est impossible en ce moment d'utiliser la technique de thermométrie en milieu clinique. En effet, comme il n'existe aucune base de données normative, il est impossible de conclure sur la valeur de l'effet de l'ajustement chiropratique. Quant à la variabilité du rythme cardiaque, il appert que l'effet serait un réflexe d'origine supraspinale plutôt qu'un effet direct sur le système nerveux autonome. Pour ce qui est des hormones pro-inflammatoires de l'IL-6 et de la PRC, il semble que ces mesures soient similaires aux tendances des valeurs de l'indice Oswestry à la suite d'un traitement chiropratique sur des sujets en douleurs. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Thermométrie, chiropratique, diagnostoque, variabilité du rythme cardiaque, cytokines.

Hemocytes are important as storage and producers of proteins of the innate immune defence, as well as actors of the cellular immune response. Therefore the hematopoietic process is critical for survival of most invertebrates. In order to search for molecules of importance for hemocyte development in crayfish we investigated proteins in crayfish plasma, which were increased after microbial challenge. As a result we were able to identify, purify and characterize a new invertebrate cytokine named astakine, and could clearly show that this protein is important for hematopoietic development in vivo as well as in an in vitro cell culture system. Astakine contains a prokineticin (PK) domain shown for the first time in an invertebrate, however, unlike the vertebrate PKs, astakine binds to a cell surface F1 ATP synthase β subunit located on the hematopoietic tissue (hpt) cell membranes. Extracellular ATP synthases as receptors have earlier been reported in different vertebrate cells and here we show that extracellular ATP synthase β subunit acts as a receptor for an invertebrate cytokine and is involved in hematopoiesis. We also found two other groups of proteins, which were increased in plasma after microbial challenge and they were further characterized. A great number of different Kazal type proteinase inhibitors were produced by the hemocytes and this type of proteinase inhibitors have variable reactive sites determining the specificity of their inhibition. In crayfish Kazal inhibitors with similar reactive sites were found as a response to specific microorganisms suggesting that the crayfish Kazal proteinase inhibitors may provide enough variability to participate in diverse innate immune reactions against different pathogens. Antimicrobial peptides were synthesized by the hemocytes and were likewise released in high amount upon microbial infection and we have characterized the main group of cystein-rich crustin-like antimicrobial peptides and investigated their tissue distribution and expression pattern.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.