Supportato anche da finanziamenti del National Institutes of Health (CA133085 a KL) e l&#39;American Cancer Society (RSG-09-209-01-TBG a KL).

1. Modello sperimentale Metastasi mouse <ol><li> Il giorno prima della iniezioni di cellule tumorali, semi di uno T75 fiasco con un massimo di 1 x 10 7 CMS4-Met cellule in 10 ml di terreno RPMI contenente il 10% del siero per ottenere cellule tumorali in rapida crescita. Incubare per una notte a 37 ° C. </li><li> Il giorno di iniezione, rimuovere il terreno e lavare le cellule una volta con PBS e poi raccogliere le cellule tumorali con il 0,05% tripsina-EDTA a 37 ° C per 5 minuti. Arrestare la reazione con 10 ml di medium RPMI contenente 10% siero. Trasferire le cellule in una provetta conica. </li><li> Spin giù le cellule a 1300 rpm in una centrifuga Sorvall Legenda RT per 3 minuti a temperatura ambiente. Rimuovere il surnatante. Risospendere le cellule in 10 ml fresca 1X HBSS da lavare, poi girare di nuovo e risospendere nello stesso modo. </li><li> Contare le cellule tumorali in un emocitometro. Diluire le cellule in HBSS, in modo che le cellule necessarie per ogni iniezione vengono risospese in un volume totale di 100 ul. </li><li> Caldo della settimana 6-8 vecchi topi BALB / c in un bicchiere immerso in acqua calda per dilatare la vena della coda. Posizionare il mouse su un dispositivo di immobilizzazione coda vena in panchina. Utilizzare una microsiringa di iniettare 100 l di cellule tumorali nella vena della coda laterale. Utilizzare una tecnica asettica per evitare l&#39;infezione. Dopo l&#39;iniezione, applicare una leggera pressione al sito di iniezione fino a sanguinamento si è fermato. </li><li> Ritorna il mouse per la gabbia, e permettere al tumore di crescere allo stadio desiderato. </li></ol> 2. Linfociti T citotossici (CTL) Immunoterapia trasferimento adottivo <ol><li> Il giorno del trasferimento, pipetta su e giù per risospendere citotossici purificato linfociti T, o CTL, preparato come descritto nel testo. Trasferire tutte le celle da una piastra in una provetta da 15 ml conica, non lasciando che il volume di superare più di 2 / 3 del tubo. </li><li> Inserire una pipetta Pasteur sterile nel tubo conico e laici Media linfociti separazione, o LSM, sotto le cellule fino a quando il volume totale si avvicina 14 mL. </li><li> Fare attenzione a non disturbare gli strati. Centrifugare a 2000 rpm per 20 minuti a temperatura ambiente. Non utilizzare il freno per rallentare il rotore dopo la filatura. </li><li> Trasferire il CTL di un nuovo tubo 15 ml. Aggiungi HBSS ad un volume di circa 10 ml per il lavaggio. </li><li> Contare le cellule. Spin down a 1300 rpm per 5 minuti a temperatura ambiente. Risospendere in HBSS alla densità cellulare richiesto per preparazioni iniettabili, mantenendo il volume totale per iniezione a 100 ul. </li><li> Utilizzare la stessa tecnica di iniezione coda come si è visto in precedenza in questo video per iniettare il CTL in topi portatori di tumore. </li><li> Ritorna il mouse per la gabbia, e consentire al CTL di interagire con il tumore. I topi sono di solito sacrificati per l&#39;analisi di 14-21 giorni dopo il trattamento CTL. </li></ol> 3. Visualizzazione delle metastasi polmonari <ol><li> Per visualizzare metastasi polmonari, posto un topo sacrificato sulla sua schiena su una tavola di polistirolo. Pin le gambe per garantire libero accesso alla trachea. Spruzzare il lato ventrale con il 70% di etanolo. </li><li> A partire dalla metà addome, usare le forbici per tagliare lungo la linea mediana, attraverso la cassa toracica, e in alto verso le ghiandole salivari. Esporre la trachea. Usare pinze per rimuovere tessuti circostanti la trachea. </li><li> Filetto da 200 microlitri punta della pipetta sotto la trachea. Tenendo la punta con una mano, sollevare delicatamente la trachea e lontano dal corpo. </li><li> Ruotare la piattaforma 180 °. Utilizzare una siringa da 50 ml per iniettare inchiostro India nei polmoni attraverso la trachea. Completamente gonfiare i polmoni con l&#39;inchiostro fino a sentire una forte resistenza. </li><li> Usare le forbici per tagliare la trachea. Far scorrere un paio di pinze sotto i polmoni e sollevare fuori del mouse. Sciacquare i polmoni brevemente in un bicchiere da 1 litro d&#39;acqua. </li><li> In una cappa, trasferire i polmoni di una fiala di scintillazione in vetro contenente 5 ml di soluzione di Fekete. Il tessuto tumorale emergerà come noduli bianco sui polmoni nero dopo pochi minuti. I tumori possono essere contati e conservati in soluzione Fekete a tempo indeterminato. </li></ol> 4. Metastasi Rappresentante in Polmoni Visualized <ol><li> Qui, i polmoni di topi che sono stati iniettati con 4T1 carcinoma mammario linea mostrare macchie bianche che indicano i tumori. I topi che sono stati iniettati con cellule 4T1 trasfettate con un dominante negativo IRF8 K79E mutante mostrano il potenziale maggiore metastatico delle cellule tumorali. </li><li> Queste immagini mostrano una analisi istologica di efficacia del trasferimento CTL immunoterapia adottiva. Portatori di tumore nei topi iniettati con soluzione salina ha mostrato tumori multipli dopo 6 giorni, indicato da frecce gialle (A). I topi iniettati con tumore-specifiche cellule T, invece, ha mostrato una riduzione dei tumori (B). </li><li> Nello stesso esperimento, il trattamento con inchiostro di china offre un modo semplice per misurare l&#39;efficacia di CTL trasferimento adottivo. Qui, i noduli tumorali bianco su portatori di tumore dei polmoni distinguerli nettamente dai polmoni che hanno superato con successo CTL trasferimento adottivo, e contando i noduli permette un gradodi quantificazione non è possibile con istologia. </li></ol> 5. Rappresentante Risultati <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Figura 1" /> Figura 1. Schema sperimentale a fini sperimentali o metastasi tumorali e CTL modello di trasferimento di adattamento immunoterapia del mouse. I punti rossi indicano le cellule tumorali e puntini verdi indicano CTL. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Figura 2" /> Figura 2. Turbativa di IRF8 funzione migliorato il potenziale metastatico delle cellule tumorali. Carcinoma mammario del topo 4T1 linea cellulare è stata stabilmente transfettate con vettore (4T1.Vector) o vettoriale che esprime uno IRF8 dominante negativo mutante K79E (4T1.IRF8K79E) (15, 16). Le cellule tumorali sono state iniettate nelle vene iv coda di topo laterale. Portatori di tumore polmonare sono stati gonfiati con inchiostro di china di visualizzare noduli tumorali. Noduli tumorali sono facilmente visibili come macchie bianche sullo sfondo nero del tessuto polmonare. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Figura 3" /> Figura 3. Analisi istologica di efficacia del trasferimento CTL immunoterapia adottiva. Sarcoma linea di topi cellule CMS4-Met è stato somministrato per via endovenosa nelle vene coda di topo laterale. Tre giorni dopo, soluzione salina (A) o tumore-specifica delle cellule T (B) sono state iniettate in topi portatori di tumore. I polmoni sono stati analizzati sei giorni dopo il trattamento convenzionale con CTL colorazione H &amp; E istologici. Noduli tumorali sono indicati da frecce. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Figura 4" /> Figura 4. Visualizzazione di noduli tumorali dall&#39;inflazione inchiostro di china. Sarcoma linea di topi cellule CMS4-Met è stato somministrato per via endovenosa nelle vene coda di topo laterale. Tre giorni dopo, soluzione salina (controllo) o tumore-specifica delle cellule T (+ CTL) sono state iniettate in topi portatori di tumore. I polmoni sono stati analizzati quattordici giorni dopo il trattamento CTL. Le macchie bianche, che sono noduli tumorali, consentono un facile quantificazione dell&#39;efficacia del trattamento CTL.

<ol>
	<li>Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+extensive+pulmonary+metastases+in+mice+by+adoptive+transfer+of+antigen-specific+CD8(+)+CTL+reactive+against+tumor+cells+expressing+a+naturally+occurring+rejection+epitope.">Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.</a> J Immunol. 167 (8), 4286-4292 (2001).</li><li>Caldwell, S. A., Ryan, M. H., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Fas/Fas+ligand+pathway+is+important+for+optimal+tumor+regression+in+a+mouse+model+of+CTL+adoptive+immunotherapy+of+experimental+CMS4+lung+metastases.">The Fas/Fas ligand pathway is important for optimal tumor regression in a mouse model of CTL adoptive immunotherapy of experimental CMS4 lung metastases.</a> J Immunol. 171 (5), 2402-2412 (2003).</li><li>Liu, K., Caldwell, S. A., Greeneltch, K. M., Yang, D., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTL+Adoptive+Immunotherapy+Concurrently+Mediates+Tumor+Regression+and+Tumor+Escape.">CTL Adoptive Immunotherapy Concurrently Mediates Tumor Regression and Tumor Escape.</a> J Immunol. 176 (6), 3374-3382 (2006).</li><li>Yang, D., Stewart, T. J., Smith, K. K., Georgi, D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+IFN-gammaR+in+association+with+loss+of+Fas+function+is+linked+to+tumor+progression.">Downregulation of IFN-gammaR in association with loss of Fas function is linked to tumor progression.</a> International journal of cancer. 122 (2), 350-362 (2008).</li><li>Pages, F., Berger, A., Camus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+memory+T+cells,+early+metastasis,+and+survival+in+colorectal+cancer.">Effector memory T cells, early metastasis, and survival in colorectal cancer.</a> N Engl J Med. 353 (25), 2654-2666 (2005).</li><li>Galon, J., Costes, A., Sanchez-Cabo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type,+density,+and+location+of+immune+cells+within+human+colorectal+tumors+predict+clinical+outcome.">Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.</a> Science. 313 (5795), 1960-194 (2006).</li><li>Strater, J., Hinz, U., Hasel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+CD95+expression+predisposes+for+recurrence+in+curatively+resected+colon+carcinoma:+clinical+evidence+for+immunoselection+and+CD95L+mediated+control+of+minimal+residual+disease.">Impaired CD95 expression predisposes for recurrence in curatively resected colon carcinoma: clinical evidence for immunoselection and CD95L mediated control of minimal residual disease.</a> Gut. 54 (5), 661-665 (2005).</li><li>Camus, M., Tosolini, M., Mlecnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordination+of+intratumoral+immune+reaction+and+human+colorectal+cancer+recurrence.">Coordination of intratumoral immune reaction and human colorectal cancer recurrence.</a> Cancer research. 69 (6), 2685-2693 (2009).</li><li>Dudley, M. E., Wunderlich, J. R., Yang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer+therapy+following+non-myeloablative+but+lymphodepleting+chemotherapy+for+the+treatment+of+patients+with+refractory+metastatic+melanoma.">Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.</a> J Clin Oncol. 23 (10), 2346-2357 (2005).</li><li>Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+inhibit+T-cell+activation+by+depleting+cystine+and+cysteine.+Cancer+research.">Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer research.</a> 70 (1), 68-77 (2010).</li><li>Nagaraj, S., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+escape+mechanism+governed+by+myeloid-derived+suppressor+cells.">Tumor escape mechanism governed by myeloid-derived suppressor cells.</a> Cancer research. 68 (8), 2561-2563 (2008).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nature reviews. 9 (4), 274-284 (2009).</li><li>Heijstek, M. W., Kranenburg, O., Rinkes, B. o. r. e. l., H, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+colorectal+cancer+and+liver+metastases.">Mouse models of colorectal cancer and liver metastases.</a> Digestive surgery. 22 (1-2), 1-2 (2005).</li><li>Yang, D., Ud Din, N., Browning, D. D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+lymphotoxin+beta+receptor+with+tumor-specific+T+lymphocytes+for+tumor+regression.">Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.</a> Clin Cancer Res. 13 (17), 5202-5210 (2007).</li><li>Yang, D., Thangaraju, M., Browning, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN+Regulatory+Factor+8+Mediates+Apoptosis+in+Nonhemopoietic+Tumor+Cells+via+Regulation+of+Fas+Expression.">IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression.</a> J Immunol. 179 (7), 4775-4782 (2007).</li><li>Yang, D., Thangaraju, M., Greeneltch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+IFN+regulatory+factor+8+by+DNA+methylation+is+a+molecular+determinant+of+apoptotic+resistance+and+metastatic+phenotype+in+metastatic+tumor+cells.">Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells.</a> Cancer research. 67 (7), 3301-3309 (2007).</li><li>Wexler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+experimental+pulmonary+metastases.">Accurate identification of experimental pulmonary metastases.</a> Journal of the National Cancer Institute. 36 (4), 641-645 (1966).</li></ol>

Topi sono stati acquistati presso il National Cancer Institute (Friderick, MD) e ospitato nel Medical College of Georgia stabulario. Esperimenti e cura / benessere erano d&#39;accordo con le normative federali e un protocollo approvato dal Medical College of Georgia IACUC commissione.

Soluzioni: India Soluzione d&#39;inchiostro (17): <ol><li> Versare 150 ml di acqua distillata in un pallone da 250 ml. </li><li> Aggiungere 4 gocce di idrossido di ammonio in acqua distillata. </li><li> Aggiungere 30 ml di inchiostro magazzino India (cioè Sanford Black Magic impermeabile Ink Drawing 4465 Item 44011) per la miscela di ammoniaca e acqua. </li><li> Top con acqua distillata per un volume di 200 ml. Soluzione è pronta per l&#39;iniezione. </li></ol> Soluzione di Fekete (17): Fekete soluzione è usata per candeggina India ink-gonfiato portatori di tumore dei polmoni di distinguere noduli tumorali bianco sullo sfondo nero dei tessuti normali. <ol><li> Aggiungere 350ml EtOH 95% a bottiglia di vetro da 1 litro. </li><li> Aggiungere 150 ml di acqua distillata </li><li> Aggiungere 50 ml di formaldeide </li><li> Aggiungere 25 ml di acido glaciale acida </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Sperimentale modello murino metastasi è un modello metastasi semplice ma fisiologicamente rilevanti. Le cellule tumorali vengono iniettate per via endovenosa (iv) nelle vene coda di topo e colonizzare nei polmoni, quindi, che assomiglia le ultime fasi della metastasi delle cellule tumorali spontanea: la sopravvivenza nel stravaso circolazione, e la colonizzazione negli organi distali. Da un punto di vista terapeutico, il modello sperimentale metastasi è il modello più semplice e ideale, poiché l&#39;obiettivo delle terapie è spesso il punto finale di metastasi: stabilito tumore metastatico nell&#39;organo distale. In questo modello, le cellule tumorali vengono iniettate nelle vene iv coda di topo e ha permesso di colonizzare e crescere nei polmoni. CTL tumore-specifici sono poi iniettate iv in metastasi portante del mouse. Il numero e le dimensioni delle metastasi polmonari possono essere controllati in base al numero delle cellule tumorali da iniettare e il momento della crescita tumorale. Pertanto, le varie fasi di metastasi, da metastasi minimo metastasi estese, possono essere modellati. Metastasi polmonari vengono analizzati con l&#39;inflazione con l&#39;inchiostro, permettendo così più facile l&#39;osservazione visuale e la quantificazione.

Watch this Scientific Journal Video about Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model at JoVE.com

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Una metastasi al polmone sperimentale e CTL modello murino immunoterapia per l&#39;analisi delle cellule tumorali interazione cellule-T<em&gt; In vivo</em&gt;.

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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JoVE Journal

Immunology and Infection

19.3K Views.  Medical College of Georgia. Una metastasi al polmone sperimentale e CTL modello murino immunoterapia per l'analisi delle cellule tumorali interazione cellule-T In vivo.

Video: Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

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

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

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

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

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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Immunologia e infezioni

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Superare blocco nel encefalomielite autoimmune sperimentale (EAE) ceppi di topi resistenti dal trasferimento adottivo e uno stimolo antigenico

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolamento, la purificazione e della dicitura del mouse Bone Marrow neutrofili per studi funzionali ed Esperienze trasferimento adottivo

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Valutare lo sviluppo delle cellule murine plasmocitoidi dendritiche in placche del Peyer Utilizzando trasferimento adottivo di ematopoietiche Progenitori

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Trasferimento adottivo stereotactic di citotossici cellule immunitarie nei modelli murini di xenotrapianti Multiforme di Glioblastoma umano ortotopico

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Un modello di Mouse controllata per sepsi polimicrobica neonatale

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

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 ...