La ricerca è stata sostenuta dal Harry Winston Research Foundation.

1. Preparare freschi soluzioni di lavoro <ol><li> TBS: 0,05 M Tris Buffered Saline, pH 7.8 (TBS). Preparare 60 ml TBS / micropiastra. </li><li> PPE: elastasi pancreatica suina, di tipo 1 (PPE) viene fornito come sospensione. Agitare bene e diluire DPI in TBS a circa 0,01 U / ml. Preparare 6 ml soluzione di lavoro / micropiastra). </li><li> SA 3 NA: succinil-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3 NA). Preparare 0,1 M magazzino SA 3 soluzione NA in dimetilsolfossido (DMSO) e utilizzare immediatamente o conservare a -20 ° C. Preparare 6 ml di soluzione di lavoro / micropiastra diluendo magazzino SA 3 soluzione NA 1/100 in TBS. </li><li> Coomassie Blue: Preparare una soluzione stock del 10% Coomassie Brilliant Blue R-250 (Coomassie Blue) in TBS e utilizzare immediatamente o conservare a -20 ° C. Preparare 6 ml di soluzione di lavoro diluendo la soluzione stock 1/1000 in TBS. </li></ol> 2. Preparare Saliva <ol&gt; <li> Prelievo della saliva: Nella posizione eretta, stimolare essudazione siero in saliva inserendo un sialogogue come acido citrico o gomma da masticare in bocca. A sugar-free goccia di limone è utile per i pazienti HIV-1 che sono spesso edentulo e incapaci di masticare gomma. Ingoiare come necessario per i primi 3 min, e raccogliere la saliva tutta la bocca in una tazza con tappo per il prossimo 3 min. Ciò fornirà circa 2 ml di saliva. Utilizzare immediatamente o conservare a -80 ° C. </li><li> MANIPOLAZIONE SALIVA: Se congelato, campione scongelare a 37 ° C e il luogo su ghiaccio. Conservare campione fresco o scongelato a 2-4 ° C non più di 3 giorni. Sei la saliva non vortex ed evitare pipettaggio mucina che non contiene essudato siero. Usate Precauzioni standard quando si maneggia 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. Microplate Set-up <ol><li><strong&gt; BUFFER: Le misure vengono effettuate in triplice copia. A ciascun pozzetto di una piastra microtitolo viene aggiunto 130 pl TBS. </li><li> CAMPIONI: a ciascun pozzetto, si aggiungono 20 pl saliva per un volume finale di 150 pl / pozzetto. Mescolare pipettando senza introdurre bolle. </li><li> CONTROLLI: Il controllo positivo contiene PPE, ma non saliva. Il controllo negativo contiene saliva, ma non PPE.To controllo positivo viene aggiunto 20 pl TBS. Per il controllo negativo si aggiunge saliva 20 pl. </li><li> INCUBAZIONE PPE: Agitare la soluzione PPE funziona bene. Aggiungere 50 microlitri soluzione di lavoro DPI ad ogni pozzetto esclusi i pozzetti di controllo negativi. Ai pozzetti di controllo negativo, aggiungere 50 ul TBS. Incubare 10 minuti a 23 ° C. </li><li> INCUBAZIONE SUBSTRATO: Aggiungere 50 microlitri SA 3 soluzione NA lavorando per ciascun pozzetto. Incubare 45 minuti a 23 ° C. </li><li> Coomassie Blue:. Aggiungere 50 microlitri soluzione di Coomassie Blu di lavoro &lt;/ Li&gt; <li> READ-OUT: Leggere l&#39;assorbanza in lettore di micropiastre, A 405 nm per rivelare SA 3 scissione NA che rappresenta attiva α 1 PI e A 595 nm per rivelare Coomassie blu che rappresenta il contenuto proteico. </li></ol> 4. Calcolare α 1 Indice PI <ol><li> NORMALIZE DELLA MEDIA A 405 nm e 595 nm: per ogni triplice copia, calcolare la media. Sottrarre la media di controllo negativo dalla media del campione. Per ridurre al minimo piastra-to-plate variazione, normalizzare le medie dividendo ogni media per la media del campione di controllo positivo come segue: </li></ol><img alt="Equazione 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> CALCOLA IL α 1 PI INEX: Dividere la normalizzato A 405 nm dai normalizzati A 595 nm: </li></ol> 5. Risultati rappresentativi Per determinare se l&#39;α-test potrebbe essere adatto per l&#39;utilizzo come point-of-care test di screening per monitorare la conta CD4 in endemiche, risorse limitate regioni, saliva stimolata è stato raccolto da 20 femmine e 11 maschi HIV-1 ambulatorio per i soggetti partecipanti trattamento di routine in Camerun. Coerentemente con le osservazioni preliminari durante lo sviluppo del α-test con saliva raccolti in New York City, l&#39;α 1 Indice PI nella saliva raccolti in Camerun correlato con conta dei CD4 (R 2 = 0,91, p &lt;0,0001. N = 31). Il rapporto è stato definito da un 3-parametro curva sigmoidale che è coerente con una relazione dose-risposta (Figura 2A). La fiducia del 95% e intervalli di previsione sono rappresentati con linee blu e rosse, rispettivamente (Figura 2B). Inuno studio precedente, abbiamo determinato che cellule T CD4 + mostrano ciclismo sinusoidale con frequenza di 23 ± 3,5 giorni in soggetti HIV-1 e nei soggetti con la versione ereditata della α 1 PI deficit (PIZZ) 3. Qui raffigurato è un esempio rappresentativo di computer-generated analisi della curva sinusoidale di CD4 in bicicletta in un non-HIV-1 di controllo sano espone periodicità 27 giorni (Figura 3A). Computer-generated analisi curva sinusoidale delle CD4 + cambiamenti cellule T in 6 soggetti ha prodotto valori di picco-picco di ampiezza e l&#39;asse di oscillazione 3. Come sarebbe previsto, l&#39;asse di oscillazione è stata correlata con ampiezza (r 2 = 0,96, p = 0,009, n = 6) (Figura 3B). Nei pazienti a basso T CD4 + conta cellulare, i cambiamenti ciclici della conta delle cellule T CD4 erano piccoli, e nei pazienti con CD4 + alta conta di cellule T, i cambiamenti ciclici erano grandi. Poiché l&#39;linea di regressione illustrato nella figura 2 stima l&#39;asse di oscillazione e l&#39;asse di oscillazione è correlata con ampiezza, ampiezza può essere calcolato per la linea di regressione quindi stimare quanto sopra e sotto le CD4 + linea di regressione cellule T ci si aspetterebbe per variare a causa ciclismo (Figura 3C). Sovrapponendo l&#39;ampiezza calcolato in Figura 3C e l&#39;intervallo di confidenza calcolato nella figura 2B, è stato constatato che ampiezza (linea verde) si trova all&#39;interno del 95% dell&#39;intervallo di confidenza (linea blu) (Figura 3D). <img alt="Figura 1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Figura 1. Procedura sommaria: acido citrico Luogo in bocca e raccogliere la saliva. Aggiungi saliva diluito nei pozzetti di una micropiastra in triplice copia. Aggiungi PPE a ciascun pozzetto e incubare per 15 min a 23 ° C. Aggiungere il substrato PPE in ogni pozzetto e incubare per 45 min a 23 ° C. Aggiungere Coomassie Blue e letta a 405 nm per rilevare l&#39;attività di PPE e a 595 nm per rivelare proteina. <img alt="Figura 2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Figura 2. Confronto della α-test con il metodo standard di enumerazione CD4. A) L&#39;α-test è stato eseguito quantificare la α 1 Index PI in stimolato saliva intera bocca. Conta dei CD4 sono stati eseguiti da un laboratorio indipendente medica che utilizza standard di citometria a flusso. Sangue per citometria a flusso e saliva sono stati raccolti durante la visita stessa clinica. Il rapporto tra l&#39;indice α 1 PI e conta dei CD4 è stato definito da una curva a 3 parametri sigmoidale (r 2 = 0.91, p &lt;0.0001, n = 31). B) La fiducia del 95% e intervalli di previsione sono raffigurati in blu e rosso linee, rispettivamente. <img alt="Figura 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Figura 3. Precisione di calcolo CD4 counts utilizzando il Index.A α1PI) cellule CD4 + T esposto ciclismo sinusoidale con periodicità 27 giorni in un non-HIV-1 di controllo sano. L&#39;asse di oscillazione è raffigurato (rosso linea tratteggiata). B) calcolatore generati analisi sinusoide di CD4 è stato usato bicicletta cellule T per calcolare il picco-picco di ampiezza e l&#39;asse di oscillazione in 4 soggetti HIV-1, 1 non-HIV -1 Pizz soggetto, e la non-HIV-1 di controllo sano raffigurato nella parte A. Il rapporto tra T CD4 + ampiezza delle cellule e l&#39;asse di oscillazione è stato definito da 3-parametro curva sigmoidale (r 2 = 0,96, p = 0,009, n = 6). C) Il rapporto tra ampiezza e sigmoidale asse di oscillazione derivato in parte B) è stato utilizzato per calcolare l&#39;ampiezza atteso (linea verde) di punti lungo la linea di regressione (linea nera) dalla Figura 2. D) L&#39;ampiezza prevista intervallo (linee verdi) è stata significativamente diversa da l&#39;intervallo di confidenza al 95% (linee blu) come determinato dalla somma di Mann-Whitney Rank Test (basato su un confronto tra la differenza mediana tra l&#39;intervallo di confidenza 95% e la linea di regressione (63 cellule / mL) e la differenza tra la linea mediana ampiezza e la linea di regressione (37 cellule / pl); p = 0,001).

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Non diluito siero contiene mediana 36 uM α 1 PI e 3,6 uM α 2 macroglobulina (α 2 M) una differenza di 10 volte 8. Entrambe le proteine ​​competono per il legame ai DPI. Queste due caratteristiche, concentrazione e PPE affinità, sono stati sfruttati per sviluppare un metodo che sia in grado di misurare specificamente α 1 PI nel siero normale in presenza di competere α 2 M 8. Questo metodo in grado di rilevare un minimo di 50 μ nM <sub...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Sialogogue, 0,05 M di acido citrico </td><td> Eda di Caramelle Sugarfree, 4900 posteriore N.20th Street, Philadelphia, PA 19144 </td><td> Sugar-gratuiti gocce di limone </td></tr><tr><td> Suini elastasi pancreatica, di 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-nitroanilide </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> Di coltura di tessuti trattati con micropiastre, 96 e </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Lettore per micropiastre con filtri per 405 nm e 595 nm </td><td> MTX Systems Lab, 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

Vi è un urgente bisogno di conteggio CD4 a prezzi accessibili per monitorare la malattia da HIV. Conteggio CD4 è fuori portata nelle regioni a causa del tempo e le limitazioni di temperatura, sofisticazione tecnica, e il costo dei reagenti risorse limitate, in particolare per misurare gli anticorpi monoclonali CD4 sulle cellule del sangue, l&#39;unico metodo attualmente accettabile. Un laboratorio comunemente utilizzati risparmio di costi e risparmio di tempo strategia è quella di calcolare, piuttosto che i valori ematici certa misura. Per esempio, livelli di LDL sono calcolati utilizzando i livelli misurati di colesterolo totale, HDL, e trigliceridi 1. Pertanto, l&#39;identificazione di cellule libere correlati che regolano direttamente il numero di cellule T CD4 + potrebbe fornire un metodo preciso per calcolare CD4 per l&#39;importanza fisiologica dei correlati. Il numero di cellule staminali che entrano sangue e sono destinate a diventare circolanti cellule CD4 + T è determinata dalla chemiochina uno CXCL12nd suo recettore CXCR4 per la loro influenza sulla locomozione 2. Il processo di locomozione di cellule staminali nel sangue viene ulteriormente regolata dalla cellula umana superficie dei leucociti elastasi (HLE CS) e la CS-reattivo HLE attivo α 1 inibitore della proteinasi (α 1 PI, α 1 antitripsina, SerpinA1) 3. In malattia da HIV-1, α 1 PI è inattivato a causa di processi patologici 4. Nei primi categorie asintomatici di HIV-1 malattia, attiva α 1 PI è risultata inferiore normale in 100% di non trattati pazienti HIV-1 (mediana = 12 pM, e per ottenere livelli normali durante le categorie sintomatici 4, 5. Questo modello è stata attribuita a inattivazione immune, non alla sintesi insufficiente, inattivazione proteolitica, o ossigenazione. Abbiamo osservato che in HIV-1 soggetti con&gt; 220 cellule CD4 / microlitro, CD4 sono stati correlati con i livelli sierici di accesso α 1 PI (r 2 = 0.93, p &amp;lt; 0.0001, n = 26) e gli inattivi α 1 PI (r 2 = 0.91, p &lt;0.0001, n = 26) 5. La somministrazione di α 1 PI per HIV-1 i soggetti infetti e non infetti portato a un drastico incremento nella conta dei CD4 suggeriscono α 1 PI partecipa alla regolazione del numero di cellule T CD4 + nel sangue 3. Con la stimolazione, la saliva contiene tutto sufficiente essudato sieroso (plasma contenente materiale proteico che passa attraverso le pareti dei vasi sanguigni nella saliva) per consentire la misurazione delle attività α 1 PI e la correlazione di questa misura è la prova che si tratta di un metodo preciso per calcolare il numero di CD4. Brevemente, sialogogues come gomma da masticare o acido citrico stimolare l&#39;essudazione di siero in saliva intera bocca. Dopo stimolando essudazione siero, l&#39;attività di siero α 1 PI in saliva è misurata dalla sua capacità di inibire l&#39;attività elastasi. Suina elastasi pancreatica (PPE)è facilmente disponibile una fonte economica di elastasi. PPE lega α 1 PI formando uno-a-uno complesso che impedisce PPE di scindere i suoi substrati specifici, uno dei quali è il peptide colorimetriche, succinil-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3 NA). Incubazione saliva con una concentrazione di saturazione di PPE per 10 min a temperatura ambiente consente il legame di PPE a tutti attivo α 1 PI nella saliva. L&#39;inibizione di PPE risultante dalla attivo α 1 PI può essere misurata aggiungendo il substrato PPE SA 3 NA. (Figura 1). Sebbene CD4 sono misurate in termini di volume di sangue (cellule CD4 / pl), la concentrazione di α 1 PI nella saliva è correlata alla concentrazione di siero in saliva, non al volume di saliva dal volume può variare notevolmente durante il giorno e persona a persona 6. Tuttavia, quasi tutte le proteine ​​nella saliva è dovuto al contenuto di siero, e il contenuto di proteine ​​della saliva è measurable 7. Così, attivo α 1 PI in saliva viene calcolato come rapporto al contenuto proteico e saliva viene chiamata α 1 Index PI. I risultati qui presentati dimostrano che la α 1 Index PI fornisce un metodo accurato e preciso fisiologico per il calcolo 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 metodo enumerazione CD4, la α-test, viene descritto che utilizza saliva intera per fornire rapidi e precisi conta di CD4. Le α-test centesimi i costi ed elimina la necessità di una formazione tecnica, reagenti costosi come gli anticorpi monoclonali, strumentazione, refrigerazione, trasporto dei campioni, nonché di raccolta e trattamento del sangue.

La α-test: Rapid cell-free CD4 Enumeration Uso Saliva Whole

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 Views.  Weill Cornell Medical College. Un metodo enumerazione CD4, la α-test, viene descritto che utilizza saliva intera per fornire rapidi e precisi conta di CD4. Le α-test centesimi i costi ed elimina la necessità di una formazione tecnica, reagenti costosi come gli anticorpi monoclonali, strumentazione, refrigerazione, trasporto dei campioni, nonché di raccolta e trattamento del sangue.

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.

Misurazione accurata e semplice della citochina pro-infiammatoria IL-1β utilizzando un intero saggio stimolazione del sangue

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

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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 test di efficacia anticoagulante Enoxaparina nel sangue intero

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.

Campionamento umana peptidoma Indigenous Saliva Utilizzando un Lollipop-Like Probe ultrafiltrazione: Semplificare e migliorare la rilevazione Peptide per spettrometria di massa Clinica

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.

Raccolta ed estrazione di Saliva DNA per il sequenziamento di nuova generazione

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.

Frazionamento rapida e isolamento dei componenti sangue intero in campioni ottenuti da una impostazione basata su Comunità

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.

Valutazione delle proprietà anticoagulanti e anti-infiammatori delle cellule endoteliali mediante coltura cellulare 3D e Non intero anticoagulato

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.

Stima del numero di Nephron in rene intero utilizzando il metodo di macerazione acida

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.

Rapida valutazione della tossicità dei composti chimici utilizzando gli embrioni di pesce zebra

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.

Caratterizzazione viscoelastica rapida del muco delle vie aeree utilizzando un reometro da banco

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.