저자는 아이리스 Erdmann의, 바바라 폰 Beust, 줄리아 마르티네즈 마리아 고메즈의 생쥐 intralymphatic 예방 접종의 방법을 개발하는 실험적인 도움에 대해 감사합니다. 우리는이 비디오 제작에 대한 자신의 수술 극장을 사용셔서 또한 Maggy의 아라스와 니콜라 Cesarovic 감사합니다.

1. 쥐의 마취 <ol><li> 완충 식염수에 케타민 (해리 성 마취제)과 자일 라진 (진정제 및 진통제)을 혼합하여 마취제를 준비합니다. 최종 솔루션에서 케타민과 자일 라진의 농도는 각각 12.5, 2 ㎎ / ㎖이다. </li><li> 25 ~ 30 G 바늘과 주사기를 사용하여 복강 내 투여하여 마우스에 마취제를 주입한다. 체중의 0.1 ml/10 g을 사용합니다. </li><li> 각막의 건조 아웃을 방지하기 위해 마우스의 눈에 안과 연고를 적용합니다. </li><li> 마우스가 충분히 집게와의 발이나 발가락을 집어 마취 확신한다. 마우스가 핀치에 반사와 반응하지 않는 경우 수술을 진행합니다. 마우스는 반사 신경이나 근육의 수축과 핀치에 반응하는 경우, 1 ~ 2 분 정도 기다린 핀치 테스트를 반복합니다. 마우스가 여전히 반응의 경우, 새로운 동물로 마우스를 대체하고 이후 단계 1.2의 절차를 반복합니다. </li></ol> 2. 외과 I마우스의 사타구니 지역의 ncision <ol><li> 그 뒷면에 마우스를 놓습니다. </li><li> 소독 에탄올 (70 %)와 하나의 서혜부 십니다. 더 나은 소독을 제공하기 위해, 서혜부 에탄올 모피 전에 소독을 제거하는 면도 할 수있다. </li><li> 뒷다리를 가지고 고관절의 약 90 °의 각도를 생산하는 엉덩이 관절을 구부립니다. </li><li> 곡선 microdissecting 집게를 사용하여 엉덩이 관절이 구부러 서혜부에서 마우스 스킨을 약간 적신 피부를 당겨. </li><li> 집게로 피부를 누른 상태에서, 외과 용 가위를 사용하여 피부를 통해 작은 절개 (&lt;5 mm)를 절단. </li><li> 절개 부위에 폐쇄 가위의 끝을 놓고 더 절개 안쪽을 잡고 가위를 열어 절개를 엽니 다. 이 찢어 및 10 mm 이하의 직경을 생산한다 피부를 일으킬 것이다. 주 :가요 라이트 가이드 등으로 감기 광원 시인성을 향상시키기 위해 사용될 수있다. 이전에마우스, 특히 남성, 림프절의 식별 인해 사타구니 지역에있는 지방이 많은 조직에 어려울 수 있습니다. </li></ol> 3. Intralymphatic 주입 <ol><li> 28 ~ 30 G 피하 주사 바늘과 주사기 (0.5 ㎖ 이하)을 준비, 짧은 베벨 긴 베벨 것이 바람직 할 수있다. 백신의 대기음 10 ㎕를 주입 주사기가 공기 무료입니다 확신합니다. </li><li> 곡선 포셉의 도움 폐쇄 가위의 끝을 가진 서혜부 림프절 지역화. 림프절은 더 흰 지방 조직 내에서 회색 표시되고 추가로 입력하고 림프절을 떠나 심성 및 원심성 모세 혈관을 통해 확인할 수 있습니다. </li><li> 곡선 포셉의 가지 사이를 잡고 림프절을 고정시킨다. </li><li> 주사기를 타고 위로 향하게 경사와 림프절에 바늘을 삽입하고 전체 경사가 림프절을 들어 확인하고. </li><li> 백신 (10 μL)을 주입한다. 만약 림프 고개를 끄덕전자는 (폭파) 팽창되고, 주입이 성공적으로 가정 할 수있다. 바늘이 림프절에 깊은 충분히 배치되어 있지 않은 경우, 일부 또는 전체 백신은 누출 및 림프 노드를 입력 할 수 있습니다. 바늘이 너무 깊이 삽입하면, 주입 된 백신은 피하 림프 노드 아래에 발표 될 예정이다. 후자의 경우, 주사를 반복하거나 새로운 미처리 마우스로 마우스를 교체. </li></ol> 4. 봉합하여 절개 닫기 <ol><li> 무균 수술 봉합의 팩을 엽니 다. </li><li> 바늘 몸의 말단 부분에 바늘 홀더 봉합 바늘을 잡아. 첫 번째 래칫 어획량까지 짜내서 바늘 홀더를 조입니다. </li><li> (톱니 또는 취소 톱니 포셉이나 피부 후크 사용될 수있는 외과 의사의 선호도에 따라) 집게를 사용하여 절개의 한면에서 피부의 보류를 가져 가라. 바늘 CA입니까. 2-3mm을 절개의 가장자리에서, 피부의 외부로부터 입력. </li><li> 이보게집게로 피부의 유지 EN과 집게로 절개의 반대편을 파악. 바늘 CA입니까. 2-3mm을 절개의 가장자리에서, 피부의 내부로부터 입력. </li><li> 바늘 홀더를 풀고 집게로 바늘 끝을 잡고. 여러 마우스가 처리 될 경우, 짧은 끝을 떠나 (제 1 바늘 삽입 지점 외부 트레드 편리한 길이를 떠나, 피부를 통해 접지면과 바늘을 빼낼 수 있지만, 5 ~ 10 마우스는 하나의 봉합 바늘에 운영 할 수있다 /) 밟다. </li><li> 표준 외과 의사의 프로토콜에 따라 선호하는 매듭을합니다. 절개 부위가 10mm 이하인 경우 하나의 스티치가 충분하다. 큰 절개를 들어, 두 번째 스티치를합니다. </li></ol> 5. 후 운영 처리 <ol><li> 새장에 마우스를 놓고 보온하는 조직 커버. 여러 쥐를 치료​​하면, 따뜻하게 서로에게 가까이 놓습니다. 그들이 깨어 때까지 다른 방법으로는, 따뜻한 패드에 마우스를 배치최대. </li><li> 그들이 일어날 때까지 쥐를 관찰합니다. </li><li> 치유뿐만 아니라 상처 감염과 같은 다른 임상 증상을 상처에 관해서는 매일 쥐를 관찰합니다. 상처는 일반적으로 충분히 이틀 폐쇄 절차의 7 일 이내에 치유됩니다. </li></ol>

<ol>
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Immunother. 52, 438-444 (2003).</li><li>Brown, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-transduced+dendritic+cells+injected+into+skin+or+lymph+node+prime+potent+simian+immunodeficiency+virus-specific+T+cell+immunity+in+monkeys.">Adenovirus-transduced dendritic cells injected into skin or lymph node prime potent simian immunodeficiency virus-specific T cell immunity in monkeys.</a> J. Immunol. 171, 6875-6882 (2003).</li><li>Fong, L., Brockstedt, D., Benike, C., Wu, L., Engleman, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+injected+via+different+routes+induce+immunity+in+cancer+patients.">Dendritic cells injected via different routes induce immunity in cancer patients.</a> J. Immunol. 166, 4254-4259 (2001).</li></ol>

TMK는 남자 intralymphatic 면역 요법을 다루는 특허의 발명자로 지정됩니다. TMK는 과학 고문되었으며 ImVisioN GmbH를, Cytos 생명 공학, MannKind 공사 및 XBiotech 미국 주식 PJ가 공개하는 충돌의 관심이없는에서 여행 경비를 받았다.

Intralymphatic 예방 접종 및 면역 항체 반응과 T 세포 반응을 모두 자극에 적절한 것으로 표시되었습니다. 이 비디오 문서에서 알 수 있듯이, 예방 접종의 intralymphatic 절차는 생쥐에 강한 면역 반응을 자극하기위한 빠르고 쉬운 방법입니다. 숙련 된 외과 의사가 3 ~ 4 분 동안 절차를 수행 할 수 있습니다. 세션은 또한 하나가 일반적 마취와 봉합을 수행하고 제 외과 절개 및 주입을하고이 외과 의사 사?...

백신은 일반적으로 면역 반응의 자극을 위해 피하 또는 근육 주사합니다. 이 절차의 성공은 항원 제시 세포는 T-와 B-세포 반응의 생성을 위해 림프구와 상호 작용할 수있는 림프절에 백신의 효율적인 배수를 필요로합니다. 또, 강도 및 유도 면역 반응의 유형은 밀도 또는 주파수와 같은 상호 작용뿐만 아니라 미세 환경 자체, 사이토 카인, 특히 콘텐츠에 의존한다. 말초 조직에 주입 백신의 단지 작은 분획이 림프절에 도달 할 때, 림프절로 백신의 직접 주입에 의해 생쥐와 인간의 접종은, 사이트가 면역 반응을 생성하고, 수행되었다. 사람에서의 절차는 초음파, 또한 림프 시스템의 시각화 및 진단을위한 이미징 요원의 관리에 사용되는 절차에 의해 안내된다. 마우스에서, 절차는 침습적이다. 여기에, 작은 (5 ~ 10 ㎜) incisioN 마취 된 동물 1의 서혜부 제, 림프 노드는 지역화 포셉 고정화하고, 백신의 10 ~ 20 μL의 부피는 시각 제어에 주입되고, 10 ㎕를 먼저 주사에 사용되는 젊은 생쥐 작은 림프 노드와, 반면에 20 ㎕의 큰 림프절이 림프 세의 노드 또는 이미 끝났다 마우스에 주입 할 수있다. 절개 수술 봉합사를 사용하여 하나의 스티치 닫을 수 있습니다. 이 방법으로, 마우스는 플라스미드 DNA, 3, 메신저 RNA 4, 펩타이드 1,3,5,6, 단백질 7 ~ 10, 11를 입자, 박테리아 (12)뿐만 아니라 보조제 7,13, 면역의 강력한 개선과 예방 접종 백신의 모든 유형에 대한 반응이 관찰되었다. 예방 접종의 intralymphatic 방법은 기존의 예방 접종이 부족한 면역을 생산 또는 사용 가능한 백신의 양이 제한 또는 버전입니다 상황에서 특히 적합하다비용이 많이 드는 예. 인간의, 예방 접종의 intralymphatic 방법은 알레르기 환자 14, 15 또는 암 16-21 환자에 적용되었습니다. 현재의 개념이 intralymphatic 방법 등 근육 및 피하 주사 등의 주사 방법보다 더 침습적 인 것입니다 만, 고통의 인식은 정맥 천자 (15) 후보다 높은 없습니다. 그것은 intralymphatic 예방 접종은 질병 예방 및 치료, 특히 예방 접종의 다른 방법에 대한 대안 또는 보완 될 것으로 예상된다. 이 문서 intralymphatic 예방 접종의 절차가 생쥐에서 수행하는 방법을 상세하게 설명합니다. 설명 모든 절차는 취리히의 주립 수의학 기관의 승인을 스위스 연방 정부의 가이드 라인과 과학적인 목적으로 사용되는 동물의 보호에 관한 지침에 따라 수행 하였다.

<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

백신은 일반적으로 면역 반응의 자극을 위해 피하 또는 근육 주사합니다. 이것의 성공은 항원 제시 세포가 원하는 면역 반응의 생성을 위해 림프구와 상호 작용할 수있는 림프절에 백신의 효율적인 배수를 필요로합니다. 강도 및 유도 된 면역 반응의 유형은 밀도 또는 주파수의 상호 작용뿐만 아니라, 미세 환경, 사이토킨, 특히 콘텐츠에 의존한다. 말초 주입 백신만을 분 분획 림프절에 도달 할 때, 마우스 및 인간의 예방 접종 서혜부 림프절, 즉 intralymphatic 주사로 백신의 직접 주입에 의해 수행되었다. 남자에서, 절차는 초음파에 의해 안내된다. 마우스에서, 작은 (5-10 ㎜) 절개, 림프절 지역화 및 집게로 고정화는 마취 된 동물의 서혜부 영역에 만들어지고, 백신의 20 μL의 부피는 카메라 제어하에 주입한다.절개 수술 봉합사를 사용하여 하나의 스티치 닫힙니다. 마우스는 플라스미드 DNA, RNA, 펩타이드, 단백질, 입자, 박테리아뿐만 아니라 보조제 접종하고, 백신의 모든 유형에 대한 면역 반응의 강력한 개선이 관찰되었다. 예방 접종의 intralymphatic 방법은 기존의 예방 접종이 부족 면제 또는 가능한 경우 백신의 양이 한정되어 생산 상황에 특히 적합합니다.

생쥐 intralymphatic 주사의 절차는 수술 성격에도 불구하고, 정직하고 상대적으로 빠르다. 훈련을받은 사람이 3 ~ 4 분의 절차를 수행 할 수 있습니다. 한 스티치 폐쇄 절개 일반적으로 두 일 이내에 치유 (그림 1) Intralymphatic 백신 또는 면역이 mRNA를 사용하여 수행되었으며, 플라스미드 DNA, 펩타이드, 단백질, 바이러스 및 박테리아. 2 단백질 포스 포 리파?...

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

Intralymphatic Immunotherapy and Vaccination in Mice

예방 및 치료 백신은 종종 노드와 면역 세포의 결과로 가난한 참여 림프로 인해 백신의 주 배수에 강한 면역 반응을 자극하는 데 실패합니다. 림프절 백신의 직접 주사에 의해, 소위 intralymphatic 주입은 백신 효능 강하게 개선 될 수 있으며, 백신의 투여 량을 저감 할 수있다.

마우스의 Intralymphatic 면역 및 예방 접종

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 조회수.  University Hospital Zurich.  예방 및 치료 백신은 종종 노드와 면역 세포의 결과로 가난한 참여 림프로 인해 백신의 주 배수에 강한 면역 반응을 자극하는 데 실패합니다. 림프절 백신의 직접 주사에 의해, 소위 intralymphatic 주입은 백신 효능 강하게 개선 될 수 있으며, 백신의 투여 량을 저감 할 수있다. 

비디오: 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.

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 ...

연구

면역학 및 감염병학

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.

재조합 RBD 기반 SARS 백신에 대한 프로토콜 : 단백질 준비, 동물 예방 접종 및 중화 감지

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.

면역 요법에 대한 종양 항원로드 성숙 수지상 세포의 준비

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 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

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

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

마우스에 현지 아나필락시스 측정

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

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 ...

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.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

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.

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

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.

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 ...

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.

예방 접종 후 기니 돼지 모델에서 측정 T 세포 응답에 최적화 된 인터페론-감마 ELISpot 분석 결과

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, ...