Eu gostaria de agradecer a CL Richards-Zawacki ajuda conceituar o projeto experimental e auxiliando com materiais, A. Pessier ajuda estabelecer um regime de tratamento e necropsia análises, MWH Chatfield, MJ Robak, K. Tasker e D. Lenger ajuda na laboratório e apoio e M. McFadden e P. Harlow no Zoológico Taronga para o uso de seu espaço durante as filmagens. O projeto foi parcialmente financiado pela Louisiana Departamento de Vida Selvagem e Pesca através da concessão Louisiana Comissão de Educação Ambiental atribuído a LA Brannelly e pela Life Technologies.

Aprovação de Ética: Este estudo e seus métodos foram aprovadas pelo Uso de Animais e Cuidados Comitê da Universidade de Tulane (protocolo n º 0407.). 1. Testes para Bd Infecção <ol><li> Limpando para Bd <ol><li> Animais Swab com um cotonete estéril cinco vezes no ventrum, cinco vezes em cada coxa, de cada lado, e cada um dos membros de um total de 45 cursos. Limpando é um método não invasivo de amostragem para Bd na pele de anfíbios. </li><li> Rodar suavemente o cotonete durante e entre os cursos para garantir a maior quantidade de DNA é recolhida no cotonete. </li><li> Armazene cotonetes em microtubos de 1,5 ml a -20 ° C. </li></ol></li><li> Extração de DNA e ensaio qPCR <ol><li> Extrair ADN genómico a partir dos cotonetes usando um kit de isolamento de ADN comercialmente disponíveis, em conformidade com as instruções de tecido animal. </li><li> Analisar o ADN extraído utilizando PCR quantitativo em tempo real (qPCR) seguinte Boyle <em&gt; et al, 17, com as seguintes modificações.: </li><li> Diluir as amostras de extração de DNA de 1:10 com água deionizada dupla. </li><li> Adicionar 0,7 mL de albumina de soro bovino (BSA) para impedir a inibição da PCR. </li><li> Incluir um controlo positivo interno em cada amostra para garantir a inibição PCR não estava afetando os resultados. </li><li> Amostras de correr com um controle positivo e negativo e uma série de padrões de diluição para estimar zoospore (infecção) de carga. </li></ol></li></ol> 2. Inoculação <ol><li> Cultura Bd <ol><li> Prepare o caldo de triptona, adicionando 10 g de triptona e 1 L de osmose inversa ou de água desionizada, autoclavada (121 ° C durante 40 minutos) e deixa-se arrefecer. Uma vez arrefecida a 23 ° C, adicionar 1 ml de penicilina-estreptomicina. </li><li> Grow Bd tensão (a partir de J. JEL411 Longcore, isolado a partir de Guabal, Panamá de um Phyllomedusa lemon) num caldo de triptona durante sete dias a 23 ° C. </li></ol></li><li> Transfira a bRoth cultura de triptona e placas de agar. <ol><li> Para fazer placas, adicionam-se 10 g de triptona e 10 g de ágar e 1 L de osmose inversa ou de água deionizada e de autoclave (121 ° C durante 40 min). </li><li> Uma vez que a mistura autoclavada é fria o suficiente para tocar, mas antes que ele congela, despeje em placas de Petri, 1/4-1/3 íntegra, em uma capela de fluxo laminar. </li><li> Quando agar tem congelado e resfriado por completo, adicionar 0,5 ml de caldo de Bd para cada prato e espalhe uniformemente em volta do prato. Deixe secar. </li><li> Uma vez que as placas são inoculados com zoósporos bd, placas de vedação com Parafilm. </li><li> Permitir que as placas a incubar a 23 ° C durante cinco a sete dias. Verifique zoospore movimento para assegurar a viabilidade antes da inoculação. </li></ol></li><li> Placas inundação para solução inoculação zoospore <ol><li> Após os cinco a sete dias de incubação Bd e crescimento, inundar cada placa com 5 ml de água da torneira e envelhecido permitir zoósporos de introduzir os meios de comunicação durante 10 min. </li><li> Despeje a suspe zoosporension para um recipiente portátil. </li><li> Estimar concentração zoospore com um hemocitômetro. </li><li> Dilui-se a mistura uma concentração de 1 x 10 6 por 3 ml com água da torneira envelhecido. </li></ol></li><li> Inocular animais <ol><li> Em cada 50 ml recipientes, inocular cada animal por vazamento 3 ml mistura de inóculo sobre a sua ventrum. Permitir inoculo extra para escorrer para a base do recipiente de inoculação. </li><li> Cada animal irá permanecer no recipiente de inoculação durante 24 h para assegurar a infecção. </li><li> Após 24 horas da inoculação, o retorno animais para terrários desinfectados individuais. </li><li> Permitir infecção construir por duas semanas. Confirme infecção com cotonete e os resultados de qPCR, e começar o tratamento. </li><li> Para controles Bd-negativos, inundação Bd-livre placas de ágar com 5 ml com idades água da torneira, e inocular os animais simulada usando os mesmos métodos acima. </li></ol></li></ol> 3. Regime de Tratamento <ol><li> Prepare banho de tratamento <ol><li> Adicionar 2,50 ml itraconazol aquosa (1% Sporanox solução oral) para uma solução de L Anfíbio Ringer (0,0025% itraconazol - a menor concentração tratamento eficaz). <ol><li> Solução receita do Anfíbio Ringer: 1 L de osmose reversa ou água deionizada, cloreto de 6,6 g de sódio, 0,15 g de cloreto de potássio, 0,15 g de cloreto de cálcio e 0,2 g de bicarbonato de sódio. Solução de autoclave (121 ° C durante 40 minutos) e deixa-se arrefecer até à temperatura ambiente. </li><li> Para 0,01% de concentração de tratamento itraconazol, adicionar 10,00 ml itraconazol aquosa (1% Sporanox solução oral) para solução de 1 L Anfíbio Ringer </li><li> Para 0.005% de concentração tratamento itraconazol, adicionar 5,00 ml itraconazol aquosa (1% Sporanox solução oral) para solução de 1 L Anfíbio Ringer. </li></ol></li><li> Para controles Bd-positivas e Bd-negativos, banhar animais em Solução de Ringer Anfíbio sem itraconazol. </li></ol></li><li> Tratamento &lt;ol&gt; <li> Coloque anfíbios individuais em 50 ml desinfectados recipientes com 35 ml da solução de tratamento. </li><li> Assegurar animais são totalmente cobertos por cinco minutos rodando suavemente o recipiente. </li><li> Retornar animais para terrários desinfectados. Repita durante seis dias. </li></ol></li><li> Assegurar o sucesso do tratamento <ol><li> Os animais de teste para a infecção Bd sobre o tratamento dia começa. </li><li> Os animais de teste para a infecção Bd uma semana após terminar o tratamento, e uma vez por semana durante quatro semanas para garantir a infecção foi eliminada. </li></ol></li></ol> 4. Bd como Biohazard <ol><li> Desinfetar semanal terrários individual. <ol><li> Banhe os recipientes Terraria e tratamento em um alvejante banho solução a 10%. </li><li> Lavar cada recipiente branqueada duas vezes com água corrente. </li><li> Permitir recipientes para secar por 24 horas antes de sua reutilização. </li></ol></li><li> Usar luvas de borracha nitrílica limpas ao manusear animais para evitar a contaminação cruzadade Bd patógeno. Mudar de luvas entre tratamento de cada animal. </li><li> Desinfectar todos os resíduos de líquido antes do descarte por levando a solução a 10% de lixívia. </li><li> Autoclave todos os resíduos sólidos que está potencialmente exposto a Bd antes do descarte. </li></ol>

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O autor declara não interesses financeiros concorrentes.

Usando este protocolo, fomos capazes de forma segura e eficaz reduzir a dose de tratamento e tempo de tratamento itraconazol por Bd em anfíbios. A menor dose do tratamento e do tempo (0,0025% por seis dias) era um tratamento bem sucedido para I. subclinically infectados nebulifer. A eficácia de ambos uma dose mais baixa e tempo de tratamento demonstram a necessidade de ensaios clínicos para tratamento de infecção tratamentos bd, como atualmente não há tratamentos seguros e efic...

Anfíbios estão experimentando atualmente o maior declínio da biodiversidade de todos os táxons de vertebrados 1. Quitridiomicose, uma doença de pele causada pelos dendrobatidis patógeno Batrachochytrium fúngicas chytrid (bd), é uma das principais causas desses declínios dramáticos 2 Bd é o pior já registrado patógeno para causar a perda de biodiversidade:. Que infecta mais de 600 espécies de anfíbios e tem causado declínios em mais de 300 espécies, das quais cerca de 200 espécies tornaram-se criticamente ameaçadas ou extintas 2,3. O fungo anfíbio não é hospedar específico e não está claro quais são as qualidades do hospedeiro permitem infecção Bd progredir causando doença grave. Chytridiomycosis causa morte por perturbar canais iônicos na pele, resultando na perda de eletrólitos e insuficiência cardíaca 4. Além disso, Bd sobrenadante (que não contém zoósporos Bd) provoca uma resposta imune em girinos 5 leading pesquisadores a acreditar Bd produz um subproduto tóxico 6. Porque muitas espécies estão diminuindo rapidamente, colônias de garantia de cativos e programas de melhoramento são importantes medidas de conservação, e pode ser a única tábua de salvação para algumas espécies. Estabelecer e manter colônias livres de doenças é um desafio para as instalações de gestão de anfíbios. Os atuais métodos quimioterápicos para o tratamento de infecções Bd, embora eficazes, podem ser prejudiciais para os animais que estão sendo tratados, e nenhum método de tratamento único tem sido bem sucedida entre as espécies e classes de idade de anfíbios 7. Poucos estudos clínicos de tratamentos para a infecção Bd foram realizados 7. Os três métodos mais utilizados de tratamento são a terapia do calor, cloranfenicol e itraconazol. Terapia de calor é uma opção de tratamento nonchemotherapeutic que parece ter poucos efeitos secundários, pode ser utilizada em vários estágios da vida, e é Generaliado eficaz. Num ensaio de tratamento, Chatfield e Richards-Zawacki 8 constatou que 96% dos animais tratados tinha desaparecido infecções depois de ser alojados a 30 ° C durante 10 dias. Embora este estudo não foi 100% bem sucedida, ele fornece uma linha de base para os outros para avaliar a terapia de calor para sua própria espécie. Terapia de calor, embora eficaz para muitas espécies, é impraticável para muitas espécies alpine que não pode permanecer a temperaturas elevadas por um longo período de tempo. Cloranfenicol, um agente antibacteriano, foi utilizada com sucesso para tratar Bd em muitas espécies, e inibe o crescimento de fungos in vitro. Como um agente antibacteriano, o cloranfenicol não ataca o fungo especificamente mas tem sido conhecido por causar a apoptose em células eucarióticas 9. Especulou-se que a terapia de cloranfenicol provoca a apoptose na pele, matando esporângios intracelular, embora o mecanismo exacto de acção é desconhecido. Embora nenhum estudo clínico foi realizado e, portanto,a dose e tempo de tratamento não ter sido avaliado, protocolos de tratamento correntes requerem tipicamente a submersão de duas a quatro semanas 10,11, tornando impraticável o tratamento de anfíbios terrestres. Cloranfenicol foi também restrito em alguns países para uso veterinário porque a exposição humana tem sido associada com uma forma rara de anemia aplástica 12. Itraconazol, um antifúngico azóis, é o mais amplamente utilizado para o tratamento da infecção por Bd em anfíbios em cativeiro. O protocolo mais utilizado é animais de banho em uma solução de 0,01% itraconazol por cinco minutos por dia durante 11 dias consecutivos. Este protocolo foi derivado de padrões utilizados para a administração de mamíferos, e, ao mesmo tempo eficaz para anfíbios, que não se baseia em estudos clínicos em anfíbios 11,13. Este protocolo pode provocar efeitos colaterais negativos em certas espécies, especialmente no género Rana e em girinos e metamorphs últimos 11.Girino tratamento foi bem sucedido com uma concentração muito mais baixa (0,0005%) do itraconazole, mas foi associado a despigmentação 14. Esta mesma concentração foi julgado em metamorphs recentes e encontrou a ser vencida no tratamento da infecção 15. Porque não existe risco de efeitos colaterais negativos com o tratamento itraconazol, alguns profissionais têm utilizado a metade da concentração usual (0,005%) por 11 dias consecutivos e encontrei este para ser bem sucedido na cura de infecções Bd 16. O presente estudo é um resumo de um estudo tratamento clínico utilizando itraconazol para tratar a infecção Bd 15. O protocolo descreve uma tentativa para encontrar a menor dose e tempo de tratamento, que é seguro e eficaz para o tratamento de animais infectados com Bd itraconazol. Animais utilizados neste estudo foram metamorphs recentes de sapos costa do golfo, Incilius nebulifer.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Anfíbios estão experimentando o maior declínio de qualquer classe de vertebrados e uma das principais causas desses declínios é um fungo, Batrachochytrium dendrobatidis (Bd), que faz com que a quitridiomicose doença. Colônias de garantia de cativeiro são importantes em todo o mundo para as espécies de anfíbios ameaçadas e pode ser a única tábua de salvação para aqueles em perigo crítico de extinção. A manutenção de colônias livres de doença é uma prioridade dos gestores de cativeiro, ainda tratamentos seguros e eficazes para todas as espécies e entre as fases da vida ainda não foram identificados. O tratamento quimioterapêutico mais amplamente utilizado é o itraconazol, embora a dose utilizada pode ser prejudicial para alguns indivíduos e espécies. Foi realizado um ensaio tratamento clínico para avaliar se uma dose mais baixa e mais seguro, mas eficaz, de itraconazol poderia ser encontrado para curar infecções bd. Observou-se que, reduzindo a concentração de tratamento ,01-,0025% e reduzindo o tempo de tratamento de 11-6 Dias de banhos 5 min, rãs poderia ser curado de infecção Bd com menos efeitos secundários e menos mortalidade associada ao tratamento.

Itraconazol tratamento foi considerado bem sucedido se todos os animais foram Bd-negativos, quatro semanas após o fim do tratamento. Antes do tratamento iniciado duas semanas após a inoculação, todos os animais foram inoculados Bd-infectadas, com uma carga média de zoosporos de 1.121,7 (± 17,34), enquanto que quatro semanas após o fim do tratamento, os animais de controlo Bd-positivos tinham uma média de zoosporos de carga 16,765.12 ( ± 14643,08). Tratamen...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Infecção Batrachochytrium dendrobatidis em anfíbios pode ser eliminado usando uma concentração reduzida (0,0025%) e duração mais curta (banhos de cinco minutos, durante seis dias) de itraconazol do que é geralmente usado. Menos de mortalidade e menos efeitos secundários negativos foram observadas utilizando este protocolo de tratamento modificado.

Redução de Concentração e Durações Itraconazol são bem sucedidos no tratamento<em&gt; Batrachochytrium dendrobatidis</em&gt; Infecção em anfíbios

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K visualizações.  James Cook University.  Infecção Batrachochytrium dendrobatidis em anfíbios pode ser eliminado usando uma concentração reduzida (0,0025%) e duração mais curta (banhos de cinco minutos, durante seis dias) de itraconazol do que é geralmente usado. Menos de mortalidade e menos efeitos secundários negativos foram observadas utilizando este protocolo de tratamento modificado. 

Vídeo: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizando Vírus da Dengue por meio Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Imunologia e Infecção

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia cultura do Sistema Nervoso Central Neonatal e Adulto

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento de Salmonella typhimurium-contendo Phagosomes de macrófagos

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Análise de análogos de ácido Iofenoxico em pequeno mangusto indiano (Herpestes auropunctatus) sera para uso como um marcador biológico de vacinação contra a raiva oral

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infecção recorrente do trato urinário de Escherichia coli desencadeada por gardnerella vaginalis exposição bexiga em camundongos

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Separação de subpopulações de células imunes em amostras de sangue periférico de crianças com mononucleose infecciosa

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

Padronização da Transferência entre Laboratórios entre Citômetros de Fluxo para Detecção de Linfócitos em Crianças Vacinadas com Encefalite Japonesa

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...