Gli autori sono grati alle comunità in provincia di Yunnan, Repubblica Popolare Cinese e Agboville, Costa d&#39;Avorio, per la loro partecipazione negli studi sopra descritti. Siamo in debito con le squadre di campo locale che hanno assistito nella sperimentazione ed esecuzione dei protocolli. Siamo particolarmente grati ai seguenti colleghi della Salute Istituto tropicale svizzero e pubblico: Yvette Endriss per fornire le immagini microscopiche, Hanspeter Marti e il suo team per il laboratorio di supporto e Stefanie Krauth per la sua assistenza durante le riprese video. Infine, ringraziamo i due referee anonimi per una serie di utili commenti.

1. Kato-Katz Tecnica <ol><li> Posizionare uno standard Kato-Katz modello con un foro per lo svolgimento di 41,7 milligrammi (mg) di feci su un vetrino da microscopio. </li><li> Scoop 2-3 grammi (g) di un campione fresco di feci su un pezzo di giornale o un foglio di alluminio, e premere un pezzo di filo o di maglia di plastica sulla parte superiore per setaccio esso. </li><li> Con una spatola di plastica, raschiare il materiale setacciato largo della maglia e riempire completamente il foro nel Kato-Katz modello con esso. Per rimuovere i residui di materiale fecale, livellare il contenuto del foro con la spatola. </li><li> Verticale rimuovere la mascherina senza disturbare il materiale fecale ora aderente al vetrino da microscopio. Il modello e spatola possono essere puliti in acqua con detergente, e riutilizzati. </li><li> Posizionare un pezzo di cellophane, pre-imbevuto di glicerina-malachite soluzione di verde per almeno 24 ore (h), sul campione fecale sul vetrino da microscopio. </li><li> Per diffondere il materiale fecale in uno striscio di spessore, premere delicatamente un panno pulito microdiapositiva portata contro la diapositiva campione, distribuire uniformemente il materiale in un cerchio di diametro leggermente inferiore alla larghezza del vetrino da microscopio. </li><li> Lasciare il vetrino per cancellare per 30-60 minuti (min), durante la quale i vetrini devono essere tenuti lontano dalla luce diretta del sole. Poi, sistematicamente esaminare lo striscio spesso sotto un microscopio ottico (40-100x ingrandimento). Contare il numero di uova STH e stratificare loro per specie (cioè, A.lumbricoides, T.trichiura e anchilostomi). Di nota, uova di elminti altri (ad esempio Schistosoma mansoni) possono anche essere rilevate da questo metodo e dovrebbero essere contati e registrati separatamente. </li><li> Per ottenere una stima standardizzata dell&#39;intensità di infezione, convenzionalmente espressa come numero di uova per grammo di feci (EPG), moltiplicare il numero di uova da 24 (24 x 41,7 mg ≈ 1 g). </li></ol> 2. Baermann Tecnica <ol><li> Allestito uno stand che può contenere un bicchiere imbuto e collegare un tubo di gomma, chiuso spiritoha clip, sul fondo dell&#39;imbuto. </li><li> Collocare un setaccio o un pezzo di schermo di filo a forma di cono, nell&#39;imbuto e strato con una garza medica. </li><li> Riempire l&#39;imbuto con acqua del rubinetto e garantire che l&#39;intero set-up non perde. </li><li> Scoop 20-30 g di feci fresche sul centro della garza medica e assicurarsi che sia completamente immerso in acqua. Aggiungere più acqua se necessario. Piegare la garza medica sul campione. </li><li> Dal basso, brillare di luce artificiale al imbuto per 2 ore. </li><li> Drenare 50 millilitri (ml) della porzione inferiore dell&#39;acqua nell&#39;imbuto in un tubo da centrifuga delicatamente rilasciando la clip sul tubo di gomma. </li><li> Centrifugare l&#39;acqua raccolta a 600 xg (forza centrifuga relativa) per 5 min e con attenzione versare il sopranatante senza disturbare il sedimento. Conservare le ultime gocce nel tubo. </li><li> Risospendere le 2 gocce di sedimento e pipettare della soluzione su un vetrino da microscopio ed esaminare il vetrinoal microscopio ottico (ingrandimento 40x per il rilevamento e 100-400x di ingrandimento per la conferma specie). Larve di primo stadio (L1) di S. stercoralis sono vivi e attivamente muoversi sotto il microscopio. </li></ol> 3. 20-m Shuttle Test Run <ol><li> Misura 20 m di percorso piatto e dritto in esecuzione e lo contrassegna con coni. </li><li> Aiuta il partecipante al test indossare un cardiofrequenzimetro al polso e il trasmettitore corrispondente attorno al torace 18. Questa apparecchiatura permette la frequenza cardiaca del partecipante a essere misurato prima e dopo la prova e quindi controlla che il partecipante ha esercitato sforzo massimo. </li><li> Chiedere ai partecipanti di correre avanti e indietro sul campo, seguendo il ritmo della pre-registrati i segnali sonori che delimitano 20 m giri. Iniziando con una velocità di marcia di 8,5 chilometri all&#39;ora (km / h), la frequenza dei segnali indicativi 20-m aumenta intervalli di un equivalente di 0,5 km / h ogni min. </li><li> Garantire °al partecipante inizia un nuovo intervallo di 20 m ogni volta che viene emesso un segnale e termina prima che il segnale successivo arriva. </li><li> Arrestare il partecipante se lui / lei non riesce a seguire il ritmo per due consecutivi di 20 m intervalli. </li><li> Registrare la massima velocità di marcia indicato per l&#39;ultimo intervallo che il partecipante ha completato completamente, insieme con l&#39;età e il sesso del partecipante. </li><li> Verificare che la frequenza cardiaca raggiunta al termine della prova è la frequenza cardiaca massima del bambino 19. Se la frequenza cardiaca non è raggiunto, il bambino potrebbe non aver esercitato il massimo sforzo, e quindi il test deve essere ripetuto. </li><li> Stimare la capacità aerobica massima (VO2 max) del partecipante, utilizzando un&#39;equazione portata avanti da Léger e collaboratori: VO 2 max = 31,025 + 3,238 * velocità (in km / h) - 3,248 * età (in anni) + 0,1536 * velocità (in km / h) * età (in anni). </li></ol> 4. Risultati rappresentativi In termini diDiagnosi STH con la tecnica Kato-Katz, caratteristiche uniche delle uova di elminti diverse consentire l&#39;individuazione della maggior parte delle specie 20. Le uova di A.lumbricoides (circa 45-75 micrometri (micron) di lunghezza e larghezza 35-50 micron, Figura 1) presentano pareti guscio più spesso rispetto alle uova di T.trichiura (Figura 2) e hookworm (Figura 3). Un uovo fecondato di A.lumbricoides (figura 1) ha un cappotto esterno albuminosa, che lo fa apparire dorato, mentre un uovo non fecondato non ha questa cappotto esterno ed è leggermente allungata e più grande generale. Le uova di T.trichiura (circa 50 micron di lunghezza e 22 micron di larghezza, Figura 2) sono allungate e sono distinti spine polari. Hookworm uova (circa 64-76 micron di lunghezza e 36-40 micron di larghezza, Figura 3), invece, ha una parete molto sottile, seguito da un anello libero intorno un cluster denso di cellulenel centro dell&#39;uovo. S.stercoralis L 1 larve (Figura 4) sono circa 180-380 micron di lunghezza e ha una cavità buccale molto breve. Un distinto primordium genitale (evidenziata con una freccia blu in figura 4) può essere osservato e la coda delle larve (indicato da una freccia rossa in figura 4) ha una estremità affilata. Come il vero impatto delle infezioni STH sulla forma fisica dei bambini è ancora in discussione 13,14,15, non ci sono risultati rappresentativi per il loro impatto, a questo punto. Interpretazione dei risultati 20-m run navetta prova può essere eseguita in vari modi. La forma fisica dei bambini possono essere confrontati tra i soggetti infetti e non infetti, che sono simili in termini di età e sesso, altre condizioni di salute potenzialmente rilevanti, lo stato socio-economico, e le abitudini culturali. Questo può essere fatto in una sezione in cui il rilevamento 13,14,15Stato di infezione STH e forma fisica dei bambini infetti e non infetti sono valutati in un punto particolare momento. La forza delle prove sarà più alto quando si segue un disegno randomizzato e controllato in cui viene valutata l&#39;idoneità fisica dei bambini in età scolare, prima e dopo anthelminthic / placebo trattamento, consentendo sia la valutazione a breve e lungo termine. Si può inoltre cercare di identificare una possibile relazione dose-risposta tra l&#39;intensità delle infezioni da STH e forma fisica e confrontare i diversi livelli di multiparasitism 1 con la forma fisica. Nel lavoro presentato da Yap e collaboratori 15, idoneità fisica, come rappresentato da VO 2 max, è risultata essere significativamente ridotta nei bambini con infezione da T.trichiura rispetto ai bambini che non sono stati infettati con T.trichiura (Figura 5) . <img alt="Figura 1" src="/files/ftp_upload/3966/3966fig1.jpg" /> Figu<html> re 1. immagini microscopiche di uova fecondate A.lumbricoides: (A) e non fecondate (B) sotto ingrandimento 500x. Barra della scala = 50 micron. <img alt="Figura 2" src="/files/ftp_upload/3966/3966fig2.jpg" /> Figura 2. Immagine microscopica di un uovo T.trichiura sotto ingrandimento 500x. Bar Scala = 50 micron. <img alt="Figura 3" src="/files/ftp_upload/3966/3966fig3.jpg" /> Figura 3. Immagine microscopica di un uovo di hookworm sotto ingrandimento 500x. Barra della scala = 50 micron. <img alt="Figura 4" src="/files/ftp_upload/3966/3966fig4.jpg" /> Figura 4. Immagine microscopica di un L S.stercoralis 1 larva sotto ingrandimento 250x. Le frecce indicano la coda (rosso) e genitale primordio (blu). Barra di scala = 100 micron. d/3966/3966fig5.jpg &quot;/&gt; Figura 5. Grafico mostra il livello di forma fisica dei bambini con infezione da T.trichiura rispetto al livello di forma fisica dei bambini non infettati con T. trichiura.

<ol>
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Nessun conflitto di interessi dichiarati.

I tre protocolli descritti in questo documento sono stati testati ed eseguito nella parte sud-ovest della provincia dello Yunnan, Repubblica popolare cinese tra i membri della minoranza etnica Bulang 21 e in diverse parti dell&#39;Africa 6,13,14. Ci sono diversi modelli disponibili per il Kato-Katz tecnica. Ogni distinto foro-size consegnerà una quantità distinta di materiale fecale. Per il calcolo delle EPG, è quindi necessario conoscere l&#39;unità del modello, in...

Nota: I materiali in questo elenco sono generiche e possono essere ottenuti da fonti diverse. <table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Kato-Katz kit [400 modelli di plastica con un foro di 6 millimetri (mm) (diametro) in un modello di spessore 1,5 mm; 400 spatola di plastica a 20-m ruolo di schermo di nylon 80 maglia, un 20-m ruolo di cellophane idrofilo, 34 micron di spessore.] </td><td> Vestergaard Frandsen </td><td></td><td> Si prega di notare che la glicerina-malachite soluzione di verde non è inclusa in questo kit, ma possono essere acquistati da qualsiasi fornitore di prodotti chimici. </td></tr><tr><td> Imbuto di vetro [circa 8 centimetri (cm) di larghezza e 6 cm di profondità] </td><td></td><td></td><td> L&#39;imbuto di vetro per la tecnica Baermann dovrebbe essere in grado di contenere circa 60 mL di acqua. Thdimensioni e del tubo di gomma e garza dipende dal tipo di imbuto di vetro utilizzato. </td></tr><tr><td> Microscopio biologico </td><td> Olimpo </td><td> CX21LED/CX21 </td><td></td></tr><tr><td> Centrifuga </td><td> Shanghai Medical Instruments Group </td><td> 80 - 2T </td><td></td></tr><tr><td> Pre-registrati segnali </td><td> Bitworks design </td><td> Squadra Bleep Test Versione 1.3.1 </td><td> applicazione iPhone </td></tr><tr><td> Cardiofrequenzimetro </td><td> POLAR </td><td> FT1 </td><td> Guarda e trasmettitore </td></tr></tbody></table>

determining soil-transmitted helminth infection status and physical fitness of school-aged children

Suolo-trasmesse da elminti (STH), le infezioni sono comuni. In effetti, più di 1 miliardo di persone sono colpite, soprattutto nel mondo in via di sviluppo dove la povertà e il comportamento prevale l&#39;igiene, l&#39;approvvigionamento idrico, servizi igienico-sanitari e sono spesso carenti 1,2. Ascaris lumbricoides, Trichuris trichiura, e le due specie hookworm, Ancylostoma duodenale e Necator americanus, sono le più diffuse STHS 3. L&#39;onere stimato globale a causa di malattia hookworm, ascariasis, e trichuriasis è 22.1, 10.5, e 6,4 milioni di disability-adjusted anni di vita (DALY), rispettivamente 4. Inoltre, si stima che 30-100000000 persone sono infettate con Strongyloides stercoralis, la specie più trascurate STH di rilevanza mondiale che logicamente anche provoca un considerevole impatto sulla salute pubblica 5,6. Multiple-specie infezioni (ad esempio, STHS diverse nutriti in un singolo individuo) sono comuni, e le infezioni sono state lin<html> ked alla produttività abbassato e quindi prospettive economiche dei paesi in via di sviluppo 1,3. Per la diagnosi di STHS comuni, l&#39;Organizzazione Mondiale della Sanità (OMS) raccomanda l&#39;Kato-Katz tecnica di 7,8, che è un metodo relativamente semplice per determinare la prevalenza e l&#39;intensità di tali infezioni. Facilita la ricerca di uova di parassiti che i soggetti infetti passano nelle loro feci. Per quanto riguarda la diagnosi di S.stercoralis, attualmente non esiste strumento semplice e preciso disponibile. La tecnica Baermann è il metodo più largamente utilizzato per la sua diagnosi. Il principio alla base della tecnica è che le larve Baermann attiva S.stercoralis migrare su un sistema di illuminazione campione fresco di feci come le larve sono phototactic 9. Richiede materiali di laboratorio meno sofisticate e meno tempo rispetto ai metodi di coltura e immunologici 5. t &quot;&gt; patologie associate a infezioni da STH gamma di sintomi acuti, ma comune, come dolore addominale, diarrea, prurito e, di sintomi cronici, come l&#39;anemia, sotto-e malnutrizione, e deficit cognitivo 10. Poiché i sintomi sono generalmente aspecifici e sottile, che spesso passano inosservati, sono considerati una condizione normale di individui affetti, o sono trattati come sintomi di altre malattie che potrebbero essere più comune in un determinato contesto. Quindi, non si può escludere che il vero peso delle infezioni STH è sottovalutato da una valutazione basandosi su strumenti di auto-dichiarati segni e dei sintomi è di solito il caso in ricerche basate sulla popolazione. Alla fine del 1980 e all&#39;inizio del 1990, Stephenson e colleghi hanno evidenziato la possibilità di infezioni da STH abbassando la forma fisica dei ragazzi di età compresa tra 6-12 anni 11,12. Questa linea di ricerca scientifica sono state accelerate di recente 13,14,15. Il 20 metri (m) di esecuzione dei test è stato messo a punto un servizio navettad convalidato da Léger et al. 16 e viene utilizzato in tutto il mondo per misurare la capacità aerobica dei bambini 17. Il test è facile da standardizzare e può essere effettuata ovunque un 20-m lungo corso e di marcia a piatto e una sorgente audio sono disponibili, rendendo il suo uso attraente in risorse limitate impostazioni 13. Per facilitare e standardizzare i tentativi di valutare se le infezioni STH hanno un effetto sulla forma fisica dei bambini in età scolare, presentiamo metodologie per la diagnosi di infezioni da STH o idoneità misura fisica che sono semplici da eseguire e ancora, fornire risultati accurati e riproducibili. Ciò contribuirà a generare nuove prove per quanto riguarda l&#39;impatto sulla salute delle infezioni STH.

Watch this Scientific Journal Video about Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children at JoVE.com

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

L&#39;infezione cronica da elminti suolo trasmesse (STHS) provoca malassorbimento, arresto della crescita, e di sprecare nel bambino in crescita. Quindi, è plausibile che queste infezioni anche ridurre la forma fisica dei bambini. Qui, noi visualizziamo due tecniche per la diagnosi di STHS e di 20 metri di test run navetta per valutare la forma fisica dei bambini.

Determinazione del suolo-infezione trasmessa elminti Stato e la forma fisica dei bambini in età scolare

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

determining-soil-transmitted-helminth-infection-status-physical

Research

JoVE Journal

Immunology and Infection

23.1K Views.  Swiss Tropical and Public Health Institute, Basel, Switzerland. L'infezione cronica da elminti suolo trasmesse (STHS) provoca malassorbimento, arresto della crescita, e di sprecare nel bambino in crescita. Quindi, è plausibile che queste infezioni anche ridurre la forma fisica dei bambini. Qui, noi visualizziamo due tecniche per la diagnosi di STHS e di 20 metri di test run navetta per valutare la forma fisica dei bambini.

Video: Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Determinazione dell&#39;attività fagocitaria dei Campioni anticorpi clinici

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Ricerca

Immunologia e infezioni

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Con ovuli<em&gt; Schistosoma mansoni</em&gt; Come<em&gt; In vivo</em&gt; Modello di elminti-indotta infiammazione polmonare

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Analisi della maturazione polmonare delle cellule dendritiche e migrazione durante l&#39;infiammazione allergica delle vie aeree

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Microscopia a fluorescenza metodi per determinare la vitalità di batteri in associazione con cellule di mammifero

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Pairwise crescita Concorso test per la determinazione della Fitness replica del virus dell&#39;immunodeficienza umana

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

La determinazione di soppressione del sistema immunitario contro il sistema nervoso centrale di protezione per farmacologici Interventi a demielinizzazione autoimmune

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizzare gli effetti di espettorato su biofilm di sviluppo utilizzando un modello Chambered coprioggetti

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocolli per Indagare la distribuzione Host-tessuti, trasmissione-mode, e Effetto sul Fitness ospite di un Densovirus in cotone bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

High throughput sequenziamento parallelo al Fitness misura di Leptospira interrogans trasposone inserimento mutanti durante dorato siriano criceto infezione

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...