Métodos alternativos ao uso de animais para a detecção de pirogênio: oportunidades e desafios no controle da qualidade de produtos biológicos.

A necessidade de alternativas ao uso de animais no teste de pirogênio foi guiada pelo principio dos 3R’s, culminando no desenvolvimento e aceitação pela Farmacopeia Europeia do método alternativo in vitro, o Teste de Ativação de Monócitos (Monocyte Activation Test- MAT, 2.6.30/ 2010). O MAT utiliza como matriz, fontes de monócitos humanos (sangue total, PBMC, linhagem), respeitando o conceito dos 3R’s e excluindo riscos inerentes à extrapolação inter-espécies. É um método promissor e eficiente, que detecta um amplo espectro de pirogênios e supri limitações dos testes atuais preconizados pelas farmacopeias, o teste in vitro Teste de Endotoxina Bacteriana e o Teste de Pirogênio em Coelhos. Apesar das vantagens abordadas, alguns obstáculos técnico-científicos e regulatórios devem ser transpostos para a implantação efetiva do MAT na rotina industrial, em especial de produtos biológicos

Métodos alternativos para a detecção de pirogênios em produtos e ambientes sujeitos a Vigilância Sanitária: avanços e perspectivas no Brasil a partir do reconhecimento internacional do Teste de Ativação de Monócitos

Introduction: The detection of pyrogens is essential for the quality control of injectable products. The Rabbit Pyrogen Test remains widely used, despite the existence of alternative methods such as the Monocyte Activation Test (MAT). Objective: To review the use of alternative methods for pyrogen testing, pointing out advances and perspectives from the recognition of MAT by the European pharmacopoeia and its acceptance for regulatory purposes in Brazil. Method: A search was performed on the PubMed and BVS databases, with further classification, categorization by topic and critical analysis of the results. Results: Twenty-four papers were identified, addressing topics such as applications of MAT, its validation and comparisons with in vivo tests. MAT presented better results when compared to other tests, both in the evaluation of biological products and in the detection of non-endotoxin pyrogens. Limitations to diffusion include difficulties in obtaining whole human blood as a source of monocytes, for which several alternatives have been proposed. Conclusions: MAT is a promising method, with application in safety evaluation of new technologies. Its application in Brazil depends on a national implementation policy, which might include greater integration between BraCVAM, Concea and RENAMA in search for its recognition for regulatory purposes.Introdução: A detecção de pirogênios é imprescindível no controle da qualidade de produtos injetáveis. O Teste de Pirogênio em coelhos ainda tem larga aplicação, apesar da existência de métodos alternativos como o Teste de Ativação de Monócitos (MAT). Objetivo: Revisar o uso dos métodos alternativos no teste de pirogênio, apontando avanços e perspectivas a partir do reconhecimento do MAT pela Farmacopeia Europeia e sua aceitação para fins regulatórios no Brasil. Método: Uma busca foi realizada nas bases PubMed e BVS, com posterior classificação, categorização por assuntos e análise crítica dos resultados. Resultados: Foram identificados 24 trabalhos, abordando temas como as aplicações do MAT, sua validação e comparação com testes in vivo. O MAT apresentou melhores resultados quando comparado a outros testes, tanto na avaliação de produtos biológicos como na detecção de pirogênios não-endotoxinas. Limitações para sua difusão incluem a dificuldade de obtenção de sangue total humano como fonte de monócitos, para o qual diversas alternativas têm sido propostas. Conclusões: O MAT se mostra um método promissor, com aplicação na avaliação da segurança de novas tecnologias. Sua aplicação no Brasil depende de uma política nacional de implantação, que inclua maior Integração entre BraCVAM, Concea e RENAMA na busca por seu reconhecimento para fins regulatórios

Deciphering the contribution of lipid droplets in leprosy: multifunctional organelles with roles in Mycobacterium leprae pathogenesis

Submitted by Sandra Infurna ([email protected]) on 2017-01-10T10:37:03Z No. of bitstreams: 1 euzenir_sarno_etal_IOC_2012.pdf: 1054189 bytes, checksum: 82e1ee518ea948f6277c12bcb8ca94d7 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-01-10T10:44:13Z (GMT) No. of bitstreams: 1 euzenir_sarno_etal_IOC_2012.pdf: 1054189 bytes, checksum: 82e1ee518ea948f6277c12bcb8ca94d7 (MD5)Made available in DSpace on 2017-01-10T10:44:13Z (GMT). No. of bitstreams: 1 euzenir_sarno_etal_IOC_2012.pdf: 1054189 bytes, checksum: 82e1ee518ea948f6277c12bcb8ca94d7 (MD5) Previous issue date: 2012Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ. Brasil.Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves, presenting a singular clinical picture. Across the leprosy spectrum, lepromatous leprosy (LL) exhibits a classical hallmark: the presence of a collection of M. leprae-infected foamy macrophages/Schwann cells characterised by their high lipid content. The significance of this foamy aspect in mycobacterial infections has garnered renewed attention in leprosy due to the recent observation that the foamy aspect represents cells enriched in lipid droplets (LD) (also known as lipid bodies). Here, we discuss the contemporary view of LD as highly regulated organelles with key functions in M. leprae persistence in the LL end of the spectrum. The modern methods of studying this ancient disease have contributed to recent findings that describe M. leprae-triggered LD biogenesis and recruitment as effective mycobacterial intracellular strategies for acquiring lipids, sheltering and/or dampening the immune response and favouring bacterial survival, likely representing a fundamental aspect of M. leprae pathogenesis. The multifaceted functions attributed to the LD in leprosy may contribute to the development of new strategies for adjunctive anti-leprosy therapies

National collaborative study for establishing the working reference material of the vaccine against measles/mumps and rubella: evolution to the self-sufficiency in the national production of triple viral vaccine

Submitted by Alexandre Sousa ([email protected]) on 2016-07-14T16:37:14Z No. of bitstreams: 1 RIAL_74_178-189.pdf: 1027585 bytes, checksum: 8718a198c01449b67cbfdc7ab47024e5 (MD5)Approved for entry into archive by Alexandre Sousa ([email protected]) on 2016-07-14T16:49:58Z (GMT) No. of bitstreams: 1 RIAL_74_178-189.pdf: 1027585 bytes, checksum: 8718a198c01449b67cbfdc7ab47024e5 (MD5)Made available in DSpace on 2016-07-14T16:49:58Z (GMT). No. of bitstreams: 1 RIAL_74_178-189.pdf: 1027585 bytes, checksum: 8718a198c01449b67cbfdc7ab47024e5 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Toxicologia e Farmacologia. Rio de Janeiro, RJ, Brasil.A monografia farmacopeica da vacina tríplice viral (sarampo/caxumba/rubéola) exige a validação de desempenho do ensaio de potência utilizando-se apropriado material de referência (MR). Com o intuito de estabelecer o primeiro MR de trabalho (MRT) nacional para a vacina tríplice viral, foi realizado o estudo colaborativo nacional com a participação de duas únicas instituições que executam o ensaio de potência desta vacina, o Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos, produtor nacional) e o Instituto Nacional de Controle de Qualidade em Saúde. O material candidato (cMRTBio), preparado pelo produtor, foi avaliado pelos laboratórios participantes utilizando-se as respectivas metodologias in-house de determinação de potência. O cMRTBio foi considerado apropriado como MR in-house por estar em concordância com as especificações recomendadas nas normativas de compêndios, a saber: variações intra- (< 5 %), inter-ensaios (< 10%) e entre laboratórios (< 10 %) abaixo dos limites aceitáveis; e potência estimada (log10 CCID50/DH) em 3,72 para sarampo, 4,80 para caxumba e 3,70 para rubéola. Este trabalho reflete o compromisso do único produtor nacional da vacina tríplice viral com a saúde pública, descrevendo-se a expansão da tecnologia, o cumprimento às diretrizes internacionais, o cuidado com o controle da qualidade e culminância para a autossuficiência nacional na produção de vacinas.The pharmacopoeia monograph for the measles/mumps and rubella (MMR) triple vaccine demands to perform the validation of the potency assay by using the suitable reference material (MR). Aiming at establishing the first work MR (MRT) for the MMR triple vaccine, a national collaborative study was performed with the participation of the two unique national institutions working on the vaccine potency evaluation test, the Imunobiological Technology Institute (Bio-Manguinhos, national manufacturer) and the National Institute for Quality Control in Health. The candidate product (cMRT Bio) prepared by the manufacturer was evaluated by the participant laboratories by employing the respective in-house methodologies for determining the potency. The cMRT Bio was considered suitable as in-house MR, according to the specifications based on the normative compendia, being the intra-assay (< 5 %), inter-assay (< 10 %) and between laboratories variations (< 10 %) below the acceptable limits, and the estimate potency (log10 CCID50/DH) in 3.72 for measles, 4.80 for mumps and 3.70 for rubella. This study reflects the commitment of the unique national MMR vaccine producer to the public health, describing the expansion of technology, the compliance with international guidelines and the careful quality control, leading to the national self-sufficiency in the vaccine production

Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages

Submitted by sandra infurna ([email protected]) on 2016-07-02T23:45:33Z No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-03T00:00:26Z (GMT) No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5)Made available in DSpace on 2016-07-03T00:00:26Z (GMT). No. of bitstreams: 1 katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) Previous issue date: 2016Submitted by Angelo Silva ([email protected]) on 2016-07-07T11:16:57Z No. of bitstreams: 3 katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-07-07T12:20:07Z (GMT) No. of bitstreams: 3 license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5)Made available in DSpace on 2016-07-07T12:20:07Z (GMT). No. of bitstreams: 3 license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) katherine_mattos_etal_IOC_2016.pdf: 1295630 bytes, checksum: f98c045639477298b4060493549ed5f8 (MD5) katherine_mattos_etal_IOC_2016.pdf.txt: 65894 bytes, checksum: e5afd9deb877b4e5fae199967e48b1f7 (MD5) Previous issue date: 2016Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilInstituto Lauro de Souza Lima. Bauru, SP, Brasil.Instituto Lauro de Souza Lima. Bauru, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis

PGL I expression in live bacteria allows activation of a CD206/PPARγ cross-talk that may contribute to successful Mycobacterium leprae colonization of peripheral nerves.

Mycobacterium leprae, an obligate intracellular bacillus, infects Schwann cells (SCs), leading to peripheral nerve damage, the most severe leprosy symptom. In the present study, we revisited the involvement of phenolic glycolipid I (PGL I), an abundant, private, surface M. leprae molecule, in M. leprae-SC interaction by using a recombinant strain of M. bovis BCG engineered to express this glycolipid. We demonstrate that PGL I is essential for bacterial adhesion and SC internalization. We also show that live mycobacterium-producing PGL I induces the expression of the endocytic mannose receptor (MR/CD206) in infected cells in a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent manner. Of note, blocking mannose recognition decreased bacterial entry and survival, pointing to a role for this alternative recognition pathway in bacterial pathogenesis in the nerve. Moreover, an active crosstalk between CD206 and the nuclear receptor PPARγ was detected that led to the induction of lipid droplets (LDs) formation and prostaglandin E2 (PGE2), previously described as fundamental players in bacterial pathogenesis. Finally, this pathway was shown to induce IL-8 secretion. Altogether, our study provides evidence that the entry of live M. leprae through PGL I recognition modulates the SC phenotype, favoring intracellular bacterial persistence with the concomitant secretion of inflammatory mediators that may ultimately be involved in neuroinflammation

Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

<div><p>Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases.</p></div

Comparison of the relative levels^a of metabolites of the arachidonic acid pathway in sera from BT and LL patients before and after antibiotic treatment.

a<p>Absent values were substituted with the limit of detection, represented by the lowest intensity value of any given sample. Then, averaged values from the untreated BT serum samples (n = 4) were normalized to 100% and other samples were normalized accordingly. SD are shown in parentheses. PG, prostaglandin; LT, leukotriene; TX, thromboxane; EET, epoxyeicosatrienoic acid; oxo-ETE, oxoicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; DHET, dihydroxyeicosatrienoic acid.</p

Principal component analysis of the metabonomics data.

<p>Raw DI-FT-ICR-MS data in both negative and positive ionization modes were combined and PCA was performed using Multibase (<a href="http://www.numericaldynamics.com/" target="_blank">http://www.numericaldynamics.com/</a>). Plots show the separation of groups based on the pole of disease (BT, LL) and treatment status (before, after). Sample groups are indicated by the dashed lines.</p