Antígenos de alta massa molecular (PI) do extrato de Ascaris suum exercem efeito supressor sobre a resposta imune a antígenos heterólogos. PI atua diretamente sobre as DCs, diminuindo a expressão das moléculas coestimuladoras, MHC de classe II e assim, a proliferação de linfócitos T. Esse efeito é independente de TLR2, TLR4 e da molécula MyD88. Nesse trabalho estudamos a participação dos receptores DC-SIGN e MR, na modulação da atividade das DCs. PI contém oligossacarídeos N-ligados com cadeias de alta manose e do tipo complexa e contém resíduos de fosforilcolina. Os componentes do PI contendo as cadeias glicosídicas N-ligadas inibem a maturação de DCs incubadas com LPS. Receptores DC-SIGN e MR estão envolvidos no reconhecimento e internalização dos componentes do PI pelas DCs. O bloqueio desses receptores foi capaz de abolir o efeito inibitório do PI sobre as DCs e a resposta proliferativa de linfócitos T in vitro. Portanto, os resultados mostram a participação do DC-SIGN e MR no reconhecimento de componentes glicosilados do PI e na sua ação imunomoduladora. / High molecular weight components (PI) of Ascaris suum extract exert suppressive effect on the immune response to OVA. PI exert direct effect on DCs, decreasing the T lymphocyte proliferation. This effect is independent of TLR2 and 4 as well as MyD88 molecule. In this work we studied the glycoconjugates in PI and the participation of DC-SIGN and MR, in the modulation of the functional activity of DCs. PI components contain high mannose- and complex-type N-linked oligosaccharides and phosphorylcholine residues. PI components containing N-linked glycans inhibited the DCs maturation induced by LPS. The previous incubation of DCs with mannan, anti-DC-SIGN and anti-MR antibodies abolished the modulatory effect of PI on the DCs maturation. It was also observed that the blockage of DC-SIGN and MR in DCs reversed the inhibitory effect of PI in the in vitro T cells proliferative response. Taking together these results show the involvement of DC-SIGN and MR in the recognition of glycosylated components of PI by DCs and in its modulatory effect.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / InteraÃÃes entre a resposta imune inata e adquirida participam na fisiopatologia de doenÃas auto-imunes. Embora infecÃÃes estejam associadas ao desenvolvimento de artrites crÃnicas, à possÃvel que exposiÃÃo a alguns germes, como helmintos e fungos, potencialmente influencie a prevalÃncia e/ou gravidade de doenÃas imunomediadas. Lectinas derivadas de plantas, por aÃÃo em receptores de resposta inata, podem modular inflamaÃÃo. NÃs investigamos o efeito dos extratos de Ascaris suum (AS) e de Coccidioides posadasii (CP) e de uma lectina isolada da Dioclea violacea (Dviol) na artrite induzida por zymosan (AZy). Ratos Wistar e camundongos Swiss receberam 1 mg ou 0,1 mg de zymosan intra-articular (i.art.), respectivamente. Grupos foram prÃ-tratados (30 min) com os extratos de AS (0,25 - 2,5 mg/animal; i.p ou p.o.), CP (1 - 100 Âg/animal; i.art., i.p. ou p.o.) ou Dviol (0,3 - 30 Âg i.art. ou 1 - 6 mg/Kg e.v.). Grupo nÃo-tratado (NT) recebeu Zy (i.art.) e veÃculo. Animais naive receberam apenas salina (i.art.) e veÃculo. A hipernocicepÃÃo foi avaliada atravÃs do teste de incapacitaÃÃo articular em s / 1min. O lavado articular foi usado para anÃlise do influxo celular (IC), nÃveis de nitrito e citocinas. A sinÃvia foi utilizada para histopatologia. O conteÃdo de glicosaminoglicanos (GAG) da cartilagem foi quantificado para medir dano estrutural. O extrato de AS, seja i.p. ou p.o., inibiu de forma dose-dependente a hipernocicepÃÃo e o IC na AZy em relaÃÃo ao grupo NT (P<0,01), bem como reverteu o dano articular avaliado pela quantificaÃÃo de GAG e a sinovite vista à histologia. A administraÃÃo do extrato de AS, reduziu significantemente os nÃveis de nitrito, inteleucina-1&#946; (IL-1&#946;) e IL-10, mas nÃo de fator de necrose tumoral alfa (TNF-&#945;), em relaÃÃo ao NT. Em camundongos, o extrato de AS reduziu os nÃveis de IL-10, mas nÃo de IL-1&#946; ou TNF-&#945;. O tratamento com o extrato de CP, seja i.p. ou p.o., inibiu significantemente a hipernocicepÃÃo e o IC na AZy, em relaÃÃo ao NT, no entanto, nÃo reverteu a lesÃo articular medida pela quantidade de GAG e histologia. A administraÃÃo da Dviol, em animais naive promoveu IC significante, embora apenas a maior dose (30Âg) promoveu hipernocicepÃÃo. Na AZy, a injeÃÃo i.art. da Dviol reduziu o IC e hipernocicepÃÃo de forma dose-dependente, em relaÃÃo ao NT (P<0,01). A administraÃÃo da Dviol (i.v.) reduziu ambos hipernocicepÃÃo e IC na AZy, em relaÃÃo ao NT (P<0,01). O efeito da Dviol foi revertido quando essa lectina foi prÃ-incubada com manose 1 M. Os dados mostram que um extrato de AS promove melhora funcional e protege do dano estrutural na AZy, que sÃo associados com reduÃÃo na liberaÃÃo de NO e citocinas i.art. Esse efeito independe da espÃcie e ocorre por via oral. Um extrato do fungo CP tem aÃÃo anti-inflamatÃria na AZy. Uma lectina isolada da Dviol reduz IC e hipernocicepÃÃo na AZy, provavelmente por acoplamento a um receptor de manose. Em conjunto, os resultados mostram que substÃncias que agem em receptores de resposta inata modulam a inflamaÃÃo articular imunomediada. / The interactions between innate and acquired immune responses participate in the pathophysiology of the autoimmune diseases. Though infections are associate with the development of the chronic arthritis it is possible that exposure to some germs as helminthes and fungi influences potentially the prevalence and/or gravity of the immune diseases. Lectins derivate of the plants can modulate the inflammation by action in receptors of the innate response. We investigated the effect of extracts from Ascaris suum (AS), Coccidioides posadasii (CS) and a lectin isolated from Dioclea violacea (Dviol) in zymosan-induced arthritis (ZyA). Wistar rats and Swiss mice received 1 mg or 0.1 mg zymosan intra-articular (i.art.), respectively. Groups were pretreated (30 min) with AS (0.25 - 2.5 mg/animal; i.p. or p.o.) CP (1 - 100 Âg/animal; i.art. i.p. or p.o) or Dviol (0.3 - 30 Âg; i.art. or 1 - 6 mg/kg; i.v.). Non-treated group (NT) received Zy (i.art.) and the vehicle. Naive animals received just saline (i.art.) and the vehicle. The hypernociception was evaluated through articular incapacitation test in s/1min. The joint exudate was used for evaluation of cell influx (CI), nitrite and cytokine levels. The synovium was used for histopatology. The glycosaminoglycan (GAG) content of the cartilage was quantificated for the measured of the structural damage. The AS extract both i.p. and p.o. significantly and dose-dependently inhibited CI and hypernociception in ZyA as compared to NT (P<0.01) as well as reverted articular damage assessed by quantification of the GAG and by synovitis observed in the histology. The administration of the AS extract reduced significantly levels of nitrite, interleukin-1&#946; (IL-1&#946;) and IL-10, but not tumor necrosis factor alpha (TNF-&#945;) as compared to NT. In mice, it reduced IL-10 but not IL-1&#946; and TNF- &#945;. The treatment with CP extract both i.p. and p.o. inhibited hypernociception and CI in ZyA as compared to NT, but not reverted articular injury measured by GAG and histology. The administration of the Dviol in naÃve animals promoted CI significant, though just the highest dose (30 &#61549;g) promoted hypernociception. In ZyA, Dviol (i.art.) reduced the CI and hypernociception dose-dependently (P<0.01). The administration of Dviol (i.v.) significantly reduced both the hyperalgesia and CI in ZyA as compared to NT (P<0.01). The effect of the Dviol was reverted when it was pre-incubated with mannose (1M). The date show that AS extract promote functional improve and protect of the articular damage in ZyA that are associate with reduction of the NO and cytokine (i.art.) liberation. This effect is species independent and functions orally. An extract of the fungi CP has anti-inflammatory activity in ZyA. A lectin isolated of the Dviol reduces CI and hypernociception in ZyA probably by coupling the mannose receptor. Together the results show that substances that act in receptors of the innate response modulate the immunomediate articular inflammation.

Helmintos e seus produtos são potentes moduladores da resposta imune. Componentes de alta massa molecular do extrato de Ascaris suum (PI) suprimem a resposta anti-ovalbumina. Nas células apresentadoras de antígeno (APCs) como as dendríticas (DCs) os componentes do PI inibem a expressão de moléculas MHC-II e coestimuladoras e, assim a ativação de linfócitos TCD4+. Receptores de membrana de APCs como os do tipo Toll (TLRs) e lectina tipo-C (CLRs) reconhecem padrões moleculares de patógenos e modulam a resposta imune efetora. Assim sendo, foi estudada o papel de TLRs e CLRs expressos nas APCs na supressão induzida por PI. Pôde-se observar que o PI inibe a expressão de TLR1, 2 e 4 na fase de indução da resposta adaptativa. O efeito supressor do PI na resposta anti-OVA e na maturação de DCs é independente de TLR2 e 4, entretanto os CLRs parecem estar envolvidos nesse processo. Portanto, estes dados podem contribuir no esclarecimento dos mecanismos de ação de substâncias imunossupressoras. / Helminths and antigens derived from them are potent immunemodulators. High molecular weight components of Ascaris suum extract (PI) suppress the anti-ovalbumin response. The PI components inhibit in antigen-presenting cells (APCs), as dendritic cells (DCs), the expression of MHC-II and coestimulatory molecules and, thus the CD4 + T cells activation. APCs via membrane receptors, as the Toll like (TLRs) and C-type lectin (CLRs), recognize distinct pathogens and then promote the effector immune response. Therefore, it was studied the role of TLRs and CLRs expressed on APCs in immunesuppression induced by PI. It was observed that PI inhibited the expression of TLR1, 2, 4 and 9 in the induction phase of adaptive response. The suppressive effect of PI in anti-OVA response and DCs maturation was independent of TLR2 and TLR4, however the CLRs seem to be involved in this process. These data can contribute to clarify the action mechanisms of immunosuppressive molecules.

Le chat est la seule espèce animale qui développe spontanément une entité clinique similaire à bien des égards à lasthme allergique humain. Communément appelée « asthme félin » par homologie à la maladie humaine, cette entité pathologique est le résultat dune inflammation persistante des voies respiratoires, associée à des phases aiguës de bronchospasme, une hyperréactivité bronchique à des stimuli divers et dans les stades avancés à des remaniements tissulaires de la paroi bronchique (e.g., érosions épithéliales, hypertrophie de la musculature lisse, hyperplasie glandulaire). Des modèles dasthme félin, obtenus par sensibilisation expérimentale à un allergène, reproduisent la majorité des caractéristiques cliniques, fonctionnelles et lésionnelles de la maladie naturelle et sont au premier plan de la découverte de nouvelles stratégies thérapeutiques (i.e., cyclosporine A, corticostéroïdes oraux et inhalés, immunothérapie spécifique). De la pathogénie de lasthme, ressort toute limportance de dominer lobstruction récurrente des voies respiratoires. Même si les bronchodilatateurs inhalés sont recommandés dans la prise en charge des crises dasthme chez le chat, leur efficacité clinique reste incertaine et peu étayée. Ainsi, les principes actifs, les posologies et les protocoles thérapeutiques actuellement préconisés sont dans une large mesure empiriques et/ou extrapolés de la médecine humaine, et nécessitent dêtre validés dans des conditions expérimentales contrôlées. Nos travaux sinscrivent donc dans cette optique et visent à définir une approche thérapeutique tant préventive que symptomatique des crises de bronchospasme chez le chat, ainsi quune utilisation rationnelle des bronchodilatateurs à longue durée daction pour un contrôle optimal de linflammation asthmatique. Lors dune première étude, nous avons investigué lactivité in vitro, vis-à-vis du muscle lisse bronchique félin, de différentes agents bronchodilatateurs parmi lesquels des agonistes β2-adrénergiques (isoprotérénol, salbutamol [SAL], fénotérol, formotérol, salmétérol [SLM]), un anticholinergique (ipratropium bromide [IB]) et une méthylxanthine (théophylline). Tous ont produit une relaxation dose-dépendante de la musculature lisse bronchique, avec des spécificités propres en termes de puissance, defficacité ou dactivité intrinsèque. Sur une base comparative, le formotérol est le ß2-mimétique le plus puissant, et lisoprotérénol le plus efficace avec le fénotérol. Le fénotérol et le formotérol sont des agonistes complets des récepteurs ß2-adrénergiques, le SAL et le SLM des agonistes partiels. Tenant compte des résultats des essais in vitro et de la disponibilité de formulations administrables par aérosols dans lespèce féline, les effets bronchoprotecteurs (intensité, durée daction) de six médications inhalées ont été caractérisés chez le chat sain, en préambule à leur utilisation dans des conditions pathologiques. Un modèle de bronchoconstriction induite par le carbachol a été retenu dans le cadre de cette deuxième étude. Il ressort de cette étude que le SLM en aérosol-doseur (25µg) présente un effet bronchoprotecteur soutenu persistant 24 heures mais est aussi la médication la moins efficace. Le SAL et lIB (nébulisation ou aérosol-doseur) sont des bronchodilatateurs à courte durée daction (48 heures) dont lutilisation combinée en aérosol-doseur (SAL IB : 100µg/20µg) met en exergue une synergie daction. Les conditions naturelles étant souvent incompatibles avec une approche anticipative de la crise dasthme, les traitements instaurés sont davantage à visée curative que prophylactique. Dans une troisième étude, nous avons exploré les effets bronchorelaxants du SAL (100µg) et de lIB (20µg), administrés seuls ou en traitement combiné, sur une bronchoconstriction induite par un aéroallergène (Ascaris suum) chez des chats rendus expérimentalement asthmatiques. La technique dinhalation par aérosol-doseur a été privilégiée vu les effets synergiques de la combinaison « SAL IB » et lintérêt de cette voie dadministration dans la gestion à domicile des crises dasthme. Aux doses testées, les bronchodilatateurs inhalés nont exercé aucun effet notable, naffectant en rien la résolution du bronchospasme induit. Chez lhomme, les crises et les épisodes dexacerbation de lasthme sont fréquents dans le cours évolutif de la maladie. Aucune étude chez le chat asthmatique en crise aiguë (induite ou spontanée) natteste de lefficacité des corticostéroïdes inhalés ni ne mentionne dans ce contexte lintérêt scientifique dune association aux ß2-mimétiques à longue durée daction. Nous avons donc comparé les effets de la prednisolone orale (1mg/kg q12h) à ceux dune dose élevée de fluticasone inhalée, seule (500µg q12h aérosol-doseur) ou en traitement combiné avec du SLM (500µg/50µg q12h aérosol-doseur), sur la fonction et linflammation pulmonaires dans un modèle félin dasthme aigu. Cette quatrième étude a montré quune courte cure orale de prednisolone diminue significativement linflammation bronchique à éosinophiles. De fortes doses de fluticasone inhalée se sont avérées bénéfiques dans le contrôle de lhyperréactivité bronchique non spécifique, sans toutefois exercer des effets marqués sur linflammation bronchique sous-jacente. Combiner cette corticothérapie inhalée au SLM a permis dobtenir des effets anti-éosinophiliques accrus et comparables à ceux de la prednisolone orale. Aucune des médications testées na modifié significativement la réponse clinique et fonctionnelle consécutive à linhalation de lallergène. Létude précédente permet de supposer que le SLM potentialise lactivité anti-inflammatoire des corticostéroïdes inhalés et/ou est doté de propriétés anti-inflammatoires intrinsèques. Dans une cinquième étude, nous avons donc investigué les effets du SLM en monothérapie (50µg q12h aérosol-doseur) sur la fonction et linflammation pulmonaires. Il ressort de cette étude, conduite sur un modèle félin dasthme aigu, que le SLM en monothérapie nexerce pas deffet protecteur sur la survenue dun bronchospasme allergique et est dépourvu de tout effet anti-inflammatoire propre. Les résultats obtenus, dans les conditions expérimentales de nos essais, conduisent aux conclusions suivantes : 1) les bronchodilatateurs inhalés (SAL, IB, SLM) sont dune efficacité thérapeutique limitée dans la prévention et le traitement symptomatique du bronchospasme allergique chez le chat sensibilisé à Ascaris suum 2) en revanche, recourir au SLM comme thérapie adjuvante aux corticostéroïdes inhalés ouvre de nouvelles perspectives thérapeutiques dans le contrôle de linflammation et de lhyperréactivité bronchiques chez le chat asthmatique./The cat is the only animal species that spontaneously develops a clinical entity closely similar to human allergic asthma and commonly referred to as feline asthma. Feline asthma is a chronic inflammatory disease of the lower airways characterised by intermittent respiratory distress due to bronchoconstriction, non-specific bronchial hyperresponsiveness and airway remodeling at latter stages (e.g., epithelial erosions, smooth muscle hypertrophy, glandular hyperplasia). Based on experimental sensitisation to allergens, models of feline asthma mimic many clinical, functional and lesional features of the naturally developing condition. Moreover, development and implementation of feline asthma models have greatly facilitated the search for novel therapies (i.e., cyclosporin A, oral and inhaled corticosteroids, specific immunotherapy). In considering the pathogenesis of asthma, it is of major importance to control recurrent airway obstruction. Although inhaled bronchodilators are recommended for the management of acute asthmatic exacerbations in cats, their clinical efficacy remains uncertain and poorly documented. Most currently recommended drugs, dosages and therapeutic schemes are largely empirical and extrapolated from human medicine, and need to be further validated under controlled experimental conditions. Hence, our work was aimed at determining the potential of inhaled bronchodilators for preventive and curative treatment of acute bronchospasms in asthmatic cats as well as at exploring the benefit of using long-acting bronchodilators to optimally control the airway inflammation. In the first study, we investigated in vitro effects on isolated feline bronchi of different bronchodilating agents including β2-adrenergic agonists (isoproterenol, salbutamol [SAL], fenoterol, formoterol, salmeterol [SLM]), an anticholinergic (ipratropium bromide [IB]) and a methylxanthin derivative (theophylline). All compounds caused a dose-related relaxation of bronchial smooth muscle, each exhibiting specificities in terms of potency, efficacy and intrinsic activity. On a comparative basis, isoproterenol and fenoterol are the most efficacious β2-mimetics while formoterol is the most potent one. Fenoterol and formoterol act as full agonists of β2-adrenoceptors, SAL and SLM as partial agonists. Taking into account results from in vitro testing procedures and availability of aerosol formulations suitable for lung delivery in cats, we compared the antispasmodic effects (magnitude, duration of action) of six inhaled medications against carbachol-induced bronchoconstriction in healthy cats. This second study showed that SLM by metered-dose inhaler (25µg) has a sustained activity for as long as 24 hours but is also the least efficacious medication. SAL and IB (nebulisation or metered-dose inhaler) are short-acting bronchodilators (4-8 hours) whose combination delivered with a metered-dose inhaler (SAL IB: 100µg/20µg) exhibits a synergistic antispasmodic effect. In the natural disease, asthmatic crisis are difficult to predict in terms of occurrence. Therefore, therapeutic interventions are more curative than preventive. In a third study, we explored the bronchodilating effects of SAL (100µg) and IB (20µg), delivered either alone or as a combined therapy, on allergen-induced bronchospasms in Ascaris suum-sensitised cats. The inhalation technique using a metered-dose inhaler was retained given the synergistic effects of the combination SAL IB with this method and its therapeutic interest for at-home management of asthmatic crisis. At the tested doses, these bronchodilators failed to reverse allergen-induced bronchospasms in cats with experimental asthma. In human beings, crisis and exacerbations of asthma are frequent events in the natural course of the disease. Efficacy of inhaled corticosteroids in cats with acute asthmatic exacerbations (induced or spontaneous) has not yet been determined, nor the potential benefit of adding long-acting β2-agonists in this context. Thus, the fourth study was aimed at comparing the effects of oral prednisolone (1mg/kg q12h) with those of inhaled fluticasone at high doses, alone (500µg q12h metered-dose inhaler) or combined with salmeterol (500µg/50µg q12h metered-dose inhaler), on lung function and airway inflammation in a feline model of acute asthma. This study showed that a short course of oral prednisolone significantly reduced allergen-induced bronchial eosinophilic inflammation. High doses of inhaled fluticasone proved to be efficient for decreasing non specific airway hyperresponsiveness but failed to markedly reduce the underlying airway inflammation. Adding salmeterol to inhaled fluticasone led to anti-eosinophilic effects of the same magnitude as those found for oral prednisolone. None of these treatments improved clinical and functional responses to allergen exposure. According to the previous study, it may be that SLM has an anti-inflammatory effect on its own and/or functions as a steroid-potentiating agent. In a fifth study, we investigated the effects of salmeterol as monotherapy (50 µg q12h metered-dose inhaler) on lung function and airway inflammation in our feline model of acute asthma. This inhaled medication did not prevent occurrence of allergic bronchospasm in Ascaris suum-sensitised and challenged cats, nor did it possess intrinsic anti-inflammatory activity. Under our experimental conditions, the main results achieved led to the following conclusions: 1) the inhaled bronchodilators (SAL, IB, SLM) are of limited efficacy for the prevention and symptomatic treatment of allergic bronchospasm in cats sensitised to Ascaris suum; 2) in contrast, using salmeterol as adjuvant therapy to inhaled corticosteroids opens up new perspectives for the treatment of bronchial inflammation and hyperresponsiveness in asthmatic cats.

Helmintos e seus produtos são potentes moduladores da resposta imune. Componentes de alta massa molecular do extrato de Ascaris suum (PI) suprimem a resposta anti-ovalbumina. Nas células apresentadoras de antígeno (APCs) como as dendríticas (DCs) os componentes do PI inibem a expressão de moléculas MHC-II e coestimuladoras e, assim a ativação de linfócitos TCD4+. Receptores de membrana de APCs como os do tipo Toll (TLRs) e lectina tipo-C (CLRs) reconhecem padrões moleculares de patógenos e modulam a resposta imune efetora. Assim sendo, foi estudada o papel de TLRs e CLRs expressos nas APCs na supressão induzida por PI. Pôde-se observar que o PI inibe a expressão de TLR1, 2 e 4 na fase de indução da resposta adaptativa. O efeito supressor do PI na resposta anti-OVA e na maturação de DCs é independente de TLR2 e 4, entretanto os CLRs parecem estar envolvidos nesse processo. Portanto, estes dados podem contribuir no esclarecimento dos mecanismos de ação de substâncias imunossupressoras. / Helminths and antigens derived from them are potent immunemodulators. High molecular weight components of Ascaris suum extract (PI) suppress the anti-ovalbumin response. The PI components inhibit in antigen-presenting cells (APCs), as dendritic cells (DCs), the expression of MHC-II and coestimulatory molecules and, thus the CD4 + T cells activation. APCs via membrane receptors, as the Toll like (TLRs) and C-type lectin (CLRs), recognize distinct pathogens and then promote the effector immune response. Therefore, it was studied the role of TLRs and CLRs expressed on APCs in immunesuppression induced by PI. It was observed that PI inhibited the expression of TLR1, 2, 4 and 9 in the induction phase of adaptive response. The suppressive effect of PI in anti-OVA response and DCs maturation was independent of TLR2 and TLR4, however the CLRs seem to be involved in this process. These data can contribute to clarify the action mechanisms of immunosuppressive molecules.

Extraction and quantitation of myosin light chain two coupled with myograph recordings from Ascaris muscle perfused with calmodulin inhibitors and neurotransmitters in conjunction with their respective agonists and antagonists have been used to establish the regulation of contraction in this muscle. Densitometric tracings of isolectric focusing gels separating the regulatory light chain were used to quantitate phosphorylation in resting, contracted and flaccid muscle. These studies indicated that inhibitory neurostimulation is mediated by a true GABA receptor. Myosin-mediated contraction is responsible for maintaining the level of tension observed in resting actin-mediated muscle. Actin-mediated contraction is responsible for the rapid rise in tension following excitatory stimuli. Both systems function simultaneously and are independant.

Anaerobic digestion is a biochemical process in which organic carbon is biodegraded in an oxygen free environment through a microbial consortium. Engineered biological systems used for resource recovery often utilize anaerobic digestion to treat anthropogenic organic wastes by reclaiming the carbon as energy (methane gas) and a soil amendment (biosolids). Small-scale, or household, semi-continuous anaerobic digesters have been used in developed and developing countries for many decades to produce biogas from human and livestock waste, which is used for heating, lighting, and cooking. This application has been shown to improve the quality of life of the user. Although there is great potential for small-scale semi-continuous anaerobic digestion to provide much needed resource recovery functions and quality of life improvements in future development, the manner in which these systems are operated could lead to unintended consequences on human health because human waste often contains resistant pathogens. This paradigm is best demonstrated by soil-transmitted helminths that are known to be highly resilient in mesophilic anaerobic digestion environments and endemic to many developing countries. The idea that soil-transmitted helminths survive mesophilic anaerobic digestion is exacerbated when the biosolids from the digesters are land applied as a soil-amendment because this process fits perfectly into the lifecycle of soil-transmitted helminths that need soil environments to develop into infective larva. This research was divided into three sections to investigate the fate of human pathogens during semi-continuous anaerobic digestion and investigate techniques to enhance their removal. The sections were: 1) an examination into the fate (embryonation, development, inactivation, destruction) of Ascaris suum ova during mesophilic semi-continuous anaerobic digestion, with an emphasis on increased inactivation, 2) an investigation into the performance (volatile solids (VS) removal, E. coli and Salmonella destruction, methane production) of semi-continuous mesophilic anaerobic digesters and the effect of variations to solids retention time (SRT) and feeding frequency, and 3) development and application of mathematical models for pathogen inactivation kinetics and typical semi-continuous reactor residence time distributions to predict the removal efficiency of Ascaris suum ova during semi-continuous anaerobic digestion under different operating conditions. Results of these studies showed that during semi-continuous mesophilic anaerobic digestion variations in feeding frequency did not impact the fate of Ascaris suum ova or Salmonella; however it was observed that better removal of E. coli and higher methane production was achieved at the longer feeding interval (weekly). Additional results indicated that embryonated ova were destroyed faster than unembryonated ova under the experimental conditions, which suggests a potential mechanism to enhance removal of this common pathogen. Since an increased feeding interval proved to be beneficial for digester performance our findings suggest that wastes containing Ascaris suum ova could be stored in an aerated environment, for a period of time that does not negatively impact resource recovery, to lengthen the time between feedings and promote ova embryonation and ensuing destruction during digestion. Modeling results indicate that under mesophilic conditions (35oC) the ova of Ascaris suum could survive for 22 days and will not be completely removed from the effluent under typical feeding frequencies and average SRT were examined. Therefore, the use of anaerobic digestion as a resource recovery technology where soil-transmitted helminths proliferate should be applied with extra operational safeguards or be included as one step of several in a small-scale treatment train.

Lic.-avh. Uppsala : Sveriges lantbruksuniv. / Härtill 3 uppsatser.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T17:10:41Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-WhevertonRicardoCorreiaNascimento.pdf: 4035736 bytes, checksum: f33cf235cb56b67d16c3e819dbebb2a1 (MD5) / Made available in DSpace on 2017-04-06T17:10:41Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-dissertação-WhevertonRicardoCorreiaNascimento.pdf: 4035736 bytes, checksum: f33cf235cb56b67d16c3e819dbebb2a1 (MD5) Previous issue date: 2012-03-02 / O extrato de vermes adultos de Ascaris suum (Asc) possui atividade imunossupressora em modelos vacinais, de asma alérgica e doenças auto-imunes. Neste estudo, foi verificado o efeito da administração do Asc, profilática ou terapêutica, no modelo de hepatite induzida por Com A (HAI). Para isto, camundongos BALB/c, receberam ConA, i.v., (20mg/kg corpóreo) e três grupos de animais foram formados (n=6). 1)HAI: recebeu apenas ConA; 2)Protocolo profilático HAI+Asc: tratados com Asc (1mg/mL), i.p., 30 minutos antes da indução da HAI; 3)protocolo terapêutico HAI+Asc: tratados com Asc 2 horas após indução da HAI. Como grupo controle animais receberam apenas PBS. Foram dosados transaminases e imunoglobulinas totais séricas, 8, 24 horas e 7 dias após tratamento. Cultivou-se esplenócitos estimulados com Asc e ConA. Níveis de IL-4, IL-10, IL-13 e IFN-γ dos sobrenadantes foram dosados por ELISA. Os fígados foram pesados e analisados histologicamente (hematoxilina-eosina e Tricômico de Masson). No grupo HAI, ocorreu aumento no peso do fígado, dos níveis de transaminases e Imunoglobulinas totais. Estes parâmetros foram reduzidos nas 8, 24 horas e 7º dia no grupo profilático, enquanto no terapêutico apenas no 7º dia.Em ambos os tratamento houve aumento de IL-4, IL-10 e IL-13. No grupo profilático, houve redução do infiltrado celular e aumento da fibrose hepática. A administração profilática do Asc preveniu, e a terapêutica reduziu o dano hepatocelular da lesão. Ambas estimularam a fibrose hepática a longo prazo, pela possível ação da IL-13. Desta forma, o Asc teria eficiência mais acentuada na fase inicial da doença hepática. / The extract of adult Ascaris suum worms (Asc) has immunosuppressive activity in vaccine models, allergic asthma and autoimmune diseases. This study evaluated the prophylactic or therapeutic effect of administration of the Asc in a mouse model of Concanavalin A (Con A)-induced hepatitis. For this, BALB/c mice received ConA, i.v., (20mg/kg) and three groups of animals were formed (n=6). 1) IAH: received only ConA; 2) AIH+Asc prophylactic: treated with Asc (1mg/mL), i.p., 30 minutes before induction of the AIH; 3) AIH+Asc therapeutic: treated with Asc two hours after induction of AIH. The control group of animals received only PBS. Were measured transaminases and total immunoglobulins, in serum, 8, 24 hours and 7 days after treatment. Was cultivated splenocytes stimulated with ConA and Asc. IL-4, IL-10, IL-13 and IFN-γ were assayed in the supernatants by ELISA. The livers were weighed and examined histologically (hematoxylin-eosin and Masson’s trichrome). In the AIH group, there was an increase in liver weight, transaminase levels and total immunoglobulins. These parameters were reduced in 8, 24 hours and 7 days in the prophylactic group, while the therapeutic treatment only on day 7. In both treatments increased IL-4, IL-10 and IL-13. In the prophylactic group, decreased cellular infiltration and increased liver fibrosis. The prophylactic administration of Asc prevented, and treatment reduced the damage of hepatocellular injury. Both stimulated liver fibrosis in long term by IL-13 action. Thus, the efficiency of Asc would more pronounced in the initial stage of liver disease.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.