국립 보건원 (KL에 CA133085)와 미국 암 협회에서 부여 (KL에 RSG - 09 - 209-01 - TBG) 지원.

1. 실험 전이 마우스 모델 <ol><li> 당일 전에 종양 세포 주사, 최대 1과 씨앗 하나 T75 플라스크 X 10 7 CMS4 - 메트 빠르게 성장하는 종양 세포를 얻기 위해 혈청 10 %를 포함하는 RPMI 매체의 10 ML에 세포를. 37 밤새 품어 ° C. </li><li> 주사 당일, 미디어를 제거하고 PBS로 세포를 한 번 씻어 후 37 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산)으로 종양 세포를 수확 ° C를 5 분. 10 % 혈청이 포함된 RPMI 매체 10 ML로 반응을 중지합니다. 원뿔 튜브에 세포를 전송합니다. </li><li> 상온에서 3 분 Sorvall 전설 RT의 원심 분리기에서 1,300 rpm으로 세포를 스핀 다운. 뜨는을 제거합니다. 세차 10 ML 신선한 1X HBSS에있는 세포를 Resuspend, 다시 스핀 다운과 같은 방식으로 resuspend. </li><li> hemocytometer에서 종양 세포를 계산합니다. 각각의 주사에 필요한 세포 100 μL의 총 볼륨 resuspended되도록, HBSS에있는 세포를 희석. </li><li> 꼬리 정맥을 팽창하기 위해 따뜻한 물에 잠겨 비커에 6~8주 이전 BALB / C 마우스를 따뜻하게. 벤치에 꼬리 정맥 restrainer에 마우스를 놓습니다. 측면 꼬리 정맥에 종양 세포 100 μL를 주입하는 microsyringe을 사용합니다. 감염을 피하기 위해 무균 기술을 사용합니다. 출혈이 멈출 때까지 주입 후, 주사 사이트에 약간의 압력을 적용합니다. </li><li> 새장에 마우스를 반환하고, 종양이 원하는 단계로 성장할 수 있습니다. </li></ol> 2. 세포 독성 T - 림프구 (CTL) 입양 전송 Immunotherapy <ol><li> 송금 당일, 정화 T - lymphocytes 세포 독성, 또는 CTLs, 텍스트에서 설명한대로 준비를 resuspend하기 위해 아래 피펫합니다. 볼륨이 더 튜브의 2 / 3 이상을 초과 두지 않을, 15 ML 원뿔 관 한 접시의 세포를 모두 전송합니다. </li><li> 원뿔 관에 멸균 파스퇴르 피펫을 넣고 총 부피 14 ML을 nears 때까지 세포에서 림프구 분리 매체, 또는 LSM 있다고 믿는다. </li><li> 레이어를 방해하지 않도록주의하십시오. 실온에서 20 분 동안 2,000 rpm으로 원심 분리기. 회전 후 회전자을 늦추기 위해 브레이크를 사용하지 마십시오. </li><li> 새로운 15 ML 원뿔 관에 CTLs을 전송합니다. 씻어 약 10 ML의 볼륨 HBSS를 추가합니다. </li><li> 세포를 계산합니다. 실온에서 5 분간 1300 rpm으로 스핀 다운. 100 μL에 주입 당 총 볼륨을 유지, 주사에 필요한 세포 밀도에 HBSS에 Resuspend. </li><li> 종양 - 베어링 생쥐에 CTLs를 삽입하는 이전이 비디오에서 볼 같은 꼬리 사출 기술을 사용합니다. </li><li> 새장에 마우스를 반환하고, CTLs이 종양과 상호 작용 수 있습니다. 마우스는 일반적으로 14-21일 CTL 치료 후 분석을 위해 희생하고 있습니다. </li></ol> 3. 룽 Metastases의 시각화 <ol><li> 폐 metastases를 시각화하기 위해 스티로폼 보드의 뒷면에 희생 마우스를 놓으십시오. 기관에 가리지 접근을 보장하기 위해 다리를 핀. 70 % 에탄올로 복부 측면을 스프레이. </li><li> 중간 복부에서 시작, 침샘쪽으로 최대 ribcage 통해 정중선을 따라 잘라, 및 가위를 사용합니다. 기관을 폭로. 기관 주위 조직을 제거하는 집게를 사용하십시오. </li><li> 기관 밑에 200 μL 피펫 팁을 실. 한손으로 팁을 개최하는 것은 부드럽게 신체의기도를 들어갑니다. </li><li> 플랫폼 180도 회전 °. 기관을 통해 폐로 인도 잉크를 주입하기 위해 50 ML의 주사기를 사용합니다. 당신은 강한 저항을 느낄 때까지 완전히 잉크와 폐가 팽창. </li><li> 기관를 잘라 가위를 사용합니다. 폐 아래 포셉 한 쌍의 슬라이드 그들을 마우스 밖으로 리프트. 물 1L 비커에 간단히 폐가 린스. </li><li> 화학 펌 모자, Fekete의 솔루션의 5 ML를 포함하는 유리 섬광 약병에 폐을 전송하기만하면됩니다. 종양 조직은 몇 분 후에 검은 폐에 흰색 nodules로 등장할 것입니다. 종양은 이제 계산하고 무기한 Fekete의 솔루션에 저장할 수 있습니다. </li></ol> 4. 시각 폐에 대표 Metastases <ol><li> 여기, 내유 암종 세포 라인 4T1로 주입했다 생쥐의 폐 종양을 나타내는 흰 반점을 보여줍니다. IRF8 지배 - 부정적인 돌연변 K79E와 transfected 4T1 세포로 주입했다 생쥐는 종양 세포의 향상된 전이성 잠재력을 보여줍니다. </li><li> 이러한 이미지는 CTL 입양 전송 immunotherapy의 효능 histological 분석을 보여줍니다. 식염수에 주입 종양 베어링 마우스는 노란색 화살표 (A)로 표시, 6 일 후 여러 종양을 보여주었다. 종양 특정 T 세포로 주입 생쥐는 반면에, 종양의 감소 (B)를 보여주었다. </li><li> 같은 실험에서, 인도 잉크 치료 CTL 입양 전송의 효능을 측정하는 간단한 방법을 제공합니다. 여기, 종양 - 베어링 폐에 하얀 종양의 nodules 확실히 성공적으로 CTL 입양 전송받은, 그리고 nodules을 계산해야 폐가에서 그들을 구분하는 것은 학위 허용조직학과 quantitation의 수 없습니다. </li></ol> 5. 대표 결과 <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="그림 1" /> 그림 1. 실험 종양 전이 및 CTL 적응 전송 immunotherapy 마우스 모델 실험 계획. 레드 점들은 종양 세포와 녹색 점들은 CTLs를 나타냅니다 나타냅니다. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="그림 2" /> 그림 2. IRF8 기능의 장애는 종양 세포의 전이성 잠재력을 향상. 마우스 내유 암종 세포 라인 4T1은 안정 벡터 (4T1.Vector) 또는 벡터 IRF8 지배 - 부정적인 돌연변이 K79E (4T1.IRF8K79E)를 (15, 16) 표현과 transfected했다. 종양 세포는 마우스 측면 꼬리 정맥에 주입 IV했다. 종양 베어링 폐에 종양 nodules을 시각화하기 위해 인도 잉크로 비정상적으로 증가했다. 종양 nodules 쉽게 검은 폐 조직 배경에 흰색 반점으로 볼 수 있습니다. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="그림 3" /> 그림 3. CTL 입양 전송 immunotherapy의 효능 Histological 분석. 마우스 육종 세포 라인 CMS4 - 메트는 마우스 측면 꼬리 정맥에 주입 IV를했다. 사흘 후, 호수 (A) 또는 종양 특정 T 세포 (B)은 종양 베어링 생쥐에 주입했다. 폐 6 일 기존의 H &amp; E histological 얼룩하여 CTL 치료 후 분석했다. 종양 nodules는 화살표로 표시됩니다. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="그림 4" /> 그림 4. 인도 잉크 인플레이션에 의해 종양 nodules의 시각화. 마우스 육종 세포 라인 CMS4 - 메트는 마우스 측면 꼬리 정맥에 주입 IV를했다. 사흘 후, 생리 (컨트롤) 또는 종양 특정 T 세포는 (+ CTL) 종양 베어링 생쥐에 주입했다. 폐가 십사일 CTL 치료 후에 분석했다. 종양 nodules있는 하얀 명소, CTL 치료의 효능에 대해 쉽게 부량 수 있습니다.

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생쥐는 국립 암 연구소 (Friderick, MD)에서 구입 조지아 동물 시설의 메디컬 칼리지 안에 보관되어 있었다. 실험 및 의료 / 복지 연방 규정 계약 및 조지아 IACUC위원회의 메디컬 칼리지에 의해 승인된 프로토콜되었습니다.

솔루션 : 인도 잉크 솔루션 (17) : <ol><li> 250 ML 플라스크에 증류수 150 ML 하거라. </li><li> 증류수 4 방울의 암모늄 수산화을 추가합니다. </li><li> 암모니아와 물을 혼합 30 ML 인도 잉크 주식 (즉, 샌포드 블랙 매직 방수 그리기 잉크 4,465 44,011 항목) 추가합니다. </li><li> 200 ML의 볼륨에 증류수와 떨어져 톱. 솔루션 주입을위한 준비가 된 것입니다. </li></ol> Fekete의 솔루션 (17) : Fekete의 솔루션은 일반적인 조직의 검은 배경에서 흰색 종양의 nodules을 구분하기 위해 표백제를 인도 잉크 높게 종양 베어링 폐에하는 데 사용됩니다. <ol><li> 1L 유리병에 350ml 95 % EtOH를 추가합니다. </li><li> 150ml 증류수를 추가 </li><li> 50ml 포름 알데히드 추가 </li><li> 25ml 빙하 산성 산성 추가 </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

실험 전이 마우스 모델은 생리학 관련 아직 간단하고 전이 모델입니다. 종양 세포는 마우스 꼬리 정맥에 (4) 정맥 주사하고 종양 세포 자발적인 전이의 마지막 단계를 닮은, 따라서, 폐 이식과 같습니다 말초 장기의 순환, 넘쳐 흐름과 식민지에서 생존합니다. 말초 기관에서 설립 전이성 종양 : 요법의 목표는 종종 전이의 끝 지점이기 때문에보기의 치료 시점에서 실험적 전이 모델은 가장 간단한 이상적 모델입니다. 이 모델에서, 종양 세포는 마우스 꼬리 정맥에 주입 IV 및 폐의 이식과 성장을 사용할 수 있습니다. 종양 특정 CTLs은 다음 metastases 베어링 마우스에 IV를 주입하고 있습니다. 폐 metastases의 번호와 크기는 주입으로 종양 세포의 수와 종양 성장의 시간에 의해 제어하실 수 있습니다. 따라서, 전이의 여러 단계, 광범위한 전이에 최소한의 전이에서 모델 수 있습니다. 룽 metastases 따라서 쉽게 시각 관측 및 부량를 허용, 잉크와 인플레이션에 의해 분석됩니다.

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

종양 세포 - T 세포 상호 작용의 분석을 위해 실험적인 폐 전이와 CTL immunotherapy 마우스 모델<em&gt; 생체내에</em&gt;.

실험 전이 및 CTL 입양 전송 Immunotherapy 마우스 모델

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

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JoVE Journal

Immunology and Infection

19.3K 조회수.  Medical College of Georgia. 종양 세포 - T 세포 상호 작용의 분석을 위해 실험적인 폐 전이와 CTL immunotherapy 마우스 모델 생체내에.

비디오: 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.

의 실험 마우스 모델에서 질 접종 및 샘플 수집을위한 프로토콜 칸디다 질염

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

연구

면역학 및 감염병학

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.

입양 이전 및 Antigenic 챌린지에 의해 실험적 면역 Encephalomyelitis (EAE) 모성 마우스 변종에 응답하지 않는 문제를 극복

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.

Diabetogenic CD4의 입양 전송하여 마우스의 가속 제 1 형 당뇨병 유도 + T 세포

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.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

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.

조혈 전구 세포의 입양 전송을 사용 Peyer의 패치에 쥐과 Plasmacytoid 수지상 세포의 개발을 평가

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.

배려의 아교 모세포종 표준의 맥락에서 입양 치료에 대한 자동차 T 세포의 생성

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.

이펙터 CD4의 입양 전송하여 쥐과 장내 염증의 유도<sup&gt; +</sup&gt; CD45RB<sup&gt; 높은</sup&gt; 면역 결핍 마우스에게 T 세포

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.

정위 적 입양 전송 Orthotopic 인간 세포종 님 Xenografts Murine 모델에서 세포 독성 면역 세포의

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.

신생아 Polymicrobial 패 혈 증에 대 한 제어 마우스 모델

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.

HIV 관련 간 병인을 위한 이중 인간화 TK-NOG 마우스 모델 구축

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