Forschung wurde von der Harry Winston Forschungsgemeinschaft unterstützt.

1. Bereiten Sie frische Arbeitskopie Lösungen <ol><li> TBS: 0,05 M Tris-gepufferte Kochsalzlösung, pH 7,8 (TBS). Bereiten Sie 60 ml TBS / Mikroplatten. </li><li> PSA: Schweine-Pankreas-Elastase, Typ 1 (PSA) wird als Suspension zur Verfügung gestellt. Gut schütteln und verdünnen PSA in TBS auf etwa 0,01 U / ml. Bereiten Sie 6 ml Arbeitslösung / Mikroplatten). </li><li> SA 3 NA: Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilid (SA 3 NA). Bereiten 0,1 M Lager SA 3 NA Lösung in Dimethylsulfoxid (DMSO) und sofort verwenden oder bei -20 ° C lagern Bereiten 6ml Arbeitslösung / Mikrotiterplatten durch Verdünnung Lager SA 3 NA-Lösung 1/100 in TBS. </li><li> Coomassie-Blau: Bereiten Sie eine Stammlösung von 10% Coomassie Brilliant Blue R-250 (Coomassie-Blau) in TBS und sofort verwenden oder bei -20 ° C. Bereiten Sie 6 ml Arbeitslösung durch Verdünnen Stammlösung 1/1000 in TBS. </li></ol> 2. Bereiten Saliva <ol&gt; <li> Speichelsammelsystem: im Stehen, stimulieren Serum Exsudation in Speichel, indem Sie einen sialogogue wie Zitronensäure oder Kaugummi im Mund. Eine zuckerfreie Zitronen-Tropfen ist praktisch für HIV-1-Patienten, die häufig zahnlosen und nicht in der Lage, Kaugummi zu kauen sind. Schlucken, weil für die ersten 3 min benötigt, und sammeln ganzen Mund Speichel in einem verschlossenen Becher für die nächsten 3 min. Dies wird etwa 2 ml Speichel. Verwenden Sie sofort oder bei -80 ° C lagern </li><li> SPEICHEL HANDHABUNG: Wenn gefroren, auftauen Probe bei 37 ° C und auf Eis stellen. Bewahren Sie frische oder aufgetaute Probe bei 2-4 ° C nicht mehr als 3 Tage. Nicht vortexen und Speichel zu vermeiden Pipettieren Mucin, das kein Serum Exsudat enthält. Verwenden Sie Standard-Vorsichtsmaßnahmen beim Umgang mit Speichel <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. Mikroplatten Set-up <ol><li><strong&gt; BUFFER: Die Messungen werden in dreifacher Ausführung durchgeführt. In jede Vertiefung einer Mikrotiterplatte wird 130 ul TBS aufgenommen. </li><li> EXEMPLARE: Zu jedem Test gut, wird 20 ul Speichel für ein Endvolumen von 150 ul / Vertiefung zugegeben. Durch Pipettieren gründlich mischen, ohne dabei Blasen. </li><li> STEUERUNG: Die positive Kontrolle enthält PPE, aber kein Speichel. Die negative Kontrolle enthält Speichel, aber kein PPE.To die positive Kontrolle ist gut 20 ul TBS aufgenommen. Um die negative Kontrolle wird 20 ul Speichel aufgenommen. </li><li> PSA Inkubation: Schütteln Sie PSA gut funktionierende Lösung. Dann werden 50 ul PSA arbeiten Lösung in jede Vertiefung mit Ausnahme der negativen Kontrollvertiefungen. Um den negativen Kontrollvertiefungen, fügen Sie 50 ul TBS. Inkubieren 10 min bei 23 ° C </li><li> SUBSTRATINKUBATION: Je 50 ul SA 3 NA funktionierende Lösung in jede Vertiefung. Inkubieren 45 min bei 23 ° C </li><li> Coomassie-Blau:. Dann werden 50 ul Coomassie Blue Arbeitslösung &lt;/ Li&gt; <li> Read-Out: Extinktion in Mikroplatten-Reader, A 405 nm bis SA 3 NA-Spaltung die aktive α 1 PI und A 595 nm bis Coomassie Blue, die Eiweißgehalt darstellt erkennen repräsentiert zu erkennen. </li></ol> 4. Berechnen Sie α 1 PI Index <ol><li> Normalisieren der Durchschnitt eine 405 nm und einer 595 nm: Für jeden dreifacher Ausfertigung, berechnen Sie den Durchschnitt. Subtrahieren Sie die Negativ Kontrolle Durchschnitt von der Probe Durchschnitt. Um von Platte zu Platte Variation zu minimieren, normalisieren die Durchschnitte, indem jede Probe durchschnittlich durch die positive Kontrolle im Durchschnitt wie folgt: </li></ol><img alt="Gleichung 1" src="/files/ftp_upload/3999/3999eq1.jpg" /> <ol start="2"><li> BERECHNEN α 1 PI INEX: Teilen Sie den normalisierten A 405 nm durch den normierten A 595 nm: </li></ol> 5. Repräsentative Ergebnisse Um festzustellen, ob die α-Test geeignet sein könnten für die Verwendung als ein Point-of-care-Screening-Test CD4-Zellzahlen in endemischen, mit begrenzten Ressourcen zu überwachen, um Regionen, wurde angeregt Speichel aus 20 weiblichen und 11 männlichen gesammelt HIV-1 Patienten betreuende Klinik für Routineversorgung in Kamerun. In Übereinstimmung mit vorläufigen Beobachtungen während der Entwicklung des α-Test unter Verwendung von Speichel in New York City gesammelt, das α 1 PI Index im Speichel in Kamerun mit CD4-Zellzahlen (r 2 = 0,91, p &lt;0,0001. N = 31) korreliert gesammelt. Die Beziehung war von einem 3-Parameter-förmigen Kurve, die im Einklang mit einer Dosis-Wirkungs-Beziehung (Abbildung 2A) ist definiert. Die 95%-Vertrauensbereich und Vorhersage-Intervalle sind mit blauen und roten Linien (Abbildung 2B) dargestellt. InEine frühere Studie haben wir festgestellt, dass CD4 + T-Zellen sinusförmige Periodizität Radfahren mit 23 ± 3,5 Tage in der HIV-1 Patienten und bei Patienten mit der vererbten Version von α 1 PI-Mangel (PiZZ) 3 aufweisen. Abgebildet sind, ist ein repräsentatives Beispiel von Computer-generierten Sinuskurve Analyse der CD4 Radfahren in einer nicht-HIV-1-gesunden Kontrollgruppe zeigt 27 Tage Periodizität (Abbildung 3A). Computer-generierte Sinuskurve Analyse der CD4 + T-Zell-Veränderungen in 6 Fächer ergaben Werte für Spitze-Spitze-Amplitude und Schwenkachse 3. Wie erwartet, wurde die Achse der Schwingung mit der Amplitude (r 2 = 0,96, p = 0,009, n = 6) (3B) korreliert. Bei Patienten werden niedrige CD4-T-Zellzahlen waren die zyklischen Veränderungen der CD4-T-Zellzahl klein, und bei Patienten mit hoher CD4 + T-Zellzahl, waren zyklische Veränderungen groß. Da dieRegressionslinie in Abbildung 2 dargestellt schätzt die Schwingungsachse und die Achse der Schwingung mit der Amplitude korreliert die Amplitude für die Regressionslinie dadurch Abschätzen, wie weit oberhalb und unterhalb der Regressionslinie CD4 + T-Zellen zu erwarten würde, kann berechnet werden, um variieren aufgrund Radfahren (3C). Die Überlagerung der Amplitude in 3C und das Konfidenzintervall in 2B berechnet berechnet wurde festgestellt, dass Amplitude (grüne Linie) innerhalb des 95% liegt das Konfidenzintervall (blaue Linie) (3D). <img alt="1" src="/files/ftp_upload/3999/3999fig1.jpg" /> Abbildung 1. Zusammenfassung Vorgehensweise: Legen Sie Zitronensäure in den Mund und Speichel zu sammeln. In verdünnten Speichel zu Vertiefungen einer Mikrotiterplatte in dreifacher Ausfertigung. Fügen PSA in jede Vertiefung und inkubieren Sie für 15 min bei 23 ° C. Fügen Sie den PSA-Substrat in jede Vertiefung und inkubieren Sie für 45 min bei 23 ° C. In Coomassie-Blau und bei 405 nm auf PSA Aktivität zu detektieren und bei 595 nm bis Proteins nachzuweisen. <img alt="2" src="/files/ftp_upload/3999/3999fig2.jpg" /> Abbildung 2. Vergleich der α-Test mit dem Standard-CD4-Zählweise. A) Die α-Test wurde durch die Quantifizierung der α 1 PI Index in stimulierten ganzen Mund Speichel durchgeführt. CD4-Zahlen wurden von einem unabhängigen medizinischen Labor mit Hilfe der Durchflusszytometrie durchgeführt. Blut für die Durchflusszytometrie und Speichel wurden in derselben Klinik Besuch gesammelt. Die Beziehung zwischen der α 1 PI-Index und CD4-Zellzahl wurde durch eine 3-Parameter-förmigen Kurve (r 2 = 0,91, p &lt;0,0001, n = 31). B) Die 95%-Vertrauensbereich und Vorhersage-Intervalle werden in blau und rot dargestellt definiert Linien angezeigt sind. <img alt="Abbildung 3" src="/files/ftp_upload/3999/3999fig3.jpg" /> Abbildung 3. Die Genauigkeit der Berechnung CD4 counts mit dem α1PI Index.A) CD4 + T-Zellen zeigten eine sinusförmige Radfahren mit 27 Tage Periodizität in einer nicht-HIV-1-gesunden Kontrollgruppe. Die Schwenkachse ist gezeigt (rot gepunktete Linie). B) Computer festgelegt Sinuswelle Analyse von CD4-T-Zellzyklus verwendet werden, um den Spitze-Spitze-Amplitude und der Achse der Schwingung in 4 HIV-1-Patienten berechnet wurde, ein nicht-HIV -1 Pizz unterliegen, und die Nicht-HIV-1 gesunden Kontrollpersonen in Teil A. schilderte die Beziehung zwischen CD4-T-Zelle + Amplitude und Drehachse des Pendels wurde durch 3-Parameter-förmigen Kurve (r 2 = 0,96, p = 0,009, n definiert = 6). C) Die sigmoide Beziehung zwischen Amplitude und Drehachse des Pendels in Teil B) abgeleitet wurde benutzt, um die erwartete Amplitude (grüne Linien) der Punkte entlang der Regressionsgeraden (schwarze Linie) aus Abbildung 2 zu berechnen. d) die erwartete Amplitude Intervall (grüne Linien) unterschied sich signifikant von dem 95%-Konfidenzintervall (blaue Linien) wie von der Mann-Whitney-Rangsummentest (basierend auf einem Vergleich des mittleren Differenz zwischen dem 95%-Konfidenzintervall und Regressionsgerade (63 Zellen / ul) und die mediane Differenz zwischen der Amplitude Linie und Regressionsgerade (37 Zellen bestimmt / ul); 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. 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>

Keine Interessenskonflikte erklärt.

Unverdünntes Serum enthält Median 36 um α 1 PI und 3,6 um α 2 Makroglobulin (α 2 M) ein 10-facher Unterschied 8. Beide Proteine ​​um die Bindung an PSA. Diese beiden Eigenschaften, Konzentration und PPE Affinität, wurden genutzt, um ein Verfahren, das imstande ist, spezifisch Messen α 1 PI in normalem Serum in Gegenwart von konkurrierenden α 2 M 8 zu entwickeln. Diese Methode kann so wenig wie 50 nM μ 1 PI in 0,3% Serum ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Sialogogue, 0,05 M Zitronensäure </td><td> Edas Zuckerfreie Bonbons, 4900 Hintere N.20th Street, Philadelphia, PA 19144 </td><td> Sugar-Free Lemon Drops </td></tr><tr><td> Schweine-Pankreas-Elastase, Typ 1 (EC 3.4.21.36) </td><td> Sigma-Aldrich, St. Louis, MO 63103 </td><td> E1250 </td></tr><tr><td> Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilid </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> Gewebekultur-behandelt, 96 Well Mikrotiterplatten </td><td> Becton Dickinson, Franklin Lakes, NJ 07417-1886 </td><td> 35-3072 </td></tr><tr><td> Mikroplatten-Reader mit Filtern für 405 nm und 595 nm </td><td> MTX Lab Systems, 8456 Tyco Road, Building D, Vienna, Virginia 22182 </td><td> Dynex P97277 </td></tr></tbody></table>

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

Es besteht ein dringender Bedarf an erschwinglichen CD4-Enumeration, um HIV-Erkrankung zu überwachen. CD4-Zählung ist außerhalb der Reichweite in Regionen mit begrenzten Ressourcen aufgrund der Zeit und Temperatur Einschränkungen, technische Raffinesse, und die Kosten für Reagenzien, insbesondere monoklonale Antikörper gegen CD4 auf Blutzellen, die einzige derzeit akzeptable Methode zu messen. Eine häufig verwendete kostengünstige und zeitsparende Labor Strategie ist es, zu berechnen, anstatt Maßnahme bestimmte Blutwerte. Zum Beispiel werden LDL-Spiegel unter Verwendung der gemessenen Werte von Gesamtcholesterin, HDL und Triglyceride 1. Somit zählt Identifizierung von zellfreien korreliert, die direkt regulieren die Anzahl der CD4 + T-Zellen konnte eine genaue Methode zur Berechnung CD4 bereitzustellen aufgrund der physiologischen Bedeutung der korreliert. Die Anzahl der Stammzellen, die Blut eintreten und bestimmt zu werden zirkulierender CD4 + T-Zellen wird durch das Chemokin CXCL12 einer bestimmtennd seinen Rezeptor CXCR4 durch ihren Einfluss auf die Fortbewegung 2. Verfahren nach Stammzellen Bewegung in Blut wird zusätzlich durch Zelloberfläche menschliche Leukozyten-Elastase (HLE CS) und dem HLE CS-reaktiven aktiven α 1-Proteinase-Inhibitor (α 1 PI, α 1-Antitrypsin, SERPINA1) 3 geregelt. In HIV-1-Erkrankung, ist α 1 PI inaktiviert durch Krankheitsprozesse 4. In den frühen Gruppen von asymptomatischen HIV-1 Erkrankung, wurde die aktive α 1 PI zufolge unter werden in 100% der unbehandelten HIV-1-Patienten (M = 12 pM, und ein normales während der symptomatischen Kategorien 4, 5 zu erreichen normal. Diese Muster hat Immunsystem Inaktivierung zugeschrieben, nicht ausreichend Synthese proteolytischen Inaktivierung oder Sauerstoff. Wir beobachteten, dass bei HIV-1-Patienten mit&gt; 220 CD4-Zellen / ul, CD4-Zahlen mit Serum Spiegel der aktiven α 1 PI korreliert (r 2 = 0,93, p &amp;lt; 0,0001, n = 26) und inaktive α 1 PI (r 2 = 0,91, p &lt;0,0001, n = 26) 5. Die Verabreichung von α 1 PI, um HIV-1 infizierten und nicht infizierten Probanden führte zu dramatischen Anstieg der CD4-Zellzahl darauf hindeutet, α 1 PI beteiligt an der Regulation der Anzahl der CD4 + T-Zellen im Blut 3. Mit Stimulation, enthält ganze Speichel ausreichend seröse Exsudat (Plasma, das verbleibende Material, das durch Wände der Blutgefäße in den Speichel übergeht), damit die Messung von aktiven α 1 PI und die Korrelation dieser Messung gibt Hinweise, dass es eine genaue Methode zur Berechnung der CD4-Zellzahl ist. Kurz gesagt, stimulieren sialogogues wie Kaugummi oder Zitronensäure die Exsudation von Serum in ganzen Mund Speichel. Nach Stimulation Serum Exsudation, wird die Aktivität von Serum α 1 PI im Speichel durch seine Fähigkeit, Elastase-Aktivität zu hemmen gemessen. Schweine-Pankreas-Elastase (PPE)ist ein leicht verfügbares preiswerten Quelle für Elastase. PPE bindet an α 1 PI Bilden einer eins-zu-eins-Komplex, der verhindert, PPE Abspaltung ihrer spezifischen Substraten, von denen die kolorimetrische Peptid ist, Succinyl-L-Ala-L-Ala-L-Ala-p-Nitroanilid (SA 3 NA). Inkubation von Speichel mit einer sättigenden Konzentration von PSA für 10 Minuten bei Raumtemperatur ermöglicht die Bindung von PSA an alle aktiven α 1 PI im Speichel. Die daraus resultierende Hemmung der PSA, die unter aktiver α 1 PI kann durch Zugabe des PSA-Substrat SA 3 NA. (Abbildung 1) gemessen werden. Obwohl CD4 in Bezug auf das Blutvolumen (CD4-Zellen / ul) gemessen werden, wird die Konzentration an α 1 PI im Speichel zu der Konzentration von Serum im Speichel und nicht die für die Menge der Speichel da das Volumen beträchtlich variieren während des Tages und Person bis 6 Personen. Es ist praktisch das gesamte Protein im Speichel durch Serumgehalt, und das Protein von Speichel wird gemesversicherbar 7. Somit wird aktiven α 1 PI im Speichel im Verhältnis zum Speichel Proteingehalt berechnet und wird als α 1 PI-Index. Hier dargestellten Ergebnisse zeigen, dass die α 1 PI-Index eine genaue und präzise physiologische Verfahren zur Berechnung CD4 bietet.

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

Eine Aufzählung CD4-Methode, die α-Test, beschrieben, nutzt ganzen Speichel auf eine schnelle und genaue CD4-Werten geben. Die α-Test kostet Pfennige und eliminiert die Notwendigkeit für technische Schulungen, teure Reagenzien wie monoklonale Antikörper, Mess-, Kälte-, Transport der Proben, sowie Sammlung und Handhabung von Blut.

Die α-Test: Rapid Zellfreie CD4-Zählung mit ganzen Speichel

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 ...

the-test-rapid-cell-free-cd4-enumeration-using-whole-saliva

Research

JoVE Journal

Medicine

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

13.8K Aufrufe.  Weill Cornell Medical College. Eine Aufzählung CD4-Methode, die α-Test, beschrieben, nutzt ganzen Speichel auf eine schnelle und genaue CD4-Werten geben. Die α-Test kostet Pfennige und eliminiert die Notwendigkeit für technische Schulungen, teure Reagenzien wie monoklonale Antikörper, Mess-, Kälte-, Transport der Proben, sowie Sammlung und Handhabung von Blut.

Serie: 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.

Genaue und einfache Messung der pro-inflammatorischen Zytokins IL-1β mit einem Vollblut Stimulation Assay

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

Forschung

Medizin

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.

Schnelle Point-of-Care-Test von Enoxaparin Gerinnungshemmer Wirksamkeit im Vollblut

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.

Probenahme menschliche Speichel Indigene Peptidoms Mit einem Lollipop-Like Ultrafiltration Sonde: vereinfachen und erweitern Peptid-Erkennung für Klinische Massenspektrometrie

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.

Sammlung und Extraktion von DNA-Speichel für 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.

Schnelle Fraktionierung und Isolierung von Vollblutkomponenten in Proben aus einer Gemeinschaft basierende Einstellung Erhalten

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.

Bewertung der gerinnungshemmenden und entzündungshemmenden Eigenschaften von Endothelzellen mit 3D Zellkultur und nicht-Antikoagulanzien Vollblut

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.

Schätzung der Nephron-Zahl in Ganznierstein mit der Acid-Maeration-Methode

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.

Schnelle Bewertung der Toxizität chemischer Verbindungen mit Zebrafischembryonen

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.

Schnelle viskoelastische Charakterisierung von Atemwegsschleim mit einem Tischrheometer

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.