The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) Preparazione di Cell Line: <ol><li> Sviluppare la linea di cellule RBL-2H3.1 esprimere equino α FcεRI: <ol><li> Utilizzando tecniche di base di coltura di tessuti per linee cellulari monostrato, trasfezione le cellule RBL-2H3.1 genitori utilizzando il plasmide pEE6, che porta il gene equina FcεRIα (GenBank: Y18204.1) 28. Aggiungere 2 mg ml -1 del DNA plasmidico a 0,8 ml di cellule ad una densità di 1,2 x 10 7 cellule ml -1. Elettroporazione le cellule a 250 V, 960 mF utilizzando un 0,4 centimetri electrocuvette incubare immediatamente in ghiaccio per 10 min. </li><li> Selezionare le cellule trasformate utilizzando supporti contenenti 0,4 g di solfato di geneticina G418, quindi ordinare le rimanenti cellule viventi mediante FACS da loro codifica con un anticorpo fluorescente IgE. Utilizzare il conseguente RBL-2H3.1 esprimere equina linea cellulare FcεRIα per lo studio 2, 3. </li></ol></li><li> Pre-dosaggio degli anticorpi di sensibilizzazione: <ol><li&gt; Harvest RBL-2H3.1 cellule che esprimono FcεRIα equino in provenienza da una capsula di Petri confluenti. Lavare poi risospendere le cellule in coltura per una densità cellulare di 5x10 5 cellule ml -1. </li><li> Aggiungere l&#39;IgE di interesse per le cellule in sospensione ad una concentrazione finale di 1 ng ml -1, poi piastra 100 microlitri di cellule su una piastra da 96 pozzetti a colonne 1-6 e incubare a 37 ° C + 5% di CO 2 + 90% umidità relativa per 16 ore. Dopo il periodo di incubazione, e prima di eseguire il test di rilascio, controllare i pozzi sotto un microscopio per la confluenza bene e l&#39;adesione delle cellule. </li></ol></li></ol> 2) saggio di rilascio: <ol><li> Lavare le cellule: <ol><li> Buffer di stampa a caldo (PIPES 25 mM, 120 mM di cloruro di sodio, 5 mM di cloruro di potassio, 0,04 mM di cloruro di magnesio, e 1 mM di cloruro di calcio) a 37 ° C per consentire il lavaggio delle cellule delicato. </li><li> Lavare le cellule muovendo il piatto per rimuovere il supporto delle cellule e l&#39;aggiunta di 100 ml caldo, 37 ° C, buffer di stampa. Ripetere due volte. </li></ol></li><li> Sfida Antigen: <ol><li> Preparare una diluizione in serie dell&#39;antigene (NIP-HSA o DNP-HSA) di 0 ng ml -1, 0,1 ng ml -1, 1 ng ml -1, 10 ng ml -1, 100 ng ml -1, 1.000 ng ml -1, 10.000 ng ml -1 nel buffer di stampa e calda a 37 ° C. </li><li> Dopo il secondo lavaggio delle cellule, scartare i media e sostituito con 100 microlitri delle soluzioni di antigene. Assicurarsi che i pozzetti di una stessa fila (A1-6 per esempio) hanno la stessa concentrazione dell&#39;antigene aggiunto a loro. </li><li> Impostare un controllo negativo nella riga A con l&#39;aggiunta di antigene 0 ng -1 ml. Aggiungere crescente concentrazione dell&#39;antigene le righe (BG) seguiti da triton x-tampone (5% Triton X-100) in cellule H riga per lisare le cellule per essere utilizzato come controllo positivo. Incubare a 37 ° C per 20 minuti per permettere alle cellule di rilasciare i suoi mediatori. </li></ol></li><li> Impostazione di controlli e individuali: <ol><li> Dopo l&#39;incubazione, trasferire 50 ml di surnatante cellula all&#39;altra metà delle piastre (pozzi a pozzi A1-6 A7-12, ecc). Scartare i restanti 50 ml di surnatante e sostituire con 50 ml di triton x-tampone per consentire la misurazione della quantità di mediatori rilasciati in ciascun pozzetto in colonne 7-12 come percentuale dei mediatori totale all&#39;interno delle celle nella colonna 1-6 . </li></ol></li><li> Substrato enzimatico: </li><li> Aggiungere 50 ml di substrato β-dell&#39;esoaminossidasi (50 mM 4-nitrofenil N-acetil-β-D-glucosaminide preparata in DMSO diluita fino a 2 mM aggiungendolo al tampone citrato 0,2 M di acido citrico e sodio acetato 0.2 M, pH 4.5) a tutti i pozzetti facilitato la conversione del substrato 4-nitrofenolo dall&#39;enzima β-dell&#39;esoaminossidasi. Incubare le piastre a 37 ° C per 2 ore. </li></ol><img alt="Figura 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width = &quot;400&quot; /&gt; <ol start="5"><li> Terminare la reazione: <ol><li> Arrestare la reazione aggiungendo 150 microlitri del tampone Tris (1 M Tris-HCl, pH 9) a ciascun pozzetto come l&#39;alto pH del tampone arresta la reazione e trasforma il 4-nitrofenolo in un colore giallo. </li></ol></li><li> La lettura e l&#39;analisi dei risultati: <ol><li> Leggere la piastra utilizzando uno spettrofotometro piastra a 405 nm per misurare l&#39;assorbanza del colore giallo. Calcolata la percentuale di rilasciato β-dell&#39;esoaminossidasi utilizzando la seguente formula: </li></ol></li></ol><img alt="Figura 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> Applicare questa formula in ogni pozzetto, dopo di che in media è data per ogni riga. A1 e A7 rappresentano la posizione dei pozzetti della piastra da 96 pozzetti. Tracciare un grafico della percentuale di rilascio di β-esosaminidasi (che corrisponde al rilascio di mediatori totale) agaiNST antigene concentrazione di 2, 3. </li></ol>

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</ol>

The authors declare that they have no competing financial interests in this paper.

In sintesi i risultati di questa indagine hanno dimostrato che quando le cellule RBL-2H3.1 esprimono FcεRIα equini sono sensibilizzati con equina IgE e contestate da un antigene, danno un rilascio picco mediatore del 36.68% ± 4,88% del totale di mediatore all&#39;interno della cellule, rispetto alla RBL-2H3.1 cellule parentali non esprimono equina FcεRIα. Così questo test fornisce un utile strumento per indagare e studiare le risposte allergiche equini in vitro. La sua consent...

Allergia è noto da millenni. Un trattamento di asma è stato descritto nel testo medico egiziano antico noto come il Papiro di Ebers (~ 1550 aC) e discusso i rimedi a base di erbe per il trattamento di essa 7. Allergia Oggi è classificata come una risposta di ipersensibilità di tipo I, in cui il tipo cellulare T helper 2 (TH 2) braccio del sistema immunitario dirige la produzione di anticorpi immunoglobulina E (IgE) in risposta ad antigeni ambientali chiamati allergeni. Si tratta di sostanze diverse che comunemente interagiscono con le cellule del sistema immunitario e stimolano la sintesi e la secrezione di citochine pro-infiammatorie, comprese interleuchina-4 e interleuchina-13 8, 9 così come particelle in particelle di fumo di sigaretta o di scarico diesel che permettono di migliorare la sintesi di IgE 10. L&#39;aumento delle manifestazioni allergiche nei paesi industrializzati negli ultimi 50 anni è stata attribuita ad una combinazione dell&#39;effetto diinquinanti ambientali e una tendenza a un ambiente più igienizzato, che si combinano per spostare la risposta immunitaria verso un profilo predominato da TH 2 citochine, come proposto dalla &#39;ipotesi dell&#39;igiene&#39; 11. Come accennato in precedenza, gli esseri umani non sono gli unici mammiferi affetti da allergia. In particolare cavalli e cani possono anche sviluppare reazioni allergiche classiche e uno studio di 12 ha dimostrato che, come negli esseri umani, allergia equina è attribuito a fattori genetici e ambientali. Di conseguenza, questi animali presentano un buon modello per studiare l&#39;interazione tra cause genetiche e ambientali di allergia, la sua progressione da sensibilizzazione alla malattia, e le possibili strategie di intervento una volta che le manifestazioni cliniche indicate nel Nel 1887, Stömmer stato il primo a descrivere la somiglianza tra umano e asma equina 13, l&#39;effetto dell&#39;istamina sul sistema cardiovascolare equina èmolto simile a quello umano 14. I cavalli sono anche la pietra angolare del settore corse di cavalli, che vale la pena di US $ 72 miliardi, con un giro d&#39;affari delle scommesse di US $ 115 miliardi di dollari all&#39;anno 15. La maggior parte dei cavalli da corsa in stile contemporaneo sono discendenti del piccolo numero di cavalli arabi allevati da Lady Anne Blunt dal 1878 in poi. Cavalli da corsa moderne sono comunemente inbred selezionare per le abilità di performance. Essi sono inclini a malattie genetiche, uno dei quali è la loro suscettibilità a montare reazioni allergiche. Essi hanno anche 1000 volte più elevati livelli di IgE nel siero rispetto anche gli esseri umani più gravemente allergica 16. Reazioni allergiche a cavallo sono di solito manifestano come puntura d&#39;insetto ipersensibilità (IBH) 17, 18. IBH risultati nella dermatite a causa di punture di insetti formano nel genere Culicoides. Un&#39;altra forma di malattia allergica equina è ostruzioni delle vie aeree ricorrenti (RAO), questo si manifesta nei polmoni e le vie respiratorie. E &#39;caratterizzata da respiro sibilante e laboratoriorespirazione OR. RAO comunemente si verifica in risposta alla muffa spore, e alti livelli di IgE allergene-specifici sono stati registrati nei cavalli affetti da RAO in uno studio di 19 anche se un&#39;altra inchiesta non ha confermato che 20. Gli studi su allergie equina ruotavano attorno al tentativo di controllo e di neutralizzare equina IgE attraverso lo sviluppo di anticorpi anti-equino IgE monoclonali (MAK, MAB) 21, 22. Inoltre lo studio entro il 23 illustra la produzione dei domini extracellulari di α equina ad alta affinità Fc recettore catena (FcεRIα) recettori nel tentativo di rilevare e quantificare siero equino IgE. Uno studio correlato da Ledin 24 illustra un nuovo approccio volto a neutralizzare il siero IgE dal priming del sistema immunitario con un self / non-self immunogeno. Tutti questi studi, tuttavia, non avevano un test efficace per testare la sicurezza e l&#39;efficacia dei loro protocolli. In questo articolo, presentiamo ora tale prova sysTEM applicabile allo studio di strategie diagnostiche e terapeutiche relative al sistema equina, dove rilascio β-dell&#39;esoaminossidasi, come indicatore di mediatore degranulazione delle cellule, è stata valutata su cellule RBL-2H3.1 esprimono equina FcεRIα. Questo protocollo si basa su precedenti pubblicazioni 25, 4, 5, 2, 3 descrivono l&#39;ingegnerizzazione di cellule RBL trasfettate con il gene codificante il dominio di legame del recettore IgE ad alta affinità per le IgE da specie diverse. Il protocollo descritto come eseguire un saggio di rilascio β-dell&#39;esoaminossidasi, i cui risultati sono presentati come media ± deviazione standard di esperimenti in triplo. Il saggio di rilascio è stato sviluppato da Siraganian e gancio 25 per studiare l&#39;allergia umana. Il gruppo del laboratorio guidato dal dottor Reuben Siraganian ha sviluppato anche la linea di cellule RBL. Queste cellule RBL sono stati sviluppati per esprimere la FcεRIα umana e il protocollo è stato pubblicato da 4. Il pezzo finaledel dosaggio venuto con lo sviluppo del plasmide pSV nel lavoro di Neuberger 26 che descrive la produzione di un anticorpi IgE clonando suo pesante gene della catena a valle di un gene del mouse per una regione variabile di IgE che gli obiettivi l&#39;aptene 4-idrossi-3 nitro-phenacetyl (NP), l&#39;anticorpo chimerico risultante era perfettamente funzionante. La possibilità di sviluppare qualsiasi IgE rivolti stesso aptene, ma anche di clonazione suo recettore sulla superficie delle cellule RBL portato alla normalizzazione del dosaggio rendendolo un protocollo utile per misurare la degranulazione dei basofili. Il test ha i pro e contro. I pro del saggio è la sua adattabilità ad essere utilizzato in qualsiasi sistema di mammiferi, il nostro laboratorio ha quindi utilizzato per verificare la degranulazione dei sistemi umani, canini e equino, e questo è realizzabile semplicemente sintetizzando IgE dell&#39;organismo e la clonazione suo recettore sulla superficie delle cellule RBL. D&#39;altronde,i contro del saggio è che le cellule RBL sono molto sensibili alle variazioni termiche, meccaniche e PH, rendendoli dare una variazione dei livelli di degranulazione all&#39;interno dello stesso dosaggio. E &#39;quindi fortemente consigliato che i dosaggi vengano sempre ripetuti in triplicato e quindi una media è preso da loro. Inoltre, le cellule RBL tendono a spostarsi verso un fenotipo non-rilascio se lasciati in coltura per un tempo prolungati (&gt; 10 settimane) 27, rendendo la loro manutenzione ingombrante. Sono anche soggetti a infezioni da batteri micoplasma, che non sono visibili al microscopio ottico e non modificano la morfologia della cellula, ma sarebbero drasticamente cambiare i loro livelli di degranulazione. Pertanto sono necessari test micoplasma regolari.

<table><tbody><tr><td>RBL-2H3.1 Expressing Equine Fc&amp;epsilon;RI&amp;alpha;</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>Equine IgE anti NIP-HSA</td><td>-</td><td>-</td><td>Produced in the lab</td></tr><tr><td>96 Well Plate</td><td>Sigma</td><td>CLS3595</td><td>-</td></tr><tr><td>Multi Channel Pipette</td><td>Anachem</td><td>-</td><td>-</td></tr><tr><td>Incubator</td><td>Galaxy R</td><td>-</td><td>-</td></tr><tr><td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td><td>Cambridge Research Biochemicals</td><td>N-1070-1</td><td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td></tr><tr><td>Dinitrophenyl Conjugated to Human Serum Albumin</td><td>Sigma</td><td>A6661</td><td>Abbreviated DNP-HSA</td></tr><tr><td>Plate Spectrophotometer</td><td>Anthos Labtec HT2</td><td>-</td><td>-</td></tr><tr><td>Pipes</td><td>Sigma</td><td>P1851</td><td>-</td></tr><tr><td>Sodium Chloride</td><td>Sigma</td><td>S7653</td><td>-</td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9333</td><td>-</td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M2670</td><td>-</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C1016</td><td>-</td></tr><tr><td>Triton x100</td><td>Sigma</td><td>X100</td><td>-</td></tr><tr><td>4-nitrophenyl N-acetyl-&amp;beta;-D-glucosaminide</td><td>Sigma</td><td>N9376</td><td>Stock solution called &amp;beta;-hexosaminidase substrate was 50mM prepared in DMSO</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D2650</td><td>-</td></tr><tr><td>Citric Acid</td><td>Sigma</td><td>251275</td><td>-</td></tr><tr><td>Sodium Acetate</td><td>Sigma</td><td>S7670</td><td>-</td></tr><tr><td>Tris</td><td>Sigma</td><td>T5941</td><td>-</td></tr></tbody></table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

I genitori cellule RBL-2H3.1 e quelle trasfettate con il gene del recettore equina FcεRIα sono stati sensibilizzati con il mouse IgE contro DNP-HSA e sfidati con l&#39;antigene DNP-HSA. Mouse IgE si lega al recettore di ratto endogena in entrambe le linee cellulari e quindi agisce come un controllo per verificare la fattibilità rilascio di entrambe le linee cellulari di rilasciare mediatori (Figura 1 A). Questo è un controllo importante e dovrebbe essere effettuata regolarmente dal momento esteso (&...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃŽÂµRI at JoVE.com

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Sviluppo di un<em&gt; In vitro</em&gt; Sistema modello per studiare l&#39;interazione tra<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE con il suo recettore ad alta affinità FcεRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Research

JoVE Journal

Immunology and Infection

12.4K Views.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Video: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Modelli sperimentali per lo Studio di Fisiologia dell&#39;epitelio pigmentato retinico e fisiopatologia

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Ricerca

Neuroscienze

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Rilevazione di True IgE-esprimono mouse B Cells Lineage

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Immunologia e infezioni

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Applicazione di lungo termine colta interferone-γ Enzyme Linked immunospot Assay per la valutazione effettori e memoria delle cellule T risposte a Bovino

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Sviluppo di un modello colite Antigen-driven per studiare presentazione di antigeni da cellule presentanti l&#39;antigene alle cellule T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

Ottimizzazione del interferone-gamma ELISpot analisi di risposte a cellula T misura nel modello cavia dopo la vaccinazione

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Assessment of Human Natural Killer Cell Events Driven by Fc&#947;RIIIa Engagement in the Presence of Therapeutic Antibodies

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by Fc&#947;RIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the Fc&#947;RIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

A protocol for studying Fc&#947;RIIIa-driven events by therapeutic antibodies in human natural killer cells is described here. This artificial stimulation platform permits the interrogation of downstream effector functions such as degranulation, chemokine/cytokine production, and signaling pathways mediated by the Fc&#947;RIIIa and Fc portions of antibodies involved in binding.

Valutazione degli eventi delle cellule natural killer umane guidati dall'impegno di FcγRIIIa nella presenza di anticorpi terapeutici

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and ...

Istituzione di un monostrato 2D da coltura enteroide ileale bovina 3D

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Autore in primo piano: Modello enteroide avanzato per lo studio delle interazioni ospite-patogeno 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Generazione di un sistema di coltura monostrato bidimensionale di derivazione enteroide primaria bovina per applicazioni nella ricerca biomedica traslazionale

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

Sviluppo di un

Riepilogo

Esplora Altri Video

Modelli sperimentali per lo Studio di Fisiologia dell'epitelio pigmentato retinico e fisiopatologia

Rilevazione di True IgE-esprimono mouse B Cells Lineage

Applicazione di lungo termine colta interferone-γ Enzyme Linked immunospot Assay per la valutazione effettori e memoria delle cellule T risposte a Bovino

Sviluppo di un modello colite Antigen-driven per studiare presentazione di antigeni da cellule presentanti l'antigene alle cellule T

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Ottimizzazione del interferone-gamma ELISpot analisi di risposte a cellula T misura nel modello cavia dopo la vaccinazione

Valutazione degli eventi delle cellule natural killer umane guidati dall'impegno di FcγRIIIa nella presenza di anticorpi terapeutici

Generazione di un sistema di coltura monostrato bidimensionale di derivazione enteroide primaria bovina per applicazioni nella ricerca biomedica traslazionale

Generazione di un sistema di coltura monostrato bidimensionale di derivazione enteroide primaria bovina per applicazioni nella ricerca biomedica traslazionale

Generazione di un sistema di coltura monostrato bidimensionale di derivazione enteroide primaria bovina per applicazioni nella ricerca biomedica traslazionale