Gli autori ringraziano Therwa Hamza, John E. Tidwell, Nina Clovis, e Suzanne Smith per l&#39;assistenza sperimentale e Suzanne Smith per la correzione. Gli autori hanno anche ringraziare John Thomas, PhD per fornire gli isolati batterici clinici e John B. Barnett, dottorato di ricerca per il suo sostegno e l&#39;uso del laboratorio di sicurezza biologica presso il Dipartimento di Microbiologia, Immunologia e Biologia Cellulare presso la West Virginia University. Gli autori riconoscono il sostegno finanziario della osteosintesi e Trauma Care Foundation e la National Science Foundation (# 1003907). Esperimenti di microscopia e analisi di immagine sono stati effettuati nel Fondo per la West Virginia University Imaging, che è supportato in parte dal Babb Mary Randolph Cancer Center e sovvenzione del NIH P20 RR016440. Uso degli animali per i prelievi di sangue sono stati approvati dalla cura degli animali Occidentale Virginia University e del Comitato Istituzionale uso. Tutti gli esperimenti sono stati eseguiti in conformità a tutte le Linee guida per l&#39;pertinenties, regolamenti, e agenzie di regolamentazione.

1. Preparazione e attivazione di PRP 1,1 Sangue pareggio <ol><li> Anestetizzare coniglio per inalazione di isoflurano (2% O 2 per l&#39;induzione e l&#39;1% per la manutenzione). </li><li> Disegnare 2 ml 0,129 M citrato trisodico (una soluzione anticoagulent) in una siringa da 20 ml. Il tri-sodio citrato soluzione viene preparata sciogliendo 1,897 g di citrato trisodico in 50 ml di H 2 O distillata e filtraggio con un filtro 0,22 micron sterile. </li><li> Sterilizzare l&#39;orecchio di coniglio con etanolo al 70%. </li><li> Prelevare il sangue (ad esempio 5 ml) dalla vena auricolare di coniglio attraverso un ago a farfalla (25 G) collegato alla siringa. </li><li> Mescolare il sangue con il tri-sodio citrato agitando con prudenza. Il rapporto in volume di sangue e tri-sodio citrato è 9:1. </li></ol> 1,2 PRP preparato (Figura 1) <ol><li> Trasferire il sangue con anticoagulante in un tubo da centrifuga da 50 ml in plastica. Prelevare un &#39;aliquota di 10 microlitrisangue per determinare la conta piastrinica basale, utilizzando hemocytometry. </li><li> Centrifugare il sangue a 300 xg per 10 min a temperatura ambiente (RT) in una centrifuga con rotore oscillante. Impostare la velocità di accelerazione e frenata a bassa (Figura 1). </li><li> Dopo centrifugazione, il sangue viene separato in tre strati. Lo strato inferiore è globuli rossi principalmente, lo strato intermedio (comunemente denominato &quot;buffy coat&quot;) è composta di piastrine e leucociti concentrati, e lo strato superiore è principalmente plasma, che è la componente liquida del sangue, e piastrine ( la figura 1). Cura il trasporto del tubo da centrifuga ad una cappa di coltura cellulare, non disturbare gli strati. Trasferire tutto il plasma, buffy coat, e 2-3 mm di spessore rosso strato di cellule del sangue in una provetta da 15 ml di plastica con una pipetta 1 ml in plastica. </li><li> Centrifugare il campione trasferita una seconda volta a 3000 xg per 15 minuti a RT. Lo strato superiore (surnatante) è considerato plasma povero di piastrine (PPP) und viene trasferito in una nuova provetta. </li><li> Ottenere PRP regolando la concentrazione piastrinica nel campione di sangue rimanente utilizzando PPP per ottenere 2,0 x 10 6 piastrine / microlitro (determinato dal hemocytometry). </li></ol> 1,3 PRP attivazione <ol><li> Preparare soluzione di attivazione PRP disciogliendo 5000 IU trombina bovina con 5 ml di cloruro di calcio al 10% alla concentrazione di lavoro di 1000 IU / ml. </li><li> Aggiungere la soluzione di attivazione di PRP e PPP, e mescolare la soluzione di pipettare ripetutamente per formare PRP e PPP-gel. Il rapporto in volume della soluzione di attivazione di PRP o PPP è 1:4. </li></ol> 2. In Test antimicrobica in vitro mediante saggio di PRP Curve Kill (Figura 2) <ol><li> Utilizzando un&#39;ansa sterile da inoculo, aggiungere numerose colonie di S. aureus dalla piastra di coltura durante la notte in 5 ml di brodo Mueller Hinton (MHB) in un tubo di plastica. Vortex brevemente e incubare il campione per 2 ore a 37 ° C. Prossimo, La densità ottica dei media batteriche è stata determinata utilizzando uno spettrofotometro e regolato ad una densità ottica pari a ~ 1 x 10 8 UFC / ml in base al predeterminato curva standard. </li><li> Effettuare una diluizione 100x utilizzando PBS per ottenere 1 x 10 6 UFC / ml e posizionare le inoculums su ghiaccio. </li><li> Impostare e etichettare sterili, monouso da 5 ml a fondo tondo provette di polistirene, e preparare i gruppi di esempio seguenti, come indicato nella Tabella 1 per un volume finale di 2 ml in ciascun tubo. </li><li> Aggiungere PRP, PPP, o PBS prima ai tubi di polistirene, seguita dalla soluzione di trombina per l&#39;attivazione (formazione di gel). Successivamente, aggiungere MHB e poi la S. inoculums aureus (1 x 10 6 CFU / ml) per ottenere la concentrazione finale di 1 x 10 5 CFU / ml. </li><li> Incubare le provette a 37 ° C con agitazione orbitale a 150 rpm. </li><li> A predeterminati intervalli di tempo (ad esempio 0, 1 e 2 ore), mescolare le soluzioni in ogni tubo attraverso pipettaggio ripetizione (questopasso è importante poiché i batteri possono essere intrappolati all&#39;interno del gel PRP). Prelevare 10 ml di campione, diluire serialmente con soluzione salina allo 0,9%, e pipettare un&#39;aliquota di 100 ml di ciascuna diluizione su un Tryptic Soy Agar (TSA, con sangue di montone al 5%), piastra per il conteggio delle CFU. </li><li> Cultura piastre di agar notte a 37 ° C, quindi contare e registrare le colonie della piastra. Traccia di dati in una scala logarithimic con il tempo (ora) sul asse x e CFU / ml l&#39;asse y. </li></ol>

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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Plasma ricco di piastrine è stato sempre più utilizzato per applicazioni cliniche grazie alle sue proprietà di favorire la guarigione-15-17. Nel presente studio, PRP è stato presentato come un nuovo approccio per la prevenzione delle infezioni. PRP è stato trovato per avere forti proprietà antimicrobiche contro MRSA, MSSA, streptococco di gruppo A, e Neisseria gonorrhoeae. I vantaggi principali di PRP, rispetto ai tradizionali trattamenti antibiotici, per la prevenzione delle infezioni...

Implant-associata infezione è una complicazione clinico significativo. Staphylococcus aureus (S. aureus) è uno dei più comuni microrganismi isolati da protesi infezioni associate. È in grado di produrre un biofilm che riveste le superfici degli impianti e può portare ad antibiotico-resistente 1,2 infezione. Trattamento delle infezioni correlate all&#39;assistenza dell&#39;impianto richiede spesso una lunga ospedalizzazione per debridements ripetuta e prolungata terapia antibiotica parenterale. In casi resistenti agli antibiotici, la rimozione dell&#39;impianto può essere necessario. La resistenza crescente dei batteri agli antibiotici è stato di cui dai Centers for Disease Control and Prevention (CDC) come &quot;uno dei problemi sanitari più urgenti del mondo.&quot; Nel tempo, senza lo sviluppo di nuovi trattamenti antimicrobici ed efficace, è possibile che agenti patogeni multiresistenti sarà non trattabile con antibiotici convenzionali. Prevenzione di impianto associatoinfezione è quindi importante e nuovi agenti profilattici o approcci sono necessari per prevenire tali infezioni. Plasma ricco di piastrine (PRP) è una concentrazione di sangue autologo che contiene più di 30 fattori di crescita che può aiutare con innesto osseo e la guarigione ossea 3-5. L&#39;applicazione di PRP per migliorare la rigenerazione ossea e maturazione dei tessuti molli è stato sempre riportato in clinica a causa della sua alta concentrazione di vari fattori di crescita rilasciati dalle piastrine. Molte caratteristiche di PRP PRP indicano che può anche avere proprietà antimicrobiche 6-9. PRP contiene un gran numero di piastrine, una elevata concentrazione di leucociti (che può possedere host-difesa azioni contro batteri e funghi), e più peptidi antimicrobici 7,8,10. In un recente studio di una coorte di pazienti chirurgici cardiaci, è stato rivelato che l&#39;uso intraoperatorio di PRP-gel durante la chiusura della ferita significativamente ridotto l&#39;incidenza di infezione dello sterno superficiale e profondo 11. Per queste ragioni e osservazioni, abbiamo ipotizzato che PRP, oltre alle sue ben studiate che promuovono la guarigione proprietà, ha proprietà antimicrobiche. I potenziali vantaggi di utilizzare PRP per prevenire l&#39;infezione possono includere: (i) PRP è meno probabile indurre resistenza rispetto ai tradizionali trattamenti antibiotici. (Ii) PRP ha anche proprietà che favoriscono la guarigione, che possono avere un effetto sinergico sulla prevenzione delle infezioni; PRP di guarigione che promuovono le proprietà potrebbe fornire una tenuta per prevenire la fissazione batterica riducendo così le probabilità di infezione da agenti patogeni e cellule umani fanno a gara per superfici implantari 12 , 13. (Iii) PRP è intrinsecamente biocompatibile e sicuro e libero dal rischio di malattie trasmissibili. Il nostro obiettivo a lungo termine è quello di utilizzare PRP come un nuovo approccio per prevenire impianto associato infections. Lo scopo di questo studio era di preparare PRP utilizzando un approccio centrifugazione due volte, per esaminare PRP in vitro proprietà antimicrobiche, e descrivere i protocolli per la valutazione di tali proprietà antimicrobiche.

<table border="1">
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 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
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 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
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 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
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 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
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 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
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</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

Impianto associata infezione sta diventando sempre più difficile per il settore sanitario in tutto il mondo a causa della crescente resistenza agli antibiotici, la trasmissione di batteri resistenti agli antibiotici tra gli animali e gli esseri umani, e gli elevati costi di trattamento delle infezioni. In questo studio, si fornisce una nuova strategia che può essere efficace nel prevenire infezioni associate impianto basato sulle proprietà antimicrobiche potenziali di plasma ricco di piastrine (PRP). Grazie alle sue ben studiate le proprietà per promuovere la guarigione, PRP (un prodotto biologico) è stato sempre più utilizzato per applicazioni cliniche compresa la chirurgia ortopedica, chirurgia parodontale e orale, interventi chirurgici maxillo-facciale, chirurgia plastica, medicina dello sport, ecc PRP potrebbe essere un&#39;alternativa avanzata ai tradizionali trattamenti antibiotici nella prevenzione impianto-infezioni associate. L&#39;uso di PRP può essere vantaggioso rispetto ai tradizionali trattamenti antibiotici SInce PRP è meno probabile di indurre resistenza agli antibiotici e PRP antimicrobica e di guarigione che promuovono le proprietà possono avere un effetto sinergico sulla prevenzione delle infezioni. È ben noto che gli agenti patogeni e cellule umane sono corse per superfici implantari, e PRP proprietà di promozione della guarigione potrebbe migliorare attaccamento cellulare umana riducendo le probabilità di infezione. Inoltre, PRP è intrinsecamente biocompatibile e sicuro e libero dal rischio di malattie trasmissibili. Per il nostro studio, abbiamo selezionato diversi ceppi clinici di batteri che si trovano comunemente nelle infezioni ortopediche e valutato se PRP ha proprietà antimicrobiche in vitro nei confronti di questi batteri. Abbiamo preparato PRP utilizzando un approccio doppio centrifugazione che permette la stessa concentrazione piastrinica da ottenere per tutti i campioni. Abbiamo raggiunto risultati consistenti antimicrobici e ha scoperto che ha una forte PRP nelle proprietà antimicrobiche in vitro contro i batteri come methicillin-sensibili e meticillino-resistente Staphylococcus aureus, Streptococcus gruppo A, e Neisseria gonorrhoeae. Pertanto, l&#39;uso di PRP può avere il potenziale per prevenire l&#39;infezione e ridurre la necessità di costosi trattamento post-operatorio di impianto infezioni associate.

PRP è riproducibile preparato utilizzando un approccio di centrifugazione due volte (Figura 1). PRP è trovato presentare una forte (fino a 100 volte riduzione CFU) in vitro proprietà antimicrobiche contro resistente alla meticillina S. aureus (MRSA) (Figura 3), che si trova comunemente negli ospedali in tutto il mondo 14. Allo stesso modo, PRP ha forti proprietà antimicrobiche contro sensibili alla meticillina S. aureus (MSSA), streptococco ...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Impianto infezioni correlate all&#39;assistenza è una complicazione clinico significativo. Questo studio descrive un approccio con plasma ricco di piastrine (PRP) per prevenire infezioni associate all&#39;impianto, presenta il protocollo per la preparazione di PRP con costante concentrazione piastrinica, e riporta le proprietà antimicrobiche recentemente identificate di PRP e protocolli correlati per esaminare tali proprietà antimicrobiche<em&gt; In vitro.</em

PRP come un nuovo approccio per prevenire l&#39;infezione: Preparazione e<em&gt; In vitro</em&gt; Proprietà antimicrobiche di PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K Views.  West Virginia University. Impianto infezioni correlate all'assistenza è una complicazione clinico significativo. Questo studio descrive un approccio con plasma ricco di piastrine (PRP) per prevenire infezioni associate all'impianto, presenta il protocollo per la preparazione di PRP con costante concentrazione piastrinica, e riporta le proprietà antimicrobiche recentemente identificate di PRP e protocolli correlati per esaminare tali proprietà antimicrobiche In vitro.

Video: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Semplice e robusta In vivo E In vitro</emApproccio> per lo Studio Assemblea Virus

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Uso di<em&gt; Galleria mellonella</em&gt; Come organismo modello per studiare<em&gt; Legionella pneumophila</em&gt; Infezione

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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

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

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

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

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

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

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un approccio semplice ed efficiente per costruire Mutant vaccinia vettori di virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Trasduzione retrovirale di cellule T helper come un approccio genetico per studiare meccanismi che controllano la loro differenziazione e funzione

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...