This work was partially supported by the National Institutes of Health (NIH) Clinical and Translational Science Awards (CTSA) Grant (UL1TR001070) to the Ohio State University's Center for Clinical and Translational Science (CCTS) and the National Library Of Medicine under Award Number T15LM011270.

1. Termos início de Consultas de Gene, drogas ou doença <ol><li> Acesse a página inicial para &quot;RE: Drogas finos&quot; no seguinte link: http://drug-repurposing.nationwidechildrens.org. Comece digitando um termo de consulta na barra de pesquisa de qualquer uma das seguintes três categorias: drogas (nome genérico), doença (nova indicação doença) ou gene (símbolo oficial do gene HGNC). </li><li> Filtrar a função de pesquisa bar para incluir apenas &quot;nome do medicamento&quot;, &quot;Doença de New Indicação&quot;, &quot;símbolo do gene&quot; ou &quot;all&quot; categorias. A barra de pesquisa inclui uma função de auto-suficiência para as entradas de consulta. </li><li> Coloque em uma palavra-chave e clique no botão &quot;Pesquisar&quot;. Classificar a tabela de resultados por qualquer uma das seguintes colunas: &quot;Drogas&quot;, &quot;Indicação Registered&quot;, &quot;P-Value&quot;, &quot;P-valor ajustado&quot;, &quot;Odds Ratio&quot;, &quot;Estudo&quot;, &quot;New Indicação&quot;, &quot;Drogas Banco Indicação &quot;,&quot; # de Medline Abstracts &quot;,&quot; # do CLITrial Registry téc- &quot;,&quot; potencial &quot;,&quot; SNP &quot;,&quot; Gene &quot;ou&quot; ação &quot;. </li><li> Navegue até a opção de busca avançada, a fim de habilitar o recurso de informação sobre a droga. Clique no ícone na coluna &quot;Info&quot; para um determinado medicamento. Observar uma página que lista a toda a informação correspondente incluindo p-Value para a associação, nome da doença, nome do medicamento, detalhes Gene (NCBI ligação Gene) e detalhes de Drogas (link drugbank). </li></ol> 2. Exploração de Opções Avançadas <ol><li> Clique no botão &quot;Avançado&quot; localizado no lado direito da página, e são fornecidos várias opções para refinar ainda mais os resultados. As opções avançadas de pesquisa incluem modificações para o seguinte: droga, associação, doença, potencial, gene e ação. </li><li> Exportação tabelas de resultados, clicando no botão &quot;Exportar&quot; no lado direito da página. Clique no botão &quot;Simples&quot;, a fim de dobrar para baixo a janela de pesquisa avançada. &lt;/ Li&gt; <li> Sob a guia Avançado opção &quot;droga&quot;, especifique uma indicação determinado medicamento ou um nome de medicamento adicional para filtrar os resultados. </li><li> Sob a guia &quot;associação&quot;, resultados do filtro por o nível de significância P-Value, P-Valor Ajustado com FDR, tamanho do efeito (Odds Ratio), e / ou estudo do tipo (GWAS, PheWAS ou ambos). </li><li> Sob a guia &quot;doença&quot;, especifique uma certa descrição da doença para a nova utilização prevista. </li><li> No separador &quot;potencial&quot;, resultados de filtro de acordo com qualquer um dos seguintes critérios: (i) se a indicação da droga está contida na base de dados drugbank, (ii) número de resumos Medline com co-ocorrência da droga e da doença, ( iii) número de entradas de banco de dados ClinicalTrials.gov com co-ocorrência de droga e a doença e (iv) Repurposing Potencial. NOTA: A opção Potencial Repurposing descreve a novidade da descoberta: (i) Conhecido: relacionamento já existe no banco de dados drugbank, (ii) Fortemente apoiada: Algum apoio, tanto do registro de ensaios clínicos e os resumos Medline, (iii) Provável: algum apoio em qualquer registro de ensaio clínico ou os resumos Medline e (iv) Novel: nenhuma evidência no registro de ensaios clínicos nem em resumos Medline. </li><li> Sob a guia &quot;gene&quot;, digite um identificador SNP ou símbolo do gene para filtrar os resultados por genes alvo de drogas específicas. </li><li> Sob a guia &quot;ação&quot;, especifique o tipo de ação de drogas contra o alvo (s) de drogas, incluindo agonista, antagonista, outro, desconhecido ou todos (fonte: Banco de dados drugbank). </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+better+drug+repositioning:+prioritizing+and+integrating+existing+methods+into+efficient+pipelines.">Toward better drug repositioning: prioritizing and integrating existing methods into efficient pipelines.</a> Drug Discov Today. 19 (5), 637-644 (2014).</li><li>Moosavinasab, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;RE:fine+Drugs&amp;#34;:+An+Interactive+Dashboard+to+Access+Drug+Repurposing+Opportunities.">34;RE:fine Drugs&amp;#34;: An Interactive Dashboard to Access Drug Repurposing Opportunities.</a> Database (Oxford). , (2016).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genome-wide+association+studies+for+cancer+research+and+drug+repositioning.">Use of genome-wide association studies for cancer research and drug repositioning.</a> PLoS One. 10 (3), e0116477 (2015).</li><li>Rastegar-Mojarad, M., Ye, Z., Kolesar, J. M., Hebbring, S. J., Lin, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+for+drug+repositioning+from+phenome-wide+association+studies.">Opportunities for drug repositioning from phenome-wide association studies.</a> Nat Biotechnol. 33 (4), 342-345 (2015).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+current+trends+in+computational+drug+repositioning.">A survey of current trends in computational drug repositioning.</a> Brief Bioinform. 17 (1), 2-12 (2016).</li><li>Denny, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+phenome-wide+association+study+of+electronic+medical+record+data+and+genome-wide+association+study+data.">Systematic comparison of phenome-wide association study of electronic medical record data and genome-wide association study data.</a> Nat Biotechnol. 31 (12), 1102-1110 (2013).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+drug-disease+relationships+as+a+complement+to+medical+genetics-based+drug+repositioning:+Where+a+recommendation+system+meets+genome-wide+association+studies.">Mining drug-disease relationships as a complement to medical genetics-based drug repositioning: Where a recommendation system meets genome-wide association studies.</a> Clin Pharmacol Ther. 97 (5), 451-454 (2015).</li><li>Sanseau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genome-wide+association+studies+for+drug+repositioning.">Use of genome-wide association studies for drug repositioning.</a> Nat Biotechnol. 30 (4), 317-320 (2012).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validating+drug+repurposing+signals+using+electronic+health+records:+a+case+study+of+metformin+associated+with+reduced+cancer+mortality.">Validating drug repurposing signals using electronic health records: a case study of metformin associated with reduced cancer mortality.</a> J Am Med Inform Assoc. 22 (1), 179-191 (2015).</li></ol>

The authors declare that they have no competing financial interests.

The protocol described here for the RE:fine Drugs interactive dashboard can be modified in different ways according to the user's preferences. This method uniquely integrates GWAS and PheWAS data as a novel paradigm underlying drug repurposing hypothesis generation. Specifically, this system provides access to both 52,966 PheWAS associations and 7,945 GWAS associations with advanced options to filter the results by the study type, effect size and/or significance level. Another advantage of this method over existing computational drug repurposing tools is that queries may be made from drug, gene or disease perspectives. 
There are several limitations to this method. Currently, the PheWAS data is limited to primarily adult patient population from five institutions contained in the Electronic Medical Records and Genomics (eMERGE) network with a mean age of 69.5 years 12. Additionally, the "repurposing potential" feature uses co-occurrence of search terms in Medline abstracts as one of its criteria. It is well known that text mining methods using co-occurrence have limitations with respect to syntactical structure and literature bias. Thus, we recommend this feature be used as a starting point to explore the potential novelty and/or evidence supporting specific drug repurposing hypotheses and recommend additional investigation into the biomedical literature and clinical trial databases. 
Future directions for this work not described here would be to extend this database to additional sources of GWAS and PheWAS data as they become available. Similar efforts to systematically translate results from large-scale GWAS studies into drug repurposing hypotheses have been previously published 9,13-14. It may be useful to compare these different workflows to predict drug candidates from GWAS data in future studies. Additionally, several other methods exist to computationally generate drug repurposing hypotheses from different data sources, including: genomics, transcriptomics, chemical structures, drug side effect profiles, as previously summarized 6,11. Future methodological advancements could also include automating drug combination predictions and providing information on drug toxicity to guide follow up studies for drug candidates. 
Furthermore, the hypotheses generated from RE:fine Drugs may be further validated using electronic health records, before initiating clinical trials 15. Finally, future studies will be needed to compare this system to other target-based drug repurposing methods.

Os processos caros e ineficientes associados com abordagens de descoberta de drogas tradicionais, incluindo drogas de alto rendimento e de triagem composto de chumbo, estão contribuindo para os atrasos na tradução de descobertas da investigação em terapias para pacientes 1,2. Uma média de 1 bilhão de dólares e 15-20 anos é necessário para trazer uma nova droga da bancada à cabeceira 3. Além disso, 52% dos medicamentos falham durante o desenvolvimento na fase 1 de ensaios clínicos, e apenas 25% de compostos que entram fase 2 prosseguir em plena fase 3 estudos clínicos 4. O objetivo do reaproveitamento de drogas ou reposicionamento de drogas é renovar drogas falharam e / ou encontrar novas indicações para medicamentos aprovados, a fim de oferecer novas terapias para pacientes mais rápido e com uma maior taxa de sucesso. Reaproveitamento de drogas pode diminuir o cronograma para tornar os medicamentos disponíveis para uso em pacientes de 3-12 anos 5. aplicações médicas importantes para reaproveitamento de drogas incluem: doenças com Prognos pobresé e baixas taxas de sobrevivência, doenças resistentes aos medicamentos, áreas de investigação das doenças subfinanciados e populações pobres e carentes do paciente. Reaproveitamento de drogas computacional é definida como o processo de criação e validação de fluxos de trabalho automatizados que podem gerar hipóteses para novas indicações para um candidato de drogas 6. Existentes métodos de reaproveitamento de drogas computacionais foram categorizados com base em alvo, baseada no conhecimento, com base em assinatura, baseado em rede, e com base segmentada-mecanismo, e pode ser orientada a partir de genes, doenças ou drogas perspectivas. Além disso, abordagens computacionais pode acelerar mais uma prova de conceito experimentos de validação e estudos clínicos de pequena escala para a droga candidatos reaproveitado 7. Temos relatado anteriormente em &quot;RE: Drogas finos&quot;, uma ferramenta disponível gratuitamente, interativa baseada na web para reaproveitamento de drogas geração de hipóteses baseado na teoria transitiva de relações Drogas Gene-doença 8. O g globaloal deste método é integrar sistematicamente diversos tipos de drogas, genéticas e clínicas de dados para permitir a droga redefinição de objetivos para os usuários de diversas comunidades, incluindo clínicas, indústria e as comunidades reguladoras. Os métodos fundamentais para este sistema foram anteriormente relatados para o uso de estudo genômico de associação (GWAS) e dados de estudo de associação do phenome (PheWAS) em drogas Repurposing pesquisas 9,10. A nova combinação destes tipos de dados distingue a WebTool de outros métodos baseados em 6,11 alvo. O RE: sistema de Drogas fina atualmente contém 60,911 hipóteses reaproveitamento de drogas cobrindo 916 drogas, 567 genes e 1.770 doenças. O WebTool fornece uma interface amigável para os investigadores a procurar interativamente hipóteses Repurposing drogas e priorizá-los usando diversos critérios. Por exemplo, os usuários podem filtrar hipóteses Repurposing droga com suporte na literatura biomédica e de ensaios clínicos Database, p-valores significativos, odds ratio de associação ou por indicações específicas. O único requisito para este sistema é o acesso à Internet.

<table border="0" cellpadding="0" cellspacing="0" width="797">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:389px;">Access the homepage for &#8220;RE:fine Drugs&#8221; at the following link: http://drug-repurposing.nationwidechildrens.org.&#160;</td>
			<td style="width:81px;">n/a</td>
			<td style="width:136px;">n/a</td>
			<td style="width:191px;">The only requirement for this system is Internet access</td>
		</tr>
	</tbody>
</table>

drug repurposing hypothesis generation using the "re:fine drugs" system

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Neste exemplo, o gene &quot;IL2RB&quot; foi introduzido como uma consulta à base de gene, e foi automaticamente reconhecido como tal pela função auto-fill (Figura 1). As hipóteses Repurposing doze drogas para o gene IL2RB são retornados, como mostrado na Figura 2. Página de informações detalhadas para uma determinada hipótese de reaproveitamento de drogas &quot;, daclizumab&quot;, neste caso, é fornecida a partir da coluna &quot;Info&quot; (Figura 3). Os resultados foram filtrados no separador de droga foi de todos os resultados correspondentes à droga &quot;daclizumab&quot;, como mostrado na Figura 4. A Figura 5 mostra apenas os medicamentos com indicação conhecida para o &quot;transplante&quot; termo doença (fonte: Banco de dados drugbank). A guia associação permite ao usuário filtrar SNP-fenótipo relações de significância estatística (P-valor) e tamanho do efeito genético (odds ratio), definido como a relação entre as chances de presence da doença em indivíduos com um genótipo específico (SNP alelo) sobre as probabilidades de presença da doença em indivíduos sem o alelo SNP. A Figura 6 mostra os resultados filtrados sob o separador associação dentro do intervalo P-Valor de 0,000001-0,05. A Figura 7 mostra hipóteses Repurposing drogas específicas para &quot;asma&quot; com base nas novas indicações que encontramos no estudo. A figura 8 mostra resultados sob a guia potencial para filtrar por um número mínimo de 5 resumos Medline contendo um co-ocorrência de termos de drogas e doenças. Neste exemplo, todas as drogas resultados sob a guia de genes são dirigidos para o gene &quot;IL2RB&quot;, correspondente à duração da consulta original (Figura 9). Finalmente, a Figura 10 mostra os resultados filtrados sob a guia &quot;ação&quot; para retornar todos os fármacos que actuam como agonistas no gene IL2RB. <img alt="figura 1"src = &quot;/ files / ftp_upload / 54948 / 54948fig1.jpg&quot; /&gt; Figura 1: A RE: Drogas finas painel interativo homepage. Os usuários podem iniciar uma consulta digitando um nome do medicamento, nova indicação doença ou símbolo do gene. Os links também são fornecidos para os documentos de referência GWAS e PheWAS que descrevem metodologias para gerar hipóteses Repurposing drogas. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54948/54948fig2.jpg" /> Figura 2: A função Auto-suficiência para as entradas de consulta. Como um exemplo, o termo gene de consulta &quot;IL2RB&quot; foi reconhecido automaticamente como um termo gene. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <pclass = &quot;jove_content&quot; fo: manter-together.within-page = &quot;1&quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/54948/54948fig3.jpg" /> Figura 3: Drogas repurposing tabela de resultados produzido a partir de uma consulta à base de gene (por exemplo, IL2RB). Doze hipóteses reaproveitamento de drogas para o gene IL2RB são produzidos. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54948/54948fig4.jpg" /> Figura 4: Página de Informações para drogas individuais de página de resultados. Ao clicar no ícone da coluna &quot;Info&quot; mostra informações detalhadas para o daclizumab drogas. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Figura 5: opção de pesquisa avançada na guia de drogas para filtrar por uma droga específica. Neste exemplo, três resultados são apresentados para o daclizumab droga. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig5large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 6" src="/files/ftp_upload/54948/54948fig6.jpg" /> Figura 6: opção de pesquisa avançada na guia de drogas para filtrar por uma indicação de doença específica do banco de dados drugbank. Todos os medicamentos com uma indicação conhecida para o termo &quot;transplante&quot; doença são mostrados. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig6large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Figura 7: opção de pesquisa avançada no separador associação para filtrar por nível de significância. Neste caso, oito resultados são fornecidos cujo nível de significância associação cai dentro do intervalo P-Valor de 0.000001 a 0,05. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig7large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 8" src="/files/ftp_upload/54948/54948fig8.jpg" /> Figura 8: opção de pesquisa avançada na guia doença para filtrar por uma indicação de doença específica extraímos neste estudo. Neste exemplo, quatro resultados são mostrados para a asma como uma nova indicação de doença utilização. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig8large.jpg" target="_blank">Por favor clique aqui para ver uma versi maiores</a>em dessa figura. <img alt="Figura 9" src="/files/ftp_upload/54948/54948fig9.jpg" /> Figura 9: opção de pesquisa avançada na guia potencial para filtrar por um co-ocorrência de termos de drogas e doenças em resumos Medline. Neste exemplo, quatro resultados são mostrados que são apoiados por um número mínimo de 5 resumos Medline contendo um co-ocorrência de termos de drogas e doenças. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig9large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 10" src="/files/ftp_upload/54948/54948fig10.jpg" /> Figura 10: opções de pesquisa avançada na guia gene para filtrar por um símbolo gene específico, onde todos os resultados correspondem ao gene IL2RB utilizado para a consulta original. Neste exemplo, os resultados de todas as drogassob a guia gene são direcionados para o gene &quot;IL2RB&quot;, correspondente à duração da consulta original. Opção de pesquisa avançada na guia ação para filtrar por apenas drogas agonistas. Neste exemplo, seis resultados são devolvidos para todos os fármacos que actuam como agonistas no gene IL2RB. <a href="http://ecsource.jove.com/files/ftp_upload/54948/54948fig10large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System at JoVE.com

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

Drogas Repurposing Hipótese Geração Usando o &quot;RE: Drogas finos&quot; System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

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9.2K visualizações.  The Ohio State University. Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options. 

Vídeo: Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Medicina

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Geração e quantificação 3-Dimensional de Arterial lesões em ratos por meio de tomografia óptica de Projeção

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Coleta e Extração de DNA de saliva para Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Quantificação do imunossupressor tacrolimus em manchas de sangue seco utilizando LC-MS / MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Um<em&gt; In Vitro</em&gt; Dormência Modelo de câncer de mama estrogênio-sensíveis na medula óssea: Uma Ferramenta para Estudos Moleculares Mecanismo e Hipótese Geração

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Geração de Microtumors Usando 3D Biogel Humano Sistema de Cultura e células de glioblastoma derivado do paciente para Kinomic Profiling e Testes resposta à droga

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Utilizando os ácidos biliares e multi-fluorados<em&gt; In Vivo</em&gt; Magnetic Resonance Imaging medir Bile transporte do ácido

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

Utilizando imagens da retina para estudar a demência

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Estudando o sinal de insulina hipotalâmico a intolerância de glicose periférica com um sistema de infusão contínua de drogas dentro do cérebro de rato

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...