Vorrei ringraziare CL Richards-Zawacki aiuto concettualizzare il disegno sperimentale e l&#39;assistenza con materiali, A. Pessier aiuto che istituisce un regime di trattamento e di necroscopia analisi, MWH Chatfield, MJ Robak, K. Tasker e D. Lenger aiuto per la laboratorio e di supporto e M. McFadden e P. Harlow allo zoo di Taronga per l&#39;utilizzo del loro spazio durante le riprese. Il progetto è stato parzialmente finanziato dalla Louisiana Department of Wildlife and Fisheries attraverso la concessione Louisiana Environmental Education Commissione assegnato a Los Angeles Brannelly e Life Technologies.

Etica di omologazione: Questo studio e dei suoi metodi sono stati approvati da Usa e cura degli animali commissione di Tulane University (prot 0407.). 1. Test per Bd infezione <ol><li> Tamponamento per Bd <ol><li> Animali tampone con un tampone di cotone sterile cinque volte sul ventre, cinque volte su ogni coscia, ogni lato e ogni arto per un totale di 45 colpi. Swabbing è un metodo non invasivo di campionamento per Bd sulla pelle degli anfibi. </li><li> Ruotare delicatamente il tampone durante e tra i tratti di assicurare la maggiore quantità di DNA viene raccolto sul tampone. </li><li> Conservare tamponi in 1,5 ml microtubi a -20 ° C. </li></ol></li><li> Estrazione del DNA e analisi qPCR <ol><li> Estrarre DNA genomico dai tamponi utilizzando un kit di isolamento di DNA disponibile in commercio, in conformità con le istruzioni per tessuti animali. </li><li> Analizzare il DNA estratto utilizzando quantitativa in tempo reale PCR (qPCR) a seguito di Boyle <em&gt; et al 17, con le seguenti modifiche.: </li><li> Diluire i campioni di estrazione del DNA 01:10 con doppio acqua deionizzata. </li><li> Aggiungere 0,7 ml di sieroalbumina bovina (BSA) per impedire l&#39;inibizione PCR. </li><li> Includere un controllo interno positivo in ogni campione per garantire l&#39;inibizione della PCR non interessano i risultati. </li><li> Campioni familiare con un controllo positivo e negativo e una serie di norme di diluizione per stimare zoospore (infezione) di carico. </li></ol></li></ol> 2. Inoculazione <ol><li> Cultura Bd <ol><li> Preparare triptone brodo aggiungendo 10 g di triptone a 1 L osmosi inversa o acqua deionizzata, in autoclave (121 ° C per 40 min) e lasciato raffreddare. Una volta raffreddata a 23 ° C, aggiungere 1 ml di penicillina-streptomicina. </li><li> Grow ceppo Bd (JEL411 da J. Longcore, isolata dal Guabal, Panama da un Phyllomedusa lemure) in un brodo triptone per sette giorni a 23 ° C. </li></ol></li><li> Trasferire il broth cultura Triptone e piastre di agar. <ol><li> Per rendere piastre, si aggiungono 10 g di triptone e 10 g di agar per 1 L osmosi inversa o acqua deionizzata e autoclave (121 ° C per 40 min). </li><li> Una volta che l&#39;impasto autoclavato è abbastanza fresco al tatto, ma prima che si rapprende, versare in piastre di Petri, 1/4-1/3 pieno, in una cappa a flusso laminare. </li><li> Quando agar ha congelato e raffreddato completamente, aggiungere 0,5 ml Bd brodo per ogni piastra e diffondere uniformemente attorno alla piastra. Lasciare asciugare. </li><li> Una volta che le piastre vengono inoculate con zoospore Bd, piastre di tenuta con parafilm. </li><li> Lasciare le piastre a incubare a 23 ° C per 5-7 giorni. Controllare zoospore movimento per garantire la vitalità prima dell&#39;inoculazione. </li></ol></li><li> Piastre di inondazione per la soluzione di inoculazione zoospore <ol><li> Dopo i 5-7 giorni di Bd incubazione e di crescita, inondare ogni piatto con 5 ml di acqua di rubinetto invecchiata e permettono zoospore di entrare media per 10 min. </li><li> Versare il Sosp zoosporension in un contenitore portatile. </li><li> Stimare la concentrazione zoospore con un emocitometro. </li><li> Diluire miscela ad una concentrazione di 1 x 10 6 per 3 ml con acqua del rubinetto invecchiata. </li></ol></li><li> Seminare animali <ol><li> In singoli 50 ml contenitori, inoculare ogni animale versando 3 miscela inoculo ml sul suo ventre. Lasciare inoculo supplementare gocciolare nella base del contenitore inoculazione. </li><li> Ogni animale rimarrà in contenitore inoculazione per 24 ore per garantire infezione. </li><li> Dopo 24 ore di inoculazione, ritorno animali ai singoli terrari disinfettati. </li><li> Lasciare l&#39;infezione di costruire per due settimane. Confermare l&#39;infezione con tampone e risultati qPCR, e iniziare il trattamento. </li><li> Per i controlli Bd-negativi, alluvioni Bd-libero piastre di agar con 5 ml di età compresa tra acqua di rubinetto, e inoculare finto gli animali con le stesse modalità di cui sopra. </li></ol></li></ol> 3. Regime di trattamento <ol><li> Prepare bagno di trattamento <ol><li> Aggiungere 2,50 ml di itraconazolo acquosa (1% Sporanox soluzione orale) per 1 Soluzione L Anfibio Ringer (0,0025% itraconazolo - la concentrazione trattamento efficace più bassa). <ol><li> Anfibio di Ringer Soluzione ricetta: 1 L osmosi inversa o acqua deionizzata, cloruro 6,6 g di sodio, 0,15 g di cloruro di potassio, 0,15 g di cloruro di calcio, e 0,2 g di bicarbonato di sodio. Soluzione autoclave (121 ° C per 40 min) e lasciarla raffreddare fino a temperatura ambiente. </li><li> Per la concentrazione trattamento itraconazolo 0,01%, aggiungere 10,00 ml di itraconazolo acquosa (1% Sporanox soluzione orale) di Soluzione 1 L Anfibio Ringer </li><li> Per il 0,005% di concentrazione trattamento itraconazolo, aggiungere 5,00 ml di itraconazolo acquosa (1% Sporanox soluzione orale) di Soluzione 1 L Anfibio di Ringer. </li></ol></li><li> Per i controlli Bd-positivi e Bd-negativi, fare il bagno animali in soluzione di Ringer Anfibio senza itraconazolo. </li></ol></li><li> Trattamento &lt;ol&gt; <li> Mettere singoli anfibi in disinfettati 50 ml contenitori con 35 ml di soluzione di trattamento. </li><li> Assicurarsi che gli animali sono completamente coperti per cinque minuti agitando delicatamente il contenitore. </li><li> Animali Ritorno a terrari disinfettati. Ripetere l&#39;operazione per sei giorni. </li></ol></li><li> Assicurare il successo del trattamento <ol><li> Animali di laboratorio per l&#39;infezione Bd sul trattamento giorno comincia. </li><li> Animali di laboratorio per l&#39;infezione Bd una settimana dopo finiture di trattamento, e una volta a settimana per quattro settimane per assicurare scomparsa dell&#39;infezione. </li></ol></li></ol> 4. Bd come Biohazard <ol><li> Disinfettare individuale terrari settimanale. <ol><li> Fare il bagno terrari e trattamento contenitori in un bagno di candeggina soluzione al 10%. </li><li> Risciacquare ogni contenitore sbiancato due volte con acqua corrente. </li><li> Lasciare contenitori asciugare per 24 ore prima di riutilizzarli. </li></ol></li><li> Indossare guanti di nitrile per maneggiare gli animali per evitare la contaminazione incrociatadi Bd patogeno. Cambiare i guanti tra gestione di ogni animale. </li><li> Disinfettare tutti i rifiuti liquidi prima dello smaltimento portando la soluzione al 10% di candeggina. </li><li> Autoclave tutti i rifiuti solidi che è potenzialmente esposto a Bd prima dello smaltimento. </li></ol>

<ol>
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L&#39;autore dichiara interessi finanziari concorrenti.

Usando questo protocollo siamo stati in grado di ridurre in modo sicuro ed efficace la dose di trattamento e il tempo di trattamento itraconazolo per Bd in anfibi. La dose di trattamento più basso e il tempo (0,0025% per sei giorni) è stato un trattamento efficace per I. subclinicamente infetti nebulifer. L&#39;efficacia di entrambi una dose più bassa e il tempo di trattamento dimostrare la necessità di studi clinici di trattamento per i trattamenti di infezione Bd, in quanto attu...

Gli anfibi sono attualmente vivendo la più grande diminuzione della biodiversità di tutti i taxa vertebrati 1. Chitridiomicosi, una malattia della pelle causata dai chytrid fungine dendrobatidis patogeno Batrachochytrium (BD), è una delle principali cause di questi cali drammatici 2 Bd è l&#39;agente patogeno peggior record per causare la perdita di biodiversità:. Che infetta oltre 600 specie di anfibi e ha causato cali oltre 300 specie, di cui ben 200 specie sono diventate in pericolo di estinzione o estinti 2,3. Il chytrid fungo anfibio non è ospite specifico e non è chiaro quali sono le qualità di accoglienza permettono di infezione Bd progredire causando gravi malattie. Chitridiomicosi provoca la morte interrompendo canali ionici nella pelle, con conseguente perdita di elettroliti e insufficienza cardiaca 4. Inoltre, Bd surnatante (contenente nessun zoospore BD) provoca una risposta immunitaria in girini 5 leading ricercatori a credere Bd produce un sottoprodotto tossico 6. Poiché molte specie sono in rapido declino, le colonie di garanzia e programmi di allevamento in cattività sono importanti misure di conservazione, e possono essere l&#39;unica ancora di salvezza per alcune specie. Stabilire e mantenere la malattia colonie liberi è una sfida per le strutture di gestione anfibi. Gli attuali metodi chemioterapici per il trattamento di infezioni Bd, anche se efficace, possono essere nocivi per gli animali in trattamento, e nessun metodo singolo trattamento ha avuto successo tra le specie e le classi di età di anfibi 7. Pochi studi clinici di trattamenti per l&#39;infezione Bd sono stati effettuati 7. I tre metodi più comunemente usati di trattamento sono la terapia del calore, cloramfenicolo e itraconazolo. Terapia del calore è un&#39;opzione di trattamento nonchemotherapeutic che sembra avere pochi effetti collaterali, possono essere utilizzati in più fasi della vita, ed è generalleato efficace. In uno studio di trattamento, Chatfield e Richards-Zawacki 8 hanno trovato che il 96% degli animali trattati aveva eliminato le infezioni dopo essere stato ospitato a 30 ° C per 10 giorni. Anche se questo studio non era al 100% di successo, fornisce una base per gli altri a valutare la terapia del calore per loro stessa specie. Terapia del calore, anche se efficace per molte specie, è impraticabile per molte specie alpina che non possono rimanere a temperature elevate per un periodo di tempo prolungato. Cloramfenicolo, un antibatterico, è stato usato con successo per trattare Bd in molte specie, e inibisce la crescita di funghi in vitro. Come antibatterico, il cloramfenicolo non attacca il fungo specificamente ma è stato conosciuto per causare apoptosi in cellule eucariotiche 9. È stato ipotizzato che la terapia cloramfenicolo provoca apoptosi nella pelle, uccidendo sporangi intracellulare, sebbene l&#39;esatto meccanismo di azione non è noto. Anche se nessuno studio clinico è stato condotto, e quindidose e la durata del trattamento non sono stati valutati i protocolli di trattamento correnti in genere richiedono l&#39;immersione per due a quattro settimane 10,11, rendendo questo trattamento impraticabile per gli anfibi terrestri. Cloramfenicolo è stato limitato in alcuni paesi per uso veterinario perché l&#39;esposizione umana è stata associata con una rara forma di anemia aplastica 12. Itraconazolo, un antimicotico azolici, è il più utilizzato per il trattamento di infezioni Bd in anfibi in cattività. Il protocollo più usato è animali balneazione in una soluzione itraconazolo 0,01% per cinque minuti al giorno per 11 giorni consecutivi. Questo protocollo è stato derivato dagli standard utilizzati per la somministrazione dei mammiferi, e, pur efficace per gli anfibi, non si basa su studi clinici in anfibi 11,13. Questo protocollo può causare effetti collaterali negativi in alcune specie, soprattutto nel genere Rana e in girini e recenti Metamorphs 11.Trattamento Tadpole ha avuto successo con una concentrazione molto più bassa (0,0005%) di itraconazolo, ma è stato associato alla depigmentazione 14. Questa stessa concentrazione è stata trialed sui recenti Metamorphs e trovato ad essere riuscita a trattare l&#39;infezione 15. Perché non vi è rischio di effetti collaterali negativi del trattamento con itraconazolo, alcuni praticanti hanno usato una metà la solita concentrazione (0,005%) per 11 giorni consecutivi e abbiamo trovato questo per avere successo nella cura delle infezioni Bd 16. Il presente studio è la sintesi di una sperimentazione clinica di trattamento con itraconazolo per trattare l&#39;infezione Bd 15. Il protocollo descrive un tentativo di trovare la dose e il tempo di trattamento più basso che sia sicuro ed efficace per il trattamento Bd animali infetti con itraconazolo. Animali utilizzati in questo studio sono stati recenti Metamorphs di rospi Gulf Coast, Incilius nebulifer.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Anfibi stanno vivendo il più grande declino di qualsiasi classe vertebrati e una delle principali cause di questi cali è un patogeno fungino, Batrachochytrium dendrobatidis (Bd), che causa la malattia chytridiomycosis. Colonie di assicurazione in cattività sono importanti a livello mondiale per minacciate specie di anfibi e possono essere l&#39;unica ancora di salvezza per chi minaccia di critico di estinzione. Mantenere malattia colonie libere è una delle priorità di manager in cattività, non sono stati identificati ancora trattamenti sicuri ed efficaci per tutte le specie e in tutta le fasi della vita. Il trattamento chemioterapico più utilizzato è itraconazolo, sebbene il dosaggio comunemente utilizzato può essere dannoso per alcuni individui e specie. Abbiamo eseguito uno studio di trattamento clinico per valutare se una dose più bassa e più sicuro, ma efficace di itraconazolo potrebbe essere trovato per curare le infezioni Bd. Abbiamo trovato che riducendo la concentrazione trattamento ,01-,0025% e ridurre la durata del trattamento da 11Giorni -6 di 5 min bagni, rane potevano essere curate di infezione Bd con meno effetti collaterali e meno la mortalità associata al trattamento.

Trattamento Itraconazolo è stato considerato di successo se tutti gli animali fossero Bd-negativi quattro settimane dopo la fine del trattamento. Prima del trattamento iniziato, due settimane dopo l&#39;inoculazione, tutti gli animali Bd-inoculati sono stati infettati, con un carico zoospore media di 1,121.7 (± 17,34), mentre quattro settimane dopo la fine del trattamento, gli animali di controllo Bd-positivi avevano carico una media zoospore di 16,765.12 ( ± 14643,08). <p class="jove_c...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Batrachochytrium dendrobatidis infezione anfibi può essere eliminato con una concentrazione ridotta (0,0025%) e di breve durata (bagni cinque minuti per sei giorni) di itraconazolo che viene generalmente utilizzato. Meno mortalità e meno effetti collaterali negativi sono stati osservati con questo protocollo di trattamento modificata.

Ridotta concentrazione Itraconazolo e Durata hanno successo nel trattamento<em&gt; Batrachochytrium dendrobatidis</em&gt; Infezione in Anfibi

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K Views.  James Cook University.  Batrachochytrium dendrobatidis infezione anfibi può essere eliminato con una concentrazione ridotta (0,0025%) e di breve durata (bagni cinque minuti per sei giorni) di itraconazolo che viene generalmente utilizzato. Meno mortalità e meno effetti collaterali negativi sono stati osservati con questo protocollo di trattamento modificata. 

Video: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Ricerca

Immunologia e infezioni

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso di un terreno dell&#39;espettorato artificiale per testare l&#39;efficacia degli antibiotici contro<em&gt; Pseudomonas aeruginosa</em&gt; In condizioni più rilevanti al polmone Fibrosi Cistica

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Coltura Microglia dal Sistema Nervoso Centrale neonatale e adulti

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Totale Estrazione di proteine ​​e 2-D elettroforesi su gel Metodi per<em&gt; Burkholderia</em&gt; Specie

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Analisi degli analoghi dell'acido iofenostico nella piccola mangusta indiana (Herpestes Auropunctatus) Sera per l'uso come marcatore biologico di vaccinazione della rabbia orale

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infezione ricorrente del tratto urinario da Escherichia coli innescata dall'esposizione alla vescica di Gardnerella vaginalis nei topi

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Separazione delle sottopopolazioni di cellule immunitarie nei campioni di sangue periferico da bambini con mononucleosi infettiva

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

Standardizzazione del trasferimento tra laboratori tra citometri a flusso per il rilevamento di linfociti in bambini vaccinati contro l'encefalite giapponese

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...