Questo lavoro è stato supportato da una sovvenzione di progetto pilota da CREH centro sponsorizzato dal NIEHS e dalla Fondazione nazionale di scienze naturali della Cina (No. 31572626).

Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con tutte le pertinenti linee guida, regolamenti e agenzie di regolamentazione. Il protocollo in questione è stato effettuato sotto la guida e l'approvazione del istituzionale Animal Care e uso Committee (IACUC) della Texas A & M. Istituto per la medicina genomica.
1. soluzione preparazione
<ol><li>Diluire la soluzione salina bilanciata di Hank di 10 x 1 x con doppio distillata H2O e conservare a 4 ° C.</li>
	<li>Preparare la soluzione di isolamento aggiungendo HEPES (10 mM), MgCl2 (1 mM) e glucosio (5 mM). Regolare il pH a 7,4 e tenere sul ghiaccio.</li>
	<li>Preparare la soluzione di collagenasi da collagenasi tipo 4. Nota: In particolare, collagenasi tipo 4 contiene 160 U per mg di peso a secco. 1 g di collagenasi tipo 4 è diluito in 5 mL doppio distillata H2O con una concentrazione di 200 mg/mL. 2 mL della collagenosi diluita viene aggiunto alla soluzione di isolamento freddo ghiaccio 30 mL e 6 mL di soluzione di collagenasi viene trasferito in una provetta 50 mL per ogni mouse.</li>
	<li>Preparare la soluzione di lavaggio aggiungendo 1 mM CaCl2 per la soluzione di isolamento.</li>
	<li>Preparare la soluzione di purificazione con soluzione di sodio polysucrose freddo diatrizoate (1,1119 g/mL, 5 mL).</li>
	<li>Preparare 500 mL di terreno di coltura dell'isolotto con l'aggiunta di L-Glutammina (20 mM), penicillina (100 U/mL), streptomicina (100 µ g/mL) e siero bovino fetale (FBS) (10%) nel medium RPMI.</li>
	<li>Preparare la soluzione di riserva di DCFH-DA sciogliendo la polvere in acqua distillata a 200 µM e conservare a-20 ° C. Nota: Il ROS macchiatura soluzione viene preparato diluendo la soluzione di riserva di DCFH-DA alla concentrazione finale di 5 µM in RPMI. Questo reagente dovrebbe essere protetto dalla luce.</li>
</ol>2. preparazione chirurgica
<ol><li>Iniettare per via intraperitoneale (I.P.) un mouse con avertin (2,5% avertin, 0,4 mL/20 g). Dopo che il mouse è completamente anestetizzato e non risponde più a posteriori del piede pizzichi, eutanasia il mouse e spostare la cappa biologica.</li>
	<li>Posizionare il mouse eutanasizzato col lato addominale rivolto verso l'alto e spruzzare sulla pelle con etanolo al 70% per la sterilizzazione.</li>
	<li>Aprire la cavità addominale con un 1 cm U-incisione sulla parte superiore dell'addome e ritrarre la pelle in direzione rostrale sopra la cassa per esporre la cavità addominale.</li>
</ol>3. rimozione e l'aspersione del pancreas
<ol><li>Come illustrato nella Figura 1, trovare la posizione del ampulla. Utilizzare un paio di pinze curve per serrare il duodeno vicina alla posizione dello sfintere di Oddi. Utilizzare un altro paio di pinzetta per serrare la parete del duodeno per bloccare la via biliare nel duodeno. Nota: L'ampolla non è stata inserita in questo passaggio. Questo passaggio è destinato a bloccare la via biliare nel duodeno.</li>
	<li>Riposizionare il mouse con la testa prossimale e distale per quanto riguarda il ricercatore piedi.</li>
	<li>Trovare il dotto biliare comune e iniettare lentamente 3 mL di soluzione della collagenosi con una siringa di ago e 5 mL di 30 G. Assicurarsi che il dotto biliare comune e il pancreas sono gonfiati dopo l'iniezione.</li>
	<li>Rimuovere il pancreas dilatato usando la pinzetta e forbice per separarla da discendente del colon, intestino, stomaco e milza. Posizionare il pancreas in un tubo da 50 mL contenente 3 mL di soluzione di collagenasi e tenerlo sul ghiaccio.</li>
</ol>4. pancreas digestione
<ol><li>Collocare ciascuna provetta da 50 mL in bagnomaria a 37 ° C. Incubare per 15-20 minuti (il tempo varia con lo sforzo e l'età dei topi).</li>
	<li>Vibrare il tubo con un vortex.</li>
	<li>Girone 4-5 pancreas digerito in un colino grande lavandino sterile e utilizzare una paletta per rendere il pancreas cadere a pezzi accuratamente. Utilizzare una siringa di ago e 10ml 23g contenente la soluzione di lavaggio per pipettare con forza le cellule digerite fuori dallo schermo. Raccogliere la soluzione di lavaggio in un tubo da 50 mL.</li>
	<li>Centrifugare le provette a 97 x g a 4 ° C per 1 min e decantare il supernatante.</li>
	<li>Aggiungere 25 mL di soluzione di lavaggio in provette e risospendere il tessuto delicato nel Vortex. Rotazione verso il basso i tessuti a 97 x g a 4 ° C per 1 min e decantare il supernatante. Ripetere per 3 volte. Fare attenzione a non spostare il tessuto pellettato.</li>
</ol>5. purificazione degli isolotti
<ol><li>Aggiungere 10 mL di soluzione di purificazione al pellet di tessuto lavato e Risospendere delicatamente il contenuto.</li>
	<li>Sovrapposizione delicatamente 5 mL di soluzione di isolamento sulla soluzione di purificazione. Prestare attenzione a mantenere un'interfaccia liquida tagliente tra le due soluzioni.</li>
	<li>Centrifugare la provetta a 560 x g a 4 ° C per 15 min con accelerazione lenta e senza freno. Inserire la pipetta nell'interfaccia e raccogliere lo strato di isolotti in nuove provette da 50 mL.</li>
	<li>Lavare gli isolotti come descritto al punto 3.5.</li>
</ol>6. placcatura e trattamento delle cellule dell'isolotto
<ol><li>Risospendere le isolette in 10 mL di soluzione di cultura di isolotti, delicatamente mescolare bene e mettere 100 µ l/pozzetto con una pipetta multicanale per una piastra a 96 pozzetti. Posizionare bene circa 5 isolotti a 96.</li>
	<li>Posizionare la piastra nell'incubatore coltura tissutale per 12 h.</li>
	<li>Centrifugare (112 x g) la piastra a 96 pozzetti e rimuovere il terreno di coltura.</li>
	<li>Diluire i prodotti chimici ambientali nel terreno di coltura di isolotti. Aggiungere 100 µ l di ciascuno dei diluenti chimici in pozzetti separati della piastra. Risospendere le isolette in ogni pozzetto e incubare per 12 ore a 37 ° C (la concentrazione varia per i diversi prodotti chimici ambientali).</li>
</ol>7. ROS macchiatura e misurazione
<ol><li>Dopo il trattamento di isolotti, lavare con PBS 1X (200 µ l per ogni 96 pozzetti). Nota: La concentrazione delle sostanze chimiche è come segue: AFB1: 3 µM, atrazina: 100 µM, BaP: 10 µM, BPA: 100 µM, β-estrogeno, PCB126: 10 µM, nonilfenolo e BPA: 100 µM.</li>
	<li>Utilizzare N-acetil cisteina (NAC) come antiossidante per placare ROS. Aggiungere 5 mM di NAC per i diluenti chimici indotta da ROS e incubare per 12 ore a 37 ° C.</li>
	<li>Centrifugare la piastra a 96 pozzetti a 112 x g e incubare con soluzione di 5 µM di sonda fluorescente (H2-DCFH-DA dissolto nei media RPMI) a 37 ° C per 60 min.</li>
	<li>Lavare le cellule dell'isolotto trattate 3 volte con PBS e misurare la fluorescenza con un lettore di micropiastre a 488 nm. Nota: L'accumulo di DCF in cellule può essere misurata tramite un aumento nella fluorescenza a 530 nm quando il campione è eccitato a 488 nm.</li>
	<li>Eseguire il glucosio-stimolata dell'insulina secrezione (GSIS) dosaggio9 su xenobiotici che inducono ROS trattati isolette e isolotti non trattati per misurare gli effetti degli xenobiotici sulla funzione fisiologica degli isolotti pancreatici.</li>
</ol>

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Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Raccogliendo la prova suggerisce che l'esposizione ai prodotti chimici ambientali svolge un ruolo importante nello sviluppo di T2DM. ROS indotta da xenobiotici è stato riconosciuto come un potenziale fattore eziologico che contribuiscono allo sviluppo di T2DM. Gli esseri umani sono esposti a una vasta gamma di prodotti chimici xenobiotici e c'è un grande bisogno di tecniche di ricerca romanzo di identificare in modo efficace le sostanze tossiche del pancreas e di studiare il meccanismo della tossicità specifica per le...

Aumento della prevalenza di T2DM è diventati una crisi sanitaria globale negli ultimi anni ponevano una seria minaccia alla salute pubblica1. Molti fattori sono stati trovati per essere causalmente collegato allo sviluppo di T2DM, tra i quali, ricorrenti risultati suggeriscono che un comune punto convergente per questi fattori è l'induzione di stress ossidativo che porta alla generazione di eccessivo ROS2 , 3.
Un ampio spettro di prodotti chimici ambientali compresi PCB, diossine e BaP è stato trovato per indurre stress ossidativo, che può alterare la funzione delle cellule β del pancreas e portare a insulino-resistenza e T2DM4. Anche se il livello fisiologico di ROS svolge un ruolo importante nelle funzioni cellulari, l'esposizione a ROS che supera la capacità del sistema antiossidante provoca il danno ai tessuti o cellule e conduce a malattie5. Le cellule β del pancreas esprimere un basso livello di enzima antiossidante e quindi sono un obiettivo sensibile per il danno ossidativo di sforzo-mediati6,7. L'esposizione cronica ad alti livelli di ROS è stato indicato per causare la disfunzione di cellula pancreatica indotta da stress5 così come la resistenza di insulina nel fegato e nel tessuto adiposo8.
L'obiettivo generale di questo progetto è quello di sviluppare un'analisi basata su celle a sostanze chimiche di schermo per loro potenziali diabetogeni basati su loro induzione del ROS in cellule pancreatiche. Il pancreas manca disintossicazione metabolica ed è un obiettivo sensibile per danni indotti da xenobiotici6,7. Pertanto, misurando direttamente i ROS generati nelle cellule del pancreas, questo test dovrebbe fornire un'approssimazione diretta della ferita indotta da prodotto nel pancreas. Per sviluppare questo metodo, abbiamo isolato isolotti pancreatici del topo, coltivate l'isolotto isolato sotto condizione di cultura delle cellule con sostanze chimiche e utilizzate la generazione di ROS indotta da sostanze chimiche come la lettura. Questa procedura è semplice ed efficace nell'identificazione di prodotti chimici che inducono ROS nell'isolato isolotto; può essere ulteriormente sviluppato per indagine dei meccanismi di tossicità che sono specifici per il pancreas in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">10&#215;Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:283px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type 4</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLS-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysucrose/sodium diatrizoate solution&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#8217;,7&#8217;-dichlorofluorescein (DCFH-DA)</td>
			<td>Sigma</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microplate reader&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptomycin</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>GIBCO</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">avertin</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat/Mouse Insulin ELISA Kit</td>
			<td>Millipore</td>
			<td>EZRMI-13K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorval</td>
			<td>Sorval RT7</td>
			<td>for 96-well plate centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>Biotek</td>
			<td>Epoch 2</td>
			<td>for fluorescence reading</td>
		</tr>
	</tbody>
</table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

L'esposizione a determinati prodotti chimici ambientali negli umani e negli animali è stato trovato per causare danno cellulare delle cellule β del pancreas che porterà allo sviluppo di diabete mellito di tipo 2 (T2DM). Anche se i meccanismi per il danno cellulare indotto da chimica β erano poco chiare e probabile che sia complessa, una scoperta ricorrente è che queste sostanze chimiche inducono lo sforzo ossidativo che porta alla generazione di specie di eccessiva reattive dell'ossigeno (ROS) che inducono danni alla la cella β. Per identificare potenziali diabetogeni prodotti chimici ambientali, abbiamo isolato le cellule di Langerhans dai topi C57BL/6 e cellule dell'isolotto coltivate in piastre per colture cellulari di 96 pozzetti; cellule dell'isolotto sono state dosate con prodotti chimici, quindi la generazione di ROS è stata rilevata da 2', 7'-diclorofluorescina (DCFH-DA) tintura fluorescente. Utilizzando questo metodo, abbiamo trovato che il bisfenolo A (BPA), Benzo [a] pirene (BaP) e policlorobifenili (PCB), potrebbe indurre elevati livelli di ROS, suggerendo che potenzialmente possono indurre danni in cellule dell'isolotto. Questo metodo dovrebbe essere utile per lo screening di xenobiotici diabetogeni. Inoltre, cellule dell'isolotto coltivate possono anche essere adattate per l'analisi in vitro della tossicità chimica-indotta in cellule pancreatiche.

Un Micrografo dell'isolotto isolato sano è illustrato nella Figura 2, in cui isolotti hanno una forma rotonda o ovale con relativamente uniforme dimensioni (anche se uniformità di dimensione può variare da ceppo a ceppo). Successivamente abbiamo studiato le funzioni pancreatiche in un'analisi in vitro isolando l'isolotto e stimolando la secrezione di insulina negli isolotti di cultura. La figura 3 Mostra la nostra ana...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Qui presentiamo un protocollo per isolare le cellule delle isole pancreatiche del mouse per lo screening di induzioni ROS dai xenobiotici al fine di identificare i potenziali prodotti chimici xenobiotici diabetogeni.

Uno Screening basati su cellule dell'isolotto pancreatico murino per prodotti chimici ambientali diabetogeni

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

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7.2K Views.  Hunan Agriculture University. Qui presentiamo un protocollo per isolare le cellule delle isole pancreatiche del mouse per lo screening di induzioni ROS dai xenobiotici al fine di identificare i potenziali prodotti chimici xenobiotici diabetogeni.

Video: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Un nuovo metodo di screening per l&#39;Evoluzione diretta di enzimi termostabili bacteriolytic

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

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Immunologia e infezioni

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificazione e purificazione di cellule murine timica epiteliali

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una strategia rapido per l&#39;isolamento di nuovi Faustoviruses da campioni ambientali Utilizzando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

Lo sviluppo di un anticorpo salivare Multiplex immunoenzimatico per misurare l&#39;esposizione umana ad agenti patogeni ambientali

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modello murino di Gruppo B<em&gt; Streptococcus</em&gt; Colonizzazione vaginale

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Una fluorescenza a base di linfociti Assay Adatto per high-throughput screening di piccole molecole

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Un metodo per testare l&#39;efficacia della lavata a mano per la rimozione di nuovi patogeni infettivi

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Saggi di elettrochemiluminescenza autoanticorpi anti-umano dell'isolotto

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una piattaforma di High-throughput per lo Screening della Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Infezioni da arbovirus come strumenti di Screening per l'identificazione di fattori virali immunomodulatori e Host antivirale

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...