研究では、ハリーウィンストン研究財団によってサポートされていました。

1。新鮮なワーキング溶液を調製<ol><li> TBS：0.05 MトリスpHは7.8（TBS）、緩衝生理食塩水。 60ミリリットルTBS /マイクロプレートを準備します。 </li><li> PPE：ブタ膵臓エラスターゼ、タイプ1（PPE）は、懸濁液として提供されています。よく振って約0.01 U / mlのTBSでPPEを希釈する。 6ミリリットルワーキング溶液/プレート）を準備します。 </li><li> SA 3 NA：スクシニル-L-Alaの-L-Alaの-L-Alaの-p-ニトロアニリド（SA 3 NA）。ジメチルスルホキシド（DMSO）に0.1 M株式SA 3 NA溶液を調製し、-20℃で直ちに使用するか、または保存する株式SA 3 NA溶液をTBSで1/100に希釈して6ミリリットルワーキングソリューション/マイクロプレートを準備します。 </li><li> クーマシーブルー：TBS 10％クーマシーブリリアントブルーR-250（シーブルー）のストック溶液を調製し、-20℃で直ちにまたはストアを使用しTBSに原液1/1000希釈して6ミリリットル作業溶液を調製します。 </li></ol> 2。唾液を準備します。 <oL&gt; <li> 唾液採取：立った姿勢では、このような口の中でクエン酸またはチューインガムとして唾液分泌促進薬を配置することによって、唾液中に血清滲出を刺激する。無糖レモンドロップは頻繁に歯と歯茎をかむことができないHIV-1患者のために便利です。最初の3分間、必要に応じて飲み込むと、次の3分間に覆われたカップに口全体に唾液を収集します。これにより、唾液の約2ミリリットルを提供します。 -80℃で直ちに使用や保管℃、 </li><li> 唾液処理：37℃で凍結、融解試料℃、氷上に置いた場合。 2月4日で新鮮なまたは融解試料を保存°C 3日間を超えない。渦唾液をないし、血清滲出液が含まれていませんムチンピペッティングは避けてください。唾液取り扱う際には標準予防策を使用して<a href="http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdf" target="_blank">http://www.cdc.gov/hicpac/pdf/isolation/Isolation2007.pdfを</a> 。 </li></ol> 3。マイクロプレートのセットアップ<ol><li><strオング&gt; BUFFER：測定は三重で行われます。マイクロタイタープレートの各ウェルに130μLTBSに追加されます。 </li><li> 検体：各テストによく、150μl/ウェルの最終容量20μLの唾液に追加されます。気泡を導入することなく、ピペッティングにより混和する。 </li><li> コントロール：陽性対照PPEが含まれていません、しかし、唾液。ネガティブコントロールは、唾液が含まれていますが、よくないPPE.To陽性対照は20μLTBSが追加されません。ネガティブコントロールによく20μLの唾液に追加されます。 </li><li> PPEのインキュベーション：PPEワーキング溶液をよく振ってください。よくネガティブコントロールのウェルを除く各50μLPPEワーキングソリューションを追加します。ネガティブコントロールのウェルに、50μLのTBSを追加します。 23℃で10分間インキュベートする。 </li><li> 基板のインキュベーション：各ウェルに50μLSA 3 NAワーキングソリューションを追加します。 23℃で45分間インキュベート</li><li> クーマシーブルー：50μLクーマシーブルーワーキングソリューションを追加する&lt;/ LI&gt; <li> 読出し：タンパク質含有量を表してクーマシーブルーを検出するためにアクティブなα1 PIおよび595 nmを表すSA 3 NAの切断を検出するためにマイクロプレートリーダー、405 nmで吸光度を測定します。 </li></ol> 4。 α1 PI指数を計算する<ol><li> 平均 405 nm と 595 nmのを正規化 ：それぞれの三重は、平均を計算します。標本平均値から陰性コントロールの平均値を減算します。プレートからプレートの変動を​​最小限に抑えるために、次のようにポジティブコントロールの平均値によって、各標本の平均値で割って平均値を正規化する。 </li></ol><img alt="式（1）" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> α1 PI INEXを計算します。正規化された595 nmの正規化405 nmで割ります 。 </li></ol> 5。代表的な結果風土病、資源の限られた地域でのCD4数を監視するためのポイントオブケアのスクリーニングテストとしてα-テストでは、使用に適している可能性があるかどうかを決定するために、刺激唾液は、20の女性から採取されたと11男性のHIV-1の被験者参加診療所のためにカメルーンのルーチンケア。ニューヨーク市で収集した唾液を用いたα-テストの開発時には予備的観察と一致し、唾液中のα1 PI指数は、CD4数（R 2 = 0.91、P &lt;0.0001、N = 31）と相関してカメルーンで収集された。関係は、用量反応関係（図2A）と一致している3パラメータシグモイド曲線によって定義されています。 95％の信頼区間と予測区間は、それぞれ青と赤の線（図2B）に描かれています。 で以前の研究で、我々は、CD4 + T細胞は周期23の正弦波サイクリング±HIV-1患者におけるα1 PIの欠乏（PIZZ 3）の継承したバージョンの被験者で3.5日を示すことが判明しました。描かれた本書は、27日周期（図3A）を示す非HIV-1健康なコントロールでサイクリングCD4のコンピュータで生成された正弦曲線分析の代表例である。 CD4 + 6科目のT細胞の変化をコンピュータで生成された正弦曲線の分析は、ピーク·ツー·ピーク振幅と振動の3軸の値が得られた。期待されるように、振動の軸は振幅と相関した（r 2 = 0.96、P = 0.009、n = 6）は（ 図3B）。患者のする低CD4 T +細胞数は、CD4 T細胞数の周期的変化は小さかった、高いCD4 + T細胞数の患者では、周期的な変化が大きかった。 から図2に示した回帰直線は、振動と振動の軸の軸は振幅がために変化することにより、回帰直線のCD4 + T細胞が予想されるどのくらいの上下に推定する回帰直線を計算することができ、振幅と相関していると推定サイクリング（ 図3C）。図3C及び図2Bに計算された信頼区間で計算された振幅重ね合わせ、その振幅は（緑線）95％以内であることが判明した信頼区間（青線）（ 図3D）。 <img alt="図1" src="/files/ftp_upload/3999/3999fig1.jpg" /> 図1。手順の概要：口の中に置き、クエン酸と唾液を収集します。三重のマイクロプレートのウェルに希釈した唾液を追加します。各ウェルにPPEを追加し、23℃で15分間インキュベート各ウェルにPPE基板を追加し、23℃で45分間インキュベート。クマシーブルーを追加して、PPEのアクティビティを検出し、595 nmでタンパク質を検出するために405nmで読み取る。 <img alt="図2" src="/files/ftp_upload/3999/3999fig2.jpg" /> 図2。標準的なCD4列挙法によるα-テストの比較。 A）α-テスト刺激全体の口の唾液中にα1 PI指数を定量することにより行った。 CD4数は、標準的なフローサイトメトリーを用いて独立した医療研究室で行った。フローサイトメトリーと唾液の血液は、同じ診療所の訪問で集めた。 B）95％信頼区間と予測区間は、青と赤で描かれているα1 PI指数とCD4数が3パラメータシグモイド曲線（R 2 = 0.91、P &lt;0.0001、N = 31）で定義されています。との関係それぞれの行、。 <img alt="図3" src="/files/ftp_upload/3999/3999fig3.jpg" /> 図3。計算CD4の精度は、Cα1PIIndex.Aを使用してounts）は、CD4 + T細胞は、非HIV-1健常対照の27日周期の正弦波サイクリングを示した。振動の軸が描かれている（赤い点線）。B）T細胞の循環が4 HIV-1の被験者において振動のピーク·ツー·ピーク振幅と軸を計算するために使用されたCD4、1非感染のコンピュータ生成された正弦波の解析-1 PIzzの件名、および部品AにCD4との関係を示した非HIV-1健常対照T +細胞の振幅と振動の軸は3つのパラメータシグモイド曲線（R 2 = 0.96、P = 0.009、nで定義されています= 6）。C）パートB）で得られた振動の振幅と軸の間にS字型の関係は図2の回帰直線（黒線）に沿った点の予想される振幅（緑線）を計算するために使用されていました。D）予想される振幅間隔（緑線）は95％信頼区間（から有意に異なっていたとしてマンホイットニー順位和検定と振幅線と回帰直線（37セル間の中央値の差（95％信頼区間と回帰直線（63個/μLの間の中央値の差の比較に基づく）によって決定された青線） /μl）を、P = 0.001）。

<ol>
	<li>Friedlander, Y., Kark, J. D., Eisenberg, S., Stein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+LDL-cholesterol+from+total+cholesterol,+triglyceride+and+HDL-cholesterol:+a+comparison+of+methods+in+the+Jerusalem+Lipid+Research+Clinic+Prevalence+Study.">Calculation of LDL-cholesterol from total cholesterol, triglyceride and HDL-cholesterol: a comparison of methods in the Jerusalem Lipid Research Clinic Prevalence Study.</a> Isr. J. Med. 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利害の衝突が宣言されません。

原液血清は、中央値36μMを含むα1 PIおよび3.6μMα2マクログロブリン（α2 M）の10倍の差が8。両方のタンパク質は、PPEへの結合について競合します。これら二つの特性、濃度、PPE親和性は、特にM 8α2、競合の存在下で正常な血清中のα1 PIを測定することができる方法を開発するために利用されています。このメソッドは、0.3％の血清中で?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>唾液分泌促進薬、0.05Mクエン酸</td><td> EDAのSugarfreeキャンディーズ、4900リアN.20thストリート、フィラデルフィア、PA 19144 </td><td>シュガーフリーレモンドロップ</td></tr><tr><td>ブタ膵臓エラスターゼ、1型（EC 3.4.21.36） </td><td> Sigma-Aldrich社、セントルイス、MO 63103 </td><td> E1250 </td></tr><tr><td>スクシニル-L-Alaの-L-Alaの-L-Alaの-p-ニトロアニリド</td><td> Sigma-Aldrich社</td><td> S4760 </td></tr><tr><td>クマシーブリリアントブルーR-250 </td><td> Sigma-Aldrich社</td><td> B7920 </td></tr><tr><td>組織培養処理し、96ウェルマイクロプレート</td><td>ベクトン·ディッキンソン、フランクリンレイクス、NJ 07417-1886 </td><td> 35から3072 </td></tr><tr><td> 405 nmと595 nmのフィルター付きマイクロプレートリーダー</td><td> MTXラボシステム、8456コネクタ、道路、ブイlding D、ウィーン、バージニア州22182 </td><td>ダイネックスP97277 </td></tr></tbody></table>

the &alpha;-test: rapid cell-free cd4 enumeration using whole 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 and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

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

CD4列挙法、α-テストは、迅速かつ正確なCD4 + Tリンパ球数を提供するために唾液を使用していますが記載されている。 α-テストコストのペニーと技術トレーニングなど、モノクローナル抗体、計測機器、冷凍、サンプルの輸送と同様に、血液の収集と処理などの高価な試薬の必要がなくなります。

α-テスト：全唾液を用いた迅速な無細胞CD4列挙

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 ビュー.  Weill Cornell Medical College. CD4列挙法、α-テストは、迅速かつ正確なCD4 + Tリンパ球数を提供するために唾液を使用していますが記載されている。 α-テストコストのペニーと技術トレーニングなど、モノクローナル抗体、計測機器、冷凍、サンプルの輸送と同様に、血液の収集と処理などの高価な試薬の必要がなくなります。

ビデオ: 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.

全血刺激のためのアッセイを用いて炎症性サイトカインIL -1βの正確かつ簡単に測定

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

医学

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.

全血におけるエノキサパリン抗凝固効果の迅速なポイントオブケアアッセイ

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.

ロリポップライク限外プローブを用いたヒト先住民唾液ペプチドーをサンプリング：臨床質量分析のためのペプチドの検出を簡素化し、強化

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.

次世代シーケンシングのための収集と唾液DNAの抽出

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.

迅速な分別とコミュニティベースの設定から得られた試​​料中の全血液成分の単離

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.

3 D 細胞培養と非高血圧症全血を用いた血管内皮細胞の抗凝固および抗炎症作用の評価

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.

酸性揉捻法を用いた全腎ネフロン数の推定

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.

ゼブラフィッシュ胚を用いる化学化合物の毒性の迅速評価

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.

ベンチトップレオメーターを使用した気道粘液の迅速な粘弾性特性評価

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.