Nanopartículas poliméricas biodegradáveis têm recebido atenção como carreadores de fármacos ao longo dos últimos anos. Em muitos casos, a segurança destes nanocarreadores não foi demonstrada e pouco se sabe sobre a relação entre as suas características físico-químicas e suas propriedades toxicocinéticas e toxicodinâmicas. A nanotoxicologia está emergindo como uma especialidade importante da nanotecnologia e/ou toxicologia, e refere-se ao estudo da interação de nanoestruturas com sistemas biológicos. Nos últimos anos, a maioria das pesquisas foi centrada em estudos in vitro, entretanto, os resultados destes estudos necessitam também ser avaliados em experimentos in vivo para o avanço na utilização de nanocarreadores na área biomédica. Com isso, o objetivo deste trabalho foi avaliar a toxicidade de nanocápsulas de núcleo lipídico (LNC), de poli(-caprolactona), após administração intraperitoneal (i.p.) e intradérmica (i.d.) em ratos Wistar. Para a avaliação toxicológica aguda, foi administrada dose única em que se observaram sinais clínicos e fisiológicos, em ambas as vias. Após 14 dias, os animais foram eutanasiados e análises macroscópicas e histopatológicas foram realizadas. Além disso, sangue e urina foram coletados para análises laboratoriais e avaliação de funções teciduais. A avaliação toxicológica subcrônica foi procedida da mesma forma, exceto pela administração de doses repetidas diárias durante 28 dias. As suspensões de nanocápsulas foram preparadas pelo método de precipitação do polímero pré-formado, as quais apresentaram tamanho médio de partícula inferior a 250 nm, índice de polidispersão (IPD) < 1, potencial zeta negativo e pH em torno de 6,7. Os animais tratados pela via i.p. (n=6/grupo) receberam para avaliação da toxicidade aguda: solução salina ou polissorbato 80 (PS80) (12 ml/kg), utilizados como controles e diferentes doses de LNC (18,03, 36,06, e 72,12 × 1012 LNC/kg); no teste de toxicidade subcrônica foram utilizados os mesmos controles porém com doses de 3mL/kg e 6,01, 12,02 ou 18,03 × 1012 LNC/kg. Nos testes de toxicidade aguda, nos animais administrados pela via i.p., foi observada diminuição significativa de peso nos grupos tratados com LNC mesmo após 14 dias da administração (p<0,05). Entretanto no teste subcrônico esta alteração foi transitória, e ocorreu apenas no grupo que recebeu a maior dose até o quinto dia de administração (p<0,05). Houve aumento no peso relativo do baço nos animais que receberam a dose mais alta de LNC (p<0,05) no tratamento agudo. A análise histopatológica em ambos os tratamentos, demonstrou a presença de um granuloma de tipo corpo estranho no fígado e no baço dos animais que receberam a dose mais alta, provavelmente devido ao volume de LNC administrado. Não houve alteração nas análises bioquímicas de dano hepático, renal, dentre outros em todos os grupos tratados. Os dados hematológicos apresentaram uma leve alteração, entretanto foi demonstrada interferência metodológica, evidenciada por testes preliminares in vitro. Além disso, foram avaliados biomarcadores do estresse oxidativo (EO), marcadores inflamatórios e de genotoxicidade. Os resultados dos biomarcadores de oxidação de proteínas e lipídios não foram suficientes para iniciar um processo oxidativo, visto que não houve peroxidação lipídica. Ainda, não houve depleção de antioxidantes, dano ao DNA ou alteração nos marcadores inflamatórios. Nos ratos tratados pela via i.d., foi utilizada solução salina 1,2 ml/kg como grupo controle e uma dose de 7,2 × 1012 LNC/kg de LNC, para um estudo preliminar agudo e solução salina ou PS 80 (0,9ml/kg) e três doses de LNC (1,8, 3,6 ou 5,4 × 1012 LNC/kg) para avaliação da toxicidade subcrônica. No teste de toxicidade aguda, não houve alteração do peso corpóreo, entretanto no teste de toxicidade subcrônica houve uma diminuição reversível do peso no grupo que recebeu PS80 (p<0,05). Os dados histopatológicos não apresentaram alteração. Não houve alteração nos parâmetros bioquímicos, exceto uma leve diminuição da atividade da butirilcolinesterase no grupo que recebeu a dose mais alta (p<0,05). Por outro lado, houve aumento nos leucócitos no grupo que recebeu LNC, no teste de toxicidade aguda e nos grupos que receberam PS 80 e 5,4 × 1012 LNC/kg (p<0,05) após doses repetidas. Em relação à avaliação sanguínea e tecidual dos biomarcadores do EO e dos marcadores inflamatórios, foi observada uma indução nos marcadores de oxidação de proteínas juntamente com uma indução enzimática nos ratos que receberam a dose mais alta, além de uma diminuição dos níveis do IL-10 nos grupos que receberam PS80 e a dose mais alta (p<0.05). Pode-se concluir que nas condições dos experimentos, tanto pela via i.p. quanto pela via i.d., não foram demonstrados danos teciduais, pois os achados laboratoriais foram condizentes com os achados histopatológicos. Além disso, os mecanismos de reparo foram suficientes para contrabalançar eventuais danos oxidativos ou inflamatórios. Assim, o presente trabalho contribui para futuras avaliações toxicológicas de nanocápsulas poliméricas, visto que foram realizadas avaliações agudas e subcrônicas sistemáticas, com marcadores de dano renal precoce e possíveis mecanismos de toxicidade envolvidos após administração por ambas as vias. O aumento na utilização destas nanocápsulas e as lacunas nas informações toxicológicas fazem com que desafios importantes devam ser superados para permitir sua incorporação segura. Com isso, estudos nesta linha podem embasar a avaliação da resposta tóxica e, consequentemente, levar ao estabelecimento de regulamentações para avaliação da toxicidade da maioria das nanopartículas poliméricas biodegradáveis utilizadas como carreadoras de fármacos. / Biodegradable polymeric nanoparticles have received attention as drug carriers over the past years. In many cases, the safety of nanocarriers has not been demonstrated and little is known about the relationship of its physicochemical characteristics and their toxicokinetic and toxicodynamic properties. Nanotoxicology is emerging as an important field of nanotechnology and toxicology, and refers to the study of the interaction of nanostructures with biological systems. In recent years, most research has focused on in vitro studies; however, the results of these studies should also be evaluated trough in vivo experiments, in order to advance in biomedical application of nanocarriers. Thus, the objective of this study was to evaluate the toxicity of lipid-core nanocapsules (LNC), prepared with poly(ɛ-caprolactone), after intraperitoneal (i.p.) and intradermal (i.d.) administration in rats. For acute toxicological evaluation, it was administered a single dose, i.p. and i.d., clinical signs and physiological effects were observed. After 14 days, animals were euthanized and macroscopic and histopathological analyses were done. In addition, blood and urine were collected for laboratory analysis and evaluation of tissue functions. Subchronic toxicological evaluation was similar, except for the administration of repeated doses for 28 days. The suspension of nanocapsules were prepared by interfacial deposition of polymer, which had particle size less than 250 nm, polydispersity index (IPD) <1, negative zeta potential and pH around 6.7. Animals were treated via i.p. (N = 6/group), the doses used for acute toxicity test were: saline or polysorbate 80 (PS80) (12 ml/kg) as controls or three different doses of LNC (18.03, 36.06, e 72.12 × 1012 LNC/kg); for subchronic toxicity test, same controls were used but the doses were 3 ml/kg and 6.01, 12.02 ou 18.03 × 1012 LNC/kg administered daily for 28 days. In acute toxicity test, with i.p. administration, groups treated with LNC presented a significant reduction in relative weight even after 14 days of administration (p<0.05); however in the subchronic test, this change was transient, and occurred only in the group receiving the highest dose until the fifth day of administration (p<0.05). There was an increase in relative weight of spleen in animals that received the highest dose of LNC (p<0.05) in acute treatment. Histopathological analysis in both the treatments, showed a granulomatous foreign body reaction in liver and spleen of animals receiving the highest dose, probably because the volume of LNC administered. There were no changes in biochemical parameters of liver or kidney damage, among all treated groups. Hematological data showed a slight change; however it was demonstrated an interference of the methodology, further evidenced by preliminary in vitro tests. Furthermore, we evaluated biomarkers of oxidative stress (OS), inflammatory and genotoxicity markers. The results of the oxidation of proteins and lipids biomarker were not sufficient to initiate an oxidative process, since no lipid peroxidation occurred. Still, no depletion of antioxidants, DNA damage or change in inflammatory markers was observed. In rats treated via i.d., saline was used as control (1.2 ml/kg) and a dose of 7.2 × 1012 LNC/kg of LNC to a preliminary acute study, and saline or PS 80 (0.9ml/kg) used as controls or three doses of LNC (1.8, 3.6 ou 5.4 × 1012 LNC/kg) for subchronic toxicity evaluation. In acute toxicity test, there was no change in relative body weight, however, a decreased was found for the group receiving PS 80 in subchronic test (p <0.05). No histopathological alteration was found. Also, there was no change in biochemical parameters, except a slight decrease of butyrylcholinesterase activity in the group receiving the highest dose (p<0.05). Moreover, in acute toxicity test, it was found an increase in white blood cells in group receiving LNC; these increasing also occurred after repeate dose test, in PS 80 and 5.4 × 1012 LNC/kg of LNC groups (p <0.05). Regarding blood and tissue biomarkers of OS and inflammatory markers, an induction in protein oxidation marker along with antioxidant induction in rats which received the highest dose were observed, also reduced levels of IL-10 in rats that received the higher dose and PS80 (p <0.05). It can be concluded that, under the experimental conditions, for i.p. and i.d. administration, tissue damage was not found, since laboratorial analysis results were consistent with histopathological findings. Furthermore, mechanisms of repair were sufficient to offset oxidative damage or inflammation.Thus, this study contributes to future toxicological evaluations of polymeric nanocapsules, since a systematic acute and subchronic evaluation with early renal damage markers and possible mechanisms of toxicity involved after ip and id routes were performed. The increase in the use of these nanocapsules and the gaps in toxicological information make important to overcome these challenges in order to allow its safe incorporation. Thus, studies in this line are important to evaluate toxic response, and lead to establishing rules for evaluating the toxicity of most biodegradable polymer nanoparticles used as carrier of drugs.

Teste intradérmico avalia reação de hipersensibilidade a diversos agentes que possam apresentar poder reacional alérgico e são comumente utilizados para complementar o diagnóstico da dermatite atópica canina (DAC). Ainda não existe consenso sobre as concentrações de histamina e extratos alergênicos a serem utilizadas. Para determinar a concentração ideal de histamina, como controle positivo, e do limiar irritativo de extratos alergênicos em teste intradérmico é necessário que diversas concentrações sejam avaliadas em uma população bem numerosa de cães hígidos. O objetivo desta pesquisa foi avaliar em 160 cães sadios submetidos a teste intradérmico, quais seriam as concentrações de histamina e de extratos alergênicos consideradas ideais. A solução contendo 0,1 mg/mL de histamina foi considerada como parâmetro ideal, provocando reações cutâneas com diâmetro médio, mediana e desvio padrão, de 15,18 mm, 14,97 mm e 2,07 mm, respectivamente. A partir do estabelecimento da concentração de histamina, foram determinadas as concentrações ótimas dos extratos alergênicos, expressas em PNU/mL: 1.000 para Dermatophagoides pteronyssinus, 500 para D. farinae, 125 para Blomia tropicalis e 2.000 para Malassezia pachydermatis. Futuros estudos devem ser conduzidos em cães atópicos para verificação da acurácia dos testes intradérmicos realizados com essas concentrações. / Intradermal testing evaluates hipersensitivity reaction to different agents that can present allergic reactivity power. It is commonly used to complement canine atopic dermatitis diagnosis. There is still no consensus about histamine concentrations and allergen extracts to be used. The determination of the histamine ideal concentration as positive control and the irritant threshold of allergen extracts for intradermal testing, requires evaluation of different concentrations on a large population of healthy dogs. The purpose of this research was to evaluate the ideal histamine and allergen extracts concentrations on 160 healthy dogs submitted to intradermal testing. A histamine solution 0,1 mg/mL was considered the ideal parameter. It caused cutaneous reactions with average diameter, median measure and standard deviation of 15.18 mm, 14.97 mm and 2.07 mm, respectively. From the histamine concentration establishment, the optimum allergen extracts concentrations were determined, expressed by PNU/mL: 1.000 for Dermatophagoides pteronyssinus, 500 for D. farinae, 125 for Blomia tropicalis and 2.000 for Malassezia pachydermatis. Future studies have to be conducted on atopic dogs to verify the accuracy of the intradermal testing with these concentrations.

The purpose of this study was to characterize a novel technique to study neurogenic control of cutaneous vasodilation. We monitored skin blood flow (SkBF) during in-vivo electrical stimulation (e-stim) intended to activate cutaneous nerves and used intradermal microdialysis to deliver receptor antagonists to characterize their contribution to cutaneous vasodilation. We examined the role of acetylcholine receptors (RACh), nitric oxide (NO), and vasoactive intestinal peptide receptors (RVIP) on the cutaneous vasodilation induced by e-stim in the absence of the sympathetic adrenergic nervous system. Six men and three women participated in the study. Three intradermal microdialysis probes were placed in the skin of the dorsal side of their forearm. The adrenergic nervous system was eliminated by delivery of a cocktail of phentolamine (0.01 mg/ml), propranolol (1 mM), and BIBP-3226 (10 µM). At one skin site atropine (0.1 mg/ml) was delivered to block RACh. At a second site we blocked nitric oxide synthase (NOS, 10 mM L-NAME) and RACh. Finally at the third site, we blocked RACh, NOS, and RVIP (0.47 mg/ml VIP10-28). The SkBF response to 1 minute stages of graded increases in frequency (0.2, 1, 2, 4, 8, and 32 Hz) at a current of 1.0 ± 0.1 mA was used to generate a stimulus-response curve before and after drug delivery. At skin site 1 RACh blockade decreased the area under curve (AUC) by 4% from 614 ± 279 to 591 ± 331 (p>0.05). Nitric oxide synthase and RACh blockade reduced the vasodilator response to e-stim by 23% from 1036 ± 457 to 801 ± 448 AUC (p>0.05). Combined NOS, RACh, and RVIP blockade reduced the vasodilator response by 48% from 802 ± 412 to 418 ± 268 AUC (p=0.07). RACh blockade had no effect on the vasodilator response to e-stim. However, in these preliminary studies both NOS and RVIP blockade provided some attenuation of the cutaneous vasodilator response associated with e-stim. Additional studies will focus on these two neurotransmitters as this novel method is refined. Advantages of e-stim include activating the sympathetic nervous system without activating local and humoral factors and studying neurotransmitters in an in-vivo setting during rest, thermal stress, or exercise.

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.

Despite Africa accounting for ~20% of the global cattle population, prevalence estimates and related risk factors of bovine tuberculosis (bTB), caused by Mycobacterium bovis, are still poorly quantified in many countries across the continent. Control of bTB in Africa is difficult due to poor monitoring of cattle movements and limited abattoir surveillance. Also M. bovis is zoonotic and risk factors for transmission include living in close contact with cattle and consumption of unpasteurised milk. Cattle keeping is integral to some rural populations in Cameroon and understanding the epidemiology of bTB in cattle populations is important both to bovine and public health. Detection of bTB in cattle is difficult due to variability of immune responses to M. bovis infection. The interferon-γ (IFN-γ) assay maybe useful to estimate bTB prevalence and identify bTB risk factors in Cameroon. However its performance can vary at different stages of bTB pathogenesis and in different cattle populations. Recently Fasciola hepatica co-infections have been reported to suppress IFN-γ responses in M. bovis infected cattle but the potential effect with F. gigantica co-infections on bTB prevalence estimates in Cameroon is unknown. An abattoir study was conducted in Cameroon to assess the performance of the IFN-γ assay. In 2012-13; 2064 slaughtered cattle were sampled from Bamenda abattoir (North West Region; NWR) and Ngaoundere abattoir (Vina Division; VD). Individual animal data was collected from routine meat inspection including identification of bTB and Fasciola pathology. Cattle were also tested for bTB using the IFN-γ assay and an M. bovis antibody ELISA. In the absence of a gold-standard diagnostic, the IFN-γ assay was compared to other diagnostic tests to assess agreement and identify factors that affected performance of the assay. Agreement between IFN-γ assay, TB lesion identification and an M. bovis antibody ELISA was poor-moderate, probably partly related to differences in immune response detected. A presence of Fasciola gigantica also increased the odds of false negative IFN-γ assay results. On further investigation co-infected cattle had increased odds of TB lesions and reduced IFN-γ responses that potentially could lead to ~20% reduction in test sensitivity. In an attempt to take into account the potential impact of F. gigantica, when estimating bTB prevalence, an antibody ELISA was developed to detect the exposure in live cattle. To highlight the awareness of disease in cattle-rearing communities, estimate prevalence and identify risk factors of bTB in cattle populations; two cross-sectional studies were conducted in 2013. A stratified clustered cross-sectional study of pastoral cattle herds, in the NWR and the VD, sampled 1448 pastoral cattle reared by 100 pastoralists. A smaller cross-sectional study sampled 60 dairy cattle from 46 small-holder co-operative dairy farmers. Individual animal data and herd-level data were collected and animals were screened by both the single comparative intradermal skin test (SCITT) and IFN-γ assay. Awareness of zoonotic TB was low yet consumption of raw milk was high in cattle-keeping communities highlighting the need for accurate bTB prevalence estimates. Despite the high awareness of the clinical presentation of bTB, clinical signs identified by pastoral herdsmen were not associated with cattle being bTB positive. The SCITT was used to compare two manufacturers cut offs for the IFN-γ assay, ≥0.05 and ≥0.1, and highlighted that these two diagnostics may detect different populations of bTB positive cattle. Using the IFN-γ assay at ≥0.1, bTB prevalence was highest in dairy cattle (21.67%) and was also present in pastoral cattle in the NWR and VD (11.33% and 6.55% respectively). Importantly, as F. gigantica is endemic in Cameroon and its influence could mean the true prevalence of bTB could be higher. Female pastoral cattle were at lower odds of being IFN-γ assay positive potentially due to immunosuppressive factors had lower odds of disease. Husbandry practices also decreased the odds of being IFN-γ assay positive such as drinking from streams, antelope and contact with herds at grazing. Age increased the odds of pastoral cattle being IFN- assay positive potentially being a confounder to chronicity of bTB and other co-infections may influence IFN-γ responses. Dairy cattle herds had different risk factors for being IFN- positive likely due to differences in husbandry practices. Considering the potential risk to public health of M. bovis this thesis highlights the extent of bTB across two major cattle keeping regions in Cameroon and the public health risk in cattle-rearing communities. Furthermore the relationship between Fasciola co-infection and IFN- responses to M. bovis described has potential implications for bTB diagnosis in cattle populations where the parasite is present across the globe.

Pythiosis is a granulomatous disease of humans and animals caused by an aquatic oomycete named Pythium insidiosum. In Brazil, equine species is the most affected and the highest occurrence of pythiosis is observed in Brazilian Pantanal. Nowadays, in Rio Grande do Sul (RS), in South of Brazil, there has seen an increase in the number of equine pythiosis. Nevertheless, there is no information about seroprevalence in Brazil. Early diagnosis is the key to success in controlling this infection. Currently, immunotherapy with proteic antigens has been studied as a promising alternative of treatment. The intradermal (ID) route to apply proteic antigen is an option to be evaluated, for both early diagnosis and pythiosis treatment, by using proteic antigens of P. insidioum. In this study it was investigated the seroprevalence of equine pythiosis in the RS State and also it was evaluated and compared the use of proteic antigens of P. insidiosum by the ID route in the diagnosis and control of pythiosis in an experimental model (Oryctolagus cuniculus). To determine the prevalence of equine pythiosis in RS it was used 1002 equine serum samples evaluated by indirect ELISA test. Of the total samples tested, 11.1% (111/1002) were seropositive for equine pythiosis. For the diagnosis it was applied 0.1mL (1.7mg) of proteic antigen by ID route in rabbits (O.cuniculus) (n = 15) divided into three groups: negative control (n = 5), with experimental pythiosis (n = 5) and animals previously immunized with the proteic antigen (n = 5). In order to verify and compare the use of proteic antigen of P. insidiosum by ID as an option of treatment it was used ten rabbits with pythiosis. Five rabbits with pythiosis were treated with 0.1mL proteic antigen applied through ID and other five rabbits by subcutaneous (SC) route with 2mL (34mg) (n = 5). The ID test was able to detect cutaneous reactions in 24h and 72h in all animals exposed to P. insidiosum, as well as it demonstrated sensitivity and specificity comparable with the pattern of diagnosis of pythiosis (indirect ELISA). The treatment proteic antigen of P. insidiosum, by ID and SC route, did not observed difference (P> 0.05) in the size of the lesions. However, the clinical cure affect as 40% of rabbits in both groups. Thus, protein antigen of P. insidiosum by ID may be used in therapeutic protocols of pythiosis in rabbits, also this route as a protocol of pythiosis has other advantages such as lower protein concentration and volume of antigen to control pythiosis. However, research is needed for evaluation of ID route for diagnosis and control of pythiosis in naturally affected species. / A pitiose é uma doença granulomatosa de humanos e animais, causada pelo oomiceto aquático Pythium insidiosum. A maior ocorrência da enfermidade no Brasil é na espécie equina, especialmente no Pantanal Mato-Grossense, porém no Rio Grande do Sul (RS) observa-se um aumento dos casos da doença. Todavia, não há dados de soroprevalência da pitiose no Brasil. O diagnóstico precoce da enfermidade é fundamental para sucesso no controle da pitiose. Atualmente, a imunoterapia com antígenos proteicos vem sendo estudada como uma alternativa promissora de tratamento. A via intradérmica (ID) é uma opção a ser avaliada tanto para um diagnóstico precoce quanto para o tratamento da pitiose empregando antígenos proteicos de P. insidiosum. Neste estudo investigou-se: a soroprevalência da pitiose equina no estado do RS, bem como avaliou-se e comparou-se o uso de antígenos proteicos de P. insidiosum pela via ID no diagnóstico e controle da pitiose em modelo experimental. Para determinar a soroprevalência da pitiose equina no RS foram utilizadas 1002 amostras de soro equino avaliadas pelo método de ELISA indireto. Do total das amostras testadas, 11,07% (111/1002) foram soropositivas para a pitiose equina. Para o diagnóstico aplicou-se 0,1 mL (1,7mg) de antígeno proteico pela via ID, em coelhos (Oryctolagus cuniculus) (n=15) separados em três grupos: controle negativo (n=5), com pitiose experimental (n=5) e animais previamente imunizados com antígeno proteico (n=5). A fim de verificar e comparar a aplicação do antígeno proteico pela via ID como opção de tratamento utilizaram-se dez coelhos com pitiose. Cinco animais foram tratados com 0,1mL de antígeno proteico aplicado via ID e outros cinco coelhos foram tratados pela via subcutânea (SC) com 2mL (34mg) do antígeno. O teste ID foi capaz de detectar reações cutâneas em 24h e 72h nos animais previamente expostos ao agente, demonstrando sensibilidade e especificidade comparáveis ao teste padrão de diagnóstico da pitiose (ELISA indireto). No tratamento com antígeno proteico pelas vias ID e SC, embora não tenha sido observada diferença (P>0,05) no tamanho das lesões, a cura clínica em 40% dos coelhos de ambos os grupos demonstrou que a via ID pode ser utilizada em protocolos terapêuticos da pitiose em modelo experimental, pois também apresenta outras vantagens como menor concentração e volume de antígeno proteico para o controle da pitiose. No entanto, são necessárias pesquisas para o emprego da via ID no diagnóstico e controle da pitiose nas espécies naturalmente acometidas.

La rage entraîne plus de 60,000 décès par an dans le Monde, dont 800 au Cambodge, pays fortement endémique pour la rage canine.La mort survient dans près de 100% des cas de rage, maladie évitable dans presque 100% des cas par l’accès à une prophylaxie post-exposition (PPE) antirabique adéquate et en temps utile. L’amélioration de l’accès à une PPE dans les zones rurales des pays endémiques permettra d’épargner des vies humaines à court terme. Cette thèse en épidémiologie a tiré parti des données collectées auprès des patients consultant au centre antirabique et les chiens testés à l’Institut Pasteur du Cambodge (IPC), Phnom Penh. Suite à un bilan épidémiologique de la situation et des obstacles auxquels sont confrontés les patients cherchant à accéder à la PPE adéquate et en temps utile, elle vise à contribuer à améliorer 1/ l’accès géographique et 2/ l’accès financier à une PPE pour les populations rurales du Cambodge. Nous avons développé une stratégie originale d’identification des poches de populations à haut risque d’incomplétude vaccinale après une exposition potentielle à la rage. Ceci devrait permettre d’améliorer l’accès géographique à la PPE et se concrétiser par l’ouverture en Juillet 2018 d’un centre périphérique de prévention de la rage dans l’Ouest du Cambodge. Cette stratégie d’identification de difficultés d’accès aux soins est applicable à d’autres thématiques de santé, sous certaines conditions. Notre rappel des patients et l’analyse des décès par rage parmi les patients n’ayant pas complété de leur propre chef le protocole PPE de 4 sessions intradermales sur 1 mois ne permettent pas de mettre en évidence une différence de mortalité par rage parmi les patients n’ayant reçu que 3 sessions sur 1 semaine, par rapport à au moins 4 sessions/1mois. Le raccourcissement du protocole à 1 semaine permet de réduire les coûts directs et indirects et l’absence de revenus pendant la durée du traitement en capitale. La mise en place de ce protocole doit s’accompagner d’un suivi d’au moins 6 mois des patients après leur prise en charge initiale. L’ensemble de ces travaux a des implications qui dépassent le cadre du Cambodge: Dans ses recommandations d’Avril 2018, l’OMS recommande désormais ce nouveau protocole IPC– le premier protocole PPE antirabique abrégé à 1 semaine. / Rabies causes more than 60,000 deaths worldwide each year, including 800 in Cambodia, where canine-mediated rabies virus circulates. Death occurs in nearly 100% of rabies cases, a disease which is nearly 100% avoidable by timely and adequate rabies post-exposure prophylaxis (PEP). Improving access to PEP in rural areas of endemic countries will spare human lives in the short term. This epidemiology PhD used the data collected in patients referred to the rabies prevention clinic and tested dogs at Institut Pasteur du Cambodge (IPC), Phnom Penh. After a baseline assessment of access to and obstacles to access timely and adequate PEP in Cambodia, this PhD aims to contribute to improving: 1/ geographical access and 2/ financial access to PEP for rural populations in Cambodia. We developed an original strategy to identify populations with a high risk of PEP noncompletion after a bite by a potentially rabid dog. This should help improve geographical access to PEP following the implementation in July 2018 of a peripheral rabies prevention center in Western Cambodia. This strategy can be applied to identify difficulties in accessing health services relevant to other health issues, under certain conditions. After patient callback and analysis of rabies deaths among those who did and did not complete the 4-sessions/1-month intradermal PEP regimen of their own accord, we were unable to demonstrate a difference in rabies mortality among patients who only received 3 vaccine sessions over the first week compared to those receiving at least 4 sessions/one month. Abridging the protocol to one week would reduce direct and indirect costs and the loss of income during PEP in the Capital. The adoption of this abridged regimen must be associated with a strengthened clinical monitoring system for at least 6 months following patients’ initial PEP.The work presented in this PhD has implications which reach beyond Cambodia: WHO recommends this new IPC regimen – the first approved one-week, abridged rabies PEP regimen – in its April 2018 guidelines.

Refrigerated Stability of Diluted Succinylcholine, Pancuronium, and Atracurium. T. Archibald1, S. Brown1, A. Gonzalez-Estrada2 1College of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, Johnson City, TN2Quillen College of Medicine, Allergy and Clinical Immunology, East Tennessee State University, Johnson City, TN The purpose of this study is to investigate the stored stability of dilutions of neuromuscular blocking agents (NMBAs), namely succinylcholine, pancuronium, and atracurium, for skin prick/intradermal testing. Concentrations of NMBAs were monitored by liquid chromatography-mass spectrometry (LC-MS/MS) for a period of 14 days. Dilutions of NMBAs were prepared in saline by factors of 10x, 100x, 1,000x, 10,000x, and 100,000x as sensitivity of the assay allowed. Each drug was prepared with an n = 5 for each dilution, using a different newly opened product for each series. Diluted drug products were stored in a laboratory refrigerator until sampling. On sampling days (day 0, 1, 2, 4, 7, and 14), one milliliter aliquots of each dilution were removed, filtered, and analyzed against freshly prepared set of reference dilutions. The results are expressed as beyond use date (BUD), defined as recovery of drug versus the reference (90-110%). Based on the LC-MS/MS data, the BUD for succinylcholine diluted by 10x and 100x is 48 and 24 hours, respectively. The1000x dilution is also stable for 24 hours.Higher dilutions of succinylcholine (10,000x to100,000x) should be used immediately following preparation (within less than 24 hours), as the potency of these dilutions had decreased below 90% at the 24 hr sampling. .Pancuronium diluted by 10x and 100x, had a BUD of 48 hours, and the1,000x dilution was stable for 24 hours.As with the succinylcholine, the 10,000x and 100,000x dilutions expressed potency of <90% at 24 hours. .Atracurium diluted to 10x had a BUD of 96 hours, the100x dilution is stable for 24 hours yet higher dilutions (1,000x to 10,000x) do not persist beyond 24 hours. . The 100,000x dilution of atracurium was unknown, given than the signal intensity was too weak to monitor by our LC-MS/MS method. With increasing dilution factors, the stability of these drugs in saline decreases, associated with an increasing deviation between samples and freshly prepared references. The most stable dilutions for each of the drugs tested were 10x and 100x. Stability of these drugs is likely compromised by hydrolysis of the ester bonds in the drug molecules.

O teste alérgico intradérmico (TID) é uma importante ferramenta no diagnóstico dos alérgenos implicados nas reações de hipersensibilidade mediada por IgE. O objetivo deste estudo foi avaliar o TID em eqüinos não alérgicos com extratos alergênicos de Cynodon dactylon, Lolium multiflorum, Paspalum notatum, Culex sp. e Aedes aegipty, e três concentrações de histamina, em dois volumes diferentes, e baseado nos resultados, verificar a acurácia do TID em eqüinos alérgicos. Foram realizados TID em 17 eqüinos não alérgicos com três concentrações de cada extrato (1:2000v/w, 1:4000v/w e 1:8000v/w) e três concentrações de histamina (0,1mg/ml, 0,05mg/ml e 0,025mg/ml), em dois volumes (0,1 e 0,05ml). Baseado nos resultados obtidos, foram realizados TID em seis eqüinos alérgicos utilizando os extratos na concentração de 1:2000v/w, a histamina na concentração de 0,025mg/ml, em um volume de 0,05ml. Nos eqüinos não alérgicos, nenhuma das três concentrações dos extratos testados nos dois volumes mostrou-se irritativa. Concentrações maiores de histamina provocaram halos maiores e levemente mais túrgidos, e quanto maior o volume injetado, maiores as reações Os TID realizados em eqüinos alérgicos mostraram confiabilidade dos resultados por evidenciar hipersensibilidade individual e não provocar reações irritativas. Em conclusão, as diferentes concentrações dos estratos testados em eqüinos não alérgicos não provocaram reações falso positivas (irritantes). O volume de 0,05ml é mais recomendado para realização dos TID pois as aplicações com 0,1ml produziram reações maiores, prejudicando a sensibilidade do teste. A concentração de 0,025mg/ml da solução de histamina provocou a formação de halos menores, permitindo maior acurácia do cut off no TID. Os extratos alergênicos testados em equinos alérgicos não provoraram reações em todos os animais, o que poderia ser considerado uma reação irritativa. Os equinos alérgicos apresentaram reações positivas diferentes, de acordo com a hipersensibilidade individual, demonstrando sensibilização alérgica verdadeira. / The intradermal test (IDT) is an important tool in the diagnosis of allergens involved in IgE-mediated hypersensitivity reactions. The aim of this study was to evaluate the IDT in nonallergic horses with allergenic extracts of Cynodon dactylon, Lolium multiflorum, Paspalum notatum, Culex sp. and Aedes aegypti, and three histamine concentrations, in two different volumes, and based on the results, verify the accuracy of IDT in allergic horses. IDT was performed on 17 nonallergic horses with three concentrations of each extract (1:2000v/w, 1:4000v/h and 18000v/w) and three histamine concentrations (0,1mg/ml, 0,05mg/ml and 0,025mg/ml) in two volumes (0,1 and 0,05ml). Based on the results obtained, IDT was performed in six allergic horses using extracts at the concentration of 1:2000v/w, histamine at a concentration of 0.025mg/ml, in a volume of 0,05ml. In nonallergic horses, none of the three concentrations of the extracts tested in the two volumes were irritant. Larger concentrations of histamine provoked larger and slightly more turgid wheals, and the larger the volume injected, the greater the reactions The IDT performed in allergic horses showed reliability of the results because they demonstrated individual hypersensitivity and did not provoke irritative reactions. In conclusion, the different concentrations of extracts tested in nonallergic horses did not provoke false positive (irritant) reactions. The volume of 0,05ml is more recommended for execution of IDT because the applications with 0,1ml produced larger reactions, impairing the sensitivity of the test. The concentration of 0,025mg/ml of the histamine solution caused the formation of smaller wheals, allowing a better accuracy of the cut off in the IDT. The allergenic extracts tested in allergic horses did not provoke reactions in all the animals, what could be considered an irritative reaction. Allergic horses presented different positive reactions, according to individual hypersensitivity, demonstrating true allergic sensitization.

O teste alérgico intradérmico (TID) é uma importante ferramenta no diagnóstico dos alérgenos implicados nas reações de hipersensibilidade mediada por IgE. O objetivo deste estudo foi avaliar o TID em eqüinos não alérgicos com extratos alergênicos de Cynodon dactylon, Lolium multiflorum, Paspalum notatum, Culex sp. e Aedes aegipty, e três concentrações de histamina, em dois volumes diferentes, e baseado nos resultados, verificar a acurácia do TID em eqüinos alérgicos. Foram realizados TID em 17 eqüinos não alérgicos com três concentrações de cada extrato (1:2000v/w, 1:4000v/w e 1:8000v/w) e três concentrações de histamina (0,1mg/ml, 0,05mg/ml e 0,025mg/ml), em dois volumes (0,1 e 0,05ml). Baseado nos resultados obtidos, foram realizados TID em seis eqüinos alérgicos utilizando os extratos na concentração de 1:2000v/w, a histamina na concentração de 0,025mg/ml, em um volume de 0,05ml. Nos eqüinos não alérgicos, nenhuma das três concentrações dos extratos testados nos dois volumes mostrou-se irritativa. Concentrações maiores de histamina provocaram halos maiores e levemente mais túrgidos, e quanto maior o volume injetado, maiores as reações Os TID realizados em eqüinos alérgicos mostraram confiabilidade dos resultados por evidenciar hipersensibilidade individual e não provocar reações irritativas. Em conclusão, as diferentes concentrações dos estratos testados em eqüinos não alérgicos não provocaram reações falso positivas (irritantes). O volume de 0,05ml é mais recomendado para realização dos TID pois as aplicações com 0,1ml produziram reações maiores, prejudicando a sensibilidade do teste. A concentração de 0,025mg/ml da solução de histamina provocou a formação de halos menores, permitindo maior acurácia do cut off no TID. Os extratos alergênicos testados em equinos alérgicos não provoraram reações em todos os animais, o que poderia ser considerado uma reação irritativa. Os equinos alérgicos apresentaram reações positivas diferentes, de acordo com a hipersensibilidade individual, demonstrando sensibilização alérgica verdadeira. / The intradermal test (IDT) is an important tool in the diagnosis of allergens involved in IgE-mediated hypersensitivity reactions. The aim of this study was to evaluate the IDT in nonallergic horses with allergenic extracts of Cynodon dactylon, Lolium multiflorum, Paspalum notatum, Culex sp. and Aedes aegypti, and three histamine concentrations, in two different volumes, and based on the results, verify the accuracy of IDT in allergic horses. IDT was performed on 17 nonallergic horses with three concentrations of each extract (1:2000v/w, 1:4000v/h and 18000v/w) and three histamine concentrations (0,1mg/ml, 0,05mg/ml and 0,025mg/ml) in two volumes (0,1 and 0,05ml). Based on the results obtained, IDT was performed in six allergic horses using extracts at the concentration of 1:2000v/w, histamine at a concentration of 0.025mg/ml, in a volume of 0,05ml. In nonallergic horses, none of the three concentrations of the extracts tested in the two volumes were irritant. Larger concentrations of histamine provoked larger and slightly more turgid wheals, and the larger the volume injected, the greater the reactions The IDT performed in allergic horses showed reliability of the results because they demonstrated individual hypersensitivity and did not provoke irritative reactions. In conclusion, the different concentrations of extracts tested in nonallergic horses did not provoke false positive (irritant) reactions. The volume of 0,05ml is more recommended for execution of IDT because the applications with 0,1ml produced larger reactions, impairing the sensitivity of the test. The concentration of 0,025mg/ml of the histamine solution caused the formation of smaller wheals, allowing a better accuracy of the cut off in the IDT. The allergenic extracts tested in allergic horses did not provoke reactions in all the animals, what could be considered an irritative reaction. Allergic horses presented different positive reactions, according to individual hypersensitivity, demonstrating true allergic sensitization.