Suportada por concessões do National Institutes of Health (CA133085 para KL) e da American Cancer Society (RSG-09-209-01-TBG para KL).

1. Modelo Experimental rato Metástase <ol><li> No dia antes da injeção das células tumorais, uma semente T75 frasco com até 1 x 10 7 CMS4-Met células em 10 mL de meio RPMI contendo 10% de soro para obter rápido crescimento de células tumorais. Incubar durante a 37 ° C. </li><li> No dia da injeção, remova a mídia e lave as células uma vez com PBS, em seguida, colher as células tumorais com 0,05% de tripsina-EDTA a 37 ° C por 5 minutos. Parar a reação com 10 mL de meio RPMI contendo 10% de soro. Transferência de células para um tubo cônico. </li><li> Girar as células a 1300 rpm em uma centrífuga Sorvall RT Legend por 3 minutos em temperatura ambiente. Remover o sobrenadante. Ressuspender as células em 10 mL fresco 1X HBSS para lavar, então girar novamente e ressuspender da mesma maneira. </li><li> Contagem de células tumorais em um hemocitômetro. Diluir as células em HBSS, de modo que as células necessárias para cada injeção são ressuspenso em um volume total de 100 mL. </li><li> Aqueça o 6-8 semanas de idade camundongos BALB / c em um copo imersos em água morna para dilatar a veia da cauda. Posicione o mouse em um limitador veia da cauda no banco. Use uma micro para injetar 100 L de células tumorais na veia da cauda lateral. Utilizar técnicas assépticas para evitar a infecção. Após a injeção, aplique uma leve pressão no local da injeção até que o sangramento parou. </li><li> Devolver o mouse para a gaiola, e permitir que o tumor crescer para a fase desejada. </li></ol> 2. Citotóxicos T-linfócitos (CTL) Imunoterapia transferência adotiva <ol><li> No dia da transferência, pipeta cima e para baixo para voltar a suspender citotóxicos purificada linfócitos T, ou CTLs, preparado como descrito no texto. Transferência de todas as células de uma placa para um tubo cônico de 15 mL, não deixando o volume exceder mais de 2 / 3 do tubo. </li><li> Inserir um estéril Pasteur pipeta no tubo cônico e leigos meio de separação de Linfócitos, ou LSM, sob as células até que o volume total se aproxima de 14 mL. </li><li> Tenha cuidado para não perturbar as camadas. Centrifugar a 2000 rpm por 20 minutos em temperatura ambiente. Não use o freio para diminuir o rotor depois de spinning. </li><li> Transferir os CTLs para um novo tubo de 15 mL cônico. Adicionar HBSS a um volume de aproximadamente 10 mL de lavar. </li><li> Contagem das células. Girar a 1300 rpm por 5 minutos em temperatura ambiente. Ressuspender em HBSS com a densidade de células necessárias para injeções, mantendo o volume total por injeção de 100 mL. </li><li> Use a técnica de injecção mesma cauda como visto anteriormente neste vídeo para injetar o CTLs em camundongos portadores de tumor. </li><li> Devolver o mouse para a gaiola, e permitir que o CTLs para interagir com o tumor. Camundongos são geralmente sacrificados para análise 14-21 dias após o tratamento CTL. </li></ol> 3. Visualização de metástases pulmonares <ol><li> Para visualizar metástases pulmonares, coloque um rato sacrificado em sua parte traseira em uma placa de isopor. Pinos nas pernas para garantir livre acesso à traquéia. Pulverizar o lado ventral com etanol 70%. </li><li> A partir de meados da década de abdômen, use uma tesoura para cortar ao longo da linha média, através da caixa torácica, e até para as glândulas salivares. Expor a traquéia. Use uma pinça para remover os tecidos em torno da traquéia. </li><li> Um fio de 200 mL ponteira debaixo da traquéia. Segurando a ponta com uma mão, levante suavemente a traqueia para cima e longe do corpo. </li><li> Gire a plataforma de 180 °. Use uma seringa de 50 ml para injetar tinta da Índia para os pulmões através da traquéia. Completamente inflar os pulmões com tinta até sentir uma forte resistência. </li><li> Use uma tesoura para cortar a traquéia. Deslize um par de fórceps sob os pulmões e tirá-los do mouse. Enxágüe os pulmões brevemente em um Copo de 1 litro de água. </li><li> Em uma Cobertura de vapores químicos, a transferência dos pulmões para um frasco de cintilação de vidro contendo 5 mL de solução de Fekete. O tecido do tumor irá emergir como nódulos brancos sobre os pulmões pretos depois de alguns minutos. Os tumores podem ser contados e armazenados em solução Fekete indefinidamente. </li></ol> 4. Metástases representante em Pulmões visualizada <ol><li> Aqui, os pulmões de ratos que foram injetados com carcinoma mamário 4T1 linha celular mostram manchas brancas indicando tumores. Ratos que foram injetados com células 4T1 transfectadas com uma IRF8 K79E dominante negativo do mutante mostrar o potencial metastático maior de células tumorais. </li><li> Estas imagens mostram uma análise histológica da eficácia da imunoterapia transferência CTL adotivos. Tumor-rolamento camundongos injetados com solução salina mostrou tumores múltiplos seis dias mais tarde, indicado por setas amarelas (A). Camundongos injetados com células tumorais específicas T, por outro lado, mostrou uma redução de tumores (B). </li><li> No mesmo experimento, Índia tratamento de tinta dá um jeito mais simples para medir a eficácia da transferência adotiva CTL. Aqui, os nódulos tumorais branco sobre portadores de tumor nos pulmões claramente distingui-los dos pulmões, que têm sido submetidos com sucesso a transferência adotiva CTL, e contando os nódulos permite um graude quantificação não é possível com a histologia. </li></ol> 5. Resultados representante <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Figura 1" /> Figura 1. Esquema experimental para a metástase do tumor experimental e CTL transferência adaptativa modelo de camundongo imunoterapia. Pontos vermelhos indicam as células tumorais e pontos verdes indicam CTLs. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Figura 2" /> Figura 2. Rompimento de IRF8 função aumentaram o potencial metastático das células tumorais. Rato carcinoma mamário 4T1 linhagem de células foi estavelmente transfectadas com vetor (4T1.Vector) ou vector expressar uma IRF8 dominante negativo do mutante K79E (4T1.IRF8K79E) (15, 16). Células tumorais foram injetadas nas veias da cauda iv do mouse lateral. Tumor-rolamento pulmões foram inflados com tinta nanquim para visualizar nódulos tumorais. Nódulos tumorais são facilmente vistos como manchas brancas sobre o fundo preto do tecido pulmonar. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Figura 3" /> Figura 3. A análise histológica da eficácia da imunoterapia transferência CTL adotivos. Rato linha celular sarcoma CMS4-Met foi injetado nas veias do mouse iv laterais da cauda. Três dias depois, salina (A) ou células tumorais específicas T (B) foram injetadas em camundongos portadores de tumor. Pulmões foram analisados ​​seis dias após o tratamento CTL pela coloração H &amp; E convencionais histológica. Nódulos tumorais são indicados por setas. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Figura 4" /> Figura 4. Visualização de nódulos tumorais pela Índia inflação de tinta. Rato linha celular sarcoma CMS4-Met foi injetado nas veias do mouse iv laterais da cauda. Três dias depois, salina (controle) ou células tumorais específicas T (CTL +) foram injetadas em camundongos portadores de tumor. Pulmões foram analisados ​​14 dias após o tratamento CTL. As manchas brancas, que são nódulos tumorais, permitem a quantificação fácil da eficácia do tratamento CTL.

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Camundongos foram comprados do National Cancer Institute (Friderick, MD) e alojado no Medical College of Georgia instalações para animais. Experimentos e cuidado / bem-estar estavam de acordo com os regulamentos federais e um protocolo aprovado pelo Medical College of Georgia IACUC comitê.

Soluções: Índia Solução de tinta (17): <ol><li> Despeje 150 ml de água destilada para um balão de 250 ml. </li><li> Adicione 4 gotas de hidróxido de amônio à água destilada. </li><li> Adicionar 30 ml de tinta India estoque (ou seja, Sanford Black Magic Ink Drawing Waterproof 4465 item 44011) para a mistura de amônia e água. </li><li> Blusa com água destilada para um volume de 200 ml. Solução está pronta para a injecção. </li></ol> Solução Fekete (17): Fekete solução é usada para branquear nanquim-inflado portadores de tumor nos pulmões para distinguir nódulos tumorais branco do fundo preto do tecido normal. <ol><li> Adicione 350 ml EtOH 95% a garrafa de vidro 1L. </li><li> Adicionar água destilada 150ml </li><li> Adicionar 50ml de formaldeído </li><li> Adicionar 25ml de ácido glacial ácido </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Modelo de camundongo experimental de metástase é um modelo simples e metástase ainda fisiologicamente relevantes. As células tumorais são injetadas por via intravenosa (iv) nas veias da cauda do rato e colonizar nos pulmões, assim, assemelhando-se os últimos passos de metástase de células tumorais espontânea: a sobrevivência na circulação extravasamento, e colonização nos órgãos distal. Do ponto de vista terapêutico, o modelo experimental de metástase é o modelo mais simples e ideal uma vez que o alvo das terapias é freqüentemente o ponto final de metástase: estabelecida tumor metastático no órgão distal. Neste modelo, as células tumorais são injetadas nas veias da cauda iv mouse e permitido para colonizar e crescer nos pulmões. Tumor-específico CTLs são injetados iv no mouse metástases de rolamento. O número eo tamanho das metástases de pulmão pode ser controlada pelo número de células tumorais para ser injetada eo tempo de crescimento do tumor. Portanto, vários estágios de metástase, metástase do mínimo para metástases extensas, pode ser modelada. Metástases pulmonares são analisados ​​pela inflação com tinta, permitindo fácil observação visual e quantificação.

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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Uma metástase pulmonar experimental e modelo de rato CTL imunoterapia para a análise da interação de células tumorais de células-T<em&gt; In vivo</em&gt;.

Metástase experimental e CTL Adoptive Transferência Modelo rato Imunoterapia

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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Immunology and Infection

19.3K visualizações.  Medical College of Georgia. Uma metástase pulmonar experimental e modelo de rato CTL imunoterapia para a análise da interação de células tumorais de células-T In vivo.

Vídeo: 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 inoculação vaginal e coleta de amostra no Modelo Experimental de Rato Candida Vaginite

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

Imunologia e Infecção

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.

Superando indiferença em Encefalomielite autoimune experimental (EAE) linhagens mais resistentes por transferência adotiva e desafio antigênico

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 Diabetes Tipo 1 Indução em Ratos por transferência adotiva de diabetogênicos TCD4 +

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, purificação e Rotulagem de neutrófilos da medula óssea do rato de Estudos funcionais e Experimentos de transferência adotiva

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.

Avaliando o Desenvolvimento da murino células dendríticas plasmocitóides em placas de Peyer Usando transferência adoptiva de Progenitores hematopoiéticos

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.

Geração de células T para CAR adoptivo Therapy no Contexto do Glioblastoma Padrão de Atendimento

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.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

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.

Estereotáxica transferência adotiva de citotóxicas células imunes em modelos murino de humanos ortotópico 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 ...

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.

Um modelo de Mouse controlado para sepse Neonatal Polymicrobial

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.

Estabelecimento do modelo humanizado duplo do rato de TK-NOG para a patogénese HIV-associada do fígado

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