Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.

Résumé confidentiel / Confidential abstract

L’infection congénitale à CMV est la première cause de retard psychomoteur et de surdité, toutes causes confondues. En France, la prévalence de l’infection congénitale à CMV en France est estimée entre 0,6 et 0,7%. Parmi les enfants présentant une infection symptomatique, 10-20% des nourrissons font une forme sévère et décèdent, et 50 à 60% développent des lésions cérébrales avec un handicap neurologique parfois lourd consécutif à une microcéphalie, des calcifications intracrâniennes, des dilatations ventriculaires ou des convulsions. Certains parmi ceux-ci développeront aussi des séquelles neurosensorielles consécutives à une atteinte visuelle, auditive ou vestibulaire. La première partie de ce travail permet de décrire l’histoire et la physiopathologie des lésions de l’oreille interne chez l’homme. Les lésions de l’oreille interne sont évaluées chez 6 fœtus ayant une infection congénitale à CMV, âgés de 21 à 35 semaines d’aménorrhée, et corrélées aux atteintes centrales et viscérales. Dans la cochlée, les lésions prédominent au niveau de la strie vasculaire alors que l’infection est moins fréquente au niveau des cellules de l’organe de Corti. Les lésions vestibulaires sont plus florides que les lésions cochléaires et prédominent au sein de l’épithélium non sensoriel, en particulier des cellules sombres du saccule et de l’utricule, et de l’épithélium des canaux semi-circulaires. Ces lésions du compartiment endolymphatique de l’oreille interne pourraient être responsables d’une altération de la sécrétion du potassium et ainsi d’une modification de l’homéostasie de la cochlée et du vestibule par atteinte de la strie vasculaire et des cellules sombres. Secondairement, cela pourrait être responsable d’une dégénérescence de l’organe de Corti et des organes sensoriels vestibulaires. La survenue de la surdité neurosensorielle et de l’atteinte vestibulaire dépend très certainement de la durée et de l’intensité de l’infection virale et de l’inflammation. La deuxième partie de ce travail analyse les atteintes du système nerveux central qui n'ont pas fait l'objet d'étude détaillée. Nous avons sélectionné 9 fœtus de 23 SA à 38 SA dont la gestation a été interrompue devant une suspicion d’infection congénitale par le CMV. Ils ont été étudiés sur le plan foetopathologique standard, neuropathologiques, immunohistochimiques et morphométriques. La réplication virale productive prédomine dans le cortex et dans la région périventriculaire. Le virus infecte préférentiellement les cellules GFAP-positive et nestine-positive, mais aussi à un moindre degré les neurones, les cellules endothéliales et la microglie. La réaction immune microgliale et cellulaire cytotoxique semble insuffisante pour contrer l’importance de la dissémination virale suggérant son immaturité. Les malformations corticales sont de mécanismes variés et il existe une atteinte préalablement méconnue des bulbes olfactifs. Une troisième partie est consacrée au développement d’un modèle murin d’infection congénitale par le CMV visant à mieux comprendre les mécanismes physiopathologiques d'atteintes auditives et vestibulaires et à définir les modalités thérapeutiques. Les souriceaux infectés par injection intra-amniotique de virus MCMV développent une surdité dont le profil évolutif s’apparente à la clinique humaine. Les lésions histologiques se caractérisent par des atteintes de la strie vasculaire, de l’organe de Corti et du ganglion spiral. Ces différents axes de recherche complémentaires permettent de comprendre les mécanismes centraux et périphériques participant aux handicaps neurosensoriels chez les enfants atteints d’infection congénitale par le CMV. Les études cliniques et physiopathologiques complémentaires qui en découlent permettront d'affiner les cascades lésionnelles mis en jeu et d'optimiser la prise en charge de ces enfants / Congenital CMV infection is the leading cause of mental retardation and neurosensory-hearing loss. In France, the prevalence of congenital CMV infection is estimated between 0.6 and 0.7%. Amongst the children presenting with a symptomatic infection,10-20% of newborns will have a severe manifestation and die and 50-60% will develop cerebral lesions with a neurological handicap due to microcephaly, intracranial calcifications, ventricular dilations or convulsions. Some amongst these will also develop neurosensory sequelae such as visual, auditory or vestibular lesions. The first part of this work describes the history and the physiopathology of the inner ear lesions in human. The lesions were evaluated in 6 fetuses presenting with a congenital CMV infection, aged 21 to 35 gestational weeks and correlated to central and visceral lesions. In the cochlea, the lesions predominated in the stria vascularis whereas the lesions were les frequent in the organ of Corti. The vestibular lesions were more florid than cochlear lesions and predominated in the non-sensory epithelium in particular the dark cells of the saccula and the utricule and the epithelium of the semi-circular canals. These lesions of the endolymphatic compartment may be responsible of the alteration of the potassium secretion in the inner ear and therefore a modification of the homeostasis of the cochlea and the vestibule by lesion of the stria vascularis and the dark cells. With time, this could be responsible of the degeneration of the organ of Corti and of the vestibular sensory organs. The onset of neurosensory hearing loss and vestibular disorder probably depend upon the duration and the intensity of viral infection and inflammation. The second part of this work analyses the central nervous system lesions that have seldom been detailed. We have selected 9 fetuses aged 23-28 gestational weeks whose pregnancy has been interrupted due to congenital CMV infection. Standard fetopathological examination was completed with neuropathological, immunohistochemical and morphometric assessment. Viral replication predominated in the cortex and in the periventricular zone. The virus infects preferentially GFAP positive and nestin positive cells, but also, to a lesser degree, neurons, endothelial cells and microglia. Immune microglial and cytotoxic cellular responses seem insufficient to counter the importance of the viral dissemination suggesting immaturity. Cortical malformations are due to several mechanisms and olfactory bulbs are frequently infected. The third part describes the development of a murine model for congenital CMV infection in order to better understand the physiopathological mechanisms of the auditory and vestibular lesions and to define the guidelines for management. Infected newborn mice developed neurosensory-hearing loss with characteristics similar to human deafness. Histological lesions were mainly observed in the stria vascularis, the organ of Corti and the spiral ganglia. These different complementary research axes led to a better understanding of the central and peripheral mechanisms participating in the neurosensory handicaps of children with congenital CMV infection. Further clinical and physipathogical studies will allow a more precise comprehension of the lesional cascades and a optimization of the medical management of these children

Os efeitos da administração de anfotericina B a camundongos suscetíveis ao P. brasiliensis foram avaliados. A L-AmB reverte o padrão de suscetibilidade para o de resistência de forma mais eficiente do que a c-AmB, como observado na quantificação de UFC, NO e IgG2b. Porém, os níveis de TNF-a, IL-12, IFN-g, GM-CSF, IgG total, IgM, IgG1, IgG2a e IgA não são significativamente alterados. Neutrófilos e macrófagos peritoneais co-cultivados com Pb e L-AmB tendem a apresentar maior capacidade fungicida, mas não maior síntese de mediadores. O melhor desempenho de L-AmB poderia se dever a sua interação com TLR4. Em TLR4-deficientes ou não, a progressão da doença é similar. A eficácia da terapia, porém, é menor nos deficientes, como observado na quantificação de UFC; os perfis leucocitários e as concentrações de NO, TNF-a, IL-12 e GM-CSF não são significativamente alterados. Logo, a droga é capaz de reverter os parâmetros micológicos, mas não os imunológicos. A interação entre TLR4, P. brasiliensis e L-AmB não parece ser importante para o estabelecimento da imunidade. / Amphotericin B effects in mice susceptible to P. brasiliensis were evaluated. L-AmB reverts the susceptibility pattern to the resistant one with more efficiency than c-AmB, as confirmed by the CFU, NO e IgG2b quantifications. However, TNF-a, IL-12, IFN-g, GM-CSF, total IgG, IgM, IgG1, IgG2a and IgA levels are not significantly altered. Neutrophils and macrophages cocultivated with Pb e L-AmB tend to present higher fungicidal ability, but not enhanced synthesis of mediators. The better performance of L-AmB could be due to its interaction with TLR4. In TLR4-deficient or sufficient mice, progression of the disease is similar. The efficiency of the therapy, however, is lower in deficient animals, as seen on CFU; leukocyte profiles and NO, TNF-a, IL-12 and GM-CSF levels are not significantly altered by L-AmB-TLR4 interaction. Therefore, the drug administration is capable of reverting mycological parameters, but not the immunological ones. Interaction between TLR4, P. brasiliensis and L-AmB does not seem to play a special role in the establishment of immunity.

L'allergie alimentaire peut avoir des effets significatifs sur la morbidité et la qualité de vie. Il existe donc un intérêt considérable à générer de nouvelles stratégies thérapeutiques capables de réduire le risque de développer une atopie. Des études cliniques et expérimentales ont montré que l’allergie était associée à une dysbiose intestinale. Une modulation du microbiote intestinal pourrait ainsi aider à prévenir et traiter les maladies allergiques, justifiant l’utilisation rationnelle de souches probiotiques. L’objectif de mon travail a été de sélectionner au sein d’un panel de 31 souches bactériennes une ou plusieurs souche(s) probiotique(s) possédant des propriétés préventives dans l'allergie, à l'aide d'une combinaison d’approches in vitro et in vivo. Les propriétés immunomodulatrices des souches bactériennes ont été étudiées sur cellules mononucléées sanguines humaines et sur splénocytes murins orientés Th2. Les six souches qui ont induit un fort rapport IL10/IL12p70 et une faible sécrétion d'IFN-γ dans ces deux modèles cellulaires ont été testées pour leur effet protecteur dans un modèle murin d’allergie alimentaire à la β-lactoglobuline (BLG). Trois de ces six souches ont montré un effet protecteur sur l'allergie avec une diminution de la dégranulation des mastocytes et sur la sensibilisation avec une diminution des IgE et des IgG1 spécifiques de la BLG. L’étude de l'impact de ces trois souches sur l'équilibre T helper a montré qu’elles possédaient différents mécanismes d’action. Au niveau systémique, la souche LA307 s’est révélée immunosuppressive, la souche LA308 a induit un profil pro-Th1 et la souche LA305 a induit une réponse à la fois pro-Th1 et régulatrice. Au niveau iléal, l’induction de tolérance pourrait être générée par anergie pour la souche LA305 et par suppression active des réponses Th2 pour les souches LA307 et LA308. L’étude de l’impact de ces trois souches sur le microbiote ne permet pas de conclure que leur effet protecteur est lié à une modulation de la composition du microbiote. Ces résultats montrent que le criblage in vitro, basé sur les propriétés immunomodulatrices des candidats probiotiques, permet une présélection efficace avec trois des six souches sélectionnées ayant un effet protecteur in vivo. / Food allergy can have significant effects on morbidity and quality of life. There, the generation of efficient approaches to reduce the risk of developing food allergy is of considerable interest. Clinical and experimental studies have shown the association of allergy with intestinal dysbiosis. Thus, a modulation of the gut microbiota may contribute to the prevention and management of allergic diseases. This notion supports the use of probiotics.The aim of my study was to select, among a panel of 31 bacterial strains, probiotic strains with preventive properties in allergy using a combination of in vitro and in vivo approaches. Immunomodulatory properties of strains were studied on human blood mononuclear cells and on Th2 skewed splenocytes. The six strains inducing a high IL10/IL12p70 ratio and a low secretion of IFN-γ on both cellular models were tested for their protective impact in a murine model of food allergy to β-lactoglobulin (BLG). Three out of six strains showed a protective impact on sensitization with a decrease in allergen specific IgE and on allergy with a decrease in mast cell degranulation. The study of the impact on the T-helper balance for these 3 strains showed that they had different mechanisms of action. At the systemic level, LA307 strain proved to be immunosuppressive, LA308 strain induced a pro-Th1 profile and LA305 strain induced both, a pro-Th1 and a regulatory profile. At the ileal level, tolerance induction resulted from anergy for LA305 strain and from active suppression of Th2 responses for LA307 and LA308 strains. This study does not enable to conclude about the relationship between the protective impact of these 3 strains and the modulation of the composition of microbiota. These results reveal that the in vitro screening, based on immunomodulatory properties of candidate probiotics, allow an efficient pre-selection with three out of the six selected strains showing an in vivo protective impact.

La leptospirose, due aux bactéries Leptospira interrogans ou leptospires, est une zoonose ré-émergente de répartition mondiale et susceptible de se répandre en France en raison du réchauffement climatique. Les rongeurs, en particulier les rats, sont le réservoir asymptomatique des leptospires. Chez l’Homme, l’infection conduit soit à un syndrome pseudo-grippal bénin, soit à la maladie de Weil, qui se traduit par des déficiences hépatiques, rénales et pulmonaires sévères pouvant entraîner la mort. Le laboratoire a montré que les leptospires échappent à la détection par certains récepteurs de l’immunité innée de la famille des Toll-like (TLR). En effet, le lipopolysaccharide (LPS) des leptospires – composant majeur de la membrane externe – est atypique et n’est pas reconnu par le TLR4 humain, contrairement au LPS de la plupart des bactéries à Gram négatif. Cependant chez la souris, le LPS des leptospires est reconnu par le TLR4 et permet une élimination de la bactérie. Au cours de cette thèse, nous avons mis en évidence un nouveau mécanisme d’échappement des leptospires à la reconnaissance par les récepteurs de l’immunité innée Nod1 et Nod2, reconnaissant les fragments de dégradation du peptidoglycane bactérien, appelés muropeptides. Nous avons montré que la liaison au PG d’une lipoprotéine, la LipL21, spécifique de la membrane externe des leptospires, bloque la digestion en muropeptides et permet ainsi l’échappement des leptospires à la reconnaissance par les récepteurs Nods. Ceci constitue une nouvelle stratégie bactérienne d’échappement au système immunitaire inné. En effet, jusqu’alors seules des modifications des sucres ou de la chaîne peptidique du peptidoglycane entraînant le blocage de la reconnaissance par les récepteurs Nods avaient été mis en évidence. Par ailleurs, nous avons développé – grâce à la construction d’une souche de leptospires pathogènes bioluminescents – un nouveau modèle d’étude de la leptospirose aiguë et chronique chez la souris, qui à l’instar des rats et contrairement à ce que l’on pensait, est un porteur asymptomatique et chronique des leptospires dans les reins. Ce modèle biphasique de la leptospirose nous a permis de mettre en évidence que les leptospires sont protégés de l’action des antibiotiques lorsqu’ils sont dans les reins, alors qu’ils y sont sensibles lors de la phase aiguë. Ces résultats suggèrent deux mécanismes différents participant à l’échappement des leptospires au système immunitaire de l’hôte : d’une part, la lipoprotéine LipL21 joue un rôle dans le blocage de la reconnaissance du peptidoglycane par les récepteurs Nods et d’autre part, les leptospires échappent aux défenses de l'hôte par la colonisation rénale chez la souris. / Leptospirosis is a widespread zoonosis caused by the bacterium Leptospira interrogans (L interrogans). In humans, infection with L. interrogans can cause either a mild disease or in more severe cases, multi-organ failure potentially leading to death. Leptospires escape from the innate immune response by evading detection by Toll-like receptors (TLR). Indeed, the lipopolysaccharide (LPS) outer membrane component of Leptospira is atypical as, unlike most Gram-negative bacteria, it is not recognized by human TLR4. However, leptospiral LPS is recognized by murine TLR4, facilitating clearance of leptospires in mice. During my doctoral thesis, we revealed a new mechanism of leptospiral escape from recognition by Nod1 and Nod2 innate immune receptors. We have shown that a specific leptospiral outer membrane lipoprotein, LipL21, impairs muropeptides digestion and therefore avoids activation of Nod1 and Nod2 receptor-mediated pathways. This constitutes a new strategy of bacterial escape from Nod1 and Nod2 receptor recognition. Indeed, until now, only modification of the peptidoglycan sugar or peptide side-chain chemistry was shown to participate in evasion of Nod receptor recognition. Furthermore, through the construction of a bioluminescent pathogenic Leptospira strain, we were able to develop a new murine model to study acute and chronic leptospirosis. We found that, after the acute phase of leptospirosis, infected mice become asymptomatic, but exhibit chronic carriage of Leptospira in the kidneys, as in rat models of leptospirosis. This biphasic leptospirosis model allows us to highlight leptospiral renal colonization as a stealth escape strategy from the blood defenses and antibiotics. Our results reveal two new mechanisms that contribute to leptospiral escape from the host immune system: on one hand, LipL21 lipoprotein plays a role by facilitating evasion of peptidoglycan recognition through Nod1 and Nod2 receptors, and on the other hand, leptospires escape from host defenses by colonising a renal niche in mice. Together, these findings provide novel insight into the ability of Leptospira to cause serious and chronic morbidity in humans.

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

[Truncated abstract] Previous studies in the field of tumour immunology had identified regulatory T (Treg) cells as important suppressors of the anti-tumour immune response as the presence of Treg cells in the peripheral blood of cancer patients was correlated with worse disease outcomes. Other studies had identified Treg cells to be active at sites of immune regulation such as the gut of colitis patients. It was therefore hypothesised that Treg cells would be present and active within tumours to suppress the cellular antitumour immune response. ... This treatment targeting multiple pathways of Treg cell mediated immuno-suppression and resulted in tumour regression in 50% of treated animals. Finally, the anti-tumour immune response is complex and a potentially synergistic multi-modality treatment designed to inactivate intra-tumoural Treg cells but to also induce apoptosis in tumour cells themselves was investigated. Alpha-tocopheryl succinate (α-TOS), an analogue of vitamin E, had previously been shown to induce apoptosis in human MM xenografts implanted into immuno-deficient (nude) mice. Administration of α-TOS was therefore examined as a potentially synergistic treatment to be coupled with Treg cell inactivation. At the previously published doses used to treat immuno-deficient mice, α-TOS was found to be toxic to the immuno-competent mice used in this study. A marked depleting effect on total T cells was seen in the treated animals. The results of this thesis demonstrated the high potential for an adjunct immunotherapy of MM. They did however also highlight the importance of future studies into anticancer therapies to be conducted using clinically relevant tumour models and clinically relevant treatment regimes. The need to consider synergistic multi-modal therapies was also emphasised.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-16T19:49:06Z No. of bitstreams: 1 Monica Cardozo Rebouças. Resposta a quimioterapia...2012.pdf: 2111449 bytes, checksum: c4c3f7eeb987c9afdfca54d06568dd45 (MD5) / Made available in DSpace on 2013-10-16T19:49:06Z (GMT). No. of bitstreams: 1 Monica Cardozo Rebouças. Resposta a quimioterapia...2012.pdf: 2111449 bytes, checksum: c4c3f7eeb987c9afdfca54d06568dd45 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Populações que residem em áreas endêmicas da doença de Chagas são submetidas a múltiplas infecções pelo Trypanosoma cruzi e podem estar infectadas com cepas ou clones com diferentes graus de virulência e susceptibilidade à quimioterapia. Este é um fator importante para o desenvolvimento e morbidade da doença. As cepas de Trypanosoma cruzi representam complexas populações multiclonais, homogêneas ou heterogêneas, com predominância de um clone principal. São biologicamente classificados em Biodemas (Tipos I, II e III) que apresentam diferentes graus de resistência à quimioterapia. Cepas do Tipo I são muito suscetíveis ao tratamento; cepas do Tipo II possuem uma média susceptibilidade (cepa 21SF); cepas de Tipo III são muito resistentes (cepa Colombiana). No presente estudo, é avaliado o resultado do tratamento de camundongos triplamente infectados com clones da cepa 21SF, em comparação com os infectados com a cepa parental. 50 camundongos foram infectados com a cepa 21SF (infecção única). O inóculo foi de 5 x 104 formas tripomastigotas sanguícolas. 80 camundongos foram infectados, sucessivamente, com 3 clones da cepa 21SF (C6, C7 e C8), inóculo: 1x104 formas tripomastigotas (infecção tripla). A infecção simples com cada clone também foi feita. Os camundongos de ambos os grupos foram divididos em dois subgrupos: tratados com Benzonidazol – BZ (100 mg/kg/dia - 60 doses) e controles não tratados. Após 60 dias do final do tratamento, os camundongos sobreviventes foram eutanasiados, por exsanguinação, pós-anestesia; o sangue foi coletado para o exame sorológico de imunofluorescência indireta; testes de cura foram realizados (parasitemia, xenodiagnósticos, hemocultura) e seções do coração e músculo esquelético foram coletadas, fixadas e processadas para o estudo histopatológico em cortes corados com Hematoxilina & Eosina. A PCR foi também usada como uma técnica diagnóstica complementar. Os testes parasitológicos mostraram uma positividade variando de 54,4% nos camundongos infectados com a cepa parental e tratados; 33,4 a 66,7% nos animais com infecção única pelos diversos clones e tratados e 26,7% nos camundongos com infecção tríplice, tratados. Os títulos sorológicos (TIFI) variaram de 1:20 a 1:280 nos infectados com cepa parental tratados com BZ e de 1:640 a 1:1280 para controles não tratados. Os títulos sorológicos na infecção única com cada clone variaram de 1:10 a 1:1280 em camundongos tratados e de 1:160 a 1:1280 nos controles não tratados. A PCR revelou positividade em todos os animais infectados, tratados. O resultado final foi obtido pela combinação dos testes parasitológicos com os títulos sorológicos revelando positividade de 6,6% nos camundongos infectados com a cepa parental e tratados; 0 a 18,2% nos animais com infecção única pelos diversos clones e tratados e 12% nos camundongos com infecção tríplice, tratados. Estudo histopatológico: Os camundongos infectados com a cepa parental apresentaram lesões que variavam de leves a moderadas, na maioria dos casos, predominante no miocárdio (animais tratados e controles não tratados) camundongos submetidos à infecção única por cada clone apresentaram lesões semelhantes aos demonstrados pela infecção com cepa parental. Os camundongos com infecção tripla apresentaram uma exacerbação de lesões, evoluindo para a miocardite crônica. Nestes casos, havia intensas lesões no músculo esquelético; animais tratados apresentaram uma nítida redução das lesões no miocárdio e no músculo esquelético. Os resultados da quimioterapia com Benzonidazol em animais triplamente infectados, considerando os testes parasitológicos e sorológicos, revelaram baixos índices de cura e agravamento das lesões tissulares nos camundongos submetidos a múltiplas infecções com clones obtidos da cepa 21SF. / People living in endemic areas of Chagas disease are submitted to multiple infections during their lives and could be infected with strains or clones with different virulence and susceptibility to chemotherapy. This is an important factor in the development and morbidity of the disease. Strains of Trypanosoma cruzi represent complex multiclonal populations, which can be homogeneous or heterogenous with predominance of a principal clone. The strains are biologically classified in different Biodemes (Types I, II and III) which disclose different degrees of resistance to chemotherapy. Type I strains are very susceptible to treatment; Type II strains disclose medium susceptibility (21SF strain); strains of Type III are very resistant (Colombian strain). The clones isolated from different strains can also present different degrees of resistance. In the present study the results of treatment of mice triple infected with clones of the 21SF strain is evaluated, in comparison with the infected with the parental strain. For the present study, 50 Swiss mice were infected with the 21SF strain (single infection).The inoculum was of 5 x 104 blood trypomastigotes. 80 mice were infected successively with 3 clones of the 21SF strain (C6, C7 and C8) inoculum: 1x104 trypomastigotes (triple infection). Single infection with each Clone was also done. The mice of both groups were divided into 2 sub-groups: treated with Benzonidazol –BZ (100mg/kg/day – 60 doses) and untreated controls. After 60 days of the end of treatment, surviving mice were killed by exsanguinations after anesthesia; the blood was collected for indirect immunofluorescence serological test; cure tests were performed (parasitemia, xenodiagnosis, haemoculture) and sections of the heart and skeletal muscle were collected, fixed and then processed for the histopathological study in sections stained with Hematoxilin end Eosin. PCR reaction was also performed in blood samples as a complementary diagnostic technique. Parasitological tests have shown positive results that varied from 54.4% for the group of mice infected with the parental strain and treated with BZ; 33.4 to 66.7% for the mice with single infection, with each clone, treated with BZ, and 26.7% with triple infection with the three clones, treated with BZ. Serology titers (IIFT) varied from 1:20 a 1:280 for the mice infected with parental strain treated with BZ and from 1:640 to 1:1280 for untreated controls. Serology titers in the single infection with each clone varied from 1:10 to 1:1280 in treated mice and from 1:160 to 1:1280 in the untreated controls. PCR reaction was positive in all the infected mice, treated. Parasitological tests combined with the sorological titers have shown a positivity of 6.6% for the mice infected with the parental strain and treated with BZ; 0 to 18.2% for the mice with single infection with each clone and treated an 12% for the mice with triple infection and treated with BZ. Histopathological examination revealed in mice infected with the parental strain, mild to moderate inflammatory lesions. Mice submitted to single infection with clones of the 21SF strain presented similar lesions to those determined by infection with parental strain. Mice with triple infection present an intensification of lesions, and evolution to a chronic myocarditis and intense lesions in skeletal muscle; Treated animals presented clear reduction of lesions in myocardium and in skeletal muscle. Results of chemotherapy with BZ in mice with triple infection with clones of the 21 SF T. cruzi strain, considering the results of parasitological and serological tests, revealed low cure rates and aggravation of tissue lesions in those animals submitted to triple infections with clones of the parental strain.

Os efeitos da administração de anfotericina B a camundongos suscetíveis ao P. brasiliensis foram avaliados. A L-AmB reverte o padrão de suscetibilidade para o de resistência de forma mais eficiente do que a c-AmB, como observado na quantificação de UFC, NO e IgG2b. Porém, os níveis de TNF-a, IL-12, IFN-g, GM-CSF, IgG total, IgM, IgG1, IgG2a e IgA não são significativamente alterados. Neutrófilos e macrófagos peritoneais co-cultivados com Pb e L-AmB tendem a apresentar maior capacidade fungicida, mas não maior síntese de mediadores. O melhor desempenho de L-AmB poderia se dever a sua interação com TLR4. Em TLR4-deficientes ou não, a progressão da doença é similar. A eficácia da terapia, porém, é menor nos deficientes, como observado na quantificação de UFC; os perfis leucocitários e as concentrações de NO, TNF-a, IL-12 e GM-CSF não são significativamente alterados. Logo, a droga é capaz de reverter os parâmetros micológicos, mas não os imunológicos. A interação entre TLR4, P. brasiliensis e L-AmB não parece ser importante para o estabelecimento da imunidade. / Amphotericin B effects in mice susceptible to P. brasiliensis were evaluated. L-AmB reverts the susceptibility pattern to the resistant one with more efficiency than c-AmB, as confirmed by the CFU, NO e IgG2b quantifications. However, TNF-a, IL-12, IFN-g, GM-CSF, total IgG, IgM, IgG1, IgG2a and IgA levels are not significantly altered. Neutrophils and macrophages cocultivated with Pb e L-AmB tend to present higher fungicidal ability, but not enhanced synthesis of mediators. The better performance of L-AmB could be due to its interaction with TLR4. In TLR4-deficient or sufficient mice, progression of the disease is similar. The efficiency of the therapy, however, is lower in deficient animals, as seen on CFU; leukocyte profiles and NO, TNF-a, IL-12 and GM-CSF levels are not significantly altered by L-AmB-TLR4 interaction. Therefore, the drug administration is capable of reverting mycological parameters, but not the immunological ones. Interaction between TLR4, P. brasiliensis and L-AmB does not seem to play a special role in the establishment of immunity.