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

1) Herstellung der Zelllinie: <ol><li> Die Entwicklung der RBL-2H3.1 Zelllinie, Pferde FcsRI α: <ol><li> Mit grundlegenden Gewebekulturtechniken für Monolayer-Zelllinien, Transfektion Eltern RBL-2H3.1 Zellen unter Verwendung des pEE6 Plasmid, die Durchführung der Pferde FcεRIα Gens (GenBank: Y18204.1) 28. Hinzufügen 2 ug ul -1 der Plasmid-DNA auf 0,8 ml Zellen bei einer Dichte von 1.2 x10 &amp; sup7; Zellen ml -1. Elektroporieren der Zellen bei 250 V 960 uF mit einem 0,4 cm electrocuvette dann sofort auf Eis inkubieren für 10 min. </li><li> Auswahl der transformierten Zellen unter Verwendung von Medien, die 0,4 g Geneticin G418 Sulfat, sortiert dann die verbleibenden lebenden Zellen durch FACS durch Markieren sie mit einem Fluoreszenz IgE-Antikörper. Verwenden Sie die resultierende RBL-2H3.1 ausdrücken Pferde FcεRIα Zelllinie für die Untersuchung 2, 3. </li></ol></li><li> Pre-Test Antikörper Sensibilisierung: <ol><li&gt; Ernte der RBL-2H3.1 ausdrücken Pferde FcεRIα Zellen aus einer konfluenten Petrischale. Waschen dann erneut die Zellen in Kulturmedium bis zu einer Zelldichte von 5x10 5 Zellen ml -1. </li><li> Hinzufügen des IgE von Interesse für die suspendierten Zellen zu einer Endkonzentration von 1 ng ml -1, dann die Platte 100 &amp; mgr; l der Zellen auf eine Platte mit 96 Vertiefungen in den Spalten 1-6 und bei 37 ° C + 5% CO 2 + 90% relative Luftfeuchte für 16 Stunden. Nach der Inkubationszeit und vor der Durchführung der Freisetzungstest, überprüfen Sie die Brunnen unter einem Mikroskop auf gut Konfluenz und Zellhaftung. </li></ol></li></ol> 2) Veröffentlichung Assay: <ol><li> Waschen der Zellen: <ol><li> Warm Freisetzungspuffer (25 mM PIPES, 120 mM Natriumchlorid, 5 mM Kaliumchlorid, 0,04 mM Magnesiumchlorid und 1 mM Calciumchlorid) bei 37 ° C bis zur sanften Waschen der Zellen zu ermöglichen. </li><li> Wash-Zellen durch Schwenken der Platte an Zellmedien zu entfernen und das Hinzufügen von 100 ul warm, 37 ° C, Freisetzungspuffer. Zweimal wiederholen. </li></ol></li><li> Antigen Herausforderung: <ol><li> Bereiten Sie eine serielle Verdünnung des Antigens (NIP-HSA oder DNP-HSA) von 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 in Freisetzungspuffer und warm bei 37 ° C. </li><li> Nach dem zweiten Zellwäsche, entsorgen Sie die Medien und mit 100 ul der Antigenlösungen ersetzt. Sicherzustellen, daß Vertiefungen in der gleichen Reihe (A1-6 zum Beispiel) die gleiche Antigenkonzentration zu ihnen hinzugefügt. </li><li> Eine Negativkontrolle in der Zeile A durch Zugabe von 0 ng ml -1 Antigen eingestellt. Hinzufügen zunehmender Antigenkonzentration die Reihen (BG), gefolgt von Triton-x-Puffer (5% Triton X-100) in der Reihe H-Zellen, um die Zellen zu lysieren, um als positive Kontrolle verwendet werden. Inkubieren bei 37 ° C für 20 min, um damit die Zellen ihre Mediatoren freisetzen. </li></ol></li><li> Einrichten individuelle Wohlkontrollen: <ol><li> Nach der Inkubation mit einer Pipette 50 ul Zellüberstand auf die andere Hälfte der Platte (wells A1-6 bis A7-12 Vertiefungen, etc.). Verwerfen der restlichen 50 &amp; mgr; l des Überstands und Ersetzen mit 50 ul Triton-X-Puffer, um die Messung der Menge des freigesetzten Mediatoren in jede Vertiefung in den Spalten 7-12 als Prozentsatz der Gesamtmediatoren in den Zellen in Spalte 1-6 ermöglichen . </li></ol></li><li> Enzym-Substrat: </li><li> Dann werden 50 ul der β-Hexosaminidase-Substrat (50 mM 4-Nitrophenyl-N-acetyl-β-D-glucosaminid in DMSO durch Zugabe zu Citratpuffer 0,2 M Zitronensäure und 0,2 M Natriumacetat auf 2 mM verdünnt, pH 4,5) zu allen Vertiefungen, um die Umwandlung des Substrats zu 4-Nitrophenol durch das β-Hexosaminidase Enzyms erleichtert. Die Inkubation erfolgt bei 37 ° C für 2 Stunden. </li></ol><img alt="Abbildung 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width = &quot;400&quot; /&gt; <ol start="5"><li> Beenden der Reaktion: <ol><li> Stoppen der Reaktion durch Zugabe von 150 &amp; mgr; l Tris-Puffer (1 M Tris-HCl, pH 9) in jede Vertiefung wie die hohe pH-Wert des Puffers wird die Reaktion gestoppt und dann die 4-Nitrophenol in einer gelben Farbe. </li></ol></li><li> Das Lesen und die Analyse der Ergebnisse: <ol><li> Lesen der Platte unter Verwendung eines Platten Spektrophotometer bei 405 nm, die Absorption der gelben Farbe gemessen. Berechnet den Prozentsatz des freigesetzten β-Hexosaminidase Verwendung der folgenden Formel: </li></ol></li></ol><img alt="Abbildung 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> Wenden Sie diese Formel in jede Vertiefung, wonach ein Mittelwert für jede Zeile übernommen. A1 und A7 repräsentieren die Lage der Vertiefungen der Platte mit 96 Vertiefungen. Eine Grafik mit Prozentsatz der β-Hexosaminidase Mitteilung (die Gesamtmediatorfreisetzung entspricht) against Antigenkonzentration 2, 3. </li></ol>

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

In Zusammenfassung der Ergebnisse dieser Untersuchung zeigten, dass bei RBL-Zellen, 2H3.1 equine FcεRIα mit equinen IgE durch ein Antigen sensibilisiert und herausgefordert, geben sie einen Spitzen Mediator-Freisetzung von 36,68% ± 4,88% der Gesamtmenge des innerhalb des Vermittlers Zellen, verglichen mit dem RBL-2H3.1 Elternzellen nicht exprimiert equine FcεRIα. Somit Dieser Test liefert ein nützliches Werkzeug für die Untersuchung und das Studium Pferde allergischen Reaktionen i...

Allergie ist seit Jahrtausenden bekannt. Ein Asthma-Behandlung wurde in der antiken ägyptischen medizinischen Text als Papyrus Ebers (~ 1550 BCE) bekannt beschrieben und diskutiert pflanzliche Heilmittel, um es 7 zu behandeln. Heute Allergie ist als Typ I-Überempfindlichkeitsreaktion, wobei die T-Helferzell-Typ 2 (TH 2) Arm des Immunsystems steuert die Produktion von Immunglobulin E (IgE) Antikörper als Antwort auf Umweltantigene, Allergene genannt klassifiziert. Dies sind diverse Stoffe, die üblicherweise mit Zellen interagieren im Immunsystem und stimulieren die Synthese und Sekretion von proinflammatorischen Cytokinen, einschließlich IL-4 und Interleukin-13 8, 9 als weiß Partikel im Zigarettenrauch oder Dieselabgaspartikeln, die IgE-Synthese zu erhöhen 10. Der Anstieg der allergischen Manifestationen in Industrieländern in den letzten 50 Jahren hat sich eine Kombination der Wirkung zugeschriebenUmweltschadstoffe und ein Trend zu einer keim Umgebung, die die Immunantwort gegenüber einem Profil von TH 2 Zytokine überwog verschieben kombinieren, wie durch die &quot;Hygiene-Hypothese&quot; 11 vorgeschlagen. Wie oben erwähnt, sind Menschen nicht nur Säugern durch Allergie leidet. Insbesondere Pferden und Hunden entwickeln auch klassische allergische Reaktionen und eine Untersuchung von 12 zeigt, dass, wie beim Menschen, wird equine Allergie auf genetischen und Umweltfaktoren zurückzuführen. Als Folge präsentieren diese Tiere gute Modelle zur Untersuchung der Wechselwirkung zwischen genetischen und umweltbedingten Ursachen von Allergien, ihr Fortschreiten von Sensibilisierung gegen Krankheit und mögliche Interventionsstrategien einmal klinischen Manifestationen in gesetzt 1887 Stömmer war die erste Person, die Ähnlichkeit zwischen Mensch und Pferde-Asthma 13 zu beschreiben, ist die Wirkung von Histamin auf der Pferdeherzkreislaufsystemsehr ähnlich dem des Menschen 14. Pferde sind auch der Grundstein der Pferderennindustrie, die es wert USD 72 Milliarden mit einem Wettumsatz von US $ 115.000.000.000 jährlich 15. Die meisten zeitgenössischen Rennpferde sind Nachkommen von der geringen Zahl der arabischen Pferde von Lady Anne Blunt seit 1878 gezüchtet. Moderne Rennpferde werden häufig Inzucht für Performance Fähigkeiten auszuwählen. Sie sind anfällig für genetische Erkrankungen, von denen eine ihre Anfälligkeit für allergische Reaktionen zu montieren. Sie haben auch 1000 mal höhere Serum-IgE-Spiegel als selbst die am schwersten allergischen Menschen 16. Pferde allergischen Reaktionen sind in der Regel als Insektenstich Überempfindlichkeit (IBH) 17, 18 manifestiert. IBH Ergebnisse in Dermatitis durch Bisse bilden Insekten aus der Gattung Culicoides. Eine andere Form der Pferde-allergische Erkrankung ist rezidivierenden Atemwegsobstruktionen (RAO), dies in den Lungen und Atemwegen manifestiert. Es wird von Keuchen und Labor gekennzeichnetODER-verknüpft Atmung. RAO tritt häufig als Reaktion auf Sporen zu formen, und hohe allergenspezifische IgE-Spiegel haben bei Pferden, die an RAO in einer Studie 19 registriert worden, obwohl ein anderer Untersuchung nicht bestätigt diesen 20. Studien über equine Allergie drehte sich um die Versuch Überwachung und Neutralisieren equine IgE durch die Entwicklung anti equine IgE monoklonalen Antikörpern (mAb) 21, 22. Weiterhin die Studie von 23 diskutiert die Produktion der extrazellulären Domänen der α equinen Hochaffinitäts-Fc-Rezeptors Kette (FcεRIα) Rezeptors in einem Versuch zu detektieren und zu quantifizieren Pferdeserum IgE. Eine verwandte Studie von Ledin 24 bespricht einen neuen Ansatz bei der Neutralisierung Serum-IgE durch Priming des Immunsystems mit einem selbst / Nicht-Selbst Immunogen gerichtet. Alle diese Studien fehlte aber eine wirksame Assay, um die Sicherheit und Wirksamkeit der Testprotokolle. In diesem Artikel haben wir jetzt eine solche Assay sys präsentierentem für die Untersuchung der relevanten equinen System, in dem β-Hexosaminidase Release, als Indikator für die Zellmediator Degranulation wurde für RBL-Zellen, 2H3.1 equine FcεRIα beurteilt diagnostische und therapeutische Strategien. Dieses Protokoll basiert auf früheren Veröffentlichungen 25, 4, 5, 2, 3 das Engineering von RBL-Zellen mit den IgE-Bindungsdomäne des hochaffinen IgE-Rezeptors verschiedener Spezies kodiert, transfiziert Beschreibung basiert. Das Protokoll beschreibt, wie ein β-Hexosaminidase-Freisetzungstest durchzuführen, deren Ergebnisse sind als Mittelwert ± Standardabweichung von dreifachen Experimenten dargestellt. Der Freisetzungstest wurde zuerst von Siraganian entwickelt und Haken 25 Menschenallergie zu untersuchen. Das Labor Gruppe von Dr. Reuben Siraganian führte entwickelte auch die RBL-Zelllinie. Diese RBL Zellen wurden entwickelt, um die menschliche FcεRIα auszudrücken und das Protokoll wurde von 4 veröffentlicht. Das letzte Stückder Test wurde mit der Entwicklung des Plasmids pSV in der Veröffentlichung von Neuberger 26, die die Produktion von IgE-Antikörpern durch Klonierung ihrer Schwerketten-Gen stromabwärts von einem Maus-Gen für eine variable Region, die IgE das Hapten 4-Hydroxy-3 zielt beschrieben nitro-Phenacetyl (NP), war die resultierende chimäre Antikörper, vollständig funktionsfähig. Die Fähigkeit, jede IgE entwickeln die die gleichen Hapten, wobei auch die Klonierung seinen Rezeptor auf der Oberfläche der RBL-Zellen führte zu der Standardisierung des Assays so dass es ein nützliches Protokoll, um die Degranulation von Basophilen Zellen zu messen. Der Test funktioniert haben Vor- und Nachteile. Die Profis des Assays ist seine Anpassungsfähigkeit an irgendeinem Säugersystem verwendet werden, unser Labor ist somit benutzt, um die Degranulation der Mensch, Hund und Pferde Systeme zu testen, und dies ist erreichbar, einfach durch Synthetisieren des Organismus IgE und Klonieren seinen Rezeptor auf die Oberfläche der RBL-Zellen. Andererseits,die Nachteile des Assays ist, dass die RBL-Zellen sind sehr empfindlich gegen thermische, mechanische und PH ändert, so dass sie eine Veränderung der Degranulation Ebenen innerhalb des gleichen Assay zu ergeben. Es wird daher dringend empfohlen, dass die Tests werden immer dreifach wiederholt und dann ein Mittelwert von ihnen genommen. Darüber hinaus sind die RBL-Zellen sind in der Regel zu einer nicht-freisetzenden Phänotyp verschieben, wenn sie in der Gewebekultur für längere Zeiten (&gt; 10 Wochen) 27 übrig sind, was ihre Wartung umständlich. Sie sind auch anfällig für Infektionen durch Mycoplasma-Bakterien, die nicht von einem Lichtmikroskop sichtbar sind und nicht die Morphologie der Zelle ändern, würde aber ihre Degranulation Ebenen drastisch ändern. So regelmäßigen Mykoplasmen-Tests erforderlich.

<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.

Die Eltern RBL-2H3.1 Zellen und solche mit equinen FcεRIα Rezeptorgen transfiziert wurden zuerst mit Maus-IgE-anti-DNP-HSA sensibilisiert und mit dem DNP-HSA-Antigen herausgefordert. Maus-IgE bindet an den endogenen Rezeptor aus Ratte in beiden Zelllinien und dient als Kontrolle, um die Freisetzung Lebensfähigkeit der beiden Zelllinien zu testen, um Mediatoren (Figur 1 A) lösen damit. Dies ist eine wichtige Kontrolle und sollten routine da beim verlängert (&gt; 10 Wochen) Passage in Zellkultur durc...

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.

Entwicklung eines<em&gt; In vitro</em&gt; Modellsystem für die Untersuchung der Wechselwirkung von<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE mit seinen hochaffinen Rezeptors FcsRI

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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JoVE Journal

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 Aufrufe.  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.

Serie: 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.

Entwicklung eines Negativ Selektionsmarker für 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 ...

Forschung

Immunologie und Infektion

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

In-vitro-Bildung von Biofilmen in einem 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. ...

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.

Einrichtung einer<em&gt; In-vitro-</em&gt; System zur intrazelluläre Verhalten der Studie<em&gt; Candida glabrata</em&gt; Menschen THP-1-Makrophagen

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; Modell für die Untersuchung der Wechselwirkungen zwischen humanem Vollblut und Endothel

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-vitro-Analyse Myd88-vermittelte zelluläre Immunantwort gegen das West-Nil-Virus-Infektion Mutantenstamm

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.

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

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.

Studieren Mikrobielle Gemeinschaften<em&gt; In-vivo-</em&gt;: Ein Modell von Host-vermittelte Interaktion zwischen<em&gt; Candida albicans</em&gt; Und<em&gt; Pseudomonas aeruginosa</em&gt; In den Atemwegen

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.

Ein<em&gt; In Vitro</em&gt; Modell der Blut-Hirn-Schranke Mit Impedanzspektroskopie: Ein Fokus auf T-Zell-Endothelzellinteraktion

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.

Visualisierung der Auswirkungen von Sputum auf Biofilm-Entwicklung eines Modells mit Kammerdeckgläser

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.

Entwicklung eines wirtschaftlich DNA Delivery System von „Acufection“ und seine Anwendung auf die Hautforschung

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