Kardiyovasküler Hastalıklarda Humoral Bağışıklık Yanıtları (- Bu çalışma Deutsche Forschungsgemeinschaft (DFG) ve Sonderforschungsbereich Transregio 19 (SFB/TR19, C2), Ekonomi ve Teknoloji proje ZIM-KF 2727801MD0 Federal Bakanlığı ve İnovasyon Yetkinlik Merkezi tarafından desteklenen ZIK-Yürüyüş, Eğitim ve Araştırma, Almanya Federal Bakanlığı BMBF FKZ 03Z2CN12). Hayvanlarla Konut ve deneyler Avrupa Laboratuar Hayvan Bilimi Derneği (FELASA) Laboratuvarı Zootekni (Gesellschaft für Versuchstierkunde, GV-SOLAS) ve Federasyon için Derneği&#39;nin önerilerine göre yapıldı.

1. Hasta Örneklerden IgG İzolasyonu <ol><li> Boş Econo Pac sütuna 2 ml hastanın EDTA plazma başına 3 ml anti-IgG Sepharose doldurarak mini immunoadsorbsiyon sütunlar hazırlayın. IgG izolasyon hastanın plazma, en az 2 ml gerektirir. </li><li> Anti-IgG Sepharose üstünde filtre yerleştirin, sütunun alt ucu açık kesilmiş ve yaklaşık olarak 1 damla / sn &#39;lik bir akış hızına ulaşmak için filtre düğmesine basın. Sütunun tükendi korunması çözümü olsun. </li><li> 3 cilt plazma hacmi başına% 0.9 NaCl ile sütun yıkayın. </li><li> Sütun üzerinde plazma pipet. Plazma tamamen anti-IgG Sepharose batırılır sonra, aynı hacimde% 0.9 NaCl ile yıkanır. </li><li> Pipet bir plazma sütun üzerinde 0.2 M glisin HCl, pH 2.8 hacim ve Sepharose içine batırmayın edelim. Sütun üzerine glisin HCI hacmi başka ekleyin. Bir 15 ml tüp içinde ortaya çıkan akış toplayın ve 0.5 M Tris-HCI, pH 8.0 ile pH 7.3 &#39;e ayarlayın. </li><li> Sütun yenilemek için, 3 plazma hacimleri glisin HCl ile yıkayın. 2 hacim% 0.9 NaCl ile bir son yıkamadan sonra, sütun sonraki plazma örneği için kullanılabilir. ° C ila 4 hafta için 8 - Alternatif olarak, kolon koruma çözeltisi ile doldurulmuş ve 4 de muhafaza edilebilir. </li><li> Kardiyomiyositlerde üzerinde kullanım için, toplanan IgG fraksiyonu deneme tamponu (EB) karşı diyaliz gerekmektedir. EB hazırlanması için, 117 mM NaCI, 2.8 mM KCI, 0.6 mM MgCl2, 1.2 mM KH 2 PO 4, 1.2 mM CaCl2, 10 mM HEPES ve bir manyetik karıştırıcı üzerinde, 20 mM glikoz 1 L damıtılmış suya eklenir ve pH ayarlamak 7.3. </li><li> 100 kDa kesilmiş olan bir molekül ağırlığına sahip olan bir selüloz ester diyaliz zarı tüp için IgG fraksiyonu doldurun. 1 L EB tüp koyun ve 4&#39;e bir manyetik karıştırıcı ile yavaş yavaş karıştırılır ° C. 8 saat sonra, taze EB 3 L diyaliz tüpü içine aktarmak ve 4&#39;de diğer bir 20 saat boyunca dialyze ° C. </li><li> Inactivat için bir su banyosu içinde 30 dakika 57 ° C&#39;de diyaliz için, ısı örnek takibene kompleman faktörleri. Total IgG konsantrasyonunu belirleyin ve 330 mikrogram IgG içeren her alikotları hazırlamak. Ölçümü (adım 4.3) için kardiyomiyositlerde için örnek eklerken, EB ile 1 ml alikotları kadar doldurun. Seyreltilmemiş alikotları daha fazla kullanıma kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol> 2. Sıçan kardiyomiyositlerde izolasyonu <ol><li> Ca2 +-serbest izolasyon tampon (IB) hazırlanması için, 110 mM NaCI, 2.6 mM KCI, 1.2 mM MgSO4, 1.2 mM KH 2 PO 4, 20 mM HEPES ve bir manyetik kullanılarak 1 L damıtılmış su ile 11 mM glükoz eklemek karıştırıcı. Sterilizasyon için 0.2 mikron şişe üst filtre ile oda sıcaklığında ve filtre, 7.4 pH ayarlayın. </li><li> Termostatlı bir Langendorff perfüzyon sisteminin üst rezervuar içinde IB 80 ml doldurun. 37 ° C arasında bir sıcaklık muhafaza tampon ve% 95 O2 /% 5 30 dakika için CO2 ile tampon havalandırmak için termostat ayar ayarlayın. </li><li> Yaklaşık 10.000 tartılır - collag 12.000 Uenase tip II, 175 mg yağ asidi serbest BSA ve 100 mM CaCl2 stok solüsyonu 22.5 ul içeren 20 ml&#39;lik IB içinde çözülür. Üst rezervuar içinde tecrit tampon pH kontrolü ve gerekirse ayarlanır. </li><li> 375 mg / kg vücut ağırlığı tiyopental ile 200 gr - 175 bir Wistar sıçan Anesteziyoloji. Tiyopental çözümü 2500 IU Heparin ekleyin. Sağlam bir aort arkı ile dikkatlice torakotomi, tüketim kalp sonra ve buz gibi soğuk% 0.9 NaCl içine aktarın. Kan kalp ve çevresindeki doku ve taze% 0.9 NaCl transfer temizleyin. Aorta Açığa, 2-3/sec bir damla hızını elde etmek ve kanül için kalp eklemek Langendorff sistemin akışını düşürücü açın. </li><li> Kadar kalan kan dışarı yıkamak 1 damla / sn debi ile 3 dakikaya kadar kalp serpmek. Langendorff sisteminde IB dolaşmaya başlayın. Üst rezervuar 20 ml tampon kaldır gibi 80 ml ulaşmak için gerekli çok tampon kollajenaz çözüm ve dolum doldurun. C sirküle<html> Başka bir 27 dakika için çözelti ollagenase. Akış hızı Akış redüktör kullanarak 1 damla / sn aralıklarla kontrol ve muhafaza edilmelidir. </li><li> Kulakçıklar ve aortadan temiz kanül gelen kalp, ayırın ve küçük parçalar halinde doğrayın. Kollajenaz çözüm alt rezervuar içine çalıştırmak için izin ver, 40 ml, maksimum hacmi azaltmak ve daha 10 için kıyılmış ventriküller sindirmek -% 95 O 2 /% 5 CO 2 ile sürekli havalandırma altında 15 dk. Daha sonra, bir 50 ml tüp içinde bir 200 mikron elek gazlı bez ile hücre süspansiyonu filtre. </li><li> 200 uM CaCl2 içeren 10 ml IB içinde oda sıcaklığı ve tekrar süspansiyon hücreler 2 dakika için 43 x g&#39;da hücre süspansiyonu santrifüj: 3 adım hücrelerin Ca 2 + içerik yükler. Tekrar santrifüjlenir ve 500 uM CaCl2, 10 ml IB hücreleri tekrar süspansiyon. Son bir santrifüj 50.000 hücre / ml &#39;lik bir yoğunluğa steril filtre edilmiş EB kardiyomiyositlerde (1.8 bakınız) tekrar süspansiyon sonra. </li></ol>kardiyomiyositlerin &quot;&gt; 3 E_TITLE. Fura-2 Boyama <ol><li> Coat 10 mikrogram başına iyi laminin ve tamamen kurutulur beklemek bir 4 iyi odacıklıdır coverglass. </li><li> Her iyi bir kesim uçlu bir mikropipet kullanarak içine kardiyomiyosit süspansiyonu 1 ml doldurun. Hücreler, oda sıcaklığında 1 saat boyunca uymak için izin verin. </li><li> 10, 1 mg / ml &#39;lik ul Fura-2 5 ml EB stok çözelti AM. Seyreltilir Karanlıkta boyama çözüm tutun. </li><li> Coverglass ve her kuyuya boyama çözeltisi pipetle 0.5 ml tampon ve bekâr hücreleri çıkarınız. Sarsıntı orta altında 10 dakika için hücreler inkübe edin. Daha sonra, boyama çözüm take off 1 ml EB ile bir kez hücreleri yıkayın ve nihayet her oyuğuna 0.5 ml EB doldurun. Boyama işlemi sırasında doğrudan ışıktan sakının ve her zaman karanlıkta boyanan hücreleri tutun. </li></ol> 4. Hücre kısaltılması ve Ca 2 + Geçişlerine kaydedilmesi <ol><li> Ters floresan m odacıklı coverglass yerleştirinicroscope bir miyosit kalsiyum ve kontraktilite kayıt sistemine bağlı. De ilk akını ve akış tüpü ile özel yapılmış elektrot yerleştirin. 1 ml / dak EB ve (20 V, 1 Hz, 5 ms süre), elektrik uyarımı ile başlamak Superfuse kardiyomiyositlerde. Hücrelerin yaklaşık 2 dakika stimülasyon uyum sağlayın. </li><li> Net çizgili ve sürekli kasılma ile sağlam bir çubuk şekilli kardiyomiyosit seçin. Görüş alanının merkezinde hücre yerleştirin ve kamera kanal açılır. Hücre çerçeveleme adaptörü dönen ve mikroskop sahne hareket ettirerek videoyu alanı içinde yatay hücre yönlendirilmesi. </li><li> Başlangıç ​​hücre kısalma ve Ca 2 + geçici elde etmek için 15 kasılmalar - kenarı video alanı kontrol elemanları (Şekil 2) tespit ayarlayın, floresan kanal ve kayıt 10 açın. </li><li> Floresans leke ağartma önlemek için mikroskop ışık kanalı kapatın. Örnek EB aracılığıyla akışını değiştirmeHastanın IgG 1 ml ile 3-yollu vana ve superfuse kardiyomiyositlerde ile bypass. Hava iyi ulaşmadan hemen önce pompayı durdur. </li><li> Işık kanalı açın ve başka bir 10 kayıt - Akut hücre yanıtını elde etmek için 15 kasılmalar. Kayıt dondurularak ve tekrar ışık kanalı kapatın. 2&#39;den sonra kayıt ve 5 dk tekrarlayın. </li><li> Kuyunun elektrot çıkarın. EB ile iyice temizleyin ve tüpler odacıklı coverglass bir sonraki sıra ile devam edin. </li><li> Sırasıyla Hücre kısalma ve Ca 2 + &quot;Edge-Length/Length&quot; kullanarak IonWizard yazılımı ile geçici ve &quot;fura 2-Sayısal / Oranı çıkarılır&quot; penceresinde, analiz edin. Başlangıç ​​hücre kısalma ve Ca 2 + Akut için temel (bl% pik h) atıfta pik yüksekliği geçici, 2 dakika ve 5 dk yanıtı yüzde değişim hesaplayın. </li></ol>

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Çıkar çatışması ilan etti.

Sunulan yöntem belirsiz kökenli DCM hastalarda fonksiyonel olarak etkili kardiyak otoantikor saptamak için uygun bir yol sunar. Diğer yöntemler ile karşılaştırıldığında, cAMP seviyelerini 6 üzerindeki etkileri ile β1-adrenoseptör karşı işlevsel aktif antikorların saptanması, örneğin, bizim yöntem, belirli bir epitopa bağımsızdır. Tabii ki literatürde açıklanan diğer epitop bağımsız analizi yani yama klemp tekniği veya izole Purkinje lifleri 14 k...

<table border="1"><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Immunosorba koruma çözümü </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> Kollajenaz tip 2 </td><td> Hücre Sistemi </td><td> LS004176 </td><td></td></tr><tr><td> BSA, serbest yağ asidi </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> Laminin </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-02:00 </td><td> Sigma-Aldrich </td><td> F0888 </td><td> DMSO içinde 1 mg / ml </td></tr><tr><td> Econo Pac Kromatografi Sütunları </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech Selüloz Ester Diyaliz MembranE </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4 sıra Chambered coverglass </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> Miyosit Kalsiyum ve Kasılabilme Kayıt Sistemi </td><td> IonOptix </td><td></td><td></td></tr><tr><td> IonWizard 6.0 analiz yazılımı </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

Dilate kardiyomiyopati (DKMP) genç yetişkinler 1 kalp yetmezliği için ana nedenlerinden biridir. Toksik maddelere genetik eğilim ve fuar hastaların yaklaşık üçte birinde bu hastalığın nedenleri bilinmesine rağmen, DCM kökeni büyük ölçüde belirsizdir. Bu hastaların önemli bir kısmında, kardiyak epitoplara otoantikorların tespit edilmiş ve hastalığın 2,3 başlangıcı ve ilerlemesinde önemli bir rol oynadığından şüphe edilir. Kardiyak otoantikorların önemi immunoadsorbsiyon 3-5 ile otoantikorların ortadan kaldırıldıktan sonra DCM hastada hemodinamik iyileşme altı çizilir. Spesifik antijenlerin çeşitli zaten 2,3 tespit edilmiştir ve bu hedeflere karşı antikorlar immunoassay ile tespit edilebilir. Bununla birlikte, bu ölçümlerin uyarıcı (ve bu nedenle de fonksiyonel olarak etkin) ile bloke edici antikorlar arasında ayırt edemez. Eviden vardır artmaktadırBu ayrım önemlidir 6,7 olduğunu ce. Aynı zamanda, cardiotropic antikorların bir dizi hedeflerin yine tanımlanamayan ve bu nedenle bağışıklık deneyleri ile tespit edilemez olduğu kabul edilebilir. Bu nedenle, kendi antijeninin bağımsız işlevsel olarak aktif cardiotropic antikor tespiti için bir metod kurulmamıştır. Yöntem için arka plan genellikle memeli 8,9 arasında kardiyak proteinlerin işlevsel bölgeleri için gözlenen yüksek bir homoloji olduğu. Bu insan antijenlerine karşı, kardiyak antikorlar erişkin sıçan kardiyomiyositler üzerine DCM hastaların IgG test veriyor insan olmayan hedef hücreleri, ile çapraz reaksiyona girer göstermektedir. Önerilen yöntem, 3 adımlardan oluşur: ilk olarak, IgG immunoadsorbsiyon sütunlar (PlasmaSelect, Teterow, Almanya) dan elde edilen Sepharose birleştirilmiş anti-IgG antikorları kullanılarak hasta plazmadan izole edilir. İkinci olarak, erişkin kardiyomiyositlerde ap kullanılarak bir Langendorff perfüzyon cihaz içinde kolajenaz perfüzyon yoluyla izole edilirotokol önceki eserlerinden 10,11 güncellenmiştir. Elde edilen kardiyomiyositlerde laminin kaplanmış odacıklı coverglasses eklenmiş ve Fura-2, kolaylıkla (Ca 2 +) içindekiler 12 hücre içi kalsiyum gözlemlemek için hücre içine getirilebilir bir kalsiyum-seçici floresan boya ile boyanır. Son aşamada, hücre katı ve Ca 2 hasta IgG etki + alan uyarılan kardiyomiyositlerde geçici bir standart ters floresan bağlı olan bir ticari miyosit kalsiyum ve kasılmasını kontrol sisteminin (IonOptix, Milton, MA, ABD) kullanılarak online olarak izlenir mikroskop.

Alanda hücresel inotropiyi ölçümü için iki örnek aşağıda verilmektedir, bir miyosit Ca 2 + ve kasılma kayıt sistemi kullanılarak erişkin kardiyomiyositlerde (Şekil 2) ile alınabilir. Şekil 3, bir kontrol ölçümü arasında bir izlenimini vermektedir, Şekil 4 içinde iken cardiodepressive antikorların etkisi bir hastanın DCM gösterilmiştir. Her iki örnek, başlangıç ​​hücre kısalma ve Ca 2 +</s...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Sunulan yöntem izole sıçan kardiyomiyositlerde hücresel kısalma ve hücre içi kalsiyum geçici üzerinde izole hasta immunglobulin etkisini analiz ederek, bağımsız spesifik antijen, dilate kardiyomiyopatili hastaların plazmasında fonksiyonel etkin cardiotropic otoantikor saptamak için bir yol sunar.

İzole kardiyomiyositlerin Hücresel Fonksiyon üzerine Antikor Etkilerinin Ölçümü

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

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Immunology and Infection

11.8K Görüntüleme.  University Medicine Greifswald. Sunulan yöntem izole sıçan kardiyomiyositlerde hücresel kısalma ve hücre içi kalsiyum geçici üzerinde izole hasta immunglobulin etkisini analiz ederek, bağımsız spesifik antijen, dilate kardiyomiyopatili hastaların plazmasında fonksiyonel etkin cardiotropic otoantikor saptamak için bir yol sunar.

Video: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Sitosolik Ca ölçümü 2 + İzole Kontraktil Lenfatikler

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

İmmünoloji ve Enfeksiyon

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Organik Solvent kullanımı olmadan Triklosan Etkileri: RBL-2H3 Mast Hücre Degranülasyon üzerinde kimyasal etkileri değerlendirmek için bir Mikroplaka Testi

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Kırmızı kan hücreleri üzerinde antikor kaynaklı kompleman aktivasyon sayısal tespiti için Yöntemler

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Akış adlı lökositlerin üzerine Stromal hücreler Etkilerinin İncelenmesi

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10 parçacık bağlı Bet v 1 Allergen Akışı Sitometrik Analizi

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Peptid, bir monoklonal antikor tarafından tanınan lineer B-hücresi epitopunun belirlenmesi: Tarama destekli

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Mayi glikoz tolerans testi, akciğer işlev ölçümü ve akciğer fiksasyonu obezite ve pulmoner sonuçlar üzerine Engelli metabolizma etkisini incelemek için

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Fare kemik iliği ve Periton makrofaj harekete geçirmek antikor bazlı ilaçlara etkileri değerlendirilmesi

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Tükürük bezi işlevi farelerde ölçülmesi için bir yöntem

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Sentetik fajının inşaatı Fab Kütüphane özel çeşitliliği ile görüntülenir.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...