A pesquisa foi apoiada pela Harry Winston Research Foundation.

1. Prepare frescos Soluções de Trabalho <ol><li> TBS: 0,05 M de Tris pH tamponada, solução salina 7,8 (TBS). Preparar 60 ml de TBS / microplaca. </li><li> PPE: elastase pancreática porcina, tipo 1 (PPE) é fornecida como uma suspensão. Agitar bem e diluir PPE em TBS a cerca de 0,01 U / ml. Preparar 6 ml de solução de trabalho / microplaca). </li><li> SA 3 NA: succinil-L-Ala-L-Ala-L-Ala-p-nitroanilida (SA 3 NA). Preparar 0,1 M estoque SA 3 solução de NA em sulfóxido de dimetilo (DMSO) e usar imediatamente ou armazenar a -20 ° C. Preparar solução de trabalho 6ml / microplaca, diluindo estoque SA 3 solução de NA 1/100 em TBS. </li><li> Azul de Coomassie: Preparar uma solução-mãe de 10% de Coomassie Brilliant Blue R-250 (Azul de Coomassie) em TBS e usar imediatamente ou armazenar a -20 ° C. Preparar 6 ml de solução de trabalho por diluição de solução stock 1/1000 em TBS. </li></ol> 2. Prepare Saliva <ol&gt; <li> RECOLHA saliva: Na posição de pé, estimulam a exsudação soro em saliva colocando um sialogogo tal como ácido cítrico ou de goma de mascar na boca. Uma gota de limão sem açúcar é conveniente para os pacientes HIV-1 que são freqüentemente desdentada e incapaz de mascar chiclete. Engula como necessária para a 3 minutos em primeiro lugar, e coletar saliva boca inteira em um copo com tampa para a próxima 3 min. Isto irá fornecer cerca de 2 ml de saliva. Use imediatamente ou armazenar a -80 ° C. </li><li> MANUSEAMENTO saliva: Se a amostra descongelamento congelado, a 37 ° C e colocar em gelo. Armazenar amostra fresca ou descongeladas a 2-4 ° C, não mais de 3 dias. Fazer a saliva não vórtice e evitar pipetagem mucina que não contém qualquer exsudado soro. Use precauções padrão ao manusear saliva <a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf</a> . </li></ol> 3. Microplaca Set-up <ol><li><strong&gt; BUFFER: As medições são realizadas em triplicata. A cada poço de uma placa de microtitulação é adicionado 130 uL de TBS. </li><li> AMOSTRAS: Para cada poço de teste, é adicionado 20 uL de saliva para um volume final de 150 uL / poço. Misturar por pipetagem sem a introdução de bolhas. </li><li> CONTROLE: O controle positivo contém PPE, mas não saliva. O controlo negativo contém saliva, mas não PPE.To o controlo positivo poço é adicionado 20 uL de TBS. Para o controlo negativo poço é adicionado 20 uL de saliva. </li><li> INCUBAÇÃO PPE: Shake PPE solução funcionando bem. Adicionar 50 uL Solução de trabalho de PPE a cada poço excluindo os poços de controlo negativo. Para os poços de controlo negativo, adicionar 50 uL de TBS. Incubar 10 min a 23 ° C. </li><li> Incubação do substrato: Adicionar 50 uL SA 3 solução de NA de trabalho para cada poço. Incubar 45 min a 23 ° C. </li><li> Coomassie Blue:. Adicionar 50 mL solução Coomassie azul trabalho &lt;/ Li&gt; <li> Leitura: Ler a absorbância em leitor de microplacas, A 405 nm para detectar SA 3 NA clivagem que representa ativa α 1 PI e A 595 nm para detectar Coomassie Azul, que representa o conteúdo de proteína. </li></ol> 4. Calcule α 1 Índice PI <ol><li> NORMALIZE média, um 405 nm e um nm 595: Para cada triplicado, calcular a média. Subtrair a média Controle Negativo a partir da média da amostra. Para minimizar placa para a chapa de variação, normalizar as médias por divisão de cada média da amostra pela média Controlo positivo como se segue: </li></ol><img alt="Equação 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> CÁLCULO DO α 1 PI INEX: Divida o normalizado A 405 nm pelos normalizados A 595 nm: </li></ol> 5. Os resultados representativos Para determinar se o α-teste pode ser adequado para utilização como um teste de rastreio ponto-de-cuidado para monitorar CD4 em endémicas, de recursos limitados regiões, saliva estimulada foi recolhido a partir de 20 fêmeas e 11 machos HIV-1 clínica sujeitos assistir para cuidados de rotina em Camarões. Consistente com observações preliminares durante o desenvolvimento da α-teste usando saliva coletada em Nova York, a α 1 Índice de PI na saliva coletadas em Camarões correlacionados com contagem de CD4 (r 2 = 0,91, p &lt;0,0001. N = 31). A relação foi definida por uma curva sigmoidal 3-parâmetro que é consistente com uma relação dose-resposta (Figura 2A). A confiança de 95% e intervalos de predição são representadas com linhas azuis e vermelhas, respectivamente (Figura 2B). Emum estudo anterior, verificamos que células T CD4 + apresentam ciclismo sinusoidal com periodicidade de 23 ± 3,5 dias em indivíduos HIV-1 e em indivíduos com a versão herdada de α 1 PI deficiência (PiZZ) 3. Aqui apresentado é um exemplo representativo de computer-generated análise da curva de seno de CD4 de bicicleta em um non-HIV-1 controle saudável exibindo 27 periodicidade dia (Figura 3A). Gerado por computador análise da curva de seno de as células CD4 + alterações das células T em 6 indivíduos rendeu valores para pico-a-pico de amplitude e eixo de oscilação 3. Como seria de esperar, o eixo de oscilação foi correlacionada com a amplitude (r 2 = 0,96, p = 0,009, n = 6) (Figura 3B). Em pacientes irão CD4 + T contagens de células, as alterações cíclicas na contagem de células CD4 T eram pequenos, e em pacientes com CD4 elevada contagem de células T, alterações cíclicas eram grandes. Porque olinha de regressão representado na Figura 2 estima o eixo de oscilação eo eixo de oscilação é correlacionada com a amplitude, a amplitude pode ser calculado para a linha de regressão, assim, estimando como muito acima e abaixo das CD4 + de regressão da linha de células T que seria esperado a variar devido à ciclismo (Figura 3C). Sobrepondo a amplitude calculada na Figura 3C e do intervalo de confiança calculado na Figura 2B, verificou-se que a amplitude (linha verde) encontra-se dentro de 95% do intervalo de confiança (linha azul) (Figura 3D). <img alt="A Figura 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Figura 1. Resumo Procedimento: ácido cítrico Lugar na boca e coletar saliva. Adicionar saliva diluída a poços de uma microplaca em triplicado. Adicionar PPE a cada poço e incubar durante 15 min a 23 ° C. Adicionar o substrato PPE a cada poço e incubar durante 45 min a 23 ° C. Adicionar Azul de Coomassie e lida a 405 nm para detectar a actividade PPE e a 595 nm para a detecção da proteína. <img alt="A Figura 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Figura 2. Comparação da α-teste com o método padrão CD4 enumeração. A) O α-teste foi realizado por quantificação do α 1 Índice PI na saliva estimulada boca todo. Contagens de CD4 foram realizados por um laboratório independente médica por citometria de fluxo padrão. Sangue por citometria de fluxo e saliva foram coletadas na visita clínica mesmo. A relação entre o índice de α PI 1 e as contagens de CD4 foi definida por uma curva sigmoidal de 3 parâmetros (r 2 = 0,91, p &lt;0,0001, n = 31). B) A confiança de 95% e intervalos de predição são descritos em azul e vermelho linhas, respectivamente. <img alt="A Figura 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Figura 3. Precisão de cálculo CD4 counts usando o Index.A α1PI) células T CD4 + exibiram ciclismo sinusoidal com 27 dias periodicidade de um controlo não-HIV-1 saudável. O eixo de oscilação é descrita (linha pontilhada vermelho). B) COMPUTADOR análise onda gerada seno de CD4 T ciclo celular foi usado para calcular a amplitude pico-a-pico eo eixo de oscilação em 4 de HIV-1, 1 sujeitos não-HIV -1 sujeito PiZZ, e do não-HIV-1 de controlo saudável representado na parte A. A relação entre T CD4 + amplitude da célula e do eixo de oscilação foi definida por 3-parâmetro curva sigmoidal (r 2 = 0,96, p = 0,009, n = 6). C) A relação entre a amplitude sigmoidal e eixo de oscilação derivado, em parte B) foi utilizada para calcular a amplitude esperado (linhas verdes) de pontos ao longo da linha de regressão (linha preta) a partir da Figura 2. D) A amplitude esperada intervalo (linhas verdes) foi significativamente diferente do intervalo de confiança de 95% (linhas azuis) tal como determinado pelo Teste de Soma de Mann Whitney Rank (com base numa comparação da diferença média entre o intervalo de confiança de 95% e linha de regressão (63 células / ml) ea diferença entre a linha mediana de amplitude e da linha de regressão (37 células / uL), p = 0,001).

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Soro não diluído contém 36 mM mediana α 1 PI e 3,6 mM α macroglobulina 2 (α 2 M) uma diferença de 10 vezes 8. Ambas as proteínas competir para a ligação a PPE. Estas duas características, concentração e PPE afinidade, têm sido exploradas para desenvolver um método que é capaz de medir especificamente α 1 PI no soro normal na presença de competir α 2 M 8. Este método pode detectar tão pouco como 50 nM μ 1 PI no s...

<table border="1"><tbody><tr><td> Nome do o reagente de </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Sialogogo, 0,05 M de ácido cítrico </td><td> Doces Sugarfree AED, 4900 traseiro N.20th Street, Philadelphia, PA 19144 </td><td> Sugar-Free gotas de limão </td></tr><tr><td> Suínos elastase pancreática, tipo 1 (CE 3.4.21.36) </td><td> Sigma-Aldrich, St. Louis, MO 63103 </td><td> E1250 </td></tr><tr><td> Succinil-L-Ala-L-Ala-L-Ala-p-nitroanilida </td><td> Sigma-Aldrich </td><td> S4760 </td></tr><tr><td> Azul Brilhante de Coomassie R-250 </td><td> Sigma-Aldrich </td><td> B7920 </td></tr><tr><td> Da cultura de tecidos tratados, 96 microplacas bem </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Leitor com filtros para 405 nm e 595 nm </td><td> MTX Lab Systems, 8456 Tyco Road, Building D, Vienna, Virginia 22182 </td><td> Dynex P97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva

Há uma necessidade urgente de enumeração CD4 acessível para monitorar a doença HIV. Enumeração de CD4 está fora de alcance em regiões com recursos limitados, devido ao tempo e as restrições de temperatura, a sofisticação técnica e custo dos reagentes, em particular anticorpos monoclonais para medir o CD4 nas células sanguíneas, o único método atualmente aceitável. Uma estratégia comumente utilizada laboratório de economia de custos e economia de tempo é de calcular, em vez de valores de medidas determinadas no sangue. Por exemplo, os níveis de LDL são calculados usando os níveis medidos de colesterol total, HDL e triglicéridos 1. Assim, a identificação de células livres que se correlaciona directamente regulam o número de células T CD4 + poderiam fornecer um método preciso para calcular contagem de células CD4, devido à relevância fisiológica dos correlatos. O número de células estaminais que entram no sangue e destinam-se a tornar-se circular células T CD4 + é determinado pelo quimiocina CXCL12 umnd seu receptor CXCR4 devido à sua influência sobre a locomoção 2. O processo de locomoção de células estaminais no sangue é adicionalmente regulada por superfície celular leucocitário humano elastase (HLE CS) e da HLE CS-reactivo activo α 1 inibidor de protease (α 1 PI, α 1 antitripsina, SERPINA1) 3. Em HIV-1 da doença, α 1 PI é inactivado devido a processos de doença 4. Nas categorias iniciais assintomáticos de HIV-1 da doença, activa α 1 PI foi encontrado para ser abaixo do normal em 100% de HIV-1 não tratados (doentes mediana = 12 uM, e para conseguir os níveis normais durante as categorias sintomáticos 4, 5. Este padrão tem sido atribuído à inactivação imunitário, não para a síntese insuficiente, a inactivação proteolítica, ou oxigenação. Observou-se que em HIV-1 indivíduos com&gt; 220 CD4 células / uL, as contagens de CD4 foram correlacionados com os níveis séricos de activa α 1 PI (r 2 = 0,93, p &amp;lt; 0,0001, n = 26) e inativos α 1 PI (r 2 = 0,91, p &lt;0,0001, n = 26) 5. A administração de α 1 PI ao HIV-1 indivíduos infectados e não infectados resultou em aumentos dramáticos na contagem de CD4, sugerindo α 1 PI participa na regulação do número de células T CD4 + no sangue 3. Com a estimulação, a saliva contém todo exsudato seroso suficiente (plasma contendo material proteináceo que passa através das paredes dos vasos sanguíneos na saliva) para permitir a medição de ativos α 1 PI ea correlação dessa medida é prova de que é um método preciso para calcular os valores de CD4. Resumidamente, sialogogues tais como goma de mascar ou ácido cítrico estimular a exsudação de soro na saliva boca inteira. Depois de estimular a exsudação de soro, a actividade de soro α 1 PI na saliva é medido pela sua capacidade para inibir a actividade da elastase. Pâncreas suíno elastase (PPE)é uma fonte facilmente disponível barato de elastase. PPE liga-se a α 1 PI formação de um complexo um-para-um que impede PPE de clivar os seus substratos específicos, um dos quais é o péptido colorimétricas, succinil-L-Ala-L-Ala-L-Ala-p-nitroanilida (SA 3 NA). A incubação de saliva com uma concentração saturante de EPI durante 10 min à temperatura ambiente permite a ligação de PPE para todas activas α a 1 PI na saliva. A inibição resultante de EPI por activo α 1 PI pode ser medido através da adição do substrato PPE SA 3 NA. (Figura 1). Embora as contagens de CD4 são medidos em termos de volume de sangue (CD4 células / mL), a concentração de α PI 1 na saliva está relacionado com a concentração de soro na saliva, não a um volume de saliva desde que o volume pode variar consideravelmente durante o dia, por pessoa para pessoa 6. No entanto, virtualmente toda a proteína na saliva é devido ao teor de soro, eo teor de proteína de saliva é MEAmensuráveis ​​7. Assim, activa α 1 PI na saliva é calculada como uma razão para o conteúdo de proteína saliva e é denominado o α 1 Índice PI. Os resultados apresentados aqui demonstram que o Índice de α 1 PI fornece um método exacta e precisa fisiológica para calcular os valores de CD4.

Watch this Scientific Journal Video about The Î±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Um método de enumeração de células CD4, o α-teste, é descrito que utiliza saliva total para fornecer contagens de CD4 rápidos e precisos. As α-teste tostões custos e elimina a necessidade de formação técnica, reagentes caros, tais como anticorpos monoclonais, instrumentação, refrigeração, transporte de amostras, bem como coleta e manuseio de sangue.

O α-teste: Rapid livre de células CD4 enumeração Utilizando saliva total

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

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The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

13.8K visualizações.  Weill Cornell Medical College. Um método de enumeração de células CD4, o α-teste, é descrito que utiliza saliva total para fornecer contagens de CD4 rápidos e precisos. As α-teste tostões custos e elimina a necessidade de formação técnica, reagentes caros, tais como anticorpos monoclonais, instrumentação, refrigeração, transporte de amostras, bem como coleta e manuseio de sangue.

Vídeo: The &#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Medição precisa e simples do pró-inflamatórias IL-1β citocinas usando um ensaio Estimulação Sangue Total

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Medicina

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Ensaio de Point-of-Care rápida de eficácia anticoagulante enoxaparina em sangue total

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

Amostragem Peptidome saliva humana Indígena Usando uma sonda de Ultrafiltração Lollipop-Like: simplificar e melhorar a detecção Peptide de Espectrometria de Massa Clínica

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

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 ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Fracionamento rápido e isolamento de componentes de sangue total em amostras obtidas a partir de uma configuração de base comunitária

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

Avaliação das propriedades das células endoteliais utilizando cultura de células 3D e sangue não anticoagulado anticoagulantes e anti-inflamatório

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Estimates of whole kidney nephron number are important clinically and experimentally, as there is an inverse association between nephron number and an enhanced risk of renal and cardiovascular disease.Herein, the use of the acid maceration method, which provides fast and reliable estimates of whole kidney nephron number, is demonstrated.

Estimativa do número de néfron em rim inteiro usando o método de maceração ácida

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

Avaliação rápida da toxicidade de compostos químicos usando embriões de zebrafish

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G&#39;, G&#34;, G*, and tan &#948;) and gel point characteristics (&#947;c&#160;and &#963;c) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is &#60;10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more &#34;liquid-like&#34; behavior overall (e.g., higher tan &#948;). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

Caracterização rápida da Viscoelástica do Muco das Vias Aéreas usando um Reômetro Benchtop

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

Whole-Kidney Three-Dimensional Staining with CUBIC

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood vessels, proximal tubules, collecting ducts, glomeruli, and sympathetic nerves in the kidney due to the lack of three-dimensional (3D) information. Optical clearing is a good strategy to overcome this big hurdle. Multiple cells in a whole organ can be analyzed at single-cell resolution by combining tissue clearing and 3D imaging technique. However, cell labeling methods for whole-organ imaging remain underdeveloped. In particular, whole-mount organ staining is challenging because of the difficulty in antibody penetration. The present protocol developed a whole-mount mouse kidney staining for 3D imaging with the CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) tissue clearing method. The protocol has enabled visualizing renal sympathetic denervation after ischemia-reperfusion injury and glomerulomegaly in the early stage of diabetic kidney disease from a comprehensive viewpoint. Thus, this technique can lead to new discoveries in kidney research by providing a macroscopic perspective.

The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.