Broncoespasmo induzido por exercício em corredores brasileiros de longa distância de elite

OBJETIVO: Determinar a prevalência de broncoespasmo induzido por exercício em corredores brasileiros de longa distância de elite e se há uma diferença na carga de treinamento entre atletas com e sem broncoespasmo induzido por exercício. MÉTODOS: Estudo transversal com corredores de longa distância de elite sem sintomas atuais de asma e sem diagnóstico de broncoespasmo induzido por exercício. Todos os participantes foram submetidos ao teste de hiperventilação voluntária eucápnica e ao teste cardiopulmonar de esforço máximo e responderam a questionários sobre sintomas de asma e atividade física para monitorizar sua carga de treinamento semanal. RESULTADOS: Dos 86 atletas do sexo masculino recrutados, 20 concordaram em participar do estudo, dos quais 5 (25%) foram diagnosticados com broncoespasmo induzido por exercício. Não foram evidenciadas diferenças entre os atletas com e sem broncoespasmo induzido por exercício em relação a características antropométricas, consumo de oxigênio de pico, valores basais de função pulmonar ou sintomas de asma relatados. A carga de treinamento semanal foi significativamente menor nos atletas com broncoespasmo induzido por exercício do que naqueles sem esse diagnóstico. CONCLUSÕES: Nesta amostra de corredores de longa distância brasileiros, a prevalência de broncoespasmo induzido por exercício foi alta.

A poluição do ar e o sistema respiratório

A poluição atmosférica encontra-se presente nos mais diferentes cenários ao longo dos últimos 250 anos, desde que a Revolução Industrial acelerou o processo de emissão de poluentes que, até então, estava limitado ao uso doméstico de combustíveis vegetais e minerais e às emissões vulcânicas intermitentes. Hoje, aproximadamente 50% da população do planeta vivem em cidades e aglomerados urbanos e estão expostas a níveis progressivamente maiores de poluentes do ar. Este estudo é uma revisão não sistemática sobre os diferentes tipos e fontes de poluentes do ar e os efeitos respiratórios atribuídos à exposição a esses contaminantes. Podem ser creditados aos poluentes particulados e gasosos, emitidos por diferentes fontes, aumentos nos sintomas de doenças, na procura por atendimentos em serviços de emergência e no número de internações e de óbitos. Mais do que descompensar doenças pré-existentes, exposições crônicas têm ajudado a aumentar o número de casos novos de asma, de DPOC e de câncer de pulmão, tanto em áreas urbanas quanto em áreas rurais, fazendo com que os poluentes atmosféricos rivalizem com a fumaça do tabaco pelo papel de principal fator de risco para estas doenças. Na rotina de clínicos e pneumologistas, esperamos contribuir para consolidar a importância da investigação sobre a exposição aos poluentes do ar e o reconhecimento de que esse fator de risco merece ser levado em conta na adoção da melhor terapêutica para o controle das descompensações agudas das doenças respiratórias e para a sua manutenção entre as crises.

Hypersensitivity reactions to non beta-lactam antimicrobial agents, a statement of the WAO special committee on drug allergy

Antibiotics are used extensively in the treatment of various infections. Consequently, they can be considered among the most important agents involved in adverse reactions to drugs, including both allergic and non-allergic drug hypersensitivity [J Allergy Clin Immunol 113:832–836, 2004]. Most studies published to date deal mainly with reactions to the beta-lactam group, and information on hypersensitivity to each of the other antimicrobial agents is scarce. The present document has been produced by the Special Committee on Drug Allergy of the World Allergy Organization to present the most relevant information on the incidence, clinical manifestations, diagnosis, possible mechanisms, and management of hypersensitivity reactions to non beta-lactam antimicrobials for use by practitioners worldwide.

Mechanical Alterations in airway smooth muscle after interaction with indoor pollutant – A mathematical model.

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully understood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholine (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle ¯bers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, incorporates the phenomenology of both MCh and FA and reproduces experimental results observed with in vitro exposure of smooth muscle to FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells in a tissue level model. The model can also be used in different biological scales.

Mechanical alterations in airway smooth muscle after interaction with indoor pollutant - A mathematical model

The viscoelasticity of mammalian lung is determined by the mechanical properties and structural regulation of the airway smooth muscle (ASM). The exposure to polluted air may deteriorate these properties with harmful consequences to individual health. Formaldehyde (FA) is an important indoor pollutant found among volatile organic compounds. This pollutant permeates through the smooth muscle tissue forming covalent bonds between proteins in the extracellular matrix and intracellular protein structure changing mechanical properties of ASM and inducing asthma symptoms, such as airway hyperresponsiveness, even at low concentrations. In the experimental scenario, the mechanical effect of FA is the stiffening of the tissue, but the mechanism behind this effect is not fully w1derstood. Thus, the aim of this study is to reproduce the mechanical behavior of the ASM, such as contraction and stretching, under FA action or not. For this, it was created a two-dimensional viscoelastic network model based on Voronoi tessellation solved using Runge-Kutta method of fourth order. The equilibrium configuration was reached when the forces in different parts of the network were equal. This model simulates the mechanical behavior of ASM through of a network of dashpots and springs. This dashpot-spring mechanical coupling mimics the composition of the actomyosin machinery of ASM through the contraction of springs to a minimum length. We hypothesized that formation of covalent bonds, due to the FA action, can be represented in the model by a simple change in the elastic constant of the springs, while the action of methacholinc (MCh) reduce the equilibrium length of the spring. A sigmoid curve of tension as a function of MCh doses was obtained, showing increased tension when the muscle strip was exposed to FA. Our simulations suggest that FA, at a concentration of 0.1 ppm, can affect the elastic properties of the smooth muscle fibers by a factor of 120%. We also analyze the dynamic mechanical properties, observing the viscous and elastic behavior of the network. Finally, the proposed model, although simple, ir1corporates the phenomenology of both MCh and FA and reproduces experirnental results observed with ir1 vitro exposure of smooth muscle to .FA. Thus, this new mechanical approach incorporates several well know features of the contractile system of the cells ir1 a tissue level model. The model can also be used in different biological scales.

Formaldehyde inhalation reduces respiratory mechanics in a rat model with allergic lung inflammation by altering the nitric oxide/cyclooxygenase-derived products relationship

Bronchial hyperresponsiveness is a hallmark of asthma and many factors modulate bronchoconstriction episodes. A potential correlation of formaldehyde (FA) inhalation and asthma has been observed; however, the exact role of FA remains controversial. We investigated the effects of FA inhalation on Ovalbumin (OVA) sensitisation using a parameter of respiratory mechanics. The involvement of nitric oxide (NO) and cyclooxygenase-derived products were also evaluated. The rats were submitted, or not, to FA inhalation (1%, 90 min/day, 3 days) and were OVA-sensitised and challenged 14 days later. Our data showed that previous FA exposure in allergic rats reduced bronchial responsiveness, respiratory resistance (Rrs) and elastance (Ers) to methacholine. FA exposure in allergic rats also increased the iNOS gene expression and reduced COX-1. L-NAME treatment exacerbated the bronchial hyporesponsiveness and did not modify the Ers and Rrs, while Indomethacin partially reversed all of the parameters studied. The L-NAME and Indomethacin treatments reduced leukotriene B4 levels while they increased thromboxane B2 and prostaglandin E2. In conclusion, FA exposure prior to OVA sensitisation reduces the respiratory mechanics and the interaction of NO and PGE2 may be representing a compensatory mechanism in order to protect the lung from bronchoconstriction effects.

Endotoxin exposure during sensitization to Blomia tropicalis allergens shifts TH2 immunity towards a TH17-mediated airway neutrophilic inflammation: role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Isocyanates and Amines – Sampling and Analytical Procedures

This thesis covers sampling and analytical procedures for isocyanates (R-NCO) and amines (R-NH2), two kinds of chemicals frequently used in association with the polymeric material polyurethane (PUR). Exposure to isocyanates may result in respiratory disorders and dermal sensitisation, and they are one of the main causes of occupational asthma. Several of the aromatic diamines associated with PUR production are classified as suspected carcinogens. Hence, the presence of these chemicals in different exposure situations must be monitored. In the context of determining isocyanates in air, the methodologies included derivatisation with the reagent di-n-butylamine (DBA) upon collection and subsequent determination using liquid chromatography (LC) and mass spectrometric detection (MS). A user-friendly solvent-free sampler for collection of airborne isocyanates was developed as an alternative to a more cumbersome impinger-filter sampling technique. The combination of the DBA reagent together with MS detection techniques revealed several new exposure situations for isocyanates, such as isocyanic acid during thermal degradation of PUR and urea-based resins. Further, a method for characterising isocyanates in technical products used in the production of PUR was developed. This enabled determination of isocyanates in air for which pure analytical standards are missing. Tandem MS (MS/MS) determination of isocyanates in air below 10-6 of the threshold limit values was achieved. As for the determination of amines, the analytical methods included derivatisation into pentafluoropropionic amide or ethyl carbamate ester derivatives and subsequent MS analysis. Several amines in biological fluids, as markers of exposure for either the amines themselves or the corresponding isocyanates, were determined by LC-MS/MS at amol level. In aqueous extraction solutions of flexible PUR foam products, toluene diamine and related compounds were found. In conclusion, this thesis demonstrates the usefulness of well characterised analytical procedures and techniques for determination of hazardous compounds. Without reliable and robust methodologies there is a risk that exposure levels will be underestimated or, even worse, that relevant compounds will be completely missed.

Influence of climate changes on cross allergies: possible involvement of pollen transglutaminase

Allergies are a complex of symptoms derived from altered IgE-mediated reactions of the immune system towards substances known as allergens. Allergic sensibilization can be of food or respiratory origin and, in particular, apple and hazelnut allergens have been identified in pollens or fruits. Allergic cross-reactivity can occur in a patient reacting to similar allergens from different origins, justifying the research in both systems as in Europe a greater number of people suffers from apple fruit allergy, but little evidence exists about pollen. Apple fruit allergies are due to four different classes of allergens (Mal d 1, 2, 3, 4), whose allergenicity is related both to genotype and tissue specificity; therefore I have investigated their presence also in pollen at different time of germination to clarify the apple pollen allergenic potential. I have observed that the same four classes of allergens found in fruit are expressed at different levels also in pollen, and their presence might support that the apple pollen can be considered allergenic as the fruit, deducing that apple allergy could also be indirectly caused by sensitization to pollen. Climate changes resulting from increases in temperature and air pollution influence pollen allergenicity, responsible for the dramatic raise in respiratory allergies (hay fever, bronchial asthma, conjunctivitis). Although the link between climate change and pollen allergenicity is proven, the underlying mechanism is little understood. Transglutaminases (TGases), a class of enzymes able to post-translationally modify proteins, are activated under stress and involved in some inflammatory responses, enhancing the activity of pro-inflammatory phospholipase A2, suggesting a role in allergies. Recently, a calcium-dependent TGase activity has been identified in the pollen cell wall, raising the possibility that pollen TGase may have a role in the modification of pollen allergens reported above, thus stabilizing them against proteases. This enzyme can be involved also in the transamidation of proteins present in the human mucosa interacting with surface pollen or, finally, the enzyme itself can represent an allergen, as suggested by studies on celiac desease. I have hypothesized that this pollen enzyme can be affected by climate changes and be involved in exhacerbating allergy response. The data presented in this thesis represent a scientific basis for future development of studies devoted to verify the hypothesis set out here. First, I have demonstrated the presence of an extracellular TGase on the surface of the grain observed either at the apical or the proximal parts of the pollen-tube by laser confocal microscopy (Iorio et al., 2008), that plays an essential role in apple pollen-tube growth, as suggested by the arrest of tube elongation by TGase inhibitors, such as EGTA or R281. Its involvement in pollen tube growth is mainly confirmed by the data of activity and gene expression, because TGase showed a peak between 15 min and 30 min of germination, when this process is well established, and an optimal pH around 6.5, which is close to that recorded for the germination medium. Moreover, data show that pollen TGase can be a glycoprotein as the glycosylation profile is linked both with the activation of the enzyme and with its localization at the pollen cell wall during germination, because from the data presented seems that the active form of TGase involved in pollen tube growth and pollen-stylar interaction is more exposed and more weakly bound to the cell wall. Interestingly, TGase interacts with fibronectin (FN), a putative SAMs or psECM component, inducing possibly intracellular signal transduction during the interaction between pollen-stylar occuring in the germination process, since a protein immunorecognised by anti-FN antibody is also present in pollen, in particular at the level of pollen grain cell wall in a punctuate pattern, but also along the shank of the pollen tube wall, in a similar pattern that recalls the signal obtained with the antibody anti TGase. FN represents a good substrate for the enzyme activity, better than DMC usually used as standard substrate for animal TGase. Thus, this pollen enzyme, necessary for its germination, is exposed on the pollen surface and consequently can easily interact with mucosal proteins, as it has been found germinated pollen in studies conducted on human mucus (Forlani, personal communication). I have obtained data that TGase activity increases in a very remarkable way when pollen is exposed to stressful conditions, such as climate changes and environmental pollution. I have used two different species of pollen, an aero allergenic (hazelnut, Corylus avellana) pollen, whose allergenicity is well documented, and an enthomophylus (apple, Malus domestica) pollen, which is not yet well characterized, to compare data on their mechanism of action in response to stressors. The two pollens have been exposed to climate changes (different temperatures, relative humidity (rH), acid rain at pH 5.6 and copper pollution (3.10 µg/l)) and showed an increase in pollen surface TGase activity that is not accompanied to an induced expression of TGase immunoreactive protein with AtPNG1p. Probably, climate change induce an alteration or damage to pollen cell wall that carries the pollen grains to release their content in the medium including TGase enzyme, that can be free to carry out its function as confirmed by the immunolocalisation and by the in situ TGase activity assay data; morphological examination indicated pollen damage, viability significantly reduced and in acid rain conditions an early germination of apple pollen, thus possibly enhancing the TGase exposure on pollen surface. Several pollen proteins were post-translationally modified, as well as mammalian sPLA2 especially with Corylus pollen, which results in its activation, potentially altering pollen allergenicity and inflammation. Pollen TGase activity mimicked the behaviour of gpl TGase and AtPNG1p in the stimulation of sPLA2, even if the regulatory mechanism seems different to gpl TGase, because pollen TGase favours an intermolecular cross-linking between various molecules of sPLA2, giving rise to high-molecular protein networks normally more stable. In general, pollens exhibited a significant endogenous phospholipase activity and it has been observed differences according to the allergenic (Corylus) or not-well characterized allergenic (Malus) attitude of the pollen. However, even if with a different intensity level in activation, pollen enzyme share the ability to activate the sPLA2, thus suggesting an important regulatory role for the activation of a key enzyme of the inflammatory response, among which my interest was addressed to pollen allergy. In conclusion, from all the data presented, mainly presence of allergens, presence of an extracellular TGase, increasing in its activity following exposure to environmental pollution and PLA2 activation, I can conclude that also Malus pollen can behave as potentially allergenic. The mechanisms described here that could affect the allergenicity of pollen, maybe could be the same occurring in fruit, paving the way for future studies in the identification of hyper- and hypo- allergenic cultivars, in preventing environmental stressor effects and, possibly, in the production of transgenic plants.

Molekulare Reaktionsmuster in dendritischen Zellen nach Allergenexposition

Allergische Erkrankungen, wie zum Beispiel die allergische Rhinitis oder das allergische Asthma haben im Verlauf der letzten vier Jahrzehnte stark zugenommen. So leidet heute jeder vierte bis fünfte Mensch an einer Allergie. Ausgelöst wird diese IgE-vermittelte Hypersensibilitätsreaktion des Typs I (Allergie vom Soforttyp) von Allergenen und beruht auf der Aktivierung von Mastzellen durch die Interaktion eines Antigens mit dem an eine Mastzelle über die Fc-Rezeptoren gebundenen IgE-Moleküls. Die degranulierende Mastzelle sezerniert Mediatoren, was zu einem Auftreten von allergischen Symptomen führt. Die Bildung von IgE wird durch das von TH2-Zellen produzierte Zytokin IL-4 induziert. Das von TH1-Zellen produzierte Zytokin IFN- ist in der Lage die Sekretion von IL-4 zu inhibieren, wie auch IL-4 hemmend auf die Produktion von IFN- wirkt. Dieses TH1-/ TH2-Gleichgewicht ist bei allergischen Erkrankungen in Richtung TH2 verschoben. Allergene werden von antigenpräsentierenden Zellen aufgenommen, prozessiert und auf der Zelloberfläche präsentiert. Die potentesten antigenpräsentierenden Zellen sind die dendritischen Zellen, die nach Kontakt mit einem Allergen in die benachbarten Lymphknoten wandern, ausreifen und kostimulatorische Moleküle exprimieren. Sie sind so in der Lage T-Zellen zu aktivieren und entweder in TH1- oder in TH2-Zellen differenzieren zu lassen. Die zytokinabhängige TH1- beziehungsweise TH2-Differenzierung führt zur Aktivierung der Januskinasen. Im aktiven Zustand phosphorylieren sie STAT-Moleküle, die dimerisieren und in den Zellkern translozieren, wo sie unter anderem als Transkriptionsfaktoren für Zytokingene dienen. Unreife humane dendritische Zellen von Allergikern zeigen nach Stimulation mit Proteinallergenen eine schnelle Phosphorylierung des mit der TH2-Entwicklung assoziierten STAT6. Dahingegen sind TH1-Antwort hervorrufende Kontaktallergene nicht in der Lage STAT6 oder andere STAT-Moleküle in dendritischen Zellen zu induzieren. Die Transkriptionsfaktoren T-bet und GATA3 sind ebenfalls von Bedeutung für die TH1-/TH2-Entwicklung, da T-bet ausschließlich in TH1-Zellen, GATA3 nur in TH2-Zellen exprimiert wird. Die Regulation des JAK/STAT-Weg unterliegt den Molekülen der intrazellulär vorkommenden Familie der SOCS-Proteine. SOCS3 ist in TH2-Zellen höher exprimiert als SOCS1, wohingegen SOCS1 in TH1-Zellen eine erhöhte Expression gegenüber SOCS3 aufweist. In der vorliegenden Arbeit wurde der Einfluss von Proteinallergenen auf humane dendritische Zellen untersucht. Zunächst konnte eine morphologische Veränderung der unreifen dendritischen Zellen nach Kontakt mit dem Allergenextrakt beobachtet werden. Die beginnende Ausreifung der Zellen konnte mittels Durchflußzytometrie anhand der kostimulatorischen Moleküle CD80 und CD86, insbesondere aber über den Marker für reife dendritische Zellen CD83, nachgewiesen werden. Die zu beobachtende beginnende Ausreifung scheint ein Effekt des bakteriellen Lipopolysaccharids (LPS) zu sein, das in dem Allergenextrakt vorkommt, da sich durch Zugabe des kationischen Antibiotikums Polymyxin B die beginnende Reifung verhindern ließ. Auf RNA-Ebene war es im Rahmen dieser Arbeit möglich, den Einfluss verschiedener Allergene auf unreifen humanen dendritischen Zellen näher zu charakterisieren. So weisen unreife humane dendritische Zellen nach Kontakt mit Proteinallergenextrakt ein TH2-assoziiertes Genexpressionprofil auf, was sich durch eine erhöhte relative Expression der Gene SOCS3 und GATA3 auszeichnet. Im Gegensatz hierzu zeigen unreife humane dendritische Zellen nach Inkubation mit dem Kontaktallergen MCI/MI eine erhöhte relative Expression des Gens T-bet, was mit einer TH1-Antwort assoziiert ist. Nach Zugabe des „TH1-/ TH2-neutralen“ Tetanustoxoids konnten erhöhte relative Expressionen der Gene GATA3, T-bet und SOCS3 gemessen werden. Die Ergebnisse in dem in dieser Arbeit benutzten humanen in vitro System geben Anlass zur Hypothese, dass die Art der Immunantwort (TH1 versus TH2) sich bereits auf Ebene der dendritischen Zellen anbahnt. GeneChip-Analysen mittels High Density Micro Arrays von unreifen humanen dendritischen Zellen, die entweder mit Proteinallergenextrakt oder mit LPS in Berührung kamen, zeigten statistisch signifikant regulierte Gene, die allerdings keine Gemeinsamkeiten aufwiesen. Es konnten für die mit Alllergenextrakt gepulsten dendritischen Zellen insgesamt 10 Gene identifiziert werden, jedoch gelang es nicht, diese näher zu deuten oder in einen Zusammenhang mit der allergischen Erkrankung oder der dendritischen Zelle zu bringen. Für die mit LPS, dem stärkeren Stimulus, gepulsten dendritischen Zellen konnten 40 Gene identifiziert werden, die unter anderem für die Maturierung der dendritischen Zelle verantwortlich sind. Zudem war es möglich, die Daten der Arrays auf Proteinebene exemplarisch anhand des Chemokins CXCL2 (Gro-β) zu verifizieren.

Analisi del profilo tossicologico e cancerogeno del particolato atmosferico e identificazione dei rischi per la salute umana correlati all'esposizione

Gli impianti di incenerimento di rifiuti solidi suscitano preoccupazione nella popolazione per i possibili effetti avversi associati all’esposizione. Gli effetti delle polveri sottili (PM2.5), generate dai processi di combustione, sulla salute umana includono l’insorgenza di patologie a carico del sistema respiratorio e cardiovascolare e l’aumento della mortalità per malattie polmonari e probabilmente cancro al polmone. Lo scopo della tesi è quello di valutare il profilo tossicologico e cancerogeno del particolato atmosferico in prossimità dell’inceneritore di Bologna rispetto alle aree adiacenti mediante l’utilizzo di test alternativi alle metodologie in vivo, come il test di trasformazione cellulare e approcci di tossicogenomica (soprattutto trascrittomica) oltre alla valutazione della variazione del rischio cancerogeno indotto dall’esposizione di PM2.5 in diversi siti (massima ricaduta, controllo, fondo urbano e fondo rurale) e in differenti periodi di campionamento (estate 2008 e inverno 2009). Gli estratti di PM2.5 relativi alla stagione invernale sono risultati più tossici rispetto ai campioni estivi, che inducono tossicità soprattutto alle alte dosi. Per i campioni invernali il numero medio di colonie di cellule BALB/c 3T3 A31-1-1 risulta ridotto in modo significativo anche per le dosi più basse saggiate indipendentemente dal sito di provenienza. Tutti i campioni analizzati sono risultati negativi nel test di trasformazione cellulare in vitro. L’analisi dell’espressione genica delle cellule BALB/c 3T3 A31-1-1, in seguito all’esposizione agli estratti di PM2.5, ha mostrato un effetto stagionale evidente. Relativamente ai campioni invernali è stato evidenziato un maggior effetto tossico da parte del sito di controllo rispetto alla massima ricaduta, poiché nel sito di controllo risultano attivati marcatori di morte cellulare per apoptosi. La valutazione del rischio cancerogeno in tutti i siti valutati non mostra situazioni preoccupanti legate alla predizione di eccessi di rischio di tumori imputabili all’attività dell’inceneritore in quanto le stime di rischio non eccedono mai il valore limite riportato in letteratura.

Rolle von NFATc2 in einem murinen Asthmamodell

Nuclear factor of activated T cells (NFAT) ist eine Familie der Transkriptionsfaktoren, welche eine wichtige Rolle bei der Regulation der T-Zellvermittelten Signalkaskade in der Lymphozytenpopulation spielt. In dieser Arbeit konnte gezeigt werden, dass Nuclear Factor of Activated T cells-2 (NFATc2) defiziente Mäuse einen erhöhten Atemwegswiderstand, einen pathologische Veränderung der Lunge und einen erhöhten IgE Spiegel im Vergleich zu den Wildtypen vorweisen. Die NFATc2 Defizienz konnte ebenfalls sowohl mit einer erhöhten Anzahl an Th2 und Th17 Zellen, die eine erhöhte Proliferation vorweisen, als auch einer erniedrigten Anzahl an CD8+ CD122- T-Zellen, die geringere Mengen an IFN-g produzieren, in Verbindung gebracht werden. Die aus den NFATc2(-/-) Mäusen isolierten CD4+ T-Zellen zeigen im Vergleich zu denen der Wildtypen neben der erhöhten Proliferation einen vermehrte Aktivierung (CD4higCD44highCD69high). Weiterhin konnte in dieser Arbeit gezeigt werden, dass in Anwesenheit eines Allergens, die NFATc2(-/-) Mäuse eine erhöhte Anzahl an regulatorischen T-Zellen (CD4+CD25+Foxp3+GITR++) in der Lunge vorweisen, die wiederum die Effektorzellen in diesen hemmen. Ein Grund für die geringere Freisetzung an IFN-g durch die CD8+ T-Zellen in den NFATc2 defizienten Mäusen ist eine erhöhte Subpopulation von CD8+CD122+ (IL-2Rb Kette) CD127hi (IL-7Ra Kette) „long-lived memory Zellen“ in den NFATc2(-/-) Mäusen. Diese besitzen einen regulatorischen Effekt, so dass immundefiziente SCID Mäuse, die in einem adoptiven Transfer mit OVA-spezifischen CD8+ und CD4+ T-Zellen, welchen aus NFATc2(-/-) Mäuse isoliert werden, behandelt wurden, eine erhöhten Atemwegswiderstand, eine erhöhte IL-17 und eine erniedrigte IFN-g Produktion vorweisen. Eine Depletion der memory CD8+CD122+IL-7Rhigh T-Zellen hebt dagegen die verringerte IFN-g Produktion der CD8+CD122- T-Zellen auf und führt zu einer Erniedrigung des Atemwegswiderstandes in einem SCID Model Zusammenfassend zeigen unsere Untersuchungen, dass sowohl die IFN-g Produktion der CD8+ Effektor T-Zellen als auch die Anzahl an CD4+CD25+Foxp3+GITR++ regulatorischen T-Zellen die Entwicklung der Th2 und Th17 als auch die Höhe des Atemwegswiderstandes unterdrückt.

Biomarkers in chronic obstructive pulmonary diseases. Clinical and functional correlations.

Asthma and chronic obstructive pulmonary disease (COPD) are two distinct lung diseases with distinctive clinical and inflammatory features. A proportion of asthmatic patients experience a fixed airflow obstruction that persists despite optimal pharmacologic treatment for reasons that are still largely unknown. We found that patients with asthma and COPD sharing a similar fixed airflow obstruction have an increased lung function decline and frequency of exacerbations. Nevertheless, the decline in lung function is associated with specific features of the underlying inflammation. Airway inflammation increases during asthma exacerbation and disease severity. Less is known about the correlations between symptoms and airway inflammation in COPD patients. We found that there is no correlation between symptoms and lung function in COPD patients. Nevertheless symptoms changes are associated with specific inflammatory changes: cough is associated with an increase of sputum neutrophils in COPD, dyspnoea is associated with an increase of eosinophils. The mechanisms of this correlation remain unknown. Neutrophils inflammation is associated with bacterial colonization in stable COPD. Is not known whether inhaled corticosteroids might facilitate bacterial colonization in COPD patients. We found that the use of inhaled corticosteroids in COPD patients is associated with an increase of airway bacterial load and with an increase of airway pathogen detection. Bacterial and viral infections are the main causes of COPD and asthma exacerbations. Impaired innate immune responses to rhinovirus infections have been described in adult patients with atopic asthma. Whether this impaired immune condition is present early in life and whether is modulated by a concomitant atopic condition is currently unknown. We found that deficient innate immune responses to rhinovirus infection are already present early in life in atopic patients without asthma and in asthmatic subjects. These findings generalize the scenario of increased susceptibility to viral infections to other Th2 oriented conditions.

Isolierung und Charakterisierung allergener Proteine aus der Amerikanischen Schabe (Periplaneta americana)

Allergie gegen Schaben ist eine der Hauptursachen für das Auftreten von allergischem Asthma und allergischer Rhinitis. Verursacht werden diese Krankheiten durch die Schabenallergene. Obwohl bisher zahlreiche Allergene identifiziert wurden, ist bisher nicht bekannt, was ein Protein tatsächlich zu einem Allergen macht. Um die intrinsischen Eigenschaften von Allergenen besser verstehen zu können, wurden in dieser Arbeit Reinigungsprotokolle für die zwei Allergenklassen Per a 3 und Per a 9 aus der Amerikanischen Schabe entwickelt und die Allergene biochemisch und immunologisch charakterisiert. Die Besonderheit der Per a 3-Allergene liegt zum einen darin, dass bisher keine kreuzreagierenden Allergene bekannt sind und zum anderen, dass es sich um -hinsichtlich Temperatur, Harnstoff und Hydrolyse- sehr stabile hexamere Proteine handelt. Zudem sind sie in ihrer nativen hexameren Oligomerisierung stärkere Allergene und in dissoziierter Form könnten sie geeignete Kandidaten für eine Immuntherapie sein. Proteinbiochemisch sind die Per a 3-Allergene als Hexamerine einzustufen. Bei Per a 9 handelt es sich um eine monomere Arginin-Kinase mit allergenem Potential. Aufgrund der weiten Verbreitung der Arginin-Kinasen, den hohen Sequenzidentitäten zu weiteren Invertebraten-Arginin-Kinasen und den bisher bekannten Kreuzreaktionen sind diese Proteine aus Invertebraten wahrscheinlich als Pan-Allergene einzustufen.