Name: the &alpha;-test: rapid cell-free cd4 enumeration using whole saliva
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Description: Watch this Scientific Journal Video about The ÃŽÂ±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

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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：</st...

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	<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. Sci. 18, 1242-1252 (1982).</li><li>Lapidot, T., Petit, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+stem+cell+mobilization:+The+roles+of+chemokines,+proteolytic+enzymes,+adhesion+molecules,+cytokines,+and+stromal+cells.">Current understanding of stem cell mobilization: The roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells.</a> Exp. Hematol. 30, 973-981 (2002).</li><li>Bristow, C. L., Alfano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;1Antitrypsin+therapy+increases+CD4++lymphocytes+to+normal+values+in+HIV-1+patients.">&#945;1Antitrypsin therapy increases CD4+ lymphocytes to normal values in HIV-1 patients.</a> Soluble factors mediating innate immune responses to HIV infection. , (2010).</li><li>Bristow, C. L., Patel, H., Arnold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+antigen+prognostic+for+human+immunodeficiency+virus+disease+progression.">Self antigen prognostic for human immunodeficiency virus disease progression.</a> Clin. Diagn. Lab. Immunol. 8, 937-942 (2001).</li><li>Bristow, C. L., Babayeva, M. A., LaBrunda, M., Mullen, M. P., Winston, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;1Proteinase+Inhibitor+regulates+CD4++lymphocyte+levels+and+is+rate+limiting+in+HIV-1+disease.">&#945;1Proteinase Inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease.</a> PLoS One. , (2011).</li><li>de Almeida, P. D. V., Gregio, A. M. T., Machado, M. A. N., de Lima, A. A. S., Azevedo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva+Composition+and+Functions:+A+Comprehensive+Review.">Saliva Composition and Functions: A Comprehensive Review.</a> J. Contemp. Dent. Pract. 9, 72-80 (2008).</li><li>Edgar, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saliva:+its+secretion,+composition+and+funcitons.">Saliva: its secretion, composition and funcitons.</a> Br. Dent. J. , 172-305 (1992).</li><li>Bristow, C. L., Meo, F. d. i., Arnold, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+activity+of+&#945;1proteinase+inhibitor+and+++2macroglobulin+in+human+serum:+Application+to+insulin-dependent+diabetes+mellitus.">Specific activity of &#945;1proteinase inhibitor and 2macroglobulin in human serum: Application to insulin-dependent diabetes mellitus.</a> Clin. Immunol. Immunopathol. 89, 247-259 (1998).</li><li>Pederson, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+levels+of+&#945;2-macroglobulin,+&#945;1-antitrypsin,+C-reactive+protein,+cathepsin+G+and+elastase+in+humans+with+or+without+destructive+periodontal+disease.">Salivary levels of &#945;2-macroglobulin, &#945;1-antitrypsin, C-reactive protein, cathepsin G and elastase in humans with or without destructive periodontal disease.</a> Arch. Oral Biol. 40, 1151-1155 (1995).</li><li>Shak, S., Capon, D. J., Hellmiss, R., Marsters, S. A., Baker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+DNase+I+reduces+the+viscosity+of+cystic+fibrosis+sputum.">Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum.</a> PNAS. 87, 9188-9192 (1990).</li><li>LeVeque, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicenter,+randomized,+double-blind,+placebo-controlled,+dose-+titration+study+of+oral+pilocarpine+for+treatment+of+radiation-induced+xerostomia+in+head+and+neck+cancer+patients.">A multicenter, randomized, double-blind, placebo-controlled, dose- titration study of oral pilocarpine for treatment of radiation-induced xerostomia in head and neck cancer patients.</a> J. Clin. Oncol. 11, 1124-1131 (1993).</li><li>Stevens, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+new+CD4+enumeration+technologies+for+resource-constrained+countries.">Evaluating new CD4 enumeration technologies for resource-constrained countries.</a> Nat. Rev. Microbiol. 6, S29-S38 (2008).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2010 ART guidelines for adults and adolescents - evidence map. , (2010).</li></ol>

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 ÃŽÂ±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva at JoVE.com

The ÃƒÅ½Ã‚Â±-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

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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1ÃƒÅ½Ã‚Â² using a Whole Blood Stimulation Assay

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

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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

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

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唾液を収集し、家族介護者を経由して高齢者を居住する虚弱コミュニティ唾液コルチゾールおよびα-アミラーゼの測定

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

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

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 ...

An Improved Method for Rapid Intubation of the Trachea in Mice

マウスの気管の迅速な挿管のための改善された方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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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

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

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 ...