In dieser Arbeit wurden Vektorimpfstoffe auf der Basis rekombinanter Vacciniaviren hinsichtlich ihrer Eignung zur Immunisierung gegen Infektionen mit Respiratorischen Synzytialviren (RSV) untersucht. Hierfür standen genetisches Material und Viruspräparationen des Respiratorischen Synzytialvirus des Rindes (BRSV, Stamm Odijk) sowie des Respiratorischen Synzytialvirus des Menschen (HRSV, Subtyp A2) sowie rekombinante Vacciniaviren MVA-HRSV-F bzw. MVA-HRSV-G zur Verfügung. Rekombinante MVA-Viren, welche die Gene der BRSV-Oberflächenproteine G und F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu), sowie Viren in welchen die Fremdgensequenzen durch Deletion wieder entfernt sind (Revertante Viren MVA-∆BRSV-F und MVA-∆BRSV-G), wurden gentechnologisch hergestellt. Alle rekombinanten MVA-Viren wurden molekular-virologisch charakterisiert und dienten zur Gewinnung und Prüfung von Testimpfstoffen im Tiermodell. Die Untersuchungen zeigen: 1. Alle neu konstruierten rekombinanten MVA-BRSV-Viren produzierten nach Infektion von Zellkulturen die erwünschten Zielantigene, die BRSV-Glykoproteine F und G. Für das durch MVA-Expression hergestellte BRSV-F-Glykoprotein konnte außerdem die biologische Funktionalität in einem Fusionstest in infizierten HeLa-Zellen nachgewiesen werden. 2. Die Charakterisierung der Genome aller MVA-BRSV- sowie MVA-HRSV-Vektorviren bestätigte die exakte Insertion der Fremdgensequenzen im anvisierten Genombereich und zeigte die genetische Stabilität der Virusisolate nach Passagierung. 3. Bei der Untersuchung des Wachstumsverhaltens von MVA-BRSV-F und MVA-BRSV-G zeigte sich die eingeschränkte Vermehrungsfähigkeit des Virus MVA-BRSV-G. Die Konstruktion und Untersuchung der revertanten Viren MVA-∆BRSV-F und MVA-∆BRSV-G belegte die Koproduktion des G-Proteins als Ursache des verminderten Replikationsvermögens. Dieser für ein mögliches Impfvirus erhebliche Nachteil konnte durch die Verwendung eines moderateren Vacciniavirus-Promotors zur Fremdgenexpression (rekombinantes Virus MVA-BRSV-Gneu) behoben werden. 4. Die Prüfung von Testimpfstoffen auf der Grundlage der rekombinanten MVA-HRSV-Viren in einem Maus-HRSV-Infektionsmodell zeigte, dass MVA-HRSV-Impfstoffe, im Gegensatz zu Impfstoffen aus mit Formalin-inaktiviertem HRSV, Immunantworten mit einem ausgewogenen TH1/TH2-assoziierten Zytokinprofil induzierten. Eine infolge von Immunisierung verstärkte Einwanderung eosinophiler Zellen (Marker für Immunpathogenese) in die Lungen HRSV-infizierter Tiere, konnte nach MVA-Impfung nicht beziehungsweise in nur sehr geringem Ausmaß festgestellt werden (OLSZEWSKA et al. 2004). 5. Wichtige erste Daten hinsichtlich der Verträglichkeit, Immunogenität und Schutzwirkung rekombinanter Impfstoffe auf der Basis von MVA-BRSV-F und MVA-BRSV-G konnten in einem Kälber BRSV-Infektionsmodell erhoben werden. Die zweimalige Immunisierung mit MVA-Impfstoff verlief bei allen Tieren ohne feststellbare Nebenwirkungen und die Anregung Vaccinia- bzw. BRSV-F-spezifischer Antikörper bestätigte die Immunogenität der Vektorvakzinen. Schließlich belegten klinische Daten, insbesondere die fehlende Fieberreaktion bei Impflingen nach BRSV-Belastungsinfektion, die Schutzwirkung der MVA-BRSV-Impfstoffe. Insgesamt unterstützen die erzielten Ergebnisse dieser Arbeiten die weitere präklinische und klinische Untersuchung von MVA-Vektorimpfstoffen zur wirksameren und sichereren Bekämpfung von Infektionen mit Respiratorischen Synzytialviren. / This study investigated vector vaccines based on recombinant vaccinia virus MVA for their suitability to immunize against infections with respiratory syncytial viruses. Genetic material and virus stocks of bovine respiratory syncytial virus (BRSV, Strain Odijk) and human respiratory syncytial virus (HRSV, Strain A2) and recombinant vaccinia viruses MVA-HRSV-F and MVA-HRSV-G were provided and used in this study. The project work included the genetical engineering of recombinant MVA expressing gene sequences encoding the BRSV surface proteins G and F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu) and the secondary generation of mutant viruses in which recombinant gene sequences have been removed (revertant viruses MVA-∆BRSV-F, MVA-∆BRSV-G). All recombinant MVA were carefully characterized in in vitro experiments and served for generation of vaccine preparations being tested in animal model systems. The investigations demonstrate: 1. All recombinant MVA-BRSV viruses produced the target antigens (BRSV-F and -G proteins) upon tissue culture infections. Functional activity of BRSV-F protein was demonstrated in a cell fusion assay using virus-infected HeLa cells. 2. The characterization of the genomes of all MVA recombinant viruses confirmed the correct insertion of foreign gene sequences into the target site of the MVA genome and demonstrated the genetic stability of the vector viruses upon tissue culture passage. 3. In vitro studies on virus growth revealed a reduced replicative capacity of the recombinant virus MVA-BRSV-G. Construction and growth analysis of revertant viruses MVA-∆BRSV-F and MVA-∆BRSV-G demonstrated that over expression of BRSV-G protein caused this replication deficiency which could be avoided by using a more moderate vaccinia virus promoter for transcriptional control of recombinant gene expression (recombinant virus MVA-BRSV-Gneu). 4. Upon characterization in a mouse-HRSV challenge model candidate vaccines based on recombinant MVA-HRSV viruses, in contrast to formalin inactivated HRSV, and induced a well balanced TH1 and TH2 cytokine profile. In addition, none of the MVA-HRSV-F vaccinated animals and only two of the MVA-HRSV-G immunized mice showed low-level eosinophilia in the lungs after HRSV challenge infection (OLSZEWSKA et al. 2004). 5. Vaccination experiments in the calf-BRSV challenge model generated first relevant data on safety, immunogenicity and protective capacity of MVA-BRSV recombinant vaccines. The repeated application of MVA vaccine was well tolerated by all vaccinated animals and the induction of vaccinia- and BRSV-F-specific antibody responses confirmed the immunogenicity of the MVA vector vaccines. Moreover, clinical data (lack of fever response in vaccines) suggested the protective capacity of MVA-BRSV immunization upon BRSV challenge. The obtained results from these studies clearly support further preclinical and clinical evaluation of recombinant MVA candidate vaccines to immunize against disease caused by RSV infections in cattle and humans.

The human hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family of viruses. Various other hepadnaviruses are used as models to study human HBV infections as all Hepadnaviridae family members have similar virus structure and replication strategies. The studies performed and described in this thesis were carried out using duck hepatitis B virus (DHBV) infection of Pekin ducks as a model system. Hepadnavirus infections can have either an acute or a chronic outcome. The factors that contribute to these outcomes include the immune response, the age of the host at the time of infection as well as size of viral inoculum. The overall aim of this project was to gain a detailed understanding of the mechanisms involved in clearance of virus and resolution of acute DHBV infections. As a first step, molecular and immunohistochemical detection methods for a range of cellular markers in ducks had to be developed as assays were not readily available. Quantitative reverse transcription PCR assays (qRT-PCR) were developed for the detection of mRNA of the duck T-lymphocyte markers, CD3, CD4, CD8, duck cytokines, IFN-α, IFN-γ, TNF-α and the duck housekeeping genes, β-actin and GAPDH. Immunohistochemistry was developed for the detection of duck CD4 + and CD8 + on T cells and for the detection of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation. These methods were then widely used throughout the project. The innate immune response during HBV infections is not well understood. Toll-like receptors (TLR) are a family of pattern recognition receptors that form part of the innate immune response and are involved in the recognition of bacterial, fungal and viral pathogens. The only TLR that have been reported to recognise viral pathogens are TLR- 2, TLR-3, TLR-4, TLR-7 and TLR-9. The possible role of TLR during hepadnavirus infections had not been well characterized to date. In this project cDNA sequences for duck TLR-2, TLR-4 and TLR-7 were identified and characterised and qRT-PCR assays were developed for their detection. Changes in duck TLR-2, TLR-4 and TLR-7 mRNA expression during hepadnavirus infection were identified following DHBV infection of primary duck hepatocytes (PDH) in vitro. The results showed increased levels of expression of duck TLR early during infection indicating an involvement of TLR and the innate immune response during DHBV infection. During the in vivo DHBV infection studies performed to date TLR mRNA expression remained unchanged. As previously mentioned hepadnavirus infection can have an acute or chronic outcome. We aimed to understand the mechanisms involved during the resolution of acute DHBV infection and to elucidate specific factors contributing to the successful resolution of infection. During acute infections immune markers were monitored by qRT-PCR and histological analysis of fixed liver sections was performed. Liver sections were analysed to detect liver inflammation, the number and size of Kupffer cells, hepatocyte apoptosis and changes in hepatocyte proliferation throughout the course of acute DHBV infection in 6-week-old ducks. By determining the percentage of DHBV-positive hepatocytes two patterns of clearance of acute DHBV infection were observed; early clearance of infected hepatocytes occurring before day 14 post infection (p.i.), and late clearance occurring after day 14, but before or on day 31 p.i. This viral clearance was seen to occur in a cell by cell pattern. Higher levels of hepatocyte proliferation and apoptotic hepatocytes were detected during the clearance phase (on day 14 p.i.) of the late clearance group. Periodic acid schiff-diastase (PAS-D) staining was used to show significant increases in both cell number and size of Kupffer cells. Levels of IFN-γ mRNA increased significantly over the uninfected age-matched control ducks on day 14 p.i. Levels of CD3, CD4 and CD8 mRNA expression also increased over the uninfected controls on days 14 and 31 p.i. In summary, we found that resolution of acute DHBV infection occurred on a cell by cell pattern of clearance, it was accompanied by increases in hepatocyte proliferation, apoptotic hepatocytes and activated Kupffer cells, indicating that T lymphocytes and cell death play important roles in the rapid clearance of DHBV infection. Following resolution of acute hepadnaviral infections residual viral DNA has been found to persist. Residual HBV DNA in humans can result in reactivation of HBV infection following liver transplantation or immunosuppressive drug treatment. This leads to possible pathogenic outcomes thus the need for further investigations. Previous studies performed in the duck model have shown that the major form of residual DNA is present as covalently closed circular DNA (cccDNA). We aimed to understand how this residual cccDNA was being maintained and if replication was involved in the process. Following resolution of infection in ducks, levels of residual DHBV DNA were monitored by quantitative PCR (qPCR). Ducks were treated with the Bristol-Myers Squibb nucleoside analogue Entecavir (ETV) in order to suppress any possible replication that might be maintaining levels of residual cccDNA. In DHBV-infected but non-ETV treated ducks, the levels of residual DHBV DNA decreased gradually when measured on days 60, 221 and 316 p.i. The observed decrease in residual DHBV DNA occurred in parallel with decreases in the rate of hepatocyte proliferation measured by PCNA staining. This finding suggests that levels of residual DHBV DNA and hepatocyte proliferation are linked and we hypothesise that hepatocyte turnover is involved in the clearance of residual DHBV DNA. ETV treatment did not have an effect on the levels of residual DHBV DNA which suggests that it is present in a subset of long-lived hepatocytes that do not support virus replication. Mathematical modelling was performed to predict the rate of hepatocyte proliferation required for the elimination of residual cccDNA. The mathematical modelling showed that the predicted rate of hepatocyte proliferation was consistent with the rate of hepatocyte proliferation measured by PCNA. Further mathematical modelling showed that residual cccDNA is most likely to survive mitosis and it decreases due to several rounds of hepatocyte proliferation required for its elimination. Altogether, this project has elucidated mechanisms involved during the resolution of acute DHBV infection and also possible mechanisms by which residual DHBV DNA is maintained following resolution of infection. Detailed understanding of the virological and immunological events that occur during the resolution of an acute hepadnavirus infection would assist in the development of new therapeutic treatments for the cure of chronic HBV infections. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345121 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Eating disorders are serious psychological disorders associated with debilitating lifestyle, multiple health problems and high rates of suicidality and mortality. Despite extensive research, the aetiology of eating disorders still remains unclear. Amongst the identified risk factors for eating disorders, stress has been frequently studied. The purpose of the present study was to explore the possibility that tail-pinch administered to rats could provide an animal model of stress-induced eating disturbances in humans, and whether environmental enrichment might ameliorate the effects of stress. In Experiment 1, we compared eating behaviours of rats that were reared in either enriched or standard environments and later exposed to tail-pinch and allowed to eat when food deprived. The study showed that a single exposure to tail-pinch induced eating disturbances in most of the rats. When rats were not food deprived, but were conditioned to eating when placed in test chamber, tail-pinch suppressed eating in all rats, but significantly more for rats reared under standard than in enriched conditions. Experiment 2 used a between-subjects design in which rats were reared in either a standard or enriched environment, and were either exposed to tail-pinch or not exposed during sessions in which they were not food deprived and allowed to eat. Tail-pinch suppressed the food intake of rats reared in enriched but not standard environments. Although this finding appeared to contradict results of Experiment 1, analysis of body weight revealed that exposure to tail pinch suppressed increases in weight gain across sessions more for rats reared in standard than enriched environments. The suppression of food intake during test sessions for enriched but not standard rats exposed to tail-pinch was attributed to differences in contextual conditioning and discrimination of the test chamber from home cages. Overall, results of the present study suggest that rats reared in enriched environments were more resilient to the effects of tail-pinch as a stressor. Implications of these findings for the understanding of human eating disorders are discussed.

No description available.

Evaluation de l'efficacité et de l'innocuité de la microcuriethérapie à base de microparticules d'holmium pour le traitement du gliobastome induit chez un modèle porcinAuteur: Seyed Mehrdad KHOSH NEVISMot clés: U87, miniporc, glioblastome, modèle animal, Yucatan, microcuriethérapie, Holmium-166, nanoparticulesRésumé: L’objectif de notre étude a été de tester une modalité thérapeutique moins invasive (microcuriethérapie), en phase préclinique sur un modèle animal porcin, pour le traitement du GB. 1) Nous avons développé un nouveau modèle de GB en implantant la lignée U87 dans le cerveau du miniporc (Yucatan). Les images scanner ont montré la présence de tumeurs chez tous les miniporcs. 2) Nous avons évalué l’injectabilité d’holmium non radioactif (ho-165) après injection stéréotaxique dans le cerveau de miniporcs sains. L’holmium était bien circonscrit aux zones d’injection et a été validée pour les essais ultérieurs d'injections intra-tumorales. 3) Nous avons réalisé des injections d’ho-165, directement dans la tumeur d’un modèle de GB U87. La faisabilité des injections d’ho-165 a été testée chez le miniporc en utilisant un système d’injection préclinique spécifique. Les résultats de cette étude ont montré que la suspension de nanoparticules d’ho-165 pouvait être injectée, avec précision, dans la tumeur. 4) L’étape finale a été d’évaluer l’efficacité du traitement à base de nanoparticules d’ho-166 en microcuriethérapie sur le modèle de GB du miniporc. Notre étude a démontré un très bon contrôle tumoral et l’absence d’effets toxiques / Evaluation of treatment efficacy and safety of microbrachytherapy with holmium microparticles in porcine induced glioblastoma model.Author: Seyed Mehrdad KHOSH NEVISKeywords: U87, minipig, gliobastoma, animal model, Yucatan, Microbrachytherapy, Holmium-166, nanoparticlesResumé: The aim of our study was to apply a less invasive therapeutic method (microbrachytherapy) for the treatment of GB in a preclinical phase on a suitable animal model.1) First, we developed a new model of GB in Yucatan minipig by implantation of U87 cell line in the brain. All implanted minipigs demonstrated macroscopic tumors on CT acquisitions. 2) In parallel, the injectability of the non-radioactive Holmium (Ho-165) in healthy brain was assessed. Holmium was circumscribed to the injection area and was found to be suitable for future intratumoral injections. 3) In the next step, the feasibility of injection of ho-165 was tested in minipigs GB model using specific injection system. Results showed that the ho-165 nanoparticles suspension can be injected precisely inside the cerebral tumor using our prototype injection system. 4) The final step was the evaluation of treatment efficacy using radioactive ho-166, as microbrachytherapy, in Yucatan GB model. Our trial demonstrated an excellent efficiency in tumor control and the absence of toxic effects

O peptídeo liberador da gastrina (GRP), homólogo mamífero da bombesina (BN), é um neuropeptídeo com múltiplas ações biológicas, incluindo um importante papel na regulação da resposta imunológica e inflamatória. Está presente em altas concentrações no líquido sinovial de pacientes com artrite reumatóide (AR), paralelamente com os níveis de interleucina-6 (IL-6) e fator de necrose tumoral (TNF). RC-3095 é um antagonista sintético do receptor da BN/GRP que demonstrou modular a liberação de citocinas pró-inflamatórias (TNF e IL-1 ) e diminuir o infiltrado inflamatório em modelos experimentais de sepse. Este estudo avaliou os efeitos do RC-3095 nos parâmetros clínicos, histopatológicos e de mediadores inflamatórios na artrite induzida por adjuvante completo de Freund (CFA) em ratos. A artrite foi induzida por uma injeção de CFA junto a superfície subplantar da pata esquerda de ratos machos de Wistar. Os animais foram divididos 4 grupos: grupo controle, controle injetado com veículo, grupo placebo (administrado salina subcutaneamente 50ml/kg, uma vez ao dia por 8 dias após estabelecimento do modelo), grupo tratado (0.3 mg/kg de RC-3095 subcutaneamente, uma vez ao dia por 8 dias após a indução). A avaliação clínica foi acompanhada diariamente, de acordo com um escore do edema para as patas posteriores. O peptídeo liberador da gastrina (GRP), homólogo mamífero da bombesina (BN), é um neuropeptídeo com múltiplas ações biológicas, incluindo um importante papel na regulação da resposta imunológica e inflamatória. Está presente em altas concentrações no líquido sinovial de pacientes com artrite reumatóide (AR), paralelamente com os níveis de interleucina-6 (IL-6) e fator de necrose tumoral (TNF). RC-3095 é um antagonista sintético do receptor da BN/GRP que demonstrou modular a liberação de citocinas pró-inflamatórias (TNF e IL-1 ) e diminuir o infiltrado inflamatório em modelos experimentais de sepse. Este estudo avaliou os efeitos do RC-3095 nos parâmetros clínicos, histopatológicos e de mediadores inflamatórios na artrite induzida por adjuvante completo de Freund (CFA) em ratos. A artrite foi induzida por uma injeção de CFA junto a superfície subplantar da pata esquerda de ratos machos de Wistar. Os animais foram divididos 4 grupos: grupo controle, controle injetado com veículo, grupo placebo (administrado salina subcutaneamente 50ml/kg, uma vez ao dia por 8 dias após estabelecimento do modelo), grupo tratado (0.3 mg/kg de RC-3095 subcutaneamente, uma vez ao dia por 8 dias após a indução). A avaliação clínica foi acompanhada diariamente, de acordo com um escore do edema para as patas posteriores. / Background: Recently, targeting of neuropeptides has been suggested to have potential therapeutic effects in RA. The gastrin-releasing peptide (GRP) is the homologous mammalian of the bombesin (BN), and its receptor signaling is involved in several functions, including cancer growth, immune cell regulation and inflammatory conditions, and has been found in RA synovial fluid in concentrations that correlate with IL-6 and TNF. RC-3095 is the antagonist of the GRP receptor, and has been demonstrated to modulate the release of proinflammatory cytokines (TNF and IL-1 ) by activated macrophages, leading to decreased inflammatory infiltration. Objective: To determine the effects of RC-3095 in clinical and histopathologic parameters and inflammatory mediators on complete Freund´s adjuvant-induced arthritis. Methods: The arthritis was induced by injection of complete Freund´s adjuvant (CFA) into the left hind footpad in males of Wistar rats. The animals were divided into control, vehicle injected control, placebo group (saline subcutaneously 50ml/kg, once daily for 8 days after modeling), treatment group (0.3 mg/kg of RC-3095 subcutaneously, once daily for 8 days after induction). Clinical evaluation was accomplished daily, through scoring of the paw edema. The animals were sacrificed 15 days after induction for collection of hind foot joints for histology. We used a histological scoring system which was previously described, and interferon (INF)-g, Interleukin (IL)-1 , tumor necrosis factor (TNF), interleukin (IL)-6 and interleukin (IL)-10 were measured by ELISA. Statistical analyses were compared by Student t-test to determine the difference between placebo and treatment groups for histological scores, and cytokines levels were compared using Mann-Whitney test (p<0.05). Results: There was significant inhibition of joint histological findings in the RC-3095 treated group, including synovial inflammatory infiltration(p<0,001), synovial hyperplasia (p=0.004), extension of pannus (p<0.001), synovial fibrosis (p<0.001), angiogenesis (p=0.033), cartilage (p 0.001) and bone erosion (p<0.001). IFN-g (p<0.001), IL-1 (p<0.001), TNF (p=0.003), IL-6 (p=0.048) and IL-10 (p=0.015) serum levels were significantly lower in the treated group. Paw swelling and subcutaneous inflammation, evaluated clinically, were not different between CFAinduced groups. Conclusions: RC-3095 was able to improve experimental arthritis, attenuate joint damage and decrease serum levels of IFN-g, IL-1 , TNF, IL-6 and IL-10. These data indicate that interference with GRP pathway is a potential new strategy for the treatment of RA that needs further investigational studies.

Esquizofrenia (SZ) é uma doença multifacetada que afeta vários domínios, incluindo sintomas positivos e negativos. Devido a isso, até o momento, não existe um modelo animal adequado de SZ, o que contribui para o fato de sua fisiopatologia ainda ser pouco conhecida. Um dos melhores modelos animais de SZ desenvolvidos usa acetato metilazoximetanol (MAM), no 17º dia embrionário (MAM-E17) de ratas prenhes, o que leva a alguns dos padrões de histopatologia observados em SZ. O objetivo deste estudo é avaliar o uso do MAM-E17 para validar achados histológicos deste modelo animal de SZ. Um total de 4 controles e 13 MAM-E17 foram estudados. Os ratos submetidos ao tratamento MAM-E17 apresentaram uma diminuição no peso do cérebro e na área do hipocampo caudal esquerdo. Não houve diferença na área do hipocampo caudal direito nem no hipocampo dorsal. Os animais submetidos ao MAM-E17 mostraram uma diminuição na espessura do córtex pré-frontal direito. O modelo de roedor MAM-E17 demonstrou reproduzir os principais aspectos dos achados histológicos altamente relevantes na SZ. Assim, a administração do MAM-E17 pode ser um modelo animal apropriado para o estudo de aspectos histológicos de SZ. Isto é de suma importância para o melhor entendimento e tratamento desta devastadora doença no futuro. / Schizophrenia (SZ) is a multifaceted illness that affects multiple domains, including negative and positive symptons. Because of this, there is no appropriate animal model of SZ, which contributes to its still is poorly understood pathophysiology. One of the bestdeveloped animal models of SZ uses methylazoxymethanol acetate (MAM) in pregnant rat dams on embryonic day 17 (MAM-E17) to procedure patterns of histopathology similar to those observed in SZ. The aim of this study was to evaluate the use of MAM-E17 to validate the histological findings of this animal model of SZ. A total of 4 controls and 13 MAM-E17 were studied. Rats subjected to MAM-E17 treatment presented a decreased in brain weight and in the left caudal hippocampus area. There were no differences in the right caudal hippocampal area or in left and right dorsal hippocampal areas. The animals subjected to MAM-E17 showed a decrease in right prefrontal cortex thickness. The MAME17 rodent model reproduced key aspects of histological findings that are highly relevant in SZ. Thus, the admistration of MAM-E17 may be an appropriate animal model for the study of histological aspects of SZ. This is of paramount importance to the better understanding and treatment of this devastating disease in the future.

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Instituto Nacional de Controle de Qualidade em Saúde / Passados quase 16 anos após o surgimento dos medicamentos genéricos no Brasil em 1999, sua aceitação por parte dos prescritores e da população ainda não chegou a níveis alcançados por países que adotaram a política há mais tempo. A principal desconfiança da população é em relação à qualidade dos produtos devido ao baixo custo e, associações que a palavra “genérico” tem conotação em determinadas frases como sendo algo de pouca qualidade ou incompleto, que não atingiu o esperado, carregando assim uma forma pejorativa de se referir a um produto. Apesar da qualidade destes produtos ser garantida pela resolução RDC n°17/2010 que regulamenta as boas práticas para fabricação de medicamentos, o controle de qualidade pós-mercado ainda deixa a desejar. A análise laboratorial do produto, salvo por motivos de renovação de registro, só é atualmente feita quando há denúncias devido a ocorrência de muitos casos de efeitos adversos ou ineficácia a cerca de um medicamento. Nestes casos, os testes realizados são relativos ao teor, características organolépticas, microbiológicas e potência. Porém, nenhuma dessas verificações gera resultados capazes de comprovar o real comportamento dos fármacos no organismo humano. Com isso, faz-se necessária a realização de estudos que comprovem a segurança e eficácia dos medicamentos genéricos disponíveis no mercado brasileiro para que haja uma maior adesão a este programa de governo. O objetivo deste trabalho foi de desenvolver um modelo animal para testar a biodisponibilidade/ bioequivalência dos medicamentos, uma vez que esses testes são feitos em humanos e possuem alto custo de realização além das implicações éticas. Os resultados obtidos confirmam que o fato dos antibióticos não apresentarem alterações na potência não pode ser associada a eficácia do tratamento. O teste de potência não reprovou nenhuma das formulações testadas, enquanto que a avaliação da bioequivalência in vivo foi negativa para um dos medicamentos. Nossos resultados comprovam a necessidade de revisão dos modelos de controle de qualidade utilizados atualmente. E, disponibiliza uma nova e importante opção para utilização no controle de qualidade de medicamentos e, que também pode ser utilizada no desenvolvimento de novos fármacos e medicamentos. / After almost 16 years since the beginning of the Brazilian generic medicines politics in 1999, the acceptance from physicians and the general population is unlike that in other countries that have adopted them for a longer time. The main distrust of consumers regarding the quality of the product lies on its lower cost and the association the word “generic” has with some expressions that infer something of poor quality or inefficient, which gives the product pejorative character. Even though the quality of the products is guaranteed by Resolution RDC No. 17/2010, which regulates good practice for the manufacturing of drugs, post-market quality control is still poor. The verification of the product, except for registration and renewal purposes, is currently only performed when there are reports or many cases of adverse events regarding a drug. In these cases, the tests made are content, organoleptic characteristics, microbiological and potency related. However, none of these tests yields results that prove the bioavailability of the drug. Thus, it is necessary to carry out studies to prove the safety and efficacy of generic drugs available in Brazilian market to promote a greater adherence to the program. The goal of this study was to develop an animal model to test the bioavailability / bioequivalence of drugs, once these tests are made in humans and have high costs to execution and ethical questions. The results confirm the fact that antibiotics that don’t present changes in potency can’t be associated with efficacy of treatment. The in vitro test didn’t reprove any of the tested compositions, whereas the in vivo bioequivalence assessment was negative for one the drugs. Our results show the need to review quality control models currently being used. And gives another and important option for the quality control of medicines and also can be used in new drugs development and medicines.

O peptídeo liberador da gastrina (GRP), homólogo mamífero da bombesina (BN), é um neuropeptídeo com múltiplas ações biológicas, incluindo um importante papel na regulação da resposta imunológica e inflamatória. Está presente em altas concentrações no líquido sinovial de pacientes com artrite reumatóide (AR), paralelamente com os níveis de interleucina-6 (IL-6) e fator de necrose tumoral (TNF). RC-3095 é um antagonista sintético do receptor da BN/GRP que demonstrou modular a liberação de citocinas pró-inflamatórias (TNF e IL-1 ) e diminuir o infiltrado inflamatório em modelos experimentais de sepse. Este estudo avaliou os efeitos do RC-3095 nos parâmetros clínicos, histopatológicos e de mediadores inflamatórios na artrite induzida por adjuvante completo de Freund (CFA) em ratos. A artrite foi induzida por uma injeção de CFA junto a superfície subplantar da pata esquerda de ratos machos de Wistar. Os animais foram divididos 4 grupos: grupo controle, controle injetado com veículo, grupo placebo (administrado salina subcutaneamente 50ml/kg, uma vez ao dia por 8 dias após estabelecimento do modelo), grupo tratado (0.3 mg/kg de RC-3095 subcutaneamente, uma vez ao dia por 8 dias após a indução). A avaliação clínica foi acompanhada diariamente, de acordo com um escore do edema para as patas posteriores. O peptídeo liberador da gastrina (GRP), homólogo mamífero da bombesina (BN), é um neuropeptídeo com múltiplas ações biológicas, incluindo um importante papel na regulação da resposta imunológica e inflamatória. Está presente em altas concentrações no líquido sinovial de pacientes com artrite reumatóide (AR), paralelamente com os níveis de interleucina-6 (IL-6) e fator de necrose tumoral (TNF). RC-3095 é um antagonista sintético do receptor da BN/GRP que demonstrou modular a liberação de citocinas pró-inflamatórias (TNF e IL-1 ) e diminuir o infiltrado inflamatório em modelos experimentais de sepse. Este estudo avaliou os efeitos do RC-3095 nos parâmetros clínicos, histopatológicos e de mediadores inflamatórios na artrite induzida por adjuvante completo de Freund (CFA) em ratos. A artrite foi induzida por uma injeção de CFA junto a superfície subplantar da pata esquerda de ratos machos de Wistar. Os animais foram divididos 4 grupos: grupo controle, controle injetado com veículo, grupo placebo (administrado salina subcutaneamente 50ml/kg, uma vez ao dia por 8 dias após estabelecimento do modelo), grupo tratado (0.3 mg/kg de RC-3095 subcutaneamente, uma vez ao dia por 8 dias após a indução). A avaliação clínica foi acompanhada diariamente, de acordo com um escore do edema para as patas posteriores. / Background: Recently, targeting of neuropeptides has been suggested to have potential therapeutic effects in RA. The gastrin-releasing peptide (GRP) is the homologous mammalian of the bombesin (BN), and its receptor signaling is involved in several functions, including cancer growth, immune cell regulation and inflammatory conditions, and has been found in RA synovial fluid in concentrations that correlate with IL-6 and TNF. RC-3095 is the antagonist of the GRP receptor, and has been demonstrated to modulate the release of proinflammatory cytokines (TNF and IL-1 ) by activated macrophages, leading to decreased inflammatory infiltration. Objective: To determine the effects of RC-3095 in clinical and histopathologic parameters and inflammatory mediators on complete Freund´s adjuvant-induced arthritis. Methods: The arthritis was induced by injection of complete Freund´s adjuvant (CFA) into the left hind footpad in males of Wistar rats. The animals were divided into control, vehicle injected control, placebo group (saline subcutaneously 50ml/kg, once daily for 8 days after modeling), treatment group (0.3 mg/kg of RC-3095 subcutaneously, once daily for 8 days after induction). Clinical evaluation was accomplished daily, through scoring of the paw edema. The animals were sacrificed 15 days after induction for collection of hind foot joints for histology. We used a histological scoring system which was previously described, and interferon (INF)-g, Interleukin (IL)-1 , tumor necrosis factor (TNF), interleukin (IL)-6 and interleukin (IL)-10 were measured by ELISA. Statistical analyses were compared by Student t-test to determine the difference between placebo and treatment groups for histological scores, and cytokines levels were compared using Mann-Whitney test (p<0.05). Results: There was significant inhibition of joint histological findings in the RC-3095 treated group, including synovial inflammatory infiltration(p<0,001), synovial hyperplasia (p=0.004), extension of pannus (p<0.001), synovial fibrosis (p<0.001), angiogenesis (p=0.033), cartilage (p 0.001) and bone erosion (p<0.001). IFN-g (p<0.001), IL-1 (p<0.001), TNF (p=0.003), IL-6 (p=0.048) and IL-10 (p=0.015) serum levels were significantly lower in the treated group. Paw swelling and subcutaneous inflammation, evaluated clinically, were not different between CFAinduced groups. Conclusions: RC-3095 was able to improve experimental arthritis, attenuate joint damage and decrease serum levels of IFN-g, IL-1 , TNF, IL-6 and IL-10. These data indicate that interference with GRP pathway is a potential new strategy for the treatment of RA that needs further investigational studies.

Esquizofrenia (SZ) é uma doença multifacetada que afeta vários domínios, incluindo sintomas positivos e negativos. Devido a isso, até o momento, não existe um modelo animal adequado de SZ, o que contribui para o fato de sua fisiopatologia ainda ser pouco conhecida. Um dos melhores modelos animais de SZ desenvolvidos usa acetato metilazoximetanol (MAM), no 17º dia embrionário (MAM-E17) de ratas prenhes, o que leva a alguns dos padrões de histopatologia observados em SZ. O objetivo deste estudo é avaliar o uso do MAM-E17 para validar achados histológicos deste modelo animal de SZ. Um total de 4 controles e 13 MAM-E17 foram estudados. Os ratos submetidos ao tratamento MAM-E17 apresentaram uma diminuição no peso do cérebro e na área do hipocampo caudal esquerdo. Não houve diferença na área do hipocampo caudal direito nem no hipocampo dorsal. Os animais submetidos ao MAM-E17 mostraram uma diminuição na espessura do córtex pré-frontal direito. O modelo de roedor MAM-E17 demonstrou reproduzir os principais aspectos dos achados histológicos altamente relevantes na SZ. Assim, a administração do MAM-E17 pode ser um modelo animal apropriado para o estudo de aspectos histológicos de SZ. Isto é de suma importância para o melhor entendimento e tratamento desta devastadora doença no futuro. / Schizophrenia (SZ) is a multifaceted illness that affects multiple domains, including negative and positive symptons. Because of this, there is no appropriate animal model of SZ, which contributes to its still is poorly understood pathophysiology. One of the bestdeveloped animal models of SZ uses methylazoxymethanol acetate (MAM) in pregnant rat dams on embryonic day 17 (MAM-E17) to procedure patterns of histopathology similar to those observed in SZ. The aim of this study was to evaluate the use of MAM-E17 to validate the histological findings of this animal model of SZ. A total of 4 controls and 13 MAM-E17 were studied. Rats subjected to MAM-E17 treatment presented a decreased in brain weight and in the left caudal hippocampus area. There were no differences in the right caudal hippocampal area or in left and right dorsal hippocampal areas. The animals subjected to MAM-E17 showed a decrease in right prefrontal cortex thickness. The MAME17 rodent model reproduced key aspects of histological findings that are highly relevant in SZ. Thus, the admistration of MAM-E17 may be an appropriate animal model for the study of histological aspects of SZ. This is of paramount importance to the better understanding and treatment of this devastating disease in the future.