La investigación fue apoyada por la Fundación de Investigación de Harry Winston.

1. Preparar soluciones frescas de Trabajo <ol><li> TBS: 0,05 M de Tris Buffered Saline pH, 7,8 (TBS). Preparar 60 ml de TBS / microplaca. </li><li> EPI: elastasa pancreática porcina, de tipo 1 (EPP) se proporciona como una suspensión. Agitar bien y diluir PPE en TBS a aproximadamente 0,01 U / ml. Preparar 6 ml de solución de trabajo / microplaca). </li><li> SA 3 NA: succinil-L-Ala-L-Ala-L-Ala-p-nitroanilida (SA 3 NA). Prepare 0,1 M de valores SA 3 solución de NA en dimetilsulfóxido (DMSO) y utilizar inmediatamente o almacenar a -20 ° C. Preparar la solución de 6 ml de trabajo / microplaca mediante la dilución de valores SA 3 solución de NA 1/100 en TBS. </li><li> Azul de Coomassie: Prepare una solución madre de Coomassie Brilliant Blue 10% de R-250 (Azul de Coomassie) en TBS y utilizar inmediatamente o almacenar a -20 ° C. Preparar 6 ml de solución de trabajo diluyendo la solución madre 1/1000 en TBS. </li></ol> 2. Preparar la saliva <ol&gt; <li> Colector de saliva: En la posición de pie, estimular la exudación de suero en la saliva mediante la colocación de un sialogogo como el ácido cítrico o goma de mascar en la boca. Una gota de limón sin azúcar es conveniente para los pacientes VIH-1 que con frecuencia son desdentados y no puede masticar goma de mascar. Swallow, según sea necesario durante los primeros 3 minutos, y recoger la saliva toda la boca en un recipiente con tapa para el próximo 3 min. Esto proporcionará aproximadamente 2 ml de saliva. Usar inmediatamente o almacenar a -80 ° C. </li><li> MANEJO DE SALIVA: Si la muestra congelada, descongelar a 37 ° C y el lugar en el hielo. Almacenar espécimen fresco o descongelado a 2-4 ° C no más de 3 días. Hacer la saliva no vórtice y evitar pipeteado mucina que no contiene suero exudado. Use precauciones básicas cuando manipulen la 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. Microplacas Set-up <ol><li><strong&gt; BUFFER: Las mediciones se realizaron por triplicado. A cada pocillo de una placa de microtitulación se añade 130 l de TBS. </li><li> MUESTRAS: Para cada pocillo de ensayo, se añadieron 20 l de saliva para un volumen final de 150 l / pocillo. Mezclar con la pipeta sin introducir burbujas. </li><li> CONTROLES: El control positivo contiene PPE, pero la saliva no. El control negativo contiene la saliva, pero no PPE.To el control positivo se añadieron 20 l de TBS. Para el control negativo y se añadieron 20 l de saliva. </li><li> INCUBACIÓN PPE: Agite la solución PPE funcionando bien. Añadir 50 l de trabajo PPE solución a cada pocillo con exclusión de los pocillos de control negativo. A los pocillos de control negativo, se añaden 50 l de TBS. Incubar 10 min a 23 ° C. </li><li> Incubación del substrato: Añadir 50 l SA 3 solución de NA de trabajo a cada pocillo. Incubar 45 min a 23 ° C. </li><li> Azul de Coomassie. Añadir 50 l de solución de azul de Coomassie de trabajo &lt;/ Li&gt; <li> Lectura: Lea la absorbancia en el lector de microplacas, una de 405 nm para detectar SA 3 división NA que representa activa α 1 PI y A 595 nm para la detección de Coomassie azul que representa el contenido de proteína. </li></ol> 4. Calcular α 1 Índice de PI <ol><li> Normalizar LA MEDIA A 405 nm y 595 nm A: Por cada tres ejemplares, calcular la media. Restar la media del control negativo de la media de la pieza. Para minimizar la placa a placa de variación, la normalización de los promedios, dividiendo cada término medio entre el promedio de muestras de control positivo de la siguiente manera: </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> CALCULAR EL α 1 PI INEX: Dividir el normalizada A 405 nm por las normalizadas A 595 nm: </li></ol> 5. Los resultados representativos Para determinar si la α-test podría ser adecuado para su uso como prueba de detección de punto de atención para vigilar los recuentos de CD4 en endémicas, las regiones con recursos limitados, la saliva estimulada se obtuvieron de 20 mujeres y 11 hombres VIH-1 clínica de los sujetos para asistir a rutina de cuidado en el Camerún. De acuerdo con observaciones preliminares durante el desarrollo de la α-prueba con la saliva recogida en Nueva York, la α 1 Índice de PI en la saliva recogida en Camerún correlaciona con recuentos de células CD4 (R 2 = 0,91, p &lt;0,0001. N = 31). La relación se define por una curva sigmoidea 3-parámetro que es consistente con una relación dosis-respuesta (Figura 2A). La confianza del 95% y los intervalos de predicción se representan con líneas azules y rojas, respectivamente (Figura 2B). Enun estudio previo, se determinó que las células CD4 + T presentan ciclos sinusoidales con una periodicidad de 23 ± 3,5 días en sujetos VIH-1 y en sujetos con la versión heredada de la deficiencia de α 1 PI (PIZZ) 3. Se muestra aquí es un ejemplo representativo de la generada por ordenador análisis de la curva sinusoidal de las células CD4 bicicleta en un no-VIH-1 exhibe 27 controles sanos periodicidad días (Figura 3). Generada por computadora análisis de la curva sinusoidal de las células CD4 + T cambios celulares en 6 sujetos dieron valores de pico a pico de amplitud y el eje de oscilación 3. Como era de esperar, el eje de oscilación se correlacionó con la amplitud (R 2 = 0,96, p = 0,009, n = 6) (Figura 3B). En los pacientes que se CD4 bajos recuentos de células T +, los cambios cíclicos en el recuento de CD4 de células T eran pequeños, y en pacientes con CD4 + altos recuentos de células T, los cambios cíclicos eran grandes. Debido a que ellínea de regresión se muestra en la Figura 2 estima que el eje de oscilación y el eje de oscilación está correlacionada con la amplitud, la amplitud puede ser calculado por la línea de regresión así estimar la distancia por encima y por debajo de los CD4 de la línea de regresión + células T se espera que variar debido a (véase la figura 3C). La superposición de la amplitud calculada en la Figura 3C y el intervalo de confianza calculado en la Figura 2B, se encontró que la amplitud (línea verde) está dentro del 95% del intervalo de confianza (línea azul) (Figura 3D). <img alt="Figura 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Figura 1. Resumen del procedimiento: El ácido cítrico Coloque en la boca y recoger la saliva. Añadir la saliva diluida a los pocillos de una microplaca por triplicado. Añadir PPE a cada pocillo y se incuba durante 15 min a 23 ° C. Añadir el sustrato PPE a cada pocillo y se incuba durante 45 min a 23 ° C. Añadir Azul de Coomassie y se leyeron a 405 nm para detectar la actividad del PPE y a 595 nm para detectar la proteína. <img alt="Figura 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Figura 2. Comparación de la α-test con el método de enumeración de CD4 estándar. A) La α-test se realizó por la cuantificación de la α 1 Índice de PI en la saliva estimulada toda la boca. Recuentos de CD4 se realizaron en un laboratorio médico independiente mediante citometría de flujo estándar. Sangre por citometría de flujo y la saliva se recogieron en la visita a la clínica misma. La relación entre la α 1 Índice de PI y los recuentos de CD4 fue definida por una curva sigmoidea de 3 parámetros (r 2 = 0,91, p &lt;0,0001, n = 31). B) La confianza del 95% y los intervalos de predicción se muestran en azul y rojo líneas, respectivamente. <img alt="Figura 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Figura 3. La precisión del cálculo de CD4 counts utilizando el Index.A α1PI) células T CD4 + mostraron ciclo sinusoidal con periodicidad de 27 días en un control no-VIH-1 saludable. El eje de oscilación se representa (línea punteada roja). B) generado por ordenador de análisis de onda sinusoidal de CD4 se usan ciclos de células T para calcular la amplitud de pico a pico y el eje de oscilación en 4 sujetos VIH-1, 1 no-VIH -1 Pizz tema, y la no-VIH-1 control sano se muestra en la parte A. La relación entre T CD4 + amplitud de la celda y el eje de oscilación fue definida por la curva sigmoidea de 3 parámetros (r 2 = 0,96, p = 0,009, n = 6). C) La relación sigmoidal entre la amplitud y el eje de oscilación deriva en parte B) se utiliza para calcular la amplitud esperada (líneas verdes) de puntos a lo largo de la línea de regresión (línea negro) de la Figura 2. D) La amplitud esperada intervalo (líneas verdes) fue significativamente diferente de la del intervalo de confianza 95% (líneas azules) como se determina por el ensayo de Mann Whitney suma de rangos (basado en una comparación de la diferencia media entre el intervalo de confianza del 95% y la línea de regresión (63 células / l) y la mediana de la diferencia entre la línea de amplitud y la línea de regresión (37 células / l), p = 0,001).

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Suero sin diluir contiene la mediana de 36 mM α 1 PI y 3,6 mM α 2 macroglobulina (α 2 M), una diferencia de 10 veces 8. Ambas proteínas competir por la unión a EPI. Estas dos características, la concentración y la afinidad del PPE, se han explotado para desarrollar un método que es capaz de medir específicamente α 1 PI en el suero normal en presencia de competir α 2 M 8. Este método puede detectar cantidades tan pequeñas como 50 μ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Sialogogo, 0,05 M de ácido cítrico </td><td> Caramelos Sugarfree EDA, 4900 trasero N.20th Street, Philadelphia, PA 19144 </td><td> Sin azúcar gotas de limón </td></tr><tr><td> Elastasa pancreática porcina, de tipo 1 (EC 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> Coomassie Brilliant Blue R-250 </td><td> Sigma-Aldrich </td><td> B7920 </td></tr><tr><td> El cultivo de tejidos tratados, microplacas de 96 pocillos </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Lector de microplacas con filtros de 405 nm y 595 nm </td><td> MTX Lab Systems, Inc. 8456 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

Hay una necesidad urgente para el recuento de CD4 asequibles para controlar la enfermedad del VIH. Recuento de CD4 está fuera del alcance de las regiones con recursos limitados, debido a la restricciones de tiempo y temperatura, la sofisticación técnica, y el costo de los reactivos, en particular los anticuerpos monoclonales para medir los CD4 en las células de la sangre, el único método aceptable. Una estrategia de uso común de ahorro de costes y ahorro de tiempo de laboratorio consiste en calcular, en lugar de medir los valores sanguíneos determinados. Por ejemplo, los niveles de LDL se calculan utilizando los niveles medidos de colesterol total, HDL y triglicéridos 1. Por lo tanto, la identificación de células libres de los correlatos que regulan directamente el número de células CD4 + T podría proporcionar un método exacto para calcular el recuento de células CD4, debido a la importancia fisiológica de las correlaciones. El número de células madre que entran a la sangre y están destinados a convertirse en circulación células T CD4 + está determinada por la quimioquina CXCL12 unª de su receptor CXCR4, debido a su influencia sobre la locomoción 2. El proceso de la locomoción de las células madre en sangre es, además, regulada por la superficie celular elastasa de leucocitos humanos (HLE CS) y la ELH CS-reactivo activo α 1 inhibidor de proteinasa (PI 1 α, α 1 antitripsina, SERPINA1) 3. En la enfermedad de VIH-1, α 1 PI está inactiva debido a procesos de la enfermedad 4. En las categorías primeros asintomáticos de VIH-1 enfermedad, activo α 1 PI se encontró que era inferior a lo normal en el 100% de VIH-1 no tratados los pacientes (mediana = 12 mM, y para lograr niveles normales durante las categorías sintomáticas 4, 5. Este patrón se ha atribuido a la inactivación inmune, no a la síntesis insuficiente, la inactivación proteolítica, o la oxigenación. Hemos observado que en VIH-1 sujetos con CD4&gt; 220 células / l, el recuento de CD4 se correlaciona con los niveles séricos de PI activo α 1 (R 2 = 0,93, p &amp;lt; 0.0001, n = 26) y los inactivos α 1 PI (R 2 = 0,91, p &lt;0,0001, n = 26) 5. La administración de α 1 PI al VIH-1 en individuos infectados y no infectados como resultado aumentos significativos en los recuentos de CD4 que sugieren α 1 PI participa en la regulación del número de células T CD4 + en la sangre 3. Con la estimulación, toda la saliva contiene exudado seroso suficiente (plasma que contiene material proteínico que pasa a través de las paredes de los vasos sanguíneos en la saliva) para permitir la medición de la actividad α 1 PI y la correlación de esta medida es evidencia de que es un método preciso para calcular los recuentos de CD4. En pocas palabras, sialogogos tales como la goma de mascar o ácido cítrico estimula la exudación de suero en la saliva toda la boca. Después de la estimulación exudación de suero, la actividad de suero α 1 PI en la saliva se mide por su capacidad para inhibir la actividad de la elastasa. Elastasa pancreática porcina (EPP)es una fuente barata fácilmente disponibles de la elastasa. PPE se une a α 1 PI formando un complejo uno-a-uno que evita PPE de escindir sus sustratos específicos, uno de los cuales es el péptido colorimétrico, succinil-L-Ala-L-Ala-L-Ala-p-nitroanilida (SA 3 NA). Incubando saliva con una concentración de saturación de PPE durante 10 min a temperatura ambiente permite la unión de PPE a toda la sesión α 1 PI en la saliva. La inhibición de PPE por activo α 1 PI puede ser medido mediante la adición del PPE sustrato SA 3 de NA. (Figura 1). Aunque los recuentos de CD4 se miden en términos de volumen de sangre (células CD4 / l), la concentración de α 1 PI en la saliva se relaciona con la concentración de suero en la saliva, no al volumen de la saliva puesto que el volumen puede variar considerablemente durante el día y persona a persona 6. Sin embargo, prácticamente toda la proteína en la saliva es debido al contenido de suero, y el contenido en proteínas de la saliva es MEAmensurable 7. Por lo tanto, activa α 1 PI en la saliva se calcula como la relación al contenido de proteínas y la saliva se denomina α 1 Índice de PI. Los resultados presentados en este documento demuestran que la α 1 Índice de PI proporciona un método fisiológico exacto y preciso para el cálculo de los recuentos 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

Un método de enumeración de CD4, la α-test, se describe que utiliza toda la saliva para proporcionar recuentos de células CD4 rápidos y precisos. Las pruebas α-cuesta centavos y elimina la necesidad de formación técnica, reactivos costosos, como los anticuerpos monoclonales, instrumentación, refrigeración, transporte de muestras, así como la recolección y el manejo de sangre.

El α-test: Rapid libre de células CD4 enumeración utilizando toda la saliva

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

13.7K vistas.  Weill Cornell Medical College. Un método de enumeración de CD4, la α-test, se describe que utiliza toda la saliva para proporcionar recuentos de células CD4 rápidos y precisos. Las pruebas α-cuesta centavos y elimina la necesidad de formación técnica, reactivos costosos, como los anticuerpos monoclonales, instrumentación, refrigeración, transporte de muestras, así como la recolección y el manejo de sangre.

Video: 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.

La medición precisa y simple de la citoquina pro-inflamatoria IL-1β mediante un análisis de sangre entera estimulación

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

Investigación

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.

Rapid Point-of-Care Ensayo de la eficacia anticoagulante enoxaparina en sangre entera

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.

Toma de muestras de saliva humana Peptidome indígenas que utilizan una sonda de ultrafiltración Lollipop-térmica: Simplificar y mejorar la detección de péptidos de Espectrometría de Masas 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.

Recolección y extracción de ADN de la saliva de 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.

Fraccionamiento rápida y aislamiento de componentes de sangre total en las muestras obtenidas a partir de una configuración basada en la comunidad

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.

Evaluación de las propiedades anticoagulantes y antiinflamatorias de las células endoteliales mediante cultivo celular 3D y sangre no anticoagulada

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.

Estimación del número de Nefrona en riñón entera utilizando el método de maceración 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.

Evaluación rápida de la toxicidad de compuestos químicos mediante embriones de pez cebra

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.

Caracterización viscoelástica rápida del moco de las vías respiratorias utilizando un reómetro de sobremesa

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.