Siamo molto grati a K. Nakagaki, Dr. H. Ishii, il Dr. K. Suzuki, A. Yamagata, K. Oofusa per i loro preziosi contributi.

1. Produzione e purificazione di sGMRα <ol><li> Amplifica cDNA di sGMRα da placenta umana biblioteca di cDNA mediante reazione a catena della polimerasi (PCR). </li><li> Aggiungere 50 di base 5&#39;-UTR sequenza di fine baculovirus polyhedrin e 27 RGS base di codifica His-tag per terminare il 3 &#39;per l&#39;amplicone PCR da un altro PCR. </li><li> Inserire il prodotto amplificato di PCR in un pMSG1.1MG plasmide. </li><li> Iniettare il psGMR/M1.1MG costruire con il vettore helper pHA3PIG in uova di baco da seta PND-w1 ceppo. </li><li> Posteriore nati G0 larve di lepidotteri a 25 ° C. Schermo embrioni G1 ottenuto con l&#39;accoppiamento tra fratelli o con PND-w1 MGFP di espressione negli occhi di ottenere bachi da seta transgenici recanti il ​​gene sGMRα. </li><li> Estratto ricombinante sGMRα da bozzoli di bachi da seta transgenici con tampone contenente fosfato-500mm NaCl e mescolare a 4 ° C per 24 ore. </li><li> Centrifugare campione a 20000 g per 15 minuti per rimuovere un precipitati. </li><li> Applicare supernatante in una nichel-affinità delle colonne. </li><li> Lavare la colonna con 10 mM imidazolo, 500mm e fosfato di sodio NaCl 20 mM, pH6.3, ed eluire con un gradiente lineare di imidazolo da 10mm a 250mm. In comune le frazioni contenenti i sGMRα purificata e Dializzare contro PBS (pH7.4) (PBS). </li></ol> 2. Biotinilazione ricombinante di GM-CSF <ol><li> Rimuovere preparazione sorbitolo con la dialisi contro PBS. </li><li> Aggiungere 1 ml di 20 mM freddo sodio meta-periodato soluzione a 4 ° C per la soluzione su ghiaccio. </li><li> Incubare il campione per 30 minuti sul ghiaccio nel buio. </li><li> Aggiungere alla soluzione di glicerolo ad una concentrazione finale di 15 mm. </li><li> Dializzare la soluzione tampone contro 100mM acetato di sodio (pH5.5) </li><li> Aggiungi idrazide biotina alla soluzione ad una concentrazione finale di 5 mm e continuamente agitare la soluzione per 2 ore a temperatura ambiente. </li><li> Rimuovere il materiale non ha reagito con la dialisi contro PBS, pH7.4. </li></ol> 3. Una cellula priva di GM-CSF recettore-ligando-binding assay <ol><li> Cappotto micropiastra con 50μl di anticorpo monoclonale anti-polyHistidine anticorpi notte a 4 ° C. </li><li> Lavare cinque volte con 500μl di PBST. </li><li> Aggiungere 100μl di una soluzione di saturazione e incubare per 3 ore a 4 ° C. </li><li> Lavare cinque volte con 500μl di PBST. </li><li> Aggiungere 50μl di sGMRα nella soluzione di blocco e incubare una notte a 4 ° C. </li><li> Lavare cinque volte con 500μl di PBST. </li><li> Aggiungere 25μl dei campioni contenenti GM-CSF autoanticorpi e poi 25μl di biotinilato GM-CSF. Incubare per 1h a 4 ° C per formare sGMRα-GM-CSF complesso. </li><li> Lavare cinque volte con 500μl di PBST. </li><li> Aggiungere 50μl di fosfatasi alcalina 0.4mm streptavidina per 1 ora a 4 ° C per rilevare la biotinilato GM-CSF legato sGMRα. </li><li> Lavare cinque volte con 500μl di PBST. </li><li> Aggiungere 50μl di un substrato chemiluminescente alla soluzione e incubati per 1h a temperatura ambiente. </li><li> Utilizzando un lettore di piastre chemiluminescenza, rilevare l&#39;attività chemiluminescenza. </li></ol> 4. Rappresentante dei risultati: Il primo passo per il GM-CSF via di trasduzione del segnale del recettore è il legame di GM-CSF a GM-CSF alfa recettore sulla superficie delle cellule. Perché GM-CSF autoanticorpi lega in modo specifico GM-CSF e bloccare il suo legame al recettore in vitro 16,17, abbiamo ipotizzato che gli autoanticorpi inibire questa reazione prima direttamente vincolanti a GM-CSF. Come descritto in precedenza (rif. 9-15), abbiamo applicato la tecnologia transgenica baco da seta per ottenere una grande quantità di sGMRα ricombinante con elevata purezza. Quando il baco da seta derivato sGMRα ricombinante è stato caricato su SDS-PAGE in condizioni non riducenti, l&#39;ha dimostrato sia sGMRα monomerico (45 kDa) e dimerica (90 kDa) le forme, mentre solo la forma monomerica è stato rilevato in condizioni di riduzione (Figura 1B) , indicando che la sGMRα ricombinante era una miscela di monomeri e disolfuro-linked dimmer 18. Utilizzando il sistema acellulare (Fig. 2A), abbiamo valutato l&#39;inibizione di GM-CSF legame sGMRα da GM-CSF autoanticorpi (Fig. 2B e fig. 2C). Questi metodi sono stati descritti in riferimento 19 da Urano et al. L&#39;inibizione della crescita è strettamente correlata con l&#39;inibizione vincolante (r = 0,988, p = 0,002) a diverse concentrazioni del GM-CSF autoanticorpi (Fig. 3A) 19. Allo stesso modo, l&#39;inibizione vincolante è risultata significativamente correlata con l&#39;inibizione della crescita. L&#39;inibizione vincolante aumentata in modo dose-dipendente da GM-CSF autoanticorpi (Fig. 3B) 19. Questi due parametri per le frazioni IgG sieriche di diversi pazienti sono stati correlati tra loro (r = 0,589 p = 0,006, fig. 3C). Né vincolante né inibizione della crescita correlata con i valori di Kd, e quindi, entrambi i parametri è stata influenzata dalla affinità di legame 19. Di conseguenza, la riproducibilità dei dati tra vincolanti e ginibizione rescita ottenuto attraverso tre esperimenti indipendenti su tre diversi campioni è stata valutata. Il coefficiente di variazione indicato che il sistema acellulare era più eccellente di quello del biologico (tabella 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Figura 1" /> Figura 1. Diagramma di flusso della procedura per la produzione di sGMRα utilizzando transgenici bachi da seta. A) Le strutture dei vettori di trasformazione 9-11. B) SDS-PAGE e Coomassie Brilliant Blue-colorazione del sGMRα purificata sotto riducendo e non riducendo le condizioni. sGMRα, GM-CSF solubile del recettore alfa; MGFP, mostro verde fluorescente proteine; BmNPVpol5&#39;-UTR, 5&#39;-non tradotta sequenza regione di Bombyx mori nucleare poliedrosi virus polyhedrin; polyA SV40, SV40 sequenza di segnali polyA P 3xP3, 3xP3 promotore; P ser1, ser1 promotore; FiBL polyA, fibroina L-catena sequenza di segnali polyA; hr3, Bombyx mori nucleare poliedrosi virus hr3 enhancer. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Figura 2" /> Figura 2. Schema del sistema acellulare test. A) Un saggio di legame competitivo con sGMRα prodotto da baco da seta. B) Effetto di anticorpi neutralizzanti e non neutralizzanti sulla inibizione vincolanti da parte delle cellule senza sistema. C) La differenza di inibizione vincolante tra diverse concentrazioni di anticorpi neutralizzanti e non-anticorpi neutralizzanti. GM-CSF, granulociti-macrofagi fattore stimolante le colonie; sGMRα, GM-CSF solubile del recettore alfa; suo, RGS-His-tag, AP, fosfatasi alcalina. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Figura 3" /> Figura 3. GM-CSF inibizione vincolante sGMRα per gli effetti di GM-CSF anticorpi policlonali o le frazioni IgG sieriche di pazienti con PAP autoimmuni. A) Relazione tra l&#39;inibizione vincolanti e inibizione della crescita di GM-CSF autoanticorpi. B) inibizione rilegatura a diverse concentrazioni di GM-CSF autoanticorpi. C) Relazione tra IC 50 per cento inibizione vincolante e inibizione della crescita per cento dal frazioni IgG sieriche 19. IC 50, 50% di concentrazione di inibizione; GM-CSF, granulociti-macrofagi fattore stimolante le colonie. <table border="1"><tbody><tr><td> Campione </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-saggio </td></tr><tr><td> # Di determinazioni </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> Il valore medio (% di inibizione vincolante) </td><td> 61,6 </td><td> 63,8 </td><td> 69,7 </td></tr><tr><td> Valore medio (inibizione della crescita%) </td><td> 21,0 </td><td> 73,4 </td><td> 82,8 </td></tr><tr><td> Coefficiente di variazione (%) (% di inibizione vincolante) </td><td> 6.0 </td><td> 5,6 </td><td> 8,3 </td></tr><tr><td> Coefficiente di variazione (%) (inibizione della crescita%) </td><td> 64,4 </td><td> 18,3 </td><td> 10,4 </td></tr></tbody></table> Entrambi i test sono stati eseguiti a 5 ng / ml di GM-CSF e una concentrazione pari a GM-CSF autoanticorpi. Tabella 1. Confronto del coefficiente di variazione tra vincolante per cento e le inibizioni di crescita ottenuto attraverso tre esperimenti indipendenti.

<ol>
	<li>Arai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+neutralizing+capacity+of+GM-CSF+reflects+disease+severity+in+a+patient+with+pulmonary+alveolar+proteinosis+successfully+treated+with+inhaled+GM-CSF.">Serum neutralizing capacity of GM-CSF reflects disease severity in a patient with pulmonary alveolar proteinosis successfully treated with inhaled GM-CSF.</a> Respir Med. 98, 1227-1230 (2004).</li><li>Tazawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+and+lung+immunity+in+pulmonary+alveolar+proteinosis.">Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis.</a> Am J Respir Crit Care Med. 171, 1142-1149 (2005).</li><li>Inoue, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+large+cohort+of+patients+with+autoimmune+pulmonary+alveolar+proteinosis+in+Japan.">Characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in Japan.</a> Am J Respir Crit Care Med. 177, 752-762 (2008).</li><li>Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+autoantibodies+specifically+eliminate+granulocyte-macrophage+colony-stimulating+factor+activity+in+the+lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis.">High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis.</a> Blood. 103, 1089-1098 (2004).</li><li>Sakagami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+GM-CSF+autoantibodies+and+reproduction+of+pulmonary+alveolar+proteinosis.">Human GM-CSF autoantibodies and reproduction of pulmonary alveolar proteinosis.</a> N Engl J Med. 361, 2679-2681 (2009).</li><li>Raines, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+cloning+of+a+soluble+human+granulocyte-macrophage+colony-stimulating+factor+receptor.">Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.</a> Proc Natl Acad Sci U S A. 88, 8203-8207 (1991).</li><li>Williams, W., VonFeldt, J., Rosenbaum, H., Ugen, K., Weiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+soluble+form+of+the+granulocyte-macrophage+colony-stimulating+factor+receptor+alpha+chain+from+a+myelomonocytic+cell+line.+Expression,+biologic+activity,+and+preliminary+analysis+of+transcript+distribution.">Molecular cloning of a soluble form of the granulocyte-macrophage colony-stimulating factor receptor alpha chain from a myelomonocytic cell line. Expression, biologic activity, and preliminary analysis of transcript distribution.</a> Arthritis Rheum. 37, 1468-1478 (1994).</li><li>Prevost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(GM-CSF)+and+inflammatory+stimuli+up-regulate+secretion+of+the+soluble+GM-CSF+receptor+in+human+monocytes:+evidence+for+ectodomain+shedding+of+the+cell+surface+GM-CSF+receptor+alpha+subunit.">Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit.</a> J Immunol. 169, 5679-5688 (2002).</li><li>Iizuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+recombinant+mouse+monoclonal+antibody+in+transgenic+silkworm+cocoons.">Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.</a> FEBS J. 276, 5806-5820 (2009).</li><li>Iizuka, M., Tomita, M., Shimizu, K., Kikuchi, Y., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+enhancement+of+recombinant+protein+synthesis+in+transgenic+silkworms+by+a+5'-untranslated+region+of+polyhedrin+gene+of+Bombyx+mori+Nucleopolyhedrovirus.">Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.</a> J Biosci Bioeng. 105, 595-603 (2008).</li><li>Zou, W., Ueda, M., Yamanaka, H., Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+combinatorial+protein+library+displayed+on+yeast+cell+surface+using+DNA+random+priming+method.">Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method.</a> J Biosci Bioeng. 92, 393-396 (2001).</li><li>Tamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nat Biotechnol. 18, 81-84 (2000).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+silkworms+produce+recombinant+human+type+III+procollagen+in+cocoons.">Transgenic silkworms produce recombinant human type III procollagen in cocoons.</a> Nat Biotechnol. 21, 52-56 (2003).</li><li>Ogawa, S., Tomita, M., Shimizu, K., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon:+production+of+recombinant+human+serum+albumin.">Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.</a> J Biotechnol. 128, 531-544 (2007).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon.">A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.</a> Transgenic Res. 16, 449-465 (2007).</li><li>Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+alveolar+proteinosis+as+an+autoimmune+disease+with+neutralizing+antibody+against+granulocyte/macrophage+colony-stimulating+factor.">Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 190, 875-880 (1999).</li><li>Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis+express+a+factor+which+neutralizes+granulocyte-macrophage+colony+stimulating+factor.">Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor.</a> FEBS Lett. 442, 246-250 (1999).</li><li>Brown, C., Pihl, C., Murray, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+soluble+granulocyte-macrophage+colony-stimulating+factor+receptor:+identification+of+the+functional+ligand-binding+species.">Oligomerization of the soluble granulocyte-macrophage colony-stimulating factor receptor: identification of the functional ligand-binding species.</a> Cytokine. 9, 219-225 (1997).</li><li>Urano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+assay+to+estimate+the+neutralizing+capacity+of+granulocyte-macrophage+colony-stimulating+factor+autoantibodies.">A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.</a> J Immunol Methods. 360, 141-148 (2010).</li><li>Hayashida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+second+subunit+of+the+receptor+for+human+granulocyte-macrophage+colony-stimulating+factor+(GM-CSF):+reconstitution+of+a+high-affinity+GM-CSF+receptor.">Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.</a> Proc Natl Acad Sci U S A. 87, 9655-9659 (1990).</li><li>Hansen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+GM-CSF+receptor+complex+reveals+a+distinct+mode+of+cytokine+receptor+activation.">The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.</a> Cell. 134, 496-507 (2008).</li></ol>

Il cell-free saggio stimato la capacità neutralizzante di GM-CSF autoanticorpi con eccellente riproducibilità e rapidità. L&#39;inibizione vincolante da GM-CSF autoanticorpi o frazioni siero del paziente IgG è stata valutata da questa analisi. I dati hanno mostrato una correlazione tra l&#39;inibizione legame della cellula senza test e l&#39;inibizione della crescita di un saggio biologico con cellule TF-1, rispettivamente. Il biologico è stato ampiamente utilizzato, ma nutrito difficoltà inerenti alla comparazion...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

SFONDI: In precedenza, abbiamo dimostrato che la capacità di neutralizzare, ma non la concentrazione di GM-CSF autoanticorpi è stata correlata con la gravità della malattia nei pazienti con autoimmuni proteinosi alveolare polmonare (PAP) 1-3. Come abrogazione di GM-CSF bioattività nel polmone è la causa probabile per PAP autoimmuni 4,5, è promettente per misurare la capacità neutralizzante di GM-CSF autoanticorpi per valutare la gravità della malattia in ogni paziente con PAP. Fino ad oggi, la capacità neutralizzante di GM-CSF autoanticorpi è stata valutata mediante la valutazione della inibizione della crescita di cellule di midollo osseo umano o TF-1 cellule stimolate con GM-CSF 6-8. Nel sistema biologico, tuttavia, è spesso problematico ottenere dati affidabili e di confrontare i dati provenienti da diversi laboratori, a causa delle difficoltà tecniche nel mantenimento delle cellule in una condizione costante. OBIETTIVO: Per simulare GM-CSF legame con GM-CSF recettore sulla superficie cellulare tramite cell-free recettoriale-test. METODI: baco da seta transgenici tecnologia è stata applicata per ottenere una grande quantità di ricombinante solubile GM-CSF recettore alfa (sGMRα) con elevata purezza 9-13. Il sGMRα ricombinante era contenuta negli strati sericina idrofila di fili di seta, senza essere fuse alle proteine ​​della seta, e quindi, si può facilmente estrarre dai bozzoli in purezza buona con neutro soluzioni acquose 14,15. Fortunatamente, le strutture oligosaccaridi, che sono critici per il legame con GM-CSF, sono più simili alle strutture di sGMRα umane rispetto a quelle prodotte da altri insetti o lieviti. RISULTATI: La cella priva di sistema di analisi utilizzando sGMRα dato i dati con elevata plasticità e affidabilità. Vincolanti per sGMRα GM-CSF è stato dose-dipendente inibita da policlonali GM-CSF autoanticorpi in maniera simile al test biologico con cellule TF-1, indicando che il nostro nuovo sistema di test cell-free utilizzando sGMRα è più utile per la misurazione di attività neutralizzante di GM-CSF autoanticorpi rispetto al sistema biologico utilizzando TF-1 di cellule umane o cellule del midollo osseo. CONCLUSIONI: Abbiamo stabilito una cella priva di test quantificare la capacità neutralizzante di GM-CSF autoanticorpi.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Abbiamo progettato una cella priva di test legame recettore al fine di valutare il legame di granulociti-macrofagi colony-stimulating factor (GM-CSF) ai recettori. Ci permette di valutare inibizione competitiva del biotinilato GM-CSF legame al recettore alfa solubili GM-CSF da GM-CSF autoanticorpi con eccellente riproducibilità.

Un saggio gratuito Cell System Stima della capacità neutralizzante di GM-CSF utilizzando anticorpi ricombinanti solubili GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

Research

JoVE Journal

Immunology and Infection

12.6K Views.  Niigata University Medical and Dental Hospital. Abbiamo progettato una cella priva di test legame recettore al fine di valutare il legame di granulociti-macrofagi colony-stimulating factor (GM-CSF) ai recettori. Ci permette di valutare inibizione competitiva del biotinilato GM-CSF legame al recettore alfa solubili GM-CSF da GM-CSF autoanticorpi con eccellente riproducibilità.

Video: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergetica e il burst ossidativo: Protocolli per l&#39;isolamento e la valutazione dei leucociti umani e piastrine

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Ricerca

Immunologia e infezioni

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generazione e identificazione di GM-CSF Derivato alveolo-come macrofagi e dendritiche cellule da midollo osseo di topo

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La valutazione della retina microglia fagocitaria Funzione<em&gt; In Vivo</em&gt; Usando un test citometria a flusso basato su

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Generazione di varianti di fuga di neutralizzare l'Influenza Virus anticorpi monoclonali

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Differenziazione dei Mouse del Granulocyte-macrofago-colony-stimulating Factor (GM-CSF)-producendo le cellule T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.