Die Autoren danken Therwa Hamza, John E. Tidwell, Nina Clovis und Suzanne Smith für experimentelle Unterstützung und Suzanne Smith für das Korrekturlesen. Die Autoren danken John Thomas, PhD für die Bereitstellung der bakteriellen klinischen Isolaten und John B. Barnett, PhD für seine Unterstützung und die Nutzung der biologischen Sicherheit Labor an der Abteilung für Mikrobiologie, Immunologie und Zellbiologie an der West Virginia University. Die Autoren danken für die finanzielle Unterstützung durch die Osteosynthese und Trauma Care Foundation und der National Science Foundation (# 1003907). Microscope Experimente und Bildanalyse wurden auch in der West Virginia University Imaging Facility, die zum Teil von der Mary Babb Randolph Cancer Center und NIH P20 RR016440 unterstützt wird durchgeführt. Verwendung von Tieren für die Blutabnahmen wurden von der West Virginia University Institutional Animal Care und Use Committee genehmigt. Alle Experimente wurden in Übereinstimmung mit allen relevanten guidelin hingerichtetes, Vorschriften und Regulierungsbehörden.

Ein. Vorbereitung und Aktivierung von PRP 1,1 Blutentnahme <ol><li> Anesthetize Kaninchen durch Inhalation von Isofluran (2% in O 2 für die Induktion und 1% für Wartung). </li><li> Ziehe 2 ml 0,129 M Tri-Natriumcitrat (ein anticoagulent Lösung) in einen 20-ml-Spritze. Die Tri-Natriumcitrat-Lösung wird durch Auflösen von 1,897 g Trinatriumcitrat in 50 ml destilliertem H 2 O und eine Filterung mit einer 0,22 um Sterilfilter hergestellt. </li><li> Sterilisieren rabbit ear mit 70% Ethanol. </li><li> Ziehen Blut (zB 5 ml) aus dem Kaninchen Ohrvene über einen Butterfly-Nadel (25 G), die mit der Spritze. </li><li> Mischen Sie das Blut mit dem tri-Natriumcitrat-Lösung durch vorsichtiges Schütteln. Das Volumenverhältnis von Blut und Tri-Natriumcitrat-Lösung ist 9:1. </li></ol> 1,2 PRP Zubereitung (Abbildung 1) <ol><li> Übertragen der antikoaguliertem Blut zu einer 50-ml-Kunststoffröhrchen Zentrifugenröhrchen. Nehmen Sie ein Aliquot von 10 ulBlut in den Thrombozytenausgangswert mit hemocytometry bestimmen. </li><li> Zentrifuge das Blut bei 300 xg für 10 min bei Raumtemperatur (RT) in einer Zentrifuge mit einem Ausschwingrotor. Stellen Sie die Beschleunigung und Bremse Geschwindigkeit zu niedrig ist (Abbildung 1). </li><li> Nach Zentrifugation wird das Blut in drei Schichten getrennt. Die untere Schicht ist hauptsächlich roten Blutkörperchen wird die mittlere Schicht (gemeinhin als &quot;Leukozytenmanschette&quot; bezeichnet) von konzentrierten Blutplättchen und Leukozyten besteht und die oberste Schicht ist hauptsächlich Plasma, welches die flüssige Komponente des Blutes ist, und Plättchen ( Abbildung 1). Vorsichtig transportieren Zentrifugenröhrchen zu einer Zellkultur Kapuze; nicht stören die Schichten. Übertragen das Plasma, Buffy-Coat und 2-3 mm dicke Schicht roter Blutkörperchen in einem 15 ml Plastikröhrchen mit einer 1 ml Kunststoff-Pipette. </li><li> Zentrifuge die übertragenen Probe ein zweites Mal bei 3000 xg für 15 min bei RT. Die obere Schicht (Überstand) wird als plättchenarmes Plasma (PPP) eind wird in ein neues Röhrchen überführt. </li><li> PRP erhalten durch Einstellen der Blutplättchenkonzentration in dem verbleibenden Blutprobe mittels PPP bis 2,0 x 10 6 Blutplättchen / ul (bestimmt durch hemocytometry) zu erhalten. </li></ol> 1,3 PRP-Aktivierung <ol><li> Planen PRP Aktivierung durch Auflösen 5.000 IU bovines Thrombin mit 5 ml 10% Calciumchlorid der Arbeitskonzentration von 1.000 IU / ml. </li><li> Fügen Sie die Aktivierung Lösung PRP und PPP, und mischen Sie die Lösung durch wiederholtes Pipettieren zu PRP und PPP-Gele bilden. Das Volumenverhältnis der Aktivierungslösung zum PRP oder PPP ist 1:4. </li></ol> 2. In vitro Antimicrobial Test der PRP mit kill Curve Assay (Abbildung 2) <ol><li> Mit einer sterilen Impföse, fügen Sie mehrere Kolonien von S. aureus von seinem Plattenkultur über Nacht in 5 ml Mueller-Hinton-Bouillon (MHB) in einem Kunststoffrohr. Vortex kurz und dann inkubieren Sie die Probe für 2 Stunden bei 37 ° C. NächsteDie optische Dichte der Bakterien-Medium wurde mit einem Spektralphotometer und auf eine optische Dichte von ~ 1 × 10 8 CFU / ml auf der vorbestimmten Standardkurve berechnet. </li><li> Machen Sie eine 100x Verdünnung mit PBS auf 1 x 10 6 CFU / ml zu erhalten und legen Sie die Inokulums auf Eis. </li><li> Einrichten und beschriften sterilen Einweg 5 ml Rundkolben Polystyrolröhrchen, und bereiten Sie die folgenden Beispieldaten Gruppen, wie in Tabelle 1 für ein Endvolumen von 2 ml in jedem Röhrchen angegeben. </li><li> Hinzufügen PRP, PPP, oder PBS ersten bis Polystyrolröhrchen, durch den Thrombin-Lösung zur Aktivierung (Gelbildung) gefolgt. Als Nächstes fügen MHB und dann die S. aureus Impfkulturen (1 x 10 6 CFU / ml), um die Endkonzentration von 1 × 10 5 CFU / ml. </li><li> Die Röhrchen bei 37 ° C unter kreisförmigen Schütteln bei 150 Upm. </li><li> Zu vorbestimmten Zeitpunkten (zB 0, 1, und 2 h), Mischen der Lösungen in jedem Rohr über Wiederholung Pipettieren (dieserSchritt ist wichtig, da Bakterien in dem PRP-Gel eingeschlossen werden kann). Nehmen Sie sich 10 ul Probe verdünnen seriell mit steriler 0,9% iger Kochsalzlösung und pipettieren 100 ul Aliquot jeder Verdünnung auf eine CASO-Agar (TSA mit 5% Schafblut) Platte für CFU Zählen. </li><li> Kultur die Agarplatten über Nacht bei 37 ° C, dann zählen und notieren Sie die Platte Kolonien. Plotdaten auf einer Skala mit logarithimic Zeit (h) auf der x-Achse und CFU / ml auf der y-Achse. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Platelet-Rich Plasma wurde zunehmend für klinische Anwendungen aufgrund seiner Heilung fördernden Eigenschaften 15-17 verwendet. In der vorliegenden Studie wurde PRP als neuer Ansatz für die Infektionsprävention vorgestellt. PRP wurde festgestellt, dass starke antimikrobielle Eigenschaften gegen MRSA, MSSA, Gruppe A Streptococcus und Neisseria gonorrhoeae haben. Die wichtigsten Vorteile von PRP im Vergleich zu konventionellen Antibiotika, für Infektionsprävention gehören: (1) Aktuelle...

Implantat-assoziierten Infektion eine signifikante klinische Komplikation. Staphylococcus aureus (S. aureus) ist eine der häufigsten Mikroorganismen aus Implantat-assoziierten Infektionen isoliert. Es ist geeignet zur Herstellung einer Biofilm, die die Oberflächen von Implantaten abdeckt und kann zu antibiotikaresistenten Infektion 1,2 führen. Behandlung von Implantat-assoziierten Infektionen erfordert häufig langfristige Krankenhausaufenthalte für wiederholte Debridements und verlängerte parenterale antibiotische Therapie. In antibiotikaresistente Fällen kann die Entfernung des Implantats erforderlich sein. Die steigende Resistenz von Bakterien gegen Antibiotika hat auch wurde von den Centers for Disease Control and Prevention (CDC) als &quot;eines der weltweit drängendsten Gesundheitsprobleme.&quot; In der Zeit, ohne die Entwicklung von neuen und wirksame antimikrobielle Behandlungen, ist es möglich, dass multiresistenten Krankheitserreger werden mit herkömmlichen Antibiotika behandelbar. Prävention von Implantat-assoziiertenInfektion ist daher wichtig und neuartige prophylaktische Mittel oder Ansätze zur Verhinderung solcher Infektionen benötigt. Platelet-Rich Plasma (PRP) ist eine Konzentration von Eigenblut, die über 30 Wachstumsfaktoren, die mit Knochen und Knochenersatzmaterialien Heilung 3-5 helfen kann enthält. Die Anwendung des PRP, um Knochenregeneration und Weichgewebe Reifung Verbesserung wurde zunehmend in Kliniken wegen seiner hohen Konzentration verschiedener Wachstumsfaktoren durch Plättchen freigesetzt gemeldet. Mehrere Merkmale des PRP zeigen, dass PRP kann auch antimikrobielle Eigenschaften 9.6. PRP enthält eine Vielzahl von Plättchen, die eine hohe Konzentration von Leukozyten (der Host-Abwehrmaßnahmen gegen Bakterien und Pilze besitzen) und mehrere antimikrobielle Peptide 7,8,10. In einer aktuellen Studie von einer großen Kohorte von herzchirurgischen Patienten zeigte sich, dass die intraoperative Anwendung von PRP-Gel während Wundverschluss significantly verringert das Auftreten von oberflächlichen und tiefen Brustbein Infektion 11. Aus diesen Gründen und Beobachtungen stellten wir die Hypothese, dass PRP, neben seiner gut untersuchten die Heilung fördernden Eigenschaften hat antimikrobielle Eigenschaften. Die potentiellen Vorteile der Verwendung von PRP eine Infektion verhindern können gehören: (i) PRP ist weniger wahrscheinlich, Resistenz zu induzieren im Vergleich zu herkömmlichen Antibiotika. (Ii) PRP hat auch Eigenschaften, Heilung, die eine synergistische Wirkung auf Infektionsprävention aufweisen kann fördern; PRP die Heilung fördernden Eigenschaften könnten eine Dichtung zu schaffen, um bakterielle Anbringung verhindern wodurch die Chancen für Infektion als Krankheitserreger und menschlichen Zellen für Implantatoberflächen 12 sind Rennen , 13. (Iii) PRP ist inhärent biokompatibel und sicher und frei von der Gefahr von übertragbaren Krankheiten. Unser langfristiges Ziel ist es, PRP als neuer Ansatz verwenden, um Implantat-assoziierten infecti verhindernons. Das Ziel dieser Studie war es, PRP Vorbereitung mit einem doppelten Zentrifugation Ansatz, PRP in vitro antimikrobielle Eigenschaften zu untersuchen und die Protokolle zur Auswertung wie antimikrobielle Eigenschaften zu beschreiben.

<table border="1">
 <tbody>
 <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>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <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>
 </tr>
 </tbody>
</table>

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

Implantat-assoziierte Infektion wird immer schwieriger, die Healthcare-Industrie weltweit durch die zunehmende Antibiotika-Resistenz, die Übertragung von Antibiotika-resistenten Bakterien zwischen Tieren und Menschen, und die hohen Kosten der Behandlung von Infektionen. In dieser Studie offenbaren wir eine neue Strategie, die wirksam bei der Verhinderung Implantat-assoziierten Infektion basierend auf den potentiellen antimikrobiellen Eigenschaften von plättchenreichem Plasma (PRP) kann. Aufgrund seiner gut untersuchten Eigenschaften zur Förderung der Heilung hat PRP (ein biologisches Produkt) zunehmend für klinische Anwendungen einschließlich der orthopädischen Operationen, parodontalen und oralen Chirurgie, Kiefer-Operationen, Schönheitsoperationen, Sportmedizin, etc. verwendet PRP könnte eine erweiterte Alternative zu herkömmlichen Antibiotika zu verhindern Implantat-assoziierten Infektionen. Der Einsatz von PRP kann vorteilhaft sein, im Vergleich zu herkömmlichen Antibiotika since PRP ist weniger wahrscheinlich zu Antibiotikaresistenz induzieren und PRP Die antimikrobielle und Heilung fördernden Eigenschaften können einen synergistischen Effekt auf die Infektionsprävention haben. Es ist wohlbekannt, daß Erreger und menschliche Zellen werden für Implantatoberflächen Renn, und PRP die Eigenschaften der Förderung der Heilung könnte menschlichen Zellanheftung zu verbessern wodurch die Chancen für Infektion. Darüber hinaus ist PRP inhärent biokompatibel und sicher und frei von der Gefahr von übertragbaren Krankheiten. Für unsere Studie haben wir verschiedene klinische Bakterienstämme, die üblicherweise in der orthopädischen Infektionen gefunden und untersucht, ob PRP in vitro antimikrobielle Eigenschaften gegen diese Bakterien ausgewählt hat. Wir haben PRP hergestellt unter Verwendung eines doppelten Zentrifugation Ansatz, der die gleiche Thrombozytenkonzentration für alle Proben erhalten werden können. Wir haben konsequente antimikrobielle Ergebnisse erreicht und festgestellt, dass PRP starke weist in vitro eine antimikrobielle Eigenschaften gegen Bakterien wie methicillin-sensitiv und Methicillin-resistenten Staphylococcus aureus, Streptococcus der Gruppe A, und Neisseria gonorrhoeae. Daher kann der Einsatz von PRP haben das Potenzial, Infektion zu verhindern und die Notwendigkeit für kostspielige Nachbehandlung von Implantat-assoziierten Infektionen zu reduzieren.

PRP wird reproduzierbar hergestellt unter Verwendung einer doppelten Zentrifugation Ansatz (Abbildung 1). PRP wird festgestellt präsentieren starke (bis zu 100-fache Verringerung der KBE) in vitro antimikrobielle Eigenschaften gegen Methicillin resistenten S. aureus (MRSA) (Abbildung 3), die üblicherweise in Krankenhäusern weltweit 14 gefunden. Ebenso hat PRP starke antimikrobielle Eigenschaften gegen Methicillin sensiblen S. aureus (MSSA), Grupp...

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

Implantat-assoziierten Infektion ist eine signifikante klinische Komplikation. Diese Studie beschreibt einen Ansatz mit plättchenreichem Plasma (PRP) zu implantieren-assoziierten Infektionen zu verhindern, stellt das Protokoll zur Herstellung PRP mit konstanter Blutplättchenkonzentration, und meldet die neu identifizierten antimikrobiellen Eigenschaften von PRP und verwandten Protokollen für die Prüfung wie antimikrobielle Eigenschaften<em&gt; In vitro.</em

PRP als neuer Ansatz, um eine Infektion zu verhindern: Vorbereitung und<em&gt; In-vitro-</em&gt; Antimikrobielle Eigenschaften von PRP

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

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Research

JoVE Journal

Immunology and Infection

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

21.2K Aufrufe.  West Virginia University. Implantat-assoziierten Infektion ist eine signifikante klinische Komplikation. Diese Studie beschreibt einen Ansatz mit plättchenreichem Plasma (PRP) zu implantieren-assoziierten Infektionen zu verhindern, stellt das Protokoll zur Herstellung PRP mit konstanter Blutplättchenkonzentration, und meldet die neu identifizierten antimikrobiellen Eigenschaften von PRP und verwandten Protokollen für die Prüfung wie antimikrobielle Eigenschaften In vitro.

Serie: 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

Retrovirale Transduktion von T-Zell-Rezeptoren in Maus-T-Zellen

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 ...

Forschung

Immunologie und Infektion

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.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

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.

Einfach und robust In-vivo- Und In-vitro- Ansatz für das Studium Virusassemblierung

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.

Einsatz von künstlichen Sputum Mittlere bis antibiotische Wirkung gegen die getestet<em&gt; Pseudomonas aeruginosa</em&gt; AGB in einen engeren Bezug zu den Mukoviszidose-Lunge

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 Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Ein<em&gt; In-vitro-</em&gt; Modell zur Heterogenität des menschlichen Makrophagen Differenzierung und Polarisierung Studieren

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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.

Die Verwendung von<em&gt; Galleria mellon</em&gt; Als Modellorganismus zum Studium<em&gt; Legionella pneumophila</em&gt; Infektion

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.

Überwachen von Änderungen in Membran-Polarity, Membrane Integrity und intrazelluläre Ionenkonzentrationen in<em&gt; Streptococcus pneumoniae</em&gt; Verwenden von Fluoreszenzfarbstoffen

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

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.

Eine einfache und effiziente Methode zu konstruieren Mutant Vacciniavirusvektoren

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.

Retrovirale Transduktion von T-Helferzellen als genetischer Ansatz zur Untersuchung Mechanismen der Kontrolle ihrer Differenzierung und Funktion

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