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>

<ol>
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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</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-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-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 FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#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 ...

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Immunology and Infection

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

12.3K 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 Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Sviluppo di un marcatore negativo selezionabile per Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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Immunologia e infezioni

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modello per lo studio delle interazioni tra l&#39;uomo di sangue intero e l&#39;endotelio

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Analisi Vitro di Myd88 mediata Cellular Immune Response to West Nile virus mutante Strain Infezione

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modello per la misurazione Risposte immunitarie Malaria nel contesto della coinfezione da HIV

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studiare comunità microbiche<em&gt; In Vivo</em&gt;: Un modello di interazione ospite-mediata tra<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nei Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modello della barriera emato-encefalica Utilizzando spettroscopia di impedenza: un focus sui T Cell-endoteliale cellulare Interazione

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Sviluppo di un sistema di consegna economico DNA da &quot;Acufection&quot; e la sua applicazione alla ricerca della pelle

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...