Questo lavoro è stato supportato da R01 AI32556 (NIAID, National Institute of Health). Questo lavoro è stato sostenuto in parte dalla Louisiana vaccino Centro e Sud della Louisiana Istituto di ricerca sulle malattie infettive promossa dal Consiglio dei Reggenti della Louisiana.

1. Inoculazione vaginale con Candida albicans <ol><li> Tre giorni prima dell&#39;inoculazione, mentre frenare l&#39;animale per esporre l&#39;addome, iniettare 100 ml di olio di sesamo contengono 0,1-0,5 mg di β-estradiolo per via sottocutanea nella parte inferiore dell&#39;addome. Avanzare l&#39;ago di circa 5 a 10 mm laterale alla pelle per ridurre la dispersione dal sito di iniezione. La somministrazione sottocutanea di estrogeni nel basso addome è ottimale in questo modello a causa della prossimità del tratto genitale. Dosi efficaci possono variare da ceppi di topi, di età o derivati ​​degli estrogeni. In studi precedenti con CBA-J (H-2 κ), C3H/HeN (H-2 κ), C57BL / 6 (H-2 b), Balb / c (H-2 d), DBA / 2 (H- 2 d), SJL (H-2 s) i topi a 6-8 settimane di età, 0,1 mg/100 microlitri è stato trovato efficace evidenziato da ispessimento della parete vaginale, riduzione muco vaginale eaumentata desquamazione delle cellule epiteliali. I topi trattati con estrogeni sopra a questa concentrazione mostra coerente colonizzazione vaginale da Candida. Per l&#39;inoculazione in topi di altri ceppi e le età, uno studio pilota si raccomanda di garantire l&#39;efficacia degli estrogeni nelle condizioni modificate e aumentare la dose di estrogeni, se necessario. La soluzione estrogeni deve essere preparata fresca ogni volta il giorno di iniezione. Per garantire una completa solubilizzazione di estrogeni in olio di sesamo, mescolare la soluzione con un vortex e calore ad intermittenza a 37 ° C. Ripetere l&#39;iniezione settimanale per tutto il periodo di studio. </li><li> Per preparare l&#39;inoculo, aggiungere un un&#39;ansata di C. albicans blastoconidia da una preparazione sottocultura recente su Sabouraud-destrosio agar (SDA) in 10 ml di brodo peptone Phytone-integrato con 0,1% di glucosio. Incubare il brodo di cultura di fase stazionaria per 18 ore a 25 ° C in un bagno d&#39;acqua agitazione. </li><li> Dopo l&#39;incubazione, raccogliere °e brodo di cultura in un tubo da 15 ml e centrifugare a 800 xg per 5 minuti. Lavare il pellet due volte con PBS sterile. </li><li> Enumerare blastoconidia possibili in un emocitometro per esclusione trypan colorante blu. Regolare la concentrazione cellulare a 2,5 x 10 6 / ml (o ad una concentrazione di inoculo desiderato) in PBS sterile. </li><li> Per stabilizzare il mouse, tenere la base della coda con due dita e sollevare l&#39;anca verso l&#39;alto in modo che l&#39;apertura vaginale volti verso di voi (Figura 1A). E &#39;ideale se il mouse è posizionato su una superficie piana grattugiato (es. gabbia in alto) in modo che il mouse in grado di fornire la resistenza contro la coda di moderazione. </li><li> Pipettare 20 l (o un volume desiderato non superiore a 20 l), della sospensione di inoculo inserendo la punta della pipetta di circa 5 mm di profondità nel lume vaginale (Figura 1B). Completare questo passaggio il più velocemente e delicatamente possibile per ridurre al minimo disagio nel topo. </li></ol> 2. Lavaggi vaginali <ol><li> A seguito di eutanasia (o anestesia), hvecchio con il mouse verso il basso dalla base della coda con due dita in modo che l&#39;apertura vaginale diventa esposti. </li><li> Lavaggio del lume vaginale con l&#39;introduzione di 100 l di PBS sterile con l&#39;aspirazione ripetute e agitazione con la punta della pipetta. La punta della pipetta può ostruirsi con le cellule. In questo caso, dispensare le cellule ostacolando e continuare con le restanti lavaging PBS nella vagina. Raccogliere il liquido di lavaggio in una provetta. </li><li> In alternativa, lavaggi vaginali può essere eseguito su topi anestetizzati con isoflurano anestesia inalanti. Per questo, esporre i topi di vaporizzato isoflurano fino a quando non sono completamente sedato (~ 30 sec.). Tenere il mouse verso il basso dalla base della coda e delicatamente lavaggio del lume vaginale usando 50 ml di PBS sterile. Assicurati di evitare l&#39;agitazione dura con un puntale per minimizzare il trauma alla vagina durante questa procedura. Topi sedati dovrebbe recuperare dall&#39;anestesia entro 30 secondi di esposizione all&#39;aria ambiente. Isofluranopossono essere vaporizzati con un vaporizzatore isoflurano e O 2 (preferito) o un sistema standard di camera chiusa goccia anestetico senza il sistema vaporizzatore (richiede uno stretto monitoraggio degli animali, mentre sedato per evitare difficoltà respiratorie). Lavaggi vaginali in topi anestetizzati dovrebbe essere il metodo di scelta per i campioni longitudinali lavanda sui topi stessi. Lavaging consecutivi non influire sulla valutazione della carica fungina nel corso del tempo 41. </li><li> Per umido-mount preparazioni, 10 ml di trasferimento del liquido di lavaggio su un vetrino e osservare con ingrandimento 400-1000x al microscopio ottico. Inoltre, le frazioni cellulari del liquido di lavaggio può essere macchiato di esaminare morfologia cellulare e nucleare. Per i preparati striscio, 10 ml di trasferimento del liquido di lavaggio su un vetrino e delicatamente diffondere tramite la parete esterna di un puntale. Conservare i campioni di striscio con fissativo CytoPrep e macchiare con la tecnica standard di Papanicolaou (Pap test). Osservare a 400 × ingrandimento al microscopio ottico. </li></ol> 3. Quantificazione del peso vaginale da funghi <ol><li> In un ben 96 round-piastra inferiore, trasferire il liquido di lavaggio in un pozzo della riga superiore e 180 ml di PBS sterile nelle seguenti 5 pozzi di quella colonna (la piastra). </li><li> Fai 1:10 diluizioni seriali di liquido di lavaggio vaginale, trasferendo i 20 ml di liquido al pozzo successivo nella colonna. Mescolare accuratamente per aspirazione ripetuta prima di ogni trasferimento. Diluizioni seriali fino a 12 campioni di lavaggio (una riga completa orizzontale) possono essere eseguite contemporaneamente, utilizzando una pipetta a 12 canali. </li><li> A partire dalla diluizione più basso, il trasferimento di 10 ml di campione su Sabouraud-destrosio agar (SDA). Placcatura di un massimo di 36 campioni possono essere eseguite su 1 piastra con SDA preparato in piazza piastre di Petri con griglia regolabile e una pipetta multicanale spaziatura. </li><li> Enumerare le unità formanti colonia (CFU), dopo incubazione a 34 ° C fo 48 h. </li></ol> 4. Estrazione del tessuto vaginale <ol><li> Seguendo la procedura lavanda vaginale, giaceva il mouse eutanasia sul dorso e saturare la zona inguinale con il 70% di etanolo. Utilizzando un paio di pinze, sollevare l&#39;orifizio urinario verso l&#39;alto in modo che l&#39;apertura vaginale diventa esposti. </li><li> Inserire un paio di pinze curve nel lume vaginale e individuare il collo dell&#39;utero. Pur mantenendo una salda presa con la pinza, estrarre il collo dell&#39;utero attraverso la cavità vaginale (Figura 2A-C). </li><li> Accise la vagina alla base dell&#39;apertura vaginale e quindi rimuovere il collo dell&#39;utero dalla vagina con le forbici chirurgiche. Tenete presente che il tessuto vaginale è involuta (lato interno epitelio della vagina è esposta verso l&#39;esterno). Il tessuto può essere invertita lateralmente per mantenere l&#39;orientamento originale della vagina o aperto in un foglio facendo una incisione laterale. Asportati tessuti vaginali possono essere utilizzati per 1) l&#39;estrazione dei linfociti collage seguentidigestione nase (~ 1 × 10 4 / mouse) 40, 2) isolamento delle cellule epiteliali dopo digestione dispasi (~ 5 × 10 4 / mouse) 28, 3) i preparati congelati o inclusi in paraffina per le analisi istologiche 25. </li></ol> 5. Lombare linfa escissione nodo <ol><li> Seguendo la procedura lavanda vaginale, giaceva il mouse eutanasia sul dorso e saturare l&#39;addome con il 70% di etanolo. Effettuare una incisione laterale a partire dal basso ventre al petto ed esporre gli organi interni. Usando un paio di pinze con entrambe le mani, spostare l&#39;intestino verso l&#39;alto in modo che i vasi sanguigni diventano visibili centrale. </li><li> Individuare la vena cava inferiore e aorta addominale. Normalmente, un paio di linfonodi lombari possono essere identificati adiacente alla aorta addominale, che si trova circa a metà strada tra l&#39;origine delle arterie iliache renali e comuni 39. Questi linfonodi possono essere visivamente distinti da tessuto adiposo dalla texture elasticae sono più leggeri e più opaco di colore rispetto al tessuto adiposo (Figura 3). Questi linfonodi sono notevolmente più grandi di animali infetti rispetto agli animali non inoculata. </li><li> Accise linfonodi mettendo microforceps sotto il nodo e tirare dolcemente fino a separarsi dal tessuto circostante. </li></ol> 6. Isolamento di cellule linfoidi in cella singola sospensioni <ol><li> Trasferimento linfonodi su uno schermo sterile rete metallica (circa 3 x 3 cm 2 di dimensioni) posto all&#39;interno di un piatto di vetro Petri sterile contenente circa 10 ml di soluzione salina bilanciata Hanks &#39;(HBSS) (Figura 4). </li><li> Con la capsula di Petri leggermente inclinato, premere i linfonodi contro lo schermo con una testa stantuffo della siringa. Assicurati di rompere tutti i linfonodi in modo che il contenuto cellulare dei nodi passa attraverso lo schermo, mentre il componenti non cellulari dei nodi (cioè le membrane, stroma, grasso) rimangono sullo schermo. </li><li> Utilizzando il pistone stesso e siringa, aspirare tha HBSS cellule contenenti. Lavare lo schermo con ~ 5 ml HBSS e raccogliere il liquido rimanente in un tubo da 15 ml. </li><li> Centrifugare a 800 xg per 10 min. Aspirare qualsiasi depositi di grasso nella parte alta del liquido con una pipetta prima di scartare il liquido. Lavare i pellet tre volte con HBSS. Risospendere il pellet in 1 ml di HBSS ed enumerare cellule vitali per esclusione trypan colorante blu. </li></ol> 7. Rappresentante dei risultati: Le frazioni cellulari del liquido di lavaggio vaginale da&gt; 4 giorni topi inoculati tipicamente costituiti da Candida, le cellule epiteliali e infiltrati cellulari (Figura 5). Da bagnato-mount microscopia, Candida può essere visivamente identificato dalla presenza di ife e lieviti (Figura 5A). Preparazioni striscio vaginale di liquido di lavaggio può essere colorato con Papanicolaou tecnica per esaminare le cellule epiteliali e leucociti infiltranti, di cui le cellule principali sono neutrofili identificati dal tri-nuclelobi ar (Figura 5B). Neutrofili molto pochi, se del caso, vengono rilevati nei topi non inoculate (Figura 5C) 41. Un esempio di peso vaginale da funghi è mostrato in Figura 6. Liquido di lavaggio vaginale raccolti in punti temporali specifici sono coltivate per il conteggio CFU (Figura 6A). Colonizzazione vaginale / infezione da Candida persiste per settimane nei topi inoculati estrogeno-trattati (Figura 6B), mentre Candida non riesce a stabilire la colonizzazione vaginale inoculati nei topi non trattati con estrogeni (Figura 6C). Non inoculate nei topi trattati con estrogeni rimane negativo per Candida per tutto il tempo (dati non riportati). Inoltre, lavaggi vaginali possono essere eseguite una sola volta sui topi separati in ogni fase o longitudinalmente nei topi stessi sotto anestesia. I linfonodi lombari sono i nodi principali linfodrenante del tratto genitale e il sito più rilevanti per la valutazione sistemica per la risposta immunitaria a una sfida vaginale. Note che questi linfonodi possono diventare allargata nei topi inoculati mentre normalmente appaiono abbastanza piccola non inoculate nei topi. Recuperi cellulare dei leucociti in genere varia da 8 × 10 5 mouse / non inoculate fino a 5 × 10 6 mouse / inoculato. Oltre ai linfonodi lombari, linfonodi inguinali, popliteo e mesenterici può anche essere usato. <img alt="Figura 1" src="/files/ftp_upload/3382/3382fig1.jpg" /> Figura 1. Inoculazione vaginale da Candida. A) Un mouse trattenuto per l&#39;inoculazione. Il mouse è posizionato su un inserto gabbietta e detenute dalla base della coda, leggermente verso l&#39;alto per sollevare le gambe ed esporre l&#39;apertura vaginale. L&#39;anca del mouse può essere stabilizzata con la stessa mano che tenta di resistere alla moderazione coda. B) Introduzione di inoculo nel lume vaginale. Il puntale è delicatamente inserito circa 5 mm di profondità nel lume vaginale. L&#39;inoculo sospensione viene poi depositato. Figura 2. Estrazione del tessuto vaginale. AB) Estrazione della cervice. La cervice si trova con una pinza ricurva ed esposta verso l&#39;esterno attraverso la cavità vaginale. Una volta fuori della cavità vaginale, il collo dell&#39;utero è ulteriormente tirato verso l&#39;esterno per esporre completamente la vagina. C) Estrazione della vagina. La vagina è asportato dalla vulva con le forbici. Una volta staccato, rimuovere il collo dell&#39;utero dalla vagina. <img alt="Figura 3" src="/files/ftp_upload/3382/3382fig3.jpg" /> Figura 3. Identificazione dei linfonodi lombari. La posizione dei linfonodi lombare tra gli organi circostanti / vasi sanguigni in prossimità del bacino è indicato. A, aorta addominale. B, della vescica urinaria. C, arteria iliaca comune. Io, intestino. L, fegato. R, retto. S, milza. U, uteri. <img alt="Figura 4" src="/files/ftp_upload/3382/3382fig4.jpg" /> Figura 4. I linfonodi lombare posto su uno schermo di rete metallica. I linfonodi sono raggruppati sullo schermo posto in una capsula di Petri con HBSS. I linfonodi sono premuti contro lo schermo con un stantuffo della siringa per ottenere cellule linfoidi in cella singola sospensioni. <img alt="Figura 5" src="/files/ftp_upload/3382/3382fig5.jpg" /> Figura 5. Frazioni cellulari di liquido di lavaggio vaginale dai topi inoculati. Pap A) Wet-mount e B) si preparano strisci di campioni lavanda vaginale raccolto 4 giorni dopo l&#39;inoculazione e C) dai topi non inoculata. Le immagini vengono visualizzate a 1000 × (A) o 400 × (B, C) ingrandimento. L&#39;inserto in B mostra la morfologia nucleare dei neutrofili vaginale a 1000 ×. Lievito Candida (Y) e ife (H), cellule epiteliali (CE) e neutrohils (N) sono indicati. <img alt="Figura 6" src="/files/ftp_upload/3382/3382fig6.jpg" /> Figura 6. Detectioni di peso vaginale da funghi. A) Rappresentante C. albicans colonie coltivate su un piatto SDA. Neat (N) campioni di lavaggio provenienti da sei diversi tipi di mouse inoculati (riga in alto) sono stati diluiti in serie e coltivate per il conteggio CFU. B) Quantificazione degli vaginale carica fungina nel estrogeno-trattati e C) non estrogeno-topi trattati. Ufc/100 ml di liquido di lavaggio da topi inoculati è stata valutata sui punti di tempo indicato.

<ol>
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Un modello murino sperimentale di vaginite da Candida è stata stabilita e storicamente usato per gli ultimi decenni a studiare la risposta mucosale ospite Candida, nonché per testare le terapie antimicotiche 3,4,11,13,16,17,19,21,24, 25,37. I protocolli presentati qui incorporare efficiente e metodi meno alta intensità di lavoro, e sembrano essere uno dei sistemi modello più ottimizzata di Candida vaginite descritto fino ad oggi. Queste tecniche consentono la quantificazione rap...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Femmina CBA / J topi </td><td> Charles River Laboratories </td><td> 01C38 </td><td> 5-6 settimane di età </td></tr><tr><td> Candida albicans (3153A) </td><td> Collezione nazionale di funghi patogeni, Regno Unito </td><td> NCPF3153 </td><td></td></tr><tr><td> Olio di sesamo </td><td> Sigma-Aldrich </td><td> S3547 </td><td> Non ha bisogno di essere pre-sterilizzati prima dell&#39;uso </td></tr><tr><td> Β-17-estradiolo valerato </td><td> Sigma-Aldrich </td><td> E1631 </td><td> 0.1-0.5mg in olio di sesamo </td></tr><tr><td> Phytone peptone </td><td> Becton Dickinson </td><td> 211906 </td><td> Supplemento con 0,1% di glucosio </td></tr><tr><td> Blu tripano soluzione </td><td> Sigma-Aldrich </td><td> T8154 </td><td></td></tr><tr><td> Sabouraud destrosio agar </td><td> Becton Dickinson </td><td> 211584 </td><td></td></tr><tr><td> Collagenasi di tipo IV </td><td> Sigma-Aldrich </td><td> C5138 </td><td> 0,25% </td></tr><tr><td> Dispasi </td><td> Invitrogen </td><td> 17105-041 </td><td> 1,7 U / ml </td></tr><tr><td> Reti metalliche schermi </td><td> TWP </td><td> 060X060S0065W36T </td><td> N. 60 mesh, inox </td></tr><tr><td> Sale Hanks &#39;soluzione equilibrata </td><td> Invitrogen </td><td> 24020-117 </td><td></td></tr><tr><td> CytoPrep fissativo </td><td> Fisher Scientific </td><td> 12-570-10 </td><td> Conserve diapositive striscio </td></tr><tr><td> Papanicolaou macchia EA-65 </td><td> EMD chimici </td><td> 7054X-85 </td><td></td></tr><tr><td> Papanicolaou macchia OG-6 </td><td> EMD chimici </td><td> 7052X-85 </td><td></td></tr><tr><td> Alum Harris &#39;ematossilina </td><td> EMD chimici </td><td> 638A-85 </td><td></td></tr><tr><td> Isoflurano </td><td> Baxter Healthcare </td><td> NDC 10019-773-60 </td><td> Utilizzato con vaporizzatore isoflurano o in un sistema chiuso goccia anestetico da camera </td></tr></table>

protocols for vaginal inoculation and sample collection in the experimental mouse model of candida vaginitis

Candidosi vulvovaginale (VVC), causate da Candida specie, è una infezione fungina del basso tratto genitale femminile che colpisce circa il 75% delle donne in buona salute durante i loro anni riproduttivi 18,32-34. I fattori predisponenti comprendono l&#39;uso di antibiotici, il diabete non controllato e disturbi dei livelli degli ormoni riproduttivi dovuti alla gravidanza, contraccettivi orali o terapia ormonale sostitutiva 33,34. VVC ricorrente (RVVC), definita come tre o più episodi l&#39;anno, colpisce un separato da 5 a 8% delle donne senza fattori predisponenti 33. Un modello murino sperimentale di VVC è stato istituito e utilizzato per studiare la patogenesi e la risposta dell&#39;ospite mucose da Candida 3,4,11,16,17,19,21,25,37. Questo modello è stato utilizzato per testare potenziali terapie antifungine in vivo 13,24. Il modello prevede che gli animali sono mantenuti in uno stato di optare per pseudoestrusIMAL Candida colonizzazione / infezione 6,14,23. In tali condizioni, gli animali vaccinati avranno rilevabili vaginale carica fungina per settimane o mesi. Studi precedenti mostrano un parallelo estremamente elevata tra il modello animale e infezione umana rispetto alla proprietà immunologiche e fisiologiche 3,16,21. Le differenze, tuttavia, sono la mancanza di Candida come normale flora vaginale e un pH neutro vaginale nei topi. Qui, dimostrano una serie di metodi chiave del modello vaginite mouse che includono l&#39;inoculazione vaginale, la raccolta rapida di campioni vaginali, la valutazione del peso vaginale da funghi, e preparati per l&#39;estrazione dei tessuti cellulari / isolamento. Questo è seguito da risultati rappresentativi per i componenti del liquido di lavaggio vaginale, carica fungina e drenaggio linfatico rendimenti dei leucociti nodo. Con l&#39;uso degli anestetici, i campioni di lavaggio possono essere prelevati in punti più tempo sui topi stessi per la valutazione longitudinaleinfezione / colonizzazione. Inoltre, questo modello non necessita di farmaci immunosoppressori per avviare l&#39;infezione, permettendo studi immunologici in condizioni definite serie. Infine, il modello e ogni tecnica introdotta qui potrebbe potenzialmente dar luogo ad utilizzare le metodologie per esaminare altre malattie infettive del basso tratto genitale femminile (batteriche, parassitarie, virali) e le rispettive difese dell&#39;ospite locale o sistemica.

Watch this Scientific Journal Video about Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis at JoVE.com

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Tecniche di chiave da utilizzare nella valutazione dei<em&gt; Candida</emVaginite&gt; in un modello animale sperimentale sono descritti. I metodi di permetterà una rapida raccolta di campioni vaginali e dei linfociti nei linfonodi di drenaggio lombare. Queste tecniche potrebbero dare origine a modelli murini di altre malattie del basso tratto genitale femminile.

Protocolli per l&#39;inoculazione vaginale e Prelievo dei campioni nel modello murino sperimentale di Candida Vaginite

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

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Immunology and Infection

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

32.7K Views.  Louisiana State University Health Sciences Center. Tecniche di chiave da utilizzare nella valutazione dei Candida in un modello animale sperimentale sono descritti. I metodi di permetterà una rapida raccolta di campioni vaginali e dei linfociti nei linfonodi di drenaggio lombare. Queste tecniche potrebbero dare origine a modelli murini di altre malattie del basso tratto genitale femminile.

Video: Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Metastasi sperimentale e CTL adottivi Immunoterapia modello di trasferimento del mouse

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

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Immunologia e infezioni

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Preparazione di Immunogeno mouse ipofisari per l&#39;induzione di ipofisite autoimmune sperimentale

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

A Murine Model of Group B Streptococcus Vaginal Colonization

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

A Murine Model of Group B Streptococcus Vaginal Colonization

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

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

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

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 ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Istituzione del modello murino TK-NOG dualizzato per la patogenesi del fegato associata all'HIV

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Rivelare la raccolta di hemolymph e l'inoculo di Metarhizium blastospores in Rhipicephalus MicroPlus zecche verso gli studi di patologia degli invertebrati

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

Mouse Footpad Modello di inoculazione per studiare risposte neuroinfiammatorie indotte da virus

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Uveite autoimmune sperimentale: un modello murino infiammatorio intraoculare

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

A Mouse Model of Chronic Liver Fibrosis for the Study of Biliary Atresia

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to the complex etiology and unknown pathogenesis, there are still no effective drug treatments. At present, the classic BA mouse model induced by rhesus rotavirus (RRV) is the most commonly used model for studying the pathogenesis of BA. This model is characterized by growth retardation, jaundice of the skin and mucosa, clay stools, and dark yellow urine. The histopathology shows severe liver inflammation and obstruction of the intrahepatic and extrahepatic bile ducts, which are similar to the symptoms of human BA. However, the livers of end-stage mice in this model lack fibrosis and cannot fully simulate the characteristics of liver fibrosis in clinical BA. The presented study developed a novel BA mouse model of chronic liver fibrosis by injecting 5-10 &#181;g of anti-Ly6G antibody four times, with gaps of 2 days after each injection. The results showed that some of the mice successfully formed chronic BA with typical fibrosis after the time lapse, meaning these mice represent a suitable animal model for the virus-induced liver fibrosis mechanistic study of BA and a platform for developing future BA treatments.

We established a mouse model of chronic liver fibrosis, which provides a suitable animal model for the virus-induced liver fibrosis mechanistic study of biliary atresia (BA) and a platform for future BA treatments.

Un modello murino di fibrosi epatica cronica per lo studio dell'atresia biliare

Biliary atresia (BA) is a fatal disease involving obstructive jaundice, and it is the most common indication for liver transplantation in children. Due to ...

Procedure for Swine Burn Wounding, Burn Wound Imaging, Wound Inoculation, and Biopsy Collection

To begin the procedure inside the properly arranged surgery room, place the anesthetized pig in the sternal recumbency position, and ensure all the connected lines and tubes are secured. Remove any residual hairs from the shaved area designated for wounding using hair removal cream. After sterilizing the wound sites, mark the area using a sterile burn template and a surgical skin marker.Set the burner by adjusting the set point on the control unit to 150 degrees celsius. The low set point to 145 degrees Celsius and the high set point to 155 degrees Celsius. Using a heated stainless steel blocks connected to the burn device, create a full thickness burn wound by placing the blocks on the skin for 60 seconds.Take a digital photo of the whole back of the pig, including a placard with the pig's identification time point and date. Then separately take images for each wound along with a ruler and a placard displaying the pig ID, wound ID and time point. To assess the wound microvascular perfusion in real time using laser speckle imaging or LSI, image all the wounds in a single recording using a LASCA blood perfusion imager.To measure trans epidermal water loss or TEWL, place a clean probe cover over the TEWL probe tip, which will be in contact with the wound tissue. Measure each wound five times, first in the center, followed by each corner. Then export all the readings to a spreadsheet.Perform a harmonic ultrasound or HUSD mapping by scanning the wound with an ultrasound probe from the midline or vertebral column, starting from normal skin and moving towards the lateral side of the pig over the wounds until the normal skin is again reached. Follow the scanning pattern for each wound in both B mode and tissue elastography mode. Change the annotation after each wound is imaged in both B mode and elasto mode, which is two recordings per wound.Once all the imaging is complete, cover the burn wounds individually with transparent film dressings or the test dressing. Then place a larger transparent film dressing over the entire wound area. Apply a second layer of roll gauze loosely around the entire trunk of the pig to absorb any fluid exuding from the wounds.Cover the gauze loosely with a layer of flexible elastic bandage. Cover the elastic bandage with a final layer of four inch elastic tape. After discontinuing the anesthetic gas upon completion of the bandaging, allow the pig to remain on oxygen for at least five minutes.To inoculate the burn wounds on day three post burn, disperse the inoculum across the surface of the exposed wounds using a pipette, and spread it evenly using a disposable spreader. To collect the tissue biopsies for analysis following imaging, infiltrate the area around the wound with 0.5%bupivacaine. Using a disposable scalpel with a size 10 blade, cut a 3-4 millimeter wide strip from one edge of the wound to the other, keeping small margins of normal skin on both sides.Cut a 6 millimeter punch biopsy from the wound either from the wound bed or wound edge. Achieve hemostasis after the samples are collected by gently pressing the wound with a sterile gauze. Cover the wound with a non-adherent dressing and bandage it as demonstrated previously.Analysis of the biopsy sample by hematoxylin and Eosin staining showed distortion and necrosis of all the histological skin layers. The established biofilm was further validated at day seven post inoculation by colony forming unit. Scan the electron microscopy imaging, and immunofluorescence staining.The burn wounds showed a thick biogenic membrane on the wound surface by day seven post inoculation, confirming the infection and establishment of biofilm. The wound area started to shrink by day 14, but incomplete healing to about 25%of the original wound size was observed by day 56 indicating wound chronicity. Wound chronicity and impaired wound healing were further confirmed by the TEWL showing high trans epidermal water loss at all measured time points.This was also confirmed by the suppression of the tight junctional proteins, ZO-1 and 2, and the impaired restoration of skin barrier function.