Ulusal Sağlık Enstitüleri (KL için CA133085) ve Amerikan Kanser Derneği (KL RSG-09-209-01-TBG) hibe ile desteklenir.

1. Deneysel metastaz Fare Modeli <ol><li> Gün önce tümör hücresi enjeksiyonları, 1&#39;e kadar tohum T75 balon x 10 7 CMS4 Met hücreleri RPMI orta hızlı büyüyen tümör hücreleri elde etmek için% 10 serum içeren 10 ml. 37 o gece inkübe ° C </li><li> Enjeksiyon gününde, orta kaldırmak ve hücreler PBS ile bir kez yıkayın, sonra 37 0.05% tripsin-EDTA ile tümör hücrelerinin hasat ° C 5 dakika. 10 ml% 10 serum içeren RPMI orta ile reaksiyon durdurun. Konik bir tüp hücreleri aktarın. </li><li> Oda sıcaklığında 3 dakika 1300 rpm&#39;de Sorvall Legend RT santrifüj hücreleri aşağı spin. Süpernatantı. Yıkamak için 10 ml taze 1X HBSS hücrelerin yeniden süspanse edin, sonra tekrar aşağı spin ve aynı şekilde tekrar süspansiyon haline getirin. </li><li> Bir hemasitometre tümör hücreleri saymak. Böylece her enjeksiyon için gerekli hücrelerin toplam hacmi 100 mcL yeniden süspanse, HBSS hücrelerin sulandırınız. </li><li> Kuyruk ven genişletmek için ılık suya batırılmış beher 6-8 haftalık BALB / c farelerde ısıtın. Fare, bankta bir kuyruk ven süzgeç koyun. 100 mcL tümör hücrelerinin lateral kuyruk damar içine enjekte etmek için bir mikroenjektör kullanın. Bulaşmasını önlemek için aseptik teknik kullanın. Kanama durduktan kadar enjeksiyondan sonra enjeksiyon yerinde hafif bir basınç uygulayın. </li><li> Fare kafesine geri dönün ve istenilen aşamada tümör büyümesine izin. </li></ol> 2. Sitotoksik T-Lenfosit (KVK) Kabul edilmiş Transferi İmmünoterapi <ol><li> Transfer gününde, saflaştırılmış T-lenfositleri sitotoksik ya da CTL, metinde açıklandığı gibi hazırlanmış tekrar süspansiyon pipet ve aşağı yukarı. Hacmi daha fazla tüpün 2 / 3 &#39;ünden fazla izin vermeyen, tüm hücreleri bir plaka 15 ml konik tüp transferi. </li><li> Steril bir Pasteur pipeti konik tüp içine yerleştirin ve toplam hacmi 14 ml yaklaştığında kadar hücrelerin altında, Lenfosit Ayırma, Orta veya LSM yatıyordu. </li><li> Katmanları rahatsız etmemek için dikkatli olun. Oda sıcaklığında 20 dakika 2000 rpm&#39;de santrifüjleyin. Sonra rotor iplik yavaş fren kullanmayın. </li><li> Yeni bir 15 ml konik tüp CTL&#39;lerin aktarın. Yıkamak için yaklaşık 10 ml hacmi HBSS ekleyin. </li><li> Hücreleri saymak. Oda sıcaklığında 5 dakika 1300 rpm&#39;de Spin. Enjeksiyon başına toplam hacmi 100 mcL tutarak, enjeksiyon için gerekli hücre yoğunluğu HBSS tekrar </li><li> CTL&#39;lerin Tümör taşıyan farelere enjekte etmek için bu video daha önce görüldüğü gibi aynı kuyruk enjeksiyon tekniği kullanın. </li><li> Fare kafesine geri dönün ve CTL tümör ile etkileşim kurmak için izin. Fareler genellikle 14-21 gün CTL tedavi sonra analiz için feda. </li></ol> 3. Akciğer metastaz, Görselleştirme <ol><li> Akciğer metastazları görselleştirmek için, plastik bir yönetim kurulu sırtını bir kurban fare yerleştirin. Trakea engelsiz erişim sağlamak için bacaklarını Pin. % 70 etanol ile ventral taraf püskürtün. </li><li> , Orta karın itibaren, göğüs kafesi, tükürük bezlerinin doğru, orta hat boyunca kesme, ve makas kullanın. Trakea Açığa. Trakea çevre dokulara çıkarmak için forseps kullanın. </li><li> Trakea altında 200 mcL pipet geçirin. Tek elle Holding ucu hafifçe vücuttan trakea yukarı kaldırın ve uzak. </li><li> Platform 180 ° döndür. Trakea yoluyla ciğerlerine Hindistan Mürekkep enjekte etmek için 50 ml şırınga kullanın. Güçlü bir direnç hissedene kadar mürekkep ile akciğerler tamamen şişirmek. </li><li> Trakea kesmek için makas kullanın. Slayt, akciğerlerin altında forseps bir çift ve onları farenin dışarı çıkarın. Akciğerlerde su 1L beher kısaca durulayın. </li><li> Kimyasal bir davlumbaz, Fekete çözüm 5 ml cam sintilasyon içeren bir flakon için akciğerlere transfer. Tümör dokusu, birkaç dakika sonra siyah akciğerler üzerindeki beyaz nodüller olarak ortaya çıkacaktır. Tümörler şimdi sayılır ve süresiz Fekete çözümü olarak saklanabilir. </li></ol> 4. Görüntülenmiştir Akciğerler Temsilcilik Metastazları <ol><li> Burada, meme karsinom hücre hattı 4T1 ile enjekte edilen farelerden alınan akciğerlerin tümörler gösteren beyaz lekeler gösteriyor. Bir IRF8 dominant-negatif mutant K79E ile transfekte 4T1 hücreleri enjekte edildi Fare, tümör hücrelerinin gelişmiş metastatik potansiyelini gösteriyor. </li><li> Bu görüntüler CTL evlatlık transfer immünoterapi etkinliği bir histolojik analiz göstermektedir. Tümör taşıyan farelere enjekte edildi sarı oklar (A) ile gösterilir, 6 gün sonra birden fazla tümörler gösterdi. Tümör spesifik T hücreleri ile enjekte edilen farelerin, diğer taraftan, tümörler (B) bir azalma gösterdi. </li><li> Aynı deneyde, Hindistan mürekkep tedavi CTL evlatlık transferi etkinliğini ölçmek için basit bir yol verir. Burada, tümör taşıyan akciğerler üzerindeki beyaz tümör nodülü CTL evlatlık transferi başarıyla geçirmiş ve nodüller sayma akciğerler açıkça ayırt bir dereceye kadar izin verirhistoloji ile kantitatif mümkün değildir. </li></ol> 5. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Şekil 1" /> Şekil 1. Deneysel deneysel tümör metastazı ve CTL adaptif transfer immünoterapi fare modeli şeması. Kırmızı nokta, tümör hücreleri ve yeşil noktalar CTL&#39;lerin göstermektedir. <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Şekil 2" /> Şekil 2 IRF8 fonksiyonunun bozulması, tümör hücrelerinin metastatik potansiyeli geliştirilmiş . Fare meme karsinom hücre hattı 4T1 stably vektör (4T1.Vector) veya vektör bir IRF8 dominant-negatif mutant K79E (4T1.IRF8K79E) (15, 16) ifade ile transfekte edildi. Tümör hücreleri fare lateral kuyruk damarlar halinde iv enjekte edildi. Tümör taşıyan akciğerleri tümör nodülü görselleştirmek Türkiye mürekkep ile şişirildi. Tümör nodüller kolaylıkla siyah akciğer dokusunda arka plan üzerinde beyaz lekeler olarak görülür. <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Şekil 3" /> Şekil 3 CTL evlatlık transfer immünoterapi etkinliğini histolojik analiz. Fare sarkom hücre hattı CMS4-met fare lateral kuyruk damarlar halinde iv enjekte edildi. Üç gün sonra, tuzlu su (A) veya tümör-spesifik T hücrelerinin (B) Tümör taşıyan farelere enjekte edildi. Akciğerler geleneksel H &amp; E histolojik boyama tarafından CTL tedavi altı gün sonra incelendi. Tümör nodüller oklarla gösterilir. <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Şekil 4" /> Şekil 4 Hindistan mürekkep enflasyon tümör nodülleri Görselleştirme. Fare sarkom hücre hattı CMS4-met fare lateral kuyruk damarlar halinde iv enjekte edildi. Üç gün sonra, serum fizyolojik (kontrol) veya tümör-spesifik T hücreleri (CTL) Tümör taşıyan farelere enjekte edildi. Akciğerler, ondört gün CTL tedavi sonra incelendi. Tümör nodülü olan beyaz noktalar, kolay CTL tedavinin etkinliğinin ölçümü için izin verir.

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	<li>Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+extensive+pulmonary+metastases+in+mice+by+adoptive+transfer+of+antigen-specific+CD8(+)+CTL+reactive+against+tumor+cells+expressing+a+naturally+occurring+rejection+epitope.">Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.</a> J Immunol. 167 (8), 4286-4292 (2001).</li><li>Caldwell, S. A., Ryan, M. H., McDuffie, E., Abrams, S. 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E., Wunderlich, J. R., Yang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer+therapy+following+non-myeloablative+but+lymphodepleting+chemotherapy+for+the+treatment+of+patients+with+refractory+metastatic+melanoma.">Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma.</a> J Clin Oncol. 23 (10), 2346-2357 (2005).</li><li>Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells+inhibit+T-cell+activation+by+depleting+cystine+and+cysteine.+Cancer+research.">Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. 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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>

Fare Ulusal Kanser Enstitüsü (Friderick MD) alınan ve Georgia hayvan tesis Tıp Koleji alır. Deneyler ve bakım / refah federal düzenlemeler ile anlaşma ve Georgia IACUC komitesinin Tıp Koleji tarafından onaylanmış bir protokol.

Çözümler: Hindistan Mürekkep Çözümü (17): <ol><li> 150 ml, 250 ml balona distile su dökün. </li><li> 4 damla amonyum hidroksit, distile su ekleyin. </li><li> 30 ml Hindistan Mürekkep stok (yani Sanford Black Magic su geçirmez Çizim Mürekkep 4465 Öğe 44.011), amonyak ve su karışımı ekleyin. </li><li> 200 ml&#39;lik bir hacim distile su ile kapalı üst. Çözüm enjeksiyon için hazırdır. </li></ol> Fekete Çözümü (17): Fekete çözümü normal dokuların siyah arka plan beyaz tümör nodülü ayırt etmek için ağartıcı Hindistan mürekkep şişirilmiş tümör taşıyan akciğerler için kullanılır. <ol><li> 1L cam şişe, 350ml 95% EtOH ekleyin. </li><li> 150ml distile su ekleyin </li><li> 50ml formaldehit </li><li> 25ml buzul asidik asit ekle </li></ol>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Deneysel metastaz fare modeli basit ve fizyolojik ilgili henüz metastaz modeli. Tümör hücrelerinin fare kuyruğu damarlar içine (iv) damar içine enjekte ve tümör hücre spontan metastaz son adımları benzeyen, böylece, akciğerlerde kolonize: distal organlarda kan dolaşımı, ekstravazasyonu ve sömürgeciliğin yaşam. Metastatik tümör distal organ yılında kurulan: tedavilerin hedef genellikle metastaz bitiş noktası bu yana tedavi edici bir açıdan bakıldığında, deneysel metastaz modeli en basit ve ideal bir model. Bu modelde, tümör hücrelerinin fare kuyruğu damarlar içine iv enjekte edilir ve akciğerlerde kolonize ve büyümeye izin. Tümör spesifik CTL sonra iv metastaz taşıyan fare içine enjekte edilir. Akciğer metastazları sayısını ve boyutunu enjekte edilecek tümör hücrelerinin sayısı ve tümör büyümesini zaman kontrol edilebilir. Bu nedenle, çeşitli aşamalarında metastaz, geniş metastaz minimal metastaz modellenmiş olabilir. Akciğer metastazı, böylece daha kolay görsel gözlem ve miktar sağlayarak, mürekkep ile enflasyon analiz edilir.

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

Tümör hücrelerinin T hücre etkileşimi analizi için deneysel bir akciğer metastazı ve CTL immünoterapi fare modeli<em&gt; In vivo</em&gt;.

Deneysel metastaz ve CTL Kabul edilmiş Transfer İmmünoterapi Fare Modeli

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 Görüntüleme.  Medical College of Georgia. Tümör hücrelerinin T hücre etkileşimi analizi için deneysel bir akciğer metastazı ve CTL immünoterapi fare modeli 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.

Deneysel Fare Modeli Vajinal Aşılama ve Örnek Toplama Protokolleri Candida Vajinit

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

İmmünoloji ve Enfeksiyon

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.

Kabul edilmiş Transfer ve antijenik Challenge Deneysel Otoimmün Encephalomyelitis (EAE) Dayanıklı Fare Suşlarında yanıtsızlık Aşmak

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.

Diyabetojenik CD4 Kabul edilmiş Transfer ile Farelerde Hızlandırılmış Tip 1 Diyabet İndüksiyon + T Hücreleri

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.

İzolasyon, Arıtma ve İşlevsel Araştırmalar ve Kabul edilmiş Transfer deneyler için Fare Kemik İliği Nötrofiller Etiketlenmesi

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.

Fare intralenfatik immünoterapi ve Aşılama

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.

Hematopoetik progenitörlerin Evlatlık Transferi kullanarak Peter Parçacıklannda Murin plazmasitoid dendritik hücreleri Gelişimini değerlendirilmesi

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.

Bakım Glioblastoma Standardı Kapsamında Adoptif Tedavisi için CAR T hücrelerinin üretilmesi

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.

Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup&gt; +</sup&gt; CD45RB<sup&gt; Yüksek</sup&gt; Bağışık yetmezliği olan farelere içine T Hücreleri

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.

Orthotopic insan Glioblastoma Multiforme Xenografts fare modelleri sitotoksik bağışıklık hücrelerinin stereotaksik evlat edinen Transfer

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 ile ilişkili Karaciğer Patogenezi için Çift İnsanize TK-NOG Fare Modelinin Oluşturulması

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