国立衛生研究所（KLへCA133085）と米国癌学会からの補助金（KLにRSG - 09 - 209 - 01 - TBG）によってサポートされています。

1。実験的転移モデルマウス<ol><li>腫瘍細胞の注射の前日に、シードone T75フラスコ上で1 × 10 7〜急成長中の腫瘍細胞を得るために、10％血清を含むRPMI培地10mLに細胞をCMS4 - MET。 37℃で一晩インキュベート℃、 </li><li>注射の日に、培地を除去し、PBSで細胞を1回洗い流した後、37℃で0.05％トリプシン- EDTAで腫瘍細胞を収穫℃で5分間。 10％血清を含むRPMI培地10mlで反応を停止します。コニカルチューブに細胞を移す。 </li><li>室温で3分間ソーバルレジェンドRT遠心機で1300 rpmで細胞をスピンダウン。上清を取り除く。洗浄に10mLの新鮮な1X HBSSで細胞を再懸濁し、再度スピンダウンと同じように再懸濁します。 </li><li>血球計算板上で腫瘍細胞を数える。それぞれの注入に必要な細胞を100μLの全体積に再懸濁しているように、HBSSで細胞を希釈する。 </li><li>尾静脈を拡張させるために温水に浸漬ビーカーに6〜8週齢のBALB / cマウスを温める。ベンチ上で尾静脈の現像抑制剤の上にマウスを置きます。外側尾静脈内に腫瘍細胞を100μLを注入するためにマイクロシリンジを使用してください。感染を避けるために、無菌操作を使用してください。出血が停止するまで注入した後、注射部位にわずかな圧力を適用する。 </li><li>ケージにマウスを返し、そして腫瘍が目的の段階に成長することができます。 </li></ol> 2。細胞傷害性Tリンパ球（CTL）の養子移入免疫療法<ol><li>移転の日に、精製されたT -リンパ球細胞傷害性、またはCTLは、テキストで説明したように調製を再懸濁するために上下にピペッティングし。つのプレートから15 mLコニカルチューブに細胞のすべてを譲渡する、ボリュームがチューブの2 / 3以上を超えてさせることではない。 </li><li>コニカルチューブに滅菌パスツールピペットを挿入し、総容量は14 mLのに近づくまで、セルの下に、リンパ球分離媒体、またはLSMを置く。 </li><li>層を乱さないように注意してください。室温で20分間2000rpmで遠心する。スピンした後ローターを減速させるブレーキを使用しないでください。 </li><li>新しい15 mLコニカルチューブにCTLを転送する。洗浄に約10mlの体積にHBSSを追加。 </li><li>細胞を数える。室温で5分間1300 rpmでスピンダウン。 100μLに注入あたりの​​総容積を保ち、注射のために必要な細胞密度にHBSSに再懸濁します。 </li><li>担癌マウスにCTLを注入する前のこのビデオで見たのと同じテールのインジェクションの手法を使用してください。 </li><li>ケージにマウスを返す、とCTLsは腫瘍と対話することができます。マウスは、通常14-21日CTL治療後の分析のために犠牲にされています。 </li></ol> 3。肺転移の可視化<ol><li>肺転移を可視化するために、発泡スチロールのボードに、その裏に犠牲にマウスを置きます。気管に遮るもののないアクセスを確保するために足を固定。 70％エタノールで腹側を吹き付けます。 </li><li>半ば腹部から始まる、正中線に沿って、胸郭を介して、とまで唾液腺に向かってカットするはさみを使用してください。気管を公開。気管の周囲の組織を削除するには、ピンセットを使用してください。 </li><li>気管の下に200μLピペットの先端を通します。片手で先端を保持する、静かに体から気管を持ち上げて。 </li><li>プラットフォームを180 °回転させる。気管から肺にインドのインクを注入するために50mLのシリンジを使用してください。あなたが強い抵抗を感じるまで、完全にインクで肺を膨らませる。 </li><li>気管をカットするはさみを使用してください。肺の下にピンセットをスライドさせ、それらをマウスから持ち上げます。水の1Lビーカーに簡単に肺をすすいでください。 </li><li>化学煙フードでは、フェケテの液5 mLを含有するガラスシンチレーションバイアルに肺を転送する。腫瘍組織は、数分後に黒の肺に白い結節として現れるだろう。腫瘍は、現在カウントし、無期限にフェケテのソリューションに格納することができます。 </li></ol> 4。視覚による肺の代表的な転移<ol><li>ここでは、乳癌細胞株4T1を注入したマウスからの肺には腫瘍を示す白い斑点を示す。 IRF8ドミナントネガティブ変異体K79Eでトランスフェクトした4T1細胞を注入したマウスは、腫瘍細胞の拡張された転移能を示す。 </li><li>これらのイメージは、CTLの養子移入免疫療法の有効性の組織学的分析を示しています。生理食塩水を注射した担癌マウスでは、黄色の矢印（）で示される、6日後に複数の腫瘍を認めた。腫瘍特異的T細胞を注射したマウスは、その一方で、腫瘍（B）の減少を示した。 </li><li>同じ実験では、インドのインクの治療は、CTL養子免疫伝達の有効性を測定する簡単な方法を提供します。ここでは、担癌肺の白い腫瘍結節は、明らかに成功したCTLの養子移入を受けた肺からそれらを区別し、結節を数える度を可能に組織学と定量の可能性ではない。 </li></ol> 5。代表的な結果<img src="/files/ftp_upload/2077/2077fig1.jpg" alt="図1" /> 図1実験的な腫瘍転移およびCTL適応転送免疫療法のマウスモデルの実験スキーム。赤い点は、腫瘍細胞と緑のドットはCTLを示して示している。 <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="図2" /> 図2。IRF8機能の破壊は、腫瘍細胞の転移能を増強した。マウス乳癌細胞株4T1は安定してベクトル（4T1.Vector）またはIRF8ドミナントネガティブ変異体K79E（4T1.IRF8K79E）（15,16）を発現ベクターでトランスフェクトした。腫瘍細胞は、マウスの外側尾静脈に静脈注射された。腫瘍のある肺は腫瘍結節を視覚化するインドのインクで膨らませていた。腫瘍結節は、簡単に黒の肺組織の背景に白い斑点として見られている。 <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="図3" /> 図3。CTLの養子移入免疫療法の有効性の組織学的分析。マウス肉腫細胞株CMS4 - Metはマウス側面尾静脈に静脈注射した。三日後、生理食塩水（）または腫瘍特異的T細胞は、（B）担癌マウスに注入した。肺は6日、従来のH＆E組織染色により、CTL治療後に分析した。腫瘍結節は、矢印で示されている。 <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="図4" /> 図4。水墨インフレによる腫瘍結節の可視化。マウス肉腫細胞株CMS4 - Metはマウス側面尾静脈に静脈注射した。三日後、生理食塩水（コントロール）または腫瘍特異的T細胞は、（+ CTL）担癌マウスに注入した。肺は14日CTL治療後に分析した。腫瘍結節ている白い点は、、CTL治療の有効性を簡単に定量化が可能になります。

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マウスは、国立癌研究所（Friderick、MD）から購入し、ジョージア州の動物施設の医科大学に収容された。実験と医療/福祉は連邦政府の規制との契約とグルジアIACUC委員会の医科大学によって承認されたプロトコルでいた。

ソリューション： インドのインク液（17）： <ol><li> 250mlのフラスコに蒸留水150mlを注ぐ。 </li><li> 4滴を蒸留水に水酸化アンモニウムを追加。 </li><li>アンモニアと水の混合物に30ミリリットルインドのインク株式（すなわちサンフォードブラックマジック防水ドローイングインク4465アイテム44011）を追加します。 </li><li> 200mlの量を蒸留水で仕上げる。解決策は、注射の準備ができています。 </li></ol>フェケテのソリューション（17）： フェケテのソリューションは、正常組織の背景を黒から白の腫瘍結節を区別するために漂白剤墨汁-水増し担癌肺に使用されます。 <ol><li> 1Lガラス瓶に350ミリリットルの95％EtOHを追加します。 </li><li> 150ミリリットルの蒸留水を追加します。 </li><li> 50ミリリットルホルムアルデヒドを追加</li><li> 25ミリリットルの氷酸性酸を追加</li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

実験的転移のマウスモデルは、生理学的に関連はまだシンプルと転移モデルです。循環の生存率、遠位臓器の血管外漏出と植民地化：腫瘍細胞は腫瘍細胞自然転移の最後のステップに似て、それによって、マウスの尾静脈に（IV）静脈内に注射し、肺に植民地化されています。遠位臓器で確立された転移性腫瘍：治療の目標はしばしば転移のエンドポイントであるので、治療的観点から、実験的転移モデルは最も単純で理想的なモデルです。このモデルでは、腫瘍細胞は、マウスの尾静脈に静脈注射し、肺に植民地化と拡張されます。腫瘍特異的CTLは、その後、転移、担癌マウスに静脈注射されています。肺転移の数と大きさが注入される腫瘍細胞の数と腫瘍の成長の時間によって制御することができます。したがって、転移の様々な段階では、広範な転移への最小限の転移から、モデル化することができます。肺転移は、このように簡単に目視観察および定量を可能に、インクでインフレによって分析されています。

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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

腫瘍細胞- T細胞相互作用の分析のための実験的肺転移とCTL免疫療法のマウスモデル<em&gt; in vivoで</em&gt;。

実験的転移とCTLの養子移入免疫療法のマウスモデル

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

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Immunology and Infection

19.3K ビュー.  Medical College of Georgia. 腫瘍細胞- T細胞相互作用の分析のための実験的肺転移とCTL免疫療法のマウスモデル in vivoで。

ビデオ: 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.

の実験的マウスモデルにおけるプッシー接種とサンプル採取のためのプロトコルカンジダ膣炎

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

免疫・感染学

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.

移入、抗原チャレンジにより実験的自己免疫性脳脊髄炎（EAE）抵抗性マウス系統の応答がないことを克服する

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.

糖尿病CD4の養子移入+ T細胞によるマウスの加速された1型糖尿病の誘導

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.

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

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

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

マウスのリンパ管内免疫療法やワクチン

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

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.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

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.

ケアの膠芽腫の標準のコンテキストにおける養子療法のためのCAR T細胞の生成

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.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

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.

同所性同種人間神経膠腫異種移植片のマウスモデルの細胞傷害性免疫細胞の定位の養子転送

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

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.

HIV関連肝病因に対する二重ヒト化TK-NOGマウスモデルの確立

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