Yazarlar Iris Erdmann&#39;a, Barbara von Beust ve Julia Maria Martínez-Gómez farelerde intralenfatik bağışıklama yöntemini gelişmekte deneysel yardım için minnettarız. Bize bu video üretimi için kendi cerrahi tiyatro bildirdiğiniz için de Maggy Arras ve Nikola Cesarovic teşekkürler.

1.. Farelerin Anestezi <ol><li> Tamponlu tuz çözeltisi içinde ketamin (dissosiyatif anestezi) ve ksilazini (sedatif ve analjezik) karıştırılarak anestezik hazırlayın. Nihai çözelti içinde ketamin ve ksilazin konsantrasyonları sırasıyla 12,5 ve 2 mg / ml &#39;dir. </li><li> 25-30 G iğnesi olan bir şırınga kullanılarak intraperitonal uygulama ile farelere anestezi enjekte edilir. Vücut ağırlığının 0.1 ml/10 g kullanın. </li><li> Kornea kurutulması Çıkış önlemek için farenin gözlerindeki bir oftalmik merhem uygulayın. </li><li> Fare yeterince forseps ile ayak veya ayak kısma anestezi sağlamalıdır. Fare tutam refleksleri ile tepki vermezse, cerrahi geçin. Fare refleksleri veya kas kasılmaları ile tutam tepki varsa, 1-2 dakika bekleyin ve tutam testi tekrarlayın. Fare hala tepki vermiyorsa, bir yeni hayvan ile fare değiştirin ve sonrası adım 1.2 &#39;den işlemleri tekrarlayın. </li></ol> 2. Cerrahi IMouse Kasık Alanı ncision <ol><li> Sırtında fare yerleştirin. </li><li> Dezenfeksiyon etanol (% 70) ile bir inguinal bölge ıslatın. Daha iyi dezenfeksiyon sağlamak için, inguinal bölge etanol ile kürk önce dezenfeksiyon kaldırmak için traş olabilir. </li><li> Arka bacak alın ve kalça ekleminin yaklaşık 90 ° açı üretmek için kalça eklemi bükün. </li><li> Kavisli bir Microdissecting forseps kullanarak, kalça eklem bükülmüş kasık bölgesinde fare deri alıp hafifçe ıslatılmış cildi yukarı çekin. </li><li> Forseps ile cilt kadar tutarken, cerrahi makas kullanılarak deri yoluyla küçük bir kesi (&lt;5 mm) kesti. </li><li> Kesi içine kapalı makas ucu yerleştirin ve daha fazla ensizyon içine tutarken makas açarak kesi açın. Bu, gözyaşı ve en az 10 mm&#39;lik bir çapa üretmelidir cilt neden olur. Not: esnek ışık kılavuzları ile bir soğuk ışık kaynağı görünürlüğünü artırmak için kullanılabilir. Eski yılındafareler, özellikle erkek, lenf düğümü kimlik nedeniyle kasık bölgesinde fazla yağ dokusu zor olabilir. </li></ol> 3. Intralenfatik Enjeksiyon <ol><li> Bir 28-30 G derialtı iğne ile bir şırınga (0.5 ml veya daha az) hazırlayın; kısa konik uzun eğimin tercih edilebilir. Aşının aspire 10 ul enjekte ve şırınga serbest hava olduğunu garanti edilecek. </li><li> Kavisli bir forseps yardımı ve kapalı makas ucu ile kasık lenf düğümü yerelleştirilmesine. Lenf düğümü daha beyazımsı yağ dokusu içindeki grimsi görünür ve daha fazla giriş ve lenf düğümü terk afferent ve efferent kılcal ile tespit edilebilir. </li><li> Kavisli bir forseps dalları arasında tutarak lenf düğümü hareketsiz. </li><li> Şırınga alın ve yukarı bakacak Eğim ile lenf düğümüne iğneyi, bütün konik lenf düğümü girer emin. </li><li> Aşısı (10 ul) enjekte edilir. Eğer lenf node (üfleme) şişme, enjeksiyon başarılı kabul edilebilir. Iğne lenf düğümüne derinliklerine yeterince yer değilse, bazı veya tüm aşı dışarı sızıntı ve lenf düğümü giremezsiniz. İğne çok derin yerleştirildiğinde ise, deri altından enjekte edilen aşı, lenf düğümü, aşağıda açıklanacak. Ikinci durumunda, tekrar enjeksiyon ya da yeni bir tedavi edilmemiş fare ile fare değiştirin. </li></ol> 4. Dikiş ile kesi Kapanış <ol><li> Steril cerrahi sütür bir paket açın. </li><li> Iğne gövdesinin distal kısmında bir iğne tutucusu ile dikiş iğnesi tutunuz. İlk mandal yakalar kadar sıkarak iğne tutucu sıkın. </li><li> (Un dişli veya dişli forseps veya deri kanca kullanılabilir cerrahın tercihine bağlı olarak) forseps kullanılarak kesi bir tarafında deri tutunuz. İğne ca yerleştirin. 2-3 mm kesi kenarından, derinin dış giren. </li><li> Loosforseps ile cilt beklemeye tr ve forseps ile kesi karşı tarafını kavrayın. İğne ca yerleştirin. 2-3 mm kesi kenarından, deri içine giren. </li><li> İğne tutucu gevşetmek ve forseps ile iğne ucu kavramak. Birkaç farenin tedavi edilecek ise, daha kısa bir uç bırakmak (birinci iğne ekleme noktası dışında taban yüzünün uygun bir uzunluğu bırakarak deri yoluyla profili olan iğne çekin, 5-10 farenin bir dikiş iğnesi ile çalıştırılabilir /) yürümek. </li><li> Standart cerrah protokollere göre tercih edilen düğüm yapın. Kesi 10 mm daha büyük değilse, tek dikiş yeterlidir. Daha büyük kesiler için, ikinci bir dikiş yapmak. </li></ol> 5. Post-operasyonel Tedavi <ol><li> Kafeste fare yerleştirin ve sıcak tutmak için dokuları ile kaplayın. Birden fareler tedavi ederken, sıcak tutmak için birbirine yakın onları yerleştirin. Onlar uyandı kadar alternatif, bir sıcak ped fareler yerleştirinkadar. </li><li> Onlar uyanmak kadar fareler gözlemleyin. </li><li> Iyileşme yanı sıra yara enfeksiyonları gibi diğer klinik belirtiler yara ile ilgili olarak günlük fareler dikkate alınmalıdır. Yara genellikle yeterince iki gün içinde kapalı ve prosedürün yedi gün içinde iyileşti. </li></ol>

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TMK adam intralenfatik immünoterapi ile ilgili patent mucidi olarak adlandırılır. TMK bilimsel danışmanı olmuştur ve ImVisioN GmbH, Cytos Biyoteknoloji, MannKind Corporation ve XBiotech USA Inc PJ ifşa hiçbir çatışma ilgi seyahat masraflarını aldı.

Lenf içine aşılama ve immünoterapi antikor karşılıkları ve T-hücresi tepkilerinin her ikisinin stimülasyonu için uygun olduğu gösterilmiştir. Bu video-makale de gösterildiği gibi, aşı intralenfatik prosedür farelerde güçlü bir immün yanıtları stimüle etmek için hızlı ve kolay bir yöntemdir. Eğitimli bir cerrah 3-4 dakika boyunca yordamı gerçekleştirebilir. Oturumda ayrıca bir genellikle anestezi ve dikiş atılmasını yapıyor ve ikinci cerrah kesi ve enjeksiyon yapıyor, iki cerrahla...

Aşılar genellikle bağışıklık yanıtlarının uyarılması için deri altından ya da kas içinden enjekte edilir. Bu prosedürün başarısı, antijen sunan hücreler, T-ve B-hücresi tepkilerinin üretilmesi için lenfositleri ile etkileşim lenf düğümlerine aşının etkili bir drenaj gerektirir. Buna ek olarak, dayanıklılık ve kaynaklı bağışıklık tepkilerinin türü de yoğunluğu ya da frekans gibi etkileşimlerin hem de mikro kendisi, sitokinlerin, özellikle içeriğine bağlıdır. Çevresel bir doku içine enjekte edilen bir aşının sadece küçük bir kısmı, bir lenf düğümü ulaştığında, lenf düğümleri içine aşı direkt enjeksiyonu ile farelerde ve insanlarda ve aşılar, site bağışıklık cevapları oluşturulur edildi gerçekleştirilmiştir. İnsanlarda, prosedür ultrason da lenfatik sistem görselleştirme ve teşhis için görüntüleme maddeleri uygulanması için kullanılan bir prosedür ile yönlendirilir. Farelerde, prosedür invaziv. Burada, bir küçük (5-10 mm) incision, anestezi uygulanmış hayvanlar 1&#39;in kasık bölgesinde yapılır, lenf düğümü ve lokalize forseps ile hareketsiz ve aşı 10-20 ul&#39;lik bir hacim görsel kontrol altında enjekte edilir, 10 ul ilk enjeksiyon için kullanılır ve genç farelerde küçük lenf düğümleri ile, oysa 20 ul büyük lenf düğümleri lenf düğümleri daha büyük ya da zaten farede, enjekte edilebilir. İnsizyon edilebilir cerrahi dikişle, bir tek dikiş ile kapatılabilir. Bu yöntem ile, farenin plazmid DNA 2,3, haberci RNA 4, 1,3,5,6 peptid, protein, 7-10, 11, parçacıklar, bakteriler 12 hem de 7,13 ilave maddeler ve bağışıklık güçlü iyileşme ile aşılanmış olan aşıların her tür karşı tepkiler gözlenmiştir. Aşılama intralenfatik yöntemi geleneksel aşılama yetersiz bağışıklık üreten veya mevcut aşının miktarı sınırlı ya da ver olduğu durumlarda özellikle uygundurpahalı y. İnsanda, lenf sistemi içi aşılama yöntemi, alerji hastalara 14,15 ya da kanserli 16-21 olan hastalara uygulanmıştır. Mevcut kavramı intralenfatik yöntem, kas içi ve deri altı enjeksiyonlar gibi diğer enjekte edilebilir yöntemlere göre daha invaziv olmasıdır, ancak ağrı algısı, damar delinerek 15 sonra daha yüksek değildir. Bu lenf sistemi içi aşı koruyucu ve tedavi edici aşıların, özellikle diğer yöntemlere bir alternatif veya tamamlayıcı olacağı beklenmektedir. Bu makalede, lenf sistemi içi aşılama prosedürü farelerde gerçekleştirilir kadar ayrıntılı olarak açıklanmaktadır. Açıklanan tüm prosedürler, Zürih Kantonu Veterinerlik Kurumu tarafından onaylanan ve İsviçre federal kuralları ve bilimsel amaçlar için kullanılan hayvanların korunması ile ilgili direktifleri göre yapılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Ketamine (Ketasol-100)</td>
			<td style="width:135px;">Graeub AG, Switzerland</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Anesthetics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Xylazine (Rompun)</td>
			<td style="width:135px;">Bayer, Germany</td>
			<td style="width:119px;"></td>
			<td>Anesthetics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Viscotears Eye-Gel</td>
			<td style="width:135px;">Novartis, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To keep eyes from drying out during anesthesia.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">BD Micro-Fine 0.5 ml</td>
			<td style="width:135px;">BD Medical, France</td>
			<td style="width:119px;"></td>
			<td>29 G Insulin syringes with permanently attached needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">6-0 Dermalon Monofilament nylon</td>
			<td style="width:135px;">Covidien, MA, USA</td>
			<td style="width:119px;"></td>
			<td>For sutures (0.7 metric, 18G, 45 cm, Blue)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Curved forceps, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision and holding of lymph node</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Straight surgical scissors, 4.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>For incision</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Needle holder, 5.5 inch</td>
			<td style="width:135px;">Polymed, Switzerland</td>
			<td style="width:119px;"></td>
			<td>To close incision with suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

intralymphatic immunotherapy and vaccination in mice

Aşılar genellikle bağışıklık yanıtlarının uyarılması için deri altından ya da kas içinden enjekte edilir. Bu başarısı, antijen sağlayan hücreler, aranan bağışıklık yanıtlarının oluşturulması için lenfositleri ile etkileşim lenf düğümlerine aşının etkili bir drenaj gerektirir. Gücü ve kaynaklı bağışıklık tepkilerinin türü de yoğunluğu veya frekans etkileşimlerin hem de mikro-, sitokinlerin, özellikle içeriğine bağlıdır. Çevresel olarak enjekte edilen aşı sadece küçük bir kısmını, lenf düğümleri ulaştığında, fareler ve insanlarda aşı kasık lenf düğümleri, örneğin lenf sistemi içi enjeksiyonu içine aşı direkt enjeksiyonu ile yapılmıştır. İnsanlarda, prosedür ultrason tarafından yönlendirilmektedir. Farelerde, bir küçük (5-10 mm) bir kesik, lenf düğümü ve lokalize forseps ile hareketsiz hale anestezi uygulanmış hayvanların kasık bölgesinde yapılır ve aşı 10-20 ul&#39;lik bir hacim görsel kontrol altında enjekte edilir.Kesik, cerrahi dikiş kullanarak tek bir dikiş ile kapatılmıştır. Fareler plasmid DNA, RNA, peptid, protein, parçacıklar ve bakteriler gibi yardımcı maddeler ile aşılandı ve aşıların her tür karşı bağışıklık karşılıklarının güçlü bir iyileşme gözlendi. Aşılama intralenfatik yöntem, geleneksel aşı yetersiz bağışıklık veya Müsait aşı miktarı sınırlıdır üreten durumlarda özellikle uygundur.

Farelerde intralenfatik enjeksiyon prosedürü, cerrahi doğasına rağmen, yalındır ve nispeten hızlı. Eğitimli bir kişi 3-4 dakika içinde işlemi gerçekleştirebilirsiniz. Bir dikiş ile kapatılmıştır kesik tipik haliyle iki gün içinde iyileştirir (Şekil 1) Lenf içine aşı veya aşı mRNA ile gerçekleştirilmiştir, plazmid DNA, peptidler, proteinler, virüs ve bakteri. 2 protein fosfolipaz A2 (PLA2), ana alerjen arı zehiri ile immüniz...

Watch this Scientific Journal Video about Intralymphatic Immunotherapy and Vaccination in Mice at JoVE.com

Intralymphatic Immunotherapy and Vaccination in Mice

Profilaktik ve terapötik aşılama çoğu zaman düğümleri ve bağışıklık hücrelerinin dolayısıyla kötü katılımını lenf nedeniyle aşının hafta drenaj güçlü bağışıklık tepkilerini uyarmak için başarısız olur. Lenf düğümlerine aşı doğrudan enjeksiyon ile, sözde lenf sistemi içi enjeksiyon, aşı etkinliği güçlü geliştirilebilir ve aşı doz azaltılabilir.

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 ...

intralymphatic-immunotherapy-and-vaccination-in-mice

Research

JoVE Journal

Immunology and Infection

23.0K Görüntüleme.  University Hospital Zurich.  Profilaktik ve terapötik aşılama çoğu zaman düğümleri ve bağışıklık hücrelerinin dolayısıyla kötü katılımını lenf nedeniyle aşının hafta drenaj güçlü bağışıklık tepkilerini uyarmak için başarısız olur. Lenf düğümlerine aşı doğrudan enjeksiyon ile, sözde lenf sistemi içi enjeksiyon, aşı etkinliği güçlü geliştirilebilir ve aşı doz azaltılabilir. 

Video: Intralymphatic Immunotherapy and Vaccination in Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

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 ...

İmmünoloji ve Enfeksiyon

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Rekombinant RBD tabanlı SARS Aşılar için Protokol: Protein hazırlanması, Hayvan Aşılama ve Nötralizasyon Algılama

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

İmmünoterapi için Tümör Antijen-yüklü Olgun dendritik hücreler hazırlanması

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA Virüslerinin geniş spektrumlu Kimyasal Önleyicileri için yüksek verimli tarama

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo Tahlil antijen spesifik T-hücre Cytolytic işlevi bir aşı modeli kullanılarak tespiti için

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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 ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

En iyi duruma getirilmiş Interferon-gamma ELISpot tahlil ölçü birimi T hücre yanıtları sonrasi hipererjik eskiden şiling şimdi domuz modelinde

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Küçük Hint firavunları (Herpestes Auropunctatus) sera bir oral kuduz aşılama biyolojik Marker olarak kullanım Için Iophenoxic asit analogları Analizi

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Methods Article

Fare intralenfatik immünoterapi ve Aşılama