Os autores são gratos pela ajuda experimental no desenvolvimento do método de imunização em camundongos intralinfática de Iris Erdmann, Barbara von Beust, e Julia Maria Martínez-Gómez. Obrigado também a Maggy Arras e Nikola Cesarovic por nos deixar usar seu teatro cirúrgico para esta produção de vídeo.

1. Anestesia dos Ratos <ol><li> Prepare os anestésicos pela mistura de quetamina (anestésico dissociativo) e xilazina (sedativo e analgésico) em salina. As concentrações de cetamina e xilazina na solução final é de 12,5 e 2 mg / ml, respectivamente. </li><li> Injectar os anestésicos para a ratinhos por via intraperitoneal usando uma seringa com uma agulha G 25-30. Use 0,1 ml/10 g de peso corporal. </li><li> Aplique uma pomada oftálmica para os olhos dos ratos, a fim de evitar que resseque das córneas. </li><li> Assegurar que o mouse é suficientemente anestesiados por beliscar o seu pé ou dedo do pé com a pinça. Se o rato não reage com reflexos para o aperto, proceder à cirurgia. Se o rato reage ao beliscão com reflexos ou contrações musculares, espere 1-2 minutos e repita o teste de pressão. Se o mouse ainda reage, substituir o mouse com um novo animal e repita os procedimentos a partir do passo 1.2 em diante. </li></ol> 2. Eu Surgicalncision da região inguinal do Rato <ol><li> Posicione o mouse sobre as suas costas. </li><li> Molhar uma região inguinal com etanol desinfecção (70%). Para proporcionar uma melhor desinfecção, a região inguinal podem ser raspada para remover pele desinfecção prévia com etanol. </li><li> Pegue a perna e dobrar a articulação do quadril para produzir um ângulo de aproximadamente 90 ° da articulação do quadril. </li><li> Usando um fórceps curvos microdissecting, tirar a pele do rato na área inguinal, onde a articulação do quadril é dobrado e puxe a pele molhada ligeiramente. </li><li> Enquanto mantém a pele com a pinça, corte uma pequena incisão (&lt;5 mm) através da pele utilizando uma tesoura cirúrgica. </li><li> Coloque a ponta da tesoura fechados na incisão e abrir ainda mais a incisão, abrindo a tesoura, mantendo-o dentro da incisão. Isto fará com que a pele se rasgar e deve produzir um diâmetro de menos de 10 mm. Nota: A fonte de luz fria, com guias de luz são flexíveis e podem ser utilizados para melhorar a visibilidade. Em mais velhosratinhos, especialmente do sexo masculino, a identificação do nó de linfa pode ser difícil devido à muito tecido adiposo na região inguinal. </li></ol> 3. Injeção intralinfática <ol><li> Prepara-se uma seringa (0,5 ml ou menos) com uma agulha hipodérmica de 28-30 G; bisel curto pode ser preferível a um bisel longo. Aspirar 10 mL da vacina a ser injectado e garantir que a seringa é livre de ar. </li><li> Localizar o nó de linfa inguinal com a ajuda da pinça curvos e a ponta da tesoura fechada. O linfonodo aparecerá acinzentado dentro do tecido adiposo mais esbranquiçada e pode ser ainda identificados através de aferentes e eferentes capilares que entram e saem do linfonodo. </li><li> Imobilizar o linfonodo, segurando-o entre os ramos das pinças curvas. </li><li> Pegue na seringa e insira a agulha no nódulo linfático com o bisel virado para cima, certificando-se de todo o bisel entra no linfonodo. </li><li> Injectar a vacina (10 ul). Se o aceno linfae é o inchaço (explodir), a injecção pode ser assumida de sucesso. Se a agulha não está colocada a uma profundidade suficiente para o linfonodo, alguns ou toda a vacina pode sair e não entrar no linfonodo. Se a agulha é inserida muito profundo, a vacina injetada será lançado via subcutânea abaixo do nó de linfa. Em caso deste último, repetir a injecção ou substituir o rato com uma nova rato não tratado. </li></ol> 4. Fechando a incisão com sutura <ol><li> Abrir um pacote de sutura cirúrgica estéril. </li><li> Segure a agulha de sutura com um suporte de agulha na porção distal do corpo da agulha. Apertar o suporte de agulha, apertando-o até que as primeiras capturas de roquete. </li><li> Segure a pele de um dos lados da incisão com a pinça (dependendo da preferência do cirurgião, dentada ou un-dentes fórceps ou ganchos na pele pode ser usado). Inserir a agulha de ca. 2-3 mm a partir da borda da incisão, que entra a partir do lado de fora da pele. </li><li> Loosen preensão da pele com a pinça e agarrar o lado contrário ao da incisão com a pinça. Inserir a agulha de ca. 2-3 mm a partir da borda da incisão, que entra a partir do interior da pele. </li><li> Soltar o suporte de agulha e agarrar a ponta da agulha com a pinça. Puxe a agulha com o piso através da pele, deixando um comprimento conveniente da banda de rodagem do lado de fora do primeiro ponto de agulha de inserção (se vários ratos estão a ser tratados, deixe uma extremidade mais curta; 5-10 camundongos pode ser operado com uma agulha de sutura / pisar). </li><li> Fazer o nó preferido de acordo com protocolos padrão cirurgião. Um ponto é suficiente se a incisão não é maior do que 10 mm. Para incisões maiores, fazer um segundo ponto. </li></ol> 5. Tratamento Pós-operacional <ol><li> Posicione o mouse na gaiola e cubra com tecidos para manter aquecido. Ao tratar vários mouses, coloque-os próximos uns dos outros para se manter aquecido. Alternativamente, coloque os ratos em uma almofada quente até que tenham acordadopara cima. </li><li> Observe os ratos até que eles acordam. </li><li> Observe os ratos diariamente com relação à cicatrização de feridas, bem como outros sintomas clínicos, tais como infecções de feridas. A ferida é geralmente suficientemente fechado dentro de dois dias e curado dentro de sete dias do procedimento. </li></ol>

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TMK é apontado como inventor de patentes relacionadas com a imunoterapia intralinfática no homem. TMK foi consultor científico e recebeu as despesas de viagem de ImVisioN GmbH, Cytos Biotechnology, MannKind Corporation, e XBiotech EUA Inc. PJ não tem interesse conflito de divulgar.

Imunização intralinfática e imunoterapia demonstraram ser apropriados para a estimulação de ambas as respostas de anticorpos e respostas de células-T. Como demonstrado neste vídeo-artigo, o procedimento de vacinação é intralinfática um método rápido e fácil para estimular fortes respostas imunitárias em ratinhos. Um cirurgião treinado pode realizar o procedimento durante 3-4 min. A sessão também pode ser compartilhada entre dois cirurgiões onde normalmente é fazer a anestesia ea sutura eo segundo cir...

As vacinas são tipicamente injectados por via subcutânea ou intramuscular para a estimulação de respostas imunitárias. O sucesso deste procedimento requer a drenagem eficiente da vacina para os nódulos linfáticos onde as células apresentadoras de antigénio pode interagir com os linfócitos para a geração de t-e as respostas de células-B. Além disso, a força e o tipo de respostas imunitárias induzidas também depender da densidade ou da frequência de tais interacções, bem como o próprio microambiente, especialmente o teor de citocinas. Como apenas uma pequena fração de uma vacina injetada em um tecido periférico atinge um gânglio linfático, a vacinação de camundongos e humanos por injeção direta de vacina para os gânglios linfáticos, o site foram gerados respostas imunes, foram realizados. No homem, o procedimento é guiada por ultra-sons, um procedimento também utilizados para a administração de agentes de imagem para visualização de diagnóstico e do sistema linfático. Em ratinhos, o procedimento é invasivo. Aqui, uma pequena (5-10 mm) incision é feita na região inguinal de animais anestesiados 1, o nó de linfa é localizada e imobilizada com uma pinça, e um volume de 10-20 mL da vacina é injectada sob controlo visual, 10 ul é usado para as primeiras injecções e em ratos jovens com linfonodos pequenos, enquanto que 20 l pode ser injetado em linfonodos do mais velho ou camundongos já vacinadas, que têm nódulos linfáticos maiores. A incisão pode ser fechada com um único ponto com suturas cirúrgicas. Por este método, os ratos foram vacinados com plasmídeo de DNA de 2,3, o RNA mensageiro 4, 1,3,5,6 péptido, proteína 7-10, as partículas 11, 12 bactérias, bem como adjuvantes de 7,13, e uma forte melhoria da imunitário respostas contra todos os tipos de vacinas têm sido observados. O método intralinfática da vacinação é especialmente adequada em situações onde a vacinação convencional produz imunidade insuficiente, ou quando a quantidade de vacina disponível é limitado ou very dispendioso. Em humanos, o método intralinfática de imunização foi aplicada a pacientes alérgicos 14,15 ou para pacientes com cancro 16-21. Embora a noção corrente de que o método é intralinfática é mais invasiva do que outros métodos de injectáveis, tais como injecções intramusculares e subcutâneas, a percepção da dor não é maior do que após a punção venosa de 15. Espera-se que a vacinação intralinfática vai se tornar uma alternativa ou complemento a outros métodos de vacinas profiláticas e terapêuticas, especialmente. Este artigo descreve em pormenor a forma como o processo de vacinação intralinfática é realizada em ratinhos. Todos os procedimentos descritos, foram aprovados pela Agência Veterinária Cantonal de Zurique e realizada de acordo com as diretrizes federais suíços e directivas relativas à protecção dos animais utilizados para fins científicos.

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			<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>
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		<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

Vacinas são tipicamente injectado subcutaneamente ou intramuscularmente para estimulação de respostas imunes. O sucesso desta requer drenagem eficiente da vacina para os nódulos linfáticos onde as células apresentadoras de antigénio pode interagir com os linfócitos para a geração das respostas imunitárias desejadas. A força e o tipo de respostas imunitárias induzidas também depender da densidade ou da frequência de interacções bem como o microambiente, especialmente o teor de citocinas. Como apenas uma fração mínima de vacinas perifericamente injetados atinge os gânglios linfáticos, a vacinação de camundongos e humanos foram realizados por injecção directa da vacina em linfonodos inguinais, ou seja, injeção intralinfática. No homem, o procedimento é guiada por ultra-sons. Nos ratos, uma pequena (5-10 mm) incisão é feita na região inguinal de animais anestesiados, o nó de linfa é localizada e imobilizada com uma pinça, e um volume de 10-20 mL da vacina é injectada sob controle visual.A incisão é fechada com um único ponto com suturas cirúrgicas. Os ratinhos foram vacinados com ADN de plasmídeo, ARN, péptidos, proteínas, partículas, e bactérias, bem como adjuvantes, e foi observada forte melhoria da resposta imune contra todos os tipos de vacinas. O método intralinfática da vacinação é especialmente adequada em situações onde a vacinação convencional produz imunidade insuficientes ou quando a quantidade de vacina disponível é limitado.

O procedimento de injeções intralinfática em ratos, apesar da natureza cirúrgica, é direto e relativamente rápido. Uma pessoa treinada pode realizar o procedimento em 3-4 min. A incisão que é fechada com um ponto de cura normalmente dentro de dois dias (Figura 1) Intralinfática vacinação ou imunização foi realizada com o ARNm, ADN plasmídeo, peptídeos, proteínas, vírus e bactérias. Figura 2 ilustra a produção de anticorpos após a imuniza...

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

Intralymphatic Immunotherapy and Vaccination in Mice

Vacinação profilática e terapêutica, muitas vezes não estimulam fortes respostas imunes, devido à semana de drenagem da vacina para os gânglios linfáticos e, consequentemente, fraco envolvimento das células do sistema imunológico. Por injecção directa de vacina para os nódulos linfáticos, assim chamada injecção intralinfática, a eficácia da vacina pode ser fortemente melhorada e as doses de vacina podem ser reduzidos.

Intralinfática imunoterapia e vacinação em camundongos

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

22.9K visualizações.  University Hospital Zurich.  Vacinação profilática e terapêutica, muitas vezes não estimulam fortes respostas imunes, devido à semana de drenagem da vacina para os gânglios linfáticos e, consequentemente, fraco envolvimento das células do sistema imunológico. Por injecção directa de vacina para os nódulos linfáticos, assim chamada injecção intralinfática, a eficácia da vacina pode ser fortemente melhorada e as doses de vacina podem ser reduzidos. 

Vídeo: 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.

Metástase experimental e CTL Adoptive Transferência Modelo rato Imunoterapia

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

Imunologia e Infecção

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.

Protocolo para vacinas recombinantes RBD baseado SARS: Preparação de proteínas, Vacinação Animal e Detecção de Neutralização

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.

Preparação do antigénio tumoral-carregados células dendríticas maduras para a imunoterapia

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.

Triagem de alta capacidade para amplo espectro químico Inibidores de Vírus RNA

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.

Geração de células T para CAR adoptivo Therapy no Contexto do Glioblastoma Padrão de Atendimento

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.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

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.

Geração dos anticorpos monoclonais humanos discriminativa de rara B antígeno-específicas células circulantes no sangue

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.

Estereotáxica transferência adotiva de citotóxicas células imunes em modelos murino de humanos ortotópico 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 ...

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.

Ensaio de ELISpot otimizado de Interferon-gama às respostas de célula T medida no modelo de porquinho da Índia após a vacinação

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.

Análise de análogos de ácido Iofenoxico em pequeno mangusto indiano (Herpestes auropunctatus) sera para uso como um marcador biológico de vacinação contra a raiva oral

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