Los autores agradecen la ayuda experimental en el desarrollo del método de inmunización intralinfática en ratones de Iris Erdmann, Barbara von Beust, y Julia María Martínez-Gómez. Gracias también a Maggy Arras y Nikola Cesarovic para dejarnos usar su teatro quirúrgico para esta producción de vídeo.

1. Anestesia de ratones <ol><li> Preparar los anestésicos mediante la mezcla de ketamina (anestésico disociativo) y xilazina (sedante y analgésico) en solución salina. Las concentraciones de ketamina y xilazina en la solución final son 12.5 y 2 mg / ml, respectivamente. </li><li> Inyectar los anestésicos a los ratones mediante administración intraperitoneal usando una jeringa con una aguja de calibre 25-30. Utilice 0,1 ml/10 g de peso corporal. </li><li> Aplique una pomada oftálmica a los ojos de los ratones con el fin de evitar que se reseque de las córneas. </li><li> Asegúrese de que el ratón esté suficientemente anestesiado pellizcando su pie o del pie con las pinzas. Si el ratón no reacciona con reflejos para la pizca, proceder a la cirugía. Si el ratón reacciona a la pizca con los reflejos o contracciones musculares, esperar 1-2 minutos y repetir la prueba del pellizco. Si el ratón sigue reacciona, sustituya el ratón con un animal nuevo y repita el procedimiento desde el paso 1.2 en adelante. </li></ol> 2. Me quirúrgicancision del Área inguinal del ratón <ol><li> Coloque el ratón en la espalda. </li><li> Humedecer una región inguinal con etanol desinfección (70%). Para proporcionar una mejor desinfección, la región inguinal puede afeitar para quitar la piel antes de la desinfección con etanol. </li><li> Tome la pata trasera y doble la articulación de la cadera para producir un ángulo de aproximadamente 90 ° de la articulación de la cadera. </li><li> El uso de un fórceps curvados microdissecting, tomar la piel de ratón en la zona inguinal, donde la articulación de la cadera está doblado y tire de la piel humedecida ligeramente. </li><li> Mientras mantiene la piel con las pinzas, corte una pequeña incisión (&lt;5 mm) a través de la piel utilizando tijeras quirúrgicas. </li><li> Coloque la punta de las tijeras cerradas en la incisión y abrir aún más la incisión mediante la apertura de las tijeras mientras lo mantiene dentro de la incisión. Esto hará que la piel a desgarrarse y debe producir un diámetro de menos de 10 mm. Nota: Una fuente de luz fría con guías de luz flexibles se puede utilizar para mejorar la visibilidad. En más viejosratones, especialmente los varones, la identificación del ganglio linfático puede ser difícil debido a la cantidad de tejido de grasa en la región inguinal. </li></ol> 3. Inyección intralinfática <ol><li> Preparar una jeringa (0,5 ml o menos) con una aguja hipodérmica 28-30 G; un bisel corto puede ser preferible a una larga bisel. Aspirar 10 l de la vacuna que se inyecta y aseguran que la jeringa esté libre de aire. </li><li> Localizar el ganglio linfático inguinal con ayuda de las pinzas curvas y la punta de la tijera cerrado. El ganglio linfático aparecerá grisáceo en el tejido graso más blanquecina y puede ser identificado más a través de aferentes y eferentes capilares que entran y salen del ganglio linfático. </li><li> Inmovilizar el ganglio linfático sosteniéndolo entre las ramas de las pinzas curvas. </li><li> Coja la jeringa e inserte la aguja en el ganglio linfático con el bisel hacia arriba, asegurándose de que todo el bisel entra en el ganglio linfático. </li><li> Se inyecta la vacuna (10 l). Si el visto bueno linfáticoe Es la hinchazón (voladura), la inyección se puede asumir con éxito. Si la aguja no está colocada lo más profundo en el ganglio linfático, una parte o la totalidad de la vacuna puede filtrarse y no entrar en el ganglio linfático. Si se inserta la aguja demasiado profunda, la vacuna se inyecta por vía subcutánea se dará a conocer a continuación el nodo linfático. En este último caso, repetir la inyección o sustituir el ratón con un nuevo ratón no tratado. </li></ol> 4. Cerrar la incisión suturando <ol><li> Abra un paquete de sutura quirúrgica estéril. </li><li> Agarre de la aguja de sutura con un soporte de aguja en la parte distal del cuerpo de aguja. Apriete el soporte de la aguja apretando hasta que las primeras capturas de trinquete. </li><li> Agarre de la piel en un lado de la incisión utilizando fórceps (dependiendo de la preferencia del cirujano, fórceps o ganchos de la piel dentada o de las Naciones Unidas de dientes puede ser utilizado). Insertar la aguja CA. 2-3 mm del borde de la incisión, que entra desde el exterior de la piel. </li><li> Loosen el asimiento de la piel con los fórceps y sujete el lado opuesto de la incisión con los fórceps. Insertar la aguja CA. 2-3 mm del borde de la incisión, que entra desde el interior de la piel. </li><li> Afloje el soporte de la aguja y sujete el extremo de la aguja con los fórceps. Tire de la aguja con la banda de rodadura a través de la piel, dejando una longitud conveniente de la banda de rodadura fuera del primer punto de inserción de aguja (si son varios ratones a tratar, dejar un extremo más corto; 5-10 ratones puede ser operado con una aguja de sutura / Paso). </li><li> Hacer el nudo preferido de acuerdo con protocolos estándar cirujano. Una puntada es suficiente si la incisión no es mayor de 10 mm. Para incisiones más grandes, hacer una segunda puntada. </li></ol> 5. Tratamiento post-operativa <ol><li> Coloque el ratón en la jaula y cubrir con los tejidos para mantener el calor. En el tratamiento de varios ratones, colocarlos cerca unos de otros para mantener el calor. Como alternativa, coloque el ratón sobre una almohadilla caliente hasta que han despertadoarriba. </li><li> Observar los ratones hasta que se despiertan. </li><li> Observar los ratones diariamente con respecto a la cicatrización de heridas, así como otros síntomas clínicos tales como infecciones de la herida. La herida suele ser lo suficientemente cerrado dentro de dos días, y sanó a los siete días del procedimiento. </li></ol>

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TMK es nombrado como inventor de patentes que tratan con inmunoterapia intralinfática en el hombre. TMK ha sido asesor científico y ha recibido los gastos de viaje desde ImVisioN GmbH, Cytos Biotecnología, MannKind Corporation, y XBiotech EE.UU. Inc. PJ no le interesa el conflicto de revelar.

La inmunización intralinfática e inmunoterapia han demostrado ser apropiados para la estimulación de las respuestas tanto de anticuerpos y respuestas de células T. Como se ha demostrado en este video-artículo, el procedimiento intralinfática de la vacunación es un método rápido y fácil para la estimulación de respuestas inmunes fuertes en ratones. Un cirujano entrenado puede realizar el procedimiento durante 3-4 min. En la sesión también se puede compartir entre dos cirujanos donde uno es típicamente hacie...

Las vacunas se inyectan típicamente por vía subcutánea o intramuscular para la estimulación de respuestas inmunes. El éxito de este procedimiento requiere un drenaje eficaz de la vacuna a los ganglios linfáticos donde las células presentadoras de antígeno pueden interactuar con linfocitos para la generación de T y las respuestas de células B. Además, la fuerza y ​​el tipo de respuesta inmune inducida también dependen de la densidad o de la frecuencia de tales interacciones, así como el propio microambiente, especialmente el contenido de citoquinas. Como sólo una pequeña fracción de una vacuna se inyecta en un tejido periférico llega a un nodo de linfa, las vacunaciones de ratones y seres humanos mediante inyección directa de la vacuna en los ganglios linfáticos, el sitio se se generan respuestas inmunes, se han realizado. En el hombre, el procedimiento es guiado por ultrasonido, un procedimiento también se utiliza para la administración de agentes de formación de imágenes para la visualización y el diagnóstico en el sistema linfático. En ratones, el procedimiento es invasivo. Aquí, una pequeña (5-10 mm) incision se realiza en la región inguinal de los animales anestesiados 1, el nodo linfático se localiza y se inmoviliza con fórceps, y un volumen de 10-20 l de la vacuna se inyecta bajo control visual; 10 l se utiliza para la primera inyecciones y en ratones jóvenes con pequeñas ganglios linfáticos, mientras que 20 l pueden ser inyectados en los ganglios linfáticos de los ratones más viejos o ya cebados, que tienen ganglios linfáticos más grandes. La incisión se puede cerrar con una sola puntada utilizando suturas quirúrgicas. Por este método, los ratones han vacunados con el plásmido de ADN de 2,3, el ARN mensajero 4, 1,3,5,6 péptido, proteína 7-10, las partículas 11, 12 bacterias, así como adyuvantes 7,13, y fuerte mejora de inmunológico Se han observado respuestas contra todo tipo de vacunas. El método intralinfática de la vacunación es especialmente apropiada en situaciones en las que la vacunación convencional produce inmunidad insuficiente o cuando la cantidad de vacuna disponible es limitado o very costoso. En humanos, el método intralinfática de la inmunización se ha aplicado a los pacientes de alergia 14,15 o para pacientes con cáncer de 16-21. Aunque la idea actual es que el método intralinfática es más invasivo que otros métodos inyectables tales como inyecciones intramusculares y subcutáneas, la percepción del dolor no es mayor que después de una punción venosa 15. Se espera que la vacunación intralinfática se convertirá en una alternativa o complemento a otros métodos de vacunas profilácticas y terapéuticas especialmente. En este artículo se describe en detalle cómo se realiza el procedimiento de vacunación intralinfática en ratones. Todos los procedimientos descritos, fueron aprobados por la Agencia Veterinaria Cantonal de Zúrich, y llevaron a cabo según las directrices y directivas sobre la protección de los animales utilizados para fines científicos federales suizos.

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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>
		</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

Las vacunas se inyectan típicamente por vía subcutánea o intramuscular para la estimulación de respuestas inmunes. El éxito de este requiere un drenaje eficaz de la vacuna a los ganglios linfáticos donde las células presentadoras de antígeno pueden interactuar con linfocitos para la generación de las respuestas inmunes buscadas. La fuerza y ​​el tipo de respuesta inmune inducida también dependen de la densidad o la frecuencia de las interacciones, así como el microambiente, especialmente el contenido de citoquinas. Como sólo una fracción de minuto de vacunas periféricamente inyectados llega a los ganglios linfáticos, las vacunaciones de ratones y seres humanos se realizaron por inyección directa de la vacuna en los ganglios linfáticos inguinales, es decir, la inyección intralinfática. En el hombre, el procedimiento es guiada por ultrasonido. En ratones, una pequeña incisión (5-10 mm) se realiza en la región inguinal de los animales anestesiados, el ganglio linfático se localiza y se inmoviliza con unas pinzas, y un volumen de 10-20 l de la vacuna se inyecta bajo control visual.La incisión se cierra con una única puntada utilizando suturas quirúrgicas. Los ratones fueron vacunados con el ADN plásmido, ARN, péptido, proteína, partículas, y bacterias, así como adyuvantes, y se observó una fuerte mejora de la respuesta inmune contra todo tipo de vacunas. El método intralinfática de la vacunación es especialmente apropiada en situaciones en las que la vacunación convencional produce inmunidad insuficiente, o cuando la cantidad de vacuna disponible es limitado.

El procedimiento de inyecciones intralinfáticas en ratones, a pesar de la cirugía, es sencillo y relativamente rápido. Una persona capacitada puede realizar el procedimiento en 3-4 min. La incisión que se cierra con una puntada típicamente se cura dentro de dos días (Figura 1) Vacunación o inmunización intralinfática se ha realizado con ARNm, ADN plásmido, péptidos, proteínas, virus y bacterias. Figura 2 ilustra la producción de anticuerpos desp...

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

Intralymphatic Immunotherapy and Vaccination in Mice

La vacunación profiláctica y terapéutica a menudo falla para estimular respuestas inmunes fuertes debido a la semana de drenaje de la vacuna a los ganglios linfáticos y en consecuencia pobres implicación de células inmunes. Por inyección directa de la vacuna a los ganglios linfáticos, así llamada inyección intralinfática, eficacia de la vacuna puede ser fuertemente mejorada y dosis de la vacuna se puede reducir.

Intralinfática inmunoterapia y vacunación en ratones

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 vistas.  University Hospital Zurich.  La vacunación profiláctica y terapéutica a menudo falla para estimular respuestas inmunes fuertes debido a la semana de drenaje de la vacuna a los ganglios linfáticos y en consecuencia pobres implicación de células inmunes. Por inyección directa de la vacuna a los ganglios linfáticos, así llamada inyección intralinfática, eficacia de la vacuna puede ser fuertemente mejorada y dosis de la vacuna se puede reducir. 

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.

La metástasis experimentales y CTL Modelo inmunoterapia adoptiva de transferencia de ratón

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

Investigación

Inmunología e Infección

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 las vacunas recombinantes para RBD basada en el SRAS: Preparación de la proteína, la vacunación de animales y la detección de neutralización

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.

Preparación de las células dendríticas maduras cargadas con antígeno del tumor para inmunoterapia

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.

Screening de alto rendimiento para los de amplio espectro químico Inhibidores de Virus ARN

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.

Generación de células T CAR para realizar la terapia adoptiva en el contexto de Glioblastoma estándar de cuidado

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.

En Vivo Ensayo para la detección de la función citolítica de células T específicas de antígeno utilizando un modelo de vacunación

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.

Generación de anticuerpos monoclonales humanos discriminativo de células B antígeno específicas raras circulan en sangre

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.

Estereotáctica transferencia adoptiva de células inmunes citotóxicas en modelos murinos de xenoinjertos de Glioblastoma Multiforme de Orthotopic humanos

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.

Optimizado de interferón-gamma ELISpot análisis a las respuestas de la célula de T de medida en el modelo de conejillo de Indias después de la vacunación

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.

Análisis de análogos de ácido iophenoxic en pequeña mangosta india (Herpestes Auropunctatus) Sera para su uso como un marcador biológico de vacunación de rabia 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 ...