Apoyado por becas de los Institutos Nacionales de Salud (CA133085 a KL) y la Sociedad Americana del Cáncer (RSG-09-209-01-TBG KL).

1. Metástasis modelo experimental de ratón <ol><li> El día antes de la inyección de células tumorales, las semillas de un frasco T75 con un máximo de 1 x 10 7 CMS4-Met células en 10 ml de medio RPMI con 10% de suero para obtener rápido crecimiento de las células tumorales. Incubar toda la noche a 37 ° C. </li><li> En el día de la inyección, se elimina el medio y lavar las células una vez con PBS, luego de la cosecha de las células tumorales con el 0,05% de tripsina-EDTA a 37 ° C durante 5 minutos. Detener la reacción con 10 ml de medio RPMI con 10% de suero. La transferencia de células a un tubo cónico. </li><li> Centrifugar las células a 1300 rpm en una centrífuga Sorvall Legend TA durante 3 minutos a temperatura ambiente. Eliminar el sobrenadante. Resuspender las células en 10 ml de 1X HBSS fresca para lavar, y luego girar de nuevo y volver a suspender la misma manera. </li><li> Contar con las células tumorales en un hemocitómetro. Diluir las células en HBSS, para que las células necesarias para cada inyección se resuspenden en un volumen total de 100 mL. </li><li> Caliente las 6-8 semanas de edad ratones Balb / c en un vaso sumergido en agua caliente para dilatar la vena de la cola. Coloque el ratón sobre un limitador de vena de la cola en el banco. El uso de una microjeringa para inyectar 100 L de células tumorales en la vena lateral de la cola. Utilice una técnica aséptica para evitar la infección. Después de la inyección, aplicar una ligera presión al sitio de la inyección hasta que la hemorragia se ha detenido. </li><li> Devolver el ratón de la jaula, y permitir el crecimiento del tumor a la etapa deseada. </li></ol> 2. Los linfocitos T citotóxicos (CTL) Inmunoterapia transferencia adoptiva <ol><li> En el día de la transferencia, la pipeta hacia arriba y abajo para volver a suspender citotóxica de los linfocitos T purificados, o CTL, preparado como se describe en el texto. La transferencia de todas las células de una placa a un tubo cónico de 15 ml, no dejar que el volumen exceda de más de 2 / 3 del tubo. </li><li> Inserte una pipeta Pasteur estéril en el tubo cónico y laicos medio de separación de linfocitos, o LSM, en las células hasta que el volumen total se acerca a 14 ml. </li><li> Tenga cuidado de no molestar a las capas. Centrifugar a 2000 rpm durante 20 minutos a temperatura ambiente. No use el freno para frenar el rotor tras hacer un trompo. </li><li> Transferir el CTL para un nuevo tubo de 15 mL cónico. Añadir HBSS a un volumen de aproximadamente 10 ml de lavar. </li><li> Contar las células. Centrifugar a 1300 rpm durante 5 minutos a temperatura ambiente. Volver a suspender en HBSS a la densidad celular necesaria para la inyección, manteniendo el volumen total por la inyección de 100 mL. </li><li> Utilice la técnica de inyección de cola igual que vimos anteriormente en este vídeo para inyectar el CTL en ratones portadores de tumores. </li><li> Devolver el ratón de la jaula, y permitir que el CTL para interactuar con el tumor. Los ratones suelen ser sacrificados para el análisis de 14-21 días después del tratamiento CTL. </li></ol> 3. Visualización de las metástasis pulmonares <ol><li> Para visualizar las metástasis pulmonares, lugar de un ratón sacrificado en su parte posterior en una tabla de espuma de poliestireno. Pin de las piernas para garantizar el acceso sin obstáculos a la tráquea. Rocíe la parte ventral con el 70% de etanol. </li><li> A partir de la mitad del abdomen, use tijeras para cortar a lo largo de la línea media, a través de la caja torácica, y hasta a las glándulas salivales. Exponer la tráquea. El uso de fórceps para extraer los tejidos que rodean la tráquea. </li><li> Enhebrar una punta de pipeta 200 l por debajo de la tráquea. Sujetando la punta con una mano, levante suavemente la tráquea y del cuerpo. </li><li> Gire la plataforma 180 º. Use una jeringa de 50 ml para inyectar tinta china en los pulmones a través de la tráquea. Inflar los pulmones por completo con tinta hasta que sienta una fuerte resistencia. </li><li> Use las tijeras para cortar la tráquea. Deslizar un par de pinzas en los pulmones y sacarlos del ratón. Enjuague los pulmones brevemente en un vaso de 1 litro de agua. </li><li> En una cabina de humos química, la transferencia de los pulmones a un vial de centelleo de vidrio que contiene 5 ml de solución de Fekete. El tejido del tumor va a surgir como nódulos blancos en los pulmones negro después de unos minutos. Los tumores pueden ahora ser contados y almacenados en una solución de Fekete de forma indefinida. </li></ol> 4. Las metástasis en los pulmones representante visualizados <ol><li> Aquí, los pulmones de los ratones que fueron inyectados con carcinoma mamario 4T1 línea celular muestran manchas blancas que indican los tumores. Los ratones que fueron inyectados con células 4T1 transfectadas con un IRF8 dominante negativo K79E mutantes muestran el mayor potencial metastásico de las células tumorales. </li><li> Estas imágenes muestran un análisis histológico de la eficacia de la inmunoterapia CTL transferencia adoptiva. Portadores de tumores de ratones inyectados con solución salina mostró múltiples tumores 6 días más tarde, indicado por las flechas amarillas (A). Los ratones inyectados con células T específicas del tumor, por su parte, mostró una reducción de los tumores (B). </li><li> En el mismo experimento, el tratamiento de la tinta china ofrece una manera más sencilla de medir la eficacia de la transferencia adoptiva CTL. Aquí, los nódulos tumorales en blanco con tumores pulmones distinguirlos claramente de los pulmones que han superado con éxito la transferencia adoptiva CTL, y contando los nódulos permite un gradode cuantificación no es posible con la histología. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Figura 1" /> Figura 1. Plan experimental para la metástasis de tumores experimentales y la transferencia de adaptación CTL modelo murino de inmunoterapia. Los puntos rojos indican las células tumorales y puntos verdes indican CTL. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Figura 2" /> Figura 2. La alteración de la función IRF8 aumentado el potencial metastásico de las células tumorales. Carcinoma mamario de ratón 4T1 línea celular se transfectadas establemente con el vector (4T1.Vector) o vector de expresión de un IRF8 dominante negativo mutante K79E (4T1.IRF8K79E) (15, 16). Las células tumorales se inyectaron en las venas iv lateral de la cola del ratón. Portadores del tumor pulmones se inflaron con tinta china para visualizar los nódulos tumorales. Nódulos tumorales son fácilmente visibles como manchas blancas sobre el fondo negro del tejido pulmonar. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Figura 3" /> Figura 3. El análisis histológico de eficacia de la inmunoterapia CTL transferencia adoptiva. Sarcoma de ratón línea celular CMS4-Met fue inyectado en las venas del ratón iv lateral de la cola. Tres días más tarde, la solución salina (A) o un tumor específico de células T (B) se inyectaron en los ratones portadores de tumores. Los pulmones se analizaron seis días después del tratamiento CTL mediante la tinción H &amp; E convencional histológico. Nódulos tumorales se indican con flechas. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Figura 4" /> Figura 4. Visualización de los nódulos tumorales por la inflación tinta china. Sarcoma de ratón línea celular CMS4-Met fue inyectado en las venas del ratón iv lateral de la cola. Tres días más tarde, la solución salina (control) o un tumor específico de células T (+ CTL) fueron inyectadas en los ratones portadores de tumores. Los pulmones se analizaron catorce días después del tratamiento CTL. Las manchas blancas, que son los nódulos tumorales, permite una sencilla cuantificación de la eficacia del tratamiento CTL.

<ol>
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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>

Los ratones fueron adquiridos por el Instituto Nacional del Cáncer (Friderick, MD) y ubicado en la Facultad de Medicina de la instalación para animales Georgia. Los experimentos y la atención / bienestar estaban de acuerdo con las regulaciones federales y un protocolo aprobado por el Colegio Médico de Georgia IACUC comité.

Soluciones: India Solución de tinta (17): <ol><li> Vierta 150 ml de agua destilada en un matraz de 250 ml. </li><li> Añadir 4 gotas de hidróxido de amonio en el agua destilada. </li><li> Añadir 30 ml de tinta India de acciones (es decir, Sanford Negro Magic dibujo de la tinta a prueba de agua Artículo 44 011 4465) a la mezcla de amoniaco y agua. </li><li> Complete con agua destilada hasta un volumen de 200 ml. Solución está lista para la inyección. </li></ol> Solución de Fekete (17): Fekete solución se utiliza para blanquear la tinta china-inflados con tumores pulmones para distinguir nódulos blancos tumorales del fondo negro de los tejidos normales. <ol><li> Añadir 350 ml de EtOH 95% de botella de vidrio de 1L. </li><li> Añadir 150 ml de agua destilada </li><li> Añadir 50 ml de formaldehído </li><li> Añadir 25 ml de ácido ácido glacial </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Modelo experimental de ratón metástasis es un modelo de metástasis simple y fisiológicamente relevantes. Las células tumorales se inyectan por vía intravenosa (iv) en las venas de la cola de ratón y colonizar en los pulmones, por lo tanto, semejante a los últimos pasos de la metástasis de células tumorales espontáneas: la supervivencia en la circulación, la extravasación y la colonización en los órganos distales. Desde el punto de vista terapéutico, el modelo experimental de metástasis es el modelo más simple y es ideal ya que el objetivo de las terapias es a menudo el punto final de la metástasis: tumor metastásico establecido en el órgano distal. En este modelo, las células tumorales se inyectan en las venas iv cola de ratón y se deja colonizar y crecer en los pulmones. Tumor-específica CTL se inyecta vía intravenosa en el ratón portador de metástasis. El número y tamaño de las metástasis pulmonares pueden ser controlados por el número de células tumorales a inyectar y el tiempo de crecimiento del tumor. Por lo tanto, las distintas etapas de la metástasis, las metástasis mínima a la metástasis extensa, se puede modelar. Las metástasis pulmonares son analizados por la inflación con la tinta, permitiendo así más fácil la observación visual y la cuantificación.

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 metástasis de pulmón y el modelo experimental de ratón para CTL inmunoterapia análisis de la interacción de las células tumorales las células-T<em&gt; In vivo</em&gt;.

La metástasis experimentales y CTL Modelo inmunoterapia adoptiva de transferencia de ratón

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

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Immunology and Infection

19.3K vistas.  Medical College of Georgia. Una metástasis de pulmón y el modelo experimental de ratón para CTL inmunoterapia análisis de la interacción de las células tumorales las células-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.

Protocolos para la inoculación dentro de la vagina y toma de muestras en el modelo experimental de ratón de Candida Vaginitis

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

Investigación

Inmunología e Infección

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.

Superar la falta de respuesta en encefalomielitis autoinmune experimental (EAE) cepas de ratones resistentes a través de transferencia adoptiva y la estimulación antigénica

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.

Tipo 1 Diabetes inducción acelerada en ratones por transferencia adoptiva de células CD4 + células T diabetogénico

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.

Aislamiento, Purificación y Etiquetado de neutrófilos de médula ósea de ratón de Estudios funcionales y experimentos de transferencia adoptivos

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.

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

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.

Generación de células T CAR para realizar la terapia adoptiva en el contexto de Glioblastoma estándar de cuidado

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.

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

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.

Estereotáctica transferencia adoptiva de células inmunes citotóxicas en modelos murinos de xenoinjertos de Glioblastoma Multiforme de Orthotopic humanos

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 modelo de ratón controlado para la Sepsis Neonatal polimicrobiana

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.

Establecimiento del modelo de ratón TK-NOG humanizado dual para la patogénesis hepática asociada al VIH

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