Unterstützt durch Mittel der National Institutes of Health (CA133085 zu KL) und der American Cancer Society (RSG-09-209 bis 01-TBG zu KL).

1. Experimentelle Metastasen Maus-Modell <ol><li> Am Tag vor der Tumorzelle Injektionen, Saatgut ein T75 Kolben mit bis zu 1 x 10 7 CMS4-Met-Zellen in 10 ml RPMI-Medium mit 10% Serum zu den schnell wachsenden Tumorzellen zu erhalten. Inkubieren über Nacht bei 37 ° C. </li><li> Am Tag der Injektion, entfernen Sie das Medium und spülen Sie die Zellen einmal mit PBS, dann ernten die Tumorzellen mit 0,05% Trypsin-EDTA bei 37 ° C für 5 Minuten. Stoppen der Reaktion mit 10 ml RPMI-Medium mit 10% Serum. Transfer-Zellen zu einem konischen Rohr. </li><li> Spin down die Zellen bei 1300 rpm in einem Sorvall Legend RT Zentrifuge für 3 Minuten bei Raumtemperatur. Entfernen Sie den Überstand. Resuspendieren der Zellen in 10 mL frisch 1X HBSS zu waschen, dann drehen sich wieder und resuspendieren in der gleichen Weise. </li><li> Zählen Sie die Tumorzellen auf einer Zählkammer. Verdünnen Sie die Zellen in HBSS, so dass die Zellen bei jeder Injektion erforderlich in einem Gesamtvolumen von 100 ul resuspendiert werden. </li><li> Warm die 6-8 Wochen alten BALB / c-Mäusen in einem Becherglas in warmes Wasser getaucht, um die Schwanzvene erweitern. Legen Sie die Maus auf eine Schwanzvene restrainer auf der Bank. Verwenden Sie einen Mikrospritze zu 100 ul von Tumorzellen in die laterale Schwanzvene injiziert. Sie aseptisch, um Infektionen zu vermeiden. Nach der Injektion mit leichtem Druck auf die Injektionsstelle, bis die Blutung aufgehört hat. </li><li> Bringen Sie die Maus, um den Käfig und lassen den Tumor, um zum gewünschten Zeitpunkt zu wachsen. </li></ol> 2. Zytotoxische T-Lymphozyten (CTL) adoptiven Transfer Immuntherapie <ol><li> Am Tag des Übergangs, Pipette auf und ab zu reinigende zytotoxischen T-Lymphozyten oder CTLs, hergestellt, wie im Text beschrieben resuspendieren. Übertragen Sie alle der Zellen von einer Platte auf eine 15 ml konischen Rohr, ließ nicht das Volumen überschreiten mehr als 2 / 3 des Rohres. </li><li> Legen Sie eine sterile Pasteur Pipette in die konischen Rohr und lag Lymphozyten Trennmedium oder LSM unter den Zellen, bis das Gesamtvolumen nähert 14 mL. </li><li> Achten Sie darauf, um die Schichten zu stören. Zentrifugation bei 2000 rpm für 20 Minuten bei Raumtemperatur. Verwenden Sie nicht die Bremse, um den Rotor nach dem Schleudern langsam. </li><li> Übertragen Sie die CTLs zu einem neuen 15 ml konischen Rohr. Add HBSS auf ein Volumen von ca. 10 mL zu waschen. </li><li> Zählen Sie die Zellen. Spin down bei 1300 rpm für 5 Minuten bei Raumtemperatur. Resuspendieren in HBSS auf die gewünschte Zelldichte für Injektionen, halten das Gesamtvolumen pro Injektion bei 100 ul. </li><li> Verwenden Sie den gleichen Schwanz Injektionstechnik wie zuvor in diesem Video die CTLs in tumortragenden Mäusen injiziert gesehen. </li><li> Bringen Sie die Maus, um den Käfig, und lassen Sie die CTLs mit dem Tumor zu interagieren. Mäuse sind in der Regel für die Analyse 14-21 Tage nach der CTL der Behandlung getötet. </li></ol> 3. Visualisierung von Lungenmetastasen <ol><li> Zur Visualisierung Lungenmetastasen, statt eines geopferten Maus auf dem Rücken auf einer Styropor-Platte. Pin den Beinen, um einen ungehinderten Zugang zur Luftröhre zu gewährleisten. Spray der Bauchseite mit 70% Ethanol. </li><li> Ab der Mitte des Bauches, mit einer Schere entlang der Mittellinie geschnitten, durch den Brustkorb, und bis zu den Speicheldrüsen. Expose der Luftröhre. Verwenden einer Pinzette, um Gewebe um die Luftröhre zu entfernen. </li><li> Thema einem 200 ul Pipettenspitze unter der Luftröhre. Halten Sie die Spitze mit einer Hand, heben die Luftröhre nach oben und weg vom Körper. </li><li> Drehen Sie die Plattform 180 °. Verwenden Sie eine 50 ml Spritze mit Tusche in die Lunge injiziert über die Luftröhre. Völlig aufblasen der Lunge mit Tinte, bis Sie einen starken Widerstand spüren. </li><li> Mit einer Schere in die Luftröhre geschnitten. Schieben einer Pinzette unter den Lungen und heben sie aus der Maus. Spülen Sie die Lunge kurz in einer 1L Becher Wasser. </li><li> In einem Laborabzug, Übertragung der Lunge zu einer Glasszintillationsröhrchen mit 5 ml Fekete-Lösung. Das Tumorgewebe wird als weiße Knötchen auf der schwarzen Lunge nach wenigen Minuten entstehen. Die Tumoren können nun gezählt und gespeichert werden Fekete-Lösung auf unbestimmte Zeit. </li></ol> 4. Vertreter Metastasen in Lunge Visualisierte <ol><li> Hier zeigen Lungen von Mäusen, die mit Mamma-Karzinom-Zelllinie 4T1 injiziert wurden weiße Flecken zeigt Tumoren. Mäuse, die mit 4T1-Zellen mit einem IRF8 dominant-negativen Mutante K79E transfizierten injiziert wurden, zeigen die verbesserten metastatische Potential von Tumorzellen. </li><li> Diese Bilder zeigen eine histologische Analyse der Wirksamkeit von CTL adoptiven Transfer Immuntherapie. Tumor-tragenden Mäusen mit Kochsalzlösung injiziert zeigten multiple Tumore 6 Tage später, angezeigt durch gelbe Pfeile (A). Mäuse mit Tumor-spezifischen T-Zellen injiziert, auf der anderen Seite, zeigten eine Reduktion von Tumoren (B). </li><li> In das gleiche Experiment, gibt Tusche Behandlung einen einfacheren Weg, um die Wirksamkeit von CTL adoptiven Transfer zu messen. Hier die weiße Tumorknötchen auf tumortragenden Lunge deutlich von Lunge, die erfolgreich CTL adoptiven Transfer erfahren haben, und das Zählen der Knoten ermöglicht einen Gradder Quantifizierung nicht möglich mit der Histologie. </li></ol> 5. Repräsentative Ergebnisse <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Experimental System für den experimentellen Metastasierung und CTL adaptive Transfer Immuntherapie Mausmodell. Rote Punkte zeigen, Tumorzellen und grüne Punkte zeigen CTLs. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Unterbrechung der IRF8 Funktion verbessert die metastatische Potential von Tumorzellen. Maus Mammakarzinom-Zelllinie 4T1 wurde stabil mit dem Vektor (4T1.Vector) oder Vektor-Ausdruck einer IRF8 dominant-negativen Mutante K79E (4T1.IRF8K79E) (15, 16) transfiziert. Tumorzellen wurden iv injiziert Maus seitliche Schwanzvene. Tumor-tragende Lungen wurden mit Tusche aufgeblasen, um Tumorknoten zu visualisieren. Tumorknoten sind leicht als weiße Flecken auf der schwarzen Lungengewebes Hintergrund zu sehen. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Histologische Analyse der Wirksamkeit von CTL adoptiven Transfer Immuntherapie. Maus-Sarkom-Zelllinie CMS4-Met wurde iv in Maus-laterale Schwanzvene injiziert. Drei Tage später wurden Kochsalzlösung (A) oder Tumor-spezifischer T-Zellen (B) in den Tumor-tragenden Mäusen injiziert. Lungen wurden sechs Tage nach der CTL der Behandlung mit konventionellen H &amp; E histologischen Färbung analysiert. Tumorknoten sind durch Pfeile angedeutet. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Visualisierung von Tumorknoten von Tusche Inflation. Maus-Sarkom-Zelllinie CMS4-Met wurde iv in Maus-laterale Schwanzvene injiziert. Drei Tage später wurden Kochsalzlösung (Control) oder Tumor-spezifischer T-Zellen (+ CTL) in den Tumor-tragenden Mäusen injiziert. Lungen wurden 14 Tage nach der CTL-Behandlung untersucht. Die weißen Flecken, die Tumorknoten sind, ermöglichen eine einfache Quantifizierung der Wirksamkeit von CTL Behandlung.

<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>

Die Mäuse wurden von der National Cancer Institute (Friderick, MD) erworben und befindet sich in der Medical College of Georgia Tierhaltung. Experimente und Pflege / Wohlfahrt wurden in Übereinstimmung mit behördlichen Vorschriften und genehmigten Protokoll vom Medical College of Georgia IACUC Ausschuss.

Solutions: Tusche-Lösung (17): <ol><li> Gießen Sie 150 ml destilliertem Wasser in einen 250 ml-Flasche. </li><li> 4 Tropfen Ammoniak zu dem destillierten Wasser. </li><li> 30 ml Tusche Lager (dh Sanford Black Magic Waterproof Drawing Ink 4465 Artikel 44011), um die Ammoniak-Wasser-Gemisch. </li><li> Top mit destilliertem Wasser auf ein Volumen von 200 ml. Lösung ist bereit für die Injektion. </li></ol> Fekete-Lösung (17): Fekete-Lösung ist zu bleichen Tusche aufgeblasenen tumortragenden Lunge verwendet werden, um weiße Tumorknoten aus dem schwarzen Hintergrund der normalen Gewebe unterscheiden kann. <ol><li> Add 350ml 95% EtOH auf 1L Glasflasche. </li><li> Add 150 ml destilliertem Wasser </li><li> Fügen Sie 50ml Formaldehyd </li><li> Fügen Sie 25ml Eisessig Essigsäure </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Experimentelle Metastasen Mausmodell ist eine einfache und dennoch physiologisch relevanten Metastasen-Modell. Die Tumorzellen werden intravenös (iv) in Maus-tail Venen gespritzt und besiedeln in der Lunge und damit, ähnlich wie die letzten Schritte der Metastasierung von Tumorzellen spontan: Überleben in der Zirkulation, Extravasation und Kolonialisierung in den distalen Organen. Aus therapeutischer Sicht ist die experimentelle Metastasierung Modell die einfachste und ideale Modell, da das Ziel der Therapien ist oft der Endpunkt der Metastasierung: etablierte metastasierten Tumor in der distalen Orgel. In diesem Modell werden Tumorzellen iv injiziert Maus Schwanzvenen und erlaubt zu kolonisieren und in der Lunge wachsen. Tumor-spezifische CTLs werden dann iv in den Metastasen tragenden Maus injiziert. Die Anzahl und Größe der Lungenmetastasen kann durch die Anzahl der Tumorzellen injiziert werden und die Zeit des Tumorwachstums gesteuert werden. Deshalb verschiedenen Stadien der Metastasierung, von minimal Metastasen zu ausgedehnter Metastasierung, werden modelliert. Lungenmetastasen werden durch die Inflation mit Tinte analysiert, so dass einfacher visueller Beobachtung und Quantifizierung.

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

Eine experimentelle Lungenmetastasen und CTL Immuntherapie Mausmodell für die Analyse von Tumorzellen-T-Zell-Interaktion<em&gt; In vivo</em&gt;.

Experimentelle Metastasierung und CTL adoptiven Transfer Immuntherapie Maus-Modell

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

experimental-metastasis-ctl-adoptive-transfer-immunotherapy-mouse

Research

JoVE Journal

Immunology and Infection

19.3K Aufrufe.  Medical College of Georgia. Eine experimentelle Lungenmetastasen und CTL Immuntherapie Mausmodell für die Analyse von Tumorzellen-T-Zell-Interaktion In vivo.

Serie: 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.

Protokolle für Vaginal Inokulation und Probenahme in der Experimental-Maus-Modell der Candida Vaginitis

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

Forschung

Immunologie und Infektion

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.

Die Überwindung der nicht mehr reagiert, in der experimentellen autoimmunen Enzephalomyelitis (EAE) Beständig Mausstämmen durch adoptiven Transfer-und Antigen-Challenge

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 Typ 1 Diabetes Induktion in Mäusen durch adoptiven Transfer von CD4 + T diabetogene Cells

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.

Isolierung, Reinigung und Kennzeichnung von Knochenmark der Maus Neutrophile für funktionelle Studien und Experimente adoptiven Transfer

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.

Die Beurteilung der Entwicklung der Murine Plasmazytoide dendritischen Zellen in Peyer-Patches mit Adoptiv Übertragung von hämatopoetischen Vorläuferzellen

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.

Generierung von CAR T-Zellen für die adoptive Therapie im Kontext von Glioblastoma Standardtherapie

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.

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Hoch</sup&gt; T-Zellen in immundefizienten Mäusen

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.

Stereotaktische Adoptiv Übertragung von zytotoxischen Immunzellen in murinen Modellen der Orthotopen Human Glioblastoma Multiforme Xenotransplantate

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.

Eine kontrollierte Mausmodell für neonatale Sepsis Biofilm

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.

Etablierung des dualen humanisierten TK-NOG-Mausmodells für HIV-assoziierte Leberpathogenese

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