Gli autori sono grati per l&#39;aiuto sperimentale nello sviluppo del metodo di immunizzazione endolinfatica nei topi da Iris Erdmann, Barbara von Beust, e Julia Maria Martínez-Gómez. Grazie anche a Maggy Arras e Nikola Cesarovic per averci permesso di usare la loro sala operatoria per questa produzione video.

1. Anestesia di topi <ol><li> Preparare i anestetici mescolando ketamina (anestetico dissociativo) e xilazina (sedativo e analgesico) in soluzione salina tamponata. Le concentrazioni di chetamina e xilazina nella soluzione finale sono 12,5 e 2 mg / ml, rispettivamente. </li><li> Iniettare anestetici ai topi per somministrazione intraperitoneale utilizzando una siringa con un ago G 25-30. Utilizzare 0,1 ml/10 g di peso corporeo. </li><li> Applicare una pomata oftalmica per gli occhi dei topi in modo da evitare l&#39;essiccazione-out delle cornee. </li><li> Assicurarsi che il mouse sia sufficientemente anestetizzato pizzicando il piede o del piede con le pinze. Se il mouse non reagisce con riflessi per il pizzico, procedere con un intervento chirurgico. Se il mouse reagisce pizzico di riflessi o contrazioni muscolari, aspettare 1-2 minuti e ripetere il test pizzico. Se il mouse reagisce ancora, sostituire il mouse con un nuovo animale e ripetere le procedure dal punto 1.2 in poi. </li></ol> 2. I chirurgicancision della zona inguinale del mouse <ol><li> Posizionare il mouse sulla sua schiena. </li><li> Bagnare una regione inguinale con etanolo disinfezione (70%). Per fornire una migliore disinfezione, la regione inguinale può essere rasata per rimuovere pelliccia prima disinfezione con etanolo. </li><li> Prendete la zampa posteriore e piegare l&#39;articolazione dell&#39;anca per produrre un angolo di circa 90 ° dell&#39;articolazione dell&#39;anca. </li><li> Utilizzando un curvo forcipe microdissecting, prendere la pelle del mouse nella zona inguinale, dove l&#39;articolazione dell&#39;anca è piegato e tirare la pelle bagnata leggermente. </li><li> Tenendo la pelle con le pinze, tagliare una piccola incisione (&lt;5 mm) attraverso la pelle con le forbici chirurgiche. </li><li> Posizionare la punta delle forbici chiuse nell&#39;incisione e aprire ulteriormente l&#39;incisione aprendo le forbici tenendolo dentro l&#39;incisione. Questo farà sì che la pelle allo strappo e dovrebbe produrre un diametro inferiore a 10 mm. Nota: Una sorgente di luce fredda con guide di luce flessibile può essere utilizzato per migliorare la visibilità. In più vecchiotopi, soprattutto maschi, l&#39;identificazione del linfonodo può essere difficile a causa tessuto molto grasso nella regione inguinale. </li></ol> 3. Iniezione endolinfatica <ol><li> Preparare una siringa (0,5 ml o meno) con un ago ipodermico G 28-30; un breve smussatura può essere preferibile per un lungo bisello. Aspirare 10 ml di vaccino da iniettare e assicurano che la siringa è privo di aria. </li><li> Localizzare il linfonodo inguinale con l&#39;aiuto delle pinze curve e la punta della forbice chiusa. Il linfonodo viene visualizzata grigiastro all&#39;interno del tessuto grasso più biancastra e può essere ulteriormente individuate attraverso afferenti ed efferenti capillari che entrano ed escono il linfonodo. </li><li> Immobilizzare il linfonodo tenendolo tra i rami delle pinze curve. </li><li> Prendere la siringa e inserire l&#39;ago nel linfonodo con lo smusso rivolto verso l&#39;alto, assicurandosi che il complesso smussatura entra nel linfonodo. </li><li> Iniettare il vaccino (10 microlitri). Se il cenno del capo linfae si gonfia (esplodere), l&#39;iniezione può essere assunto successo. Se l&#39;ago non è posizionato abbastanza in profondità nel linfonodo, una parte o l&#39;intero vaccino può fuoriuscire e non entrare nel linfonodo. Se l&#39;ago viene inserito troppo in profondità, il vaccino iniettato uscirà sottocutanea sotto del linfonodo. Nel caso di quest&#39;ultimo, ripetere l&#39;iniezione o sostituire il mouse con un nuovo mouse greggia. </li></ol> 4. Chiusura del Incision da sutura <ol><li> Aprire una confezione di sterile sutura chirurgica. </li><li> Afferrare l&#39;ago di sutura con un porta-aghi in corrispondenza della porzione distale del corpo dell&#39;ago. Serrare il supporto dell&#39;ago stringendola fino alle prime catture cricchetto. </li><li> Afferrare la pelle su un lato l&#39;incisione con pinze (a seconda delle preferenze del chirurgo, dentato o pinze o uncini cutanei non-denti possono essere utilizzati). Inserire l&#39;ago ca. 2-3 mm dal bordo dell&#39;incisione, entrando dall&#39;esterno della pelle. </li><li> Loosen stiva della pelle con la pinza e afferrare il lato opposto della incisione con le pinze. Inserire l&#39;ago ca. 2-3 mm dal bordo dell&#39;incisione, entrando dall&#39;interno della pelle. </li><li> Allentare il supporto dell&#39;ago e afferrare l&#39;ago con le pinze. Estrarre l&#39;ago con il battistrada attraverso la pelle, lasciando una conveniente lunghezza del battistrada di fuori del primo punto ago-inserimento (se diversi topi sono da trattare, lasciare un terminale più corto; 5-10 topi può funzionare con un ago di sutura / calcare). </li><li> Rendere il nodo preferito secondo protocolli chirurgo standard. Un punto è sufficiente che l&#39;incisione non è maggiore di 10 mm. Per incisioni di grandi dimensioni, effettuare un secondo punto. </li></ol> 5. Il trattamento post-operativa <ol><li> Posizionare il mouse nella gabbia e coprire con i tessuti per tenere in caldo. Durante il trattamento più topi, metterli uno vicino all&#39;altro per tenere in caldo. In alternativa, posizionare il mouse su un tappetino caldo fino a quando si sono svegliatiup. </li><li> Osservare il mouse fino a quando si svegliano. </li><li> Osservare i topi quotidiano con riferimento a guarigione delle ferite così come altri sintomi clinici, come infezioni della ferita. La ferita è in genere sufficientemente chiusa entro due giorni e guarito entro sette giorni dalla procedura. </li></ol>

<ol>
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J., 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=Route+of+administration+modulates+the+induction+of+dendritic+cell+vaccine-induced+antigen-specific+T+cells+in+advanced+melanoma+patients.">Route of administration modulates the induction of dendritic cell vaccine-induced antigen-specific T cells in advanced melanoma patients.</a> Clin. Cancer Res. 17, 5725-5735 (2011).</li><li>Fadul, C. E., 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=Immune+response+in+patients+with+newly+diagnosed+glioblastoma+multiforme+treated+with+intranodal+autologous+tumor+lysate-dendritic+cell+vaccination+after+radiation+chemotherapy.">Immune response in patients with newly diagnosed glioblastoma multiforme treated with intranodal autologous tumor lysate-dendritic cell vaccination after radiation chemotherapy.</a> J. Immunother. 34, 382-389 (2011).</li><li>Eizenberg, P., 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=Acceptance+of+Intanza(R)+9+mug+intradermal+influenza+vaccine+in+routine+clinical+practice+in+Australia+and+Argentina.">Acceptance of Intanza(R) 9 mug intradermal influenza vaccine in routine clinical practice in Australia and Argentina.</a> Adv. 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TMK è chiamato come inventore di brevetti che si occupano di immunoterapia endolinfatica nell&#39;uomo. TMK è stato consulente scientifico ed ha ricevuto le spese di viaggio da ImVisioN GmbH, Cytos Biotechnology, MannKind Corporation, e XBiotech USA Inc. PJ non ha alcun interesse conflitto di rivelare.

Immunizzazione endolinfatica e immunoterapia hanno dimostrato di essere adatto per la stimolazione di entrambe le risposte anticorpali e risposte delle cellule T. Come dimostrato in questo video-articolo, la procedura endolinfatica di vaccinazione è un metodo semplice e veloce per stimolare una forte risposta immunitaria nei topi. Un chirurgo esperto può eseguire la procedura durante 3-4 min. La sessione può anche essere condiviso tra due chirurghi in cui si è tipicamente facendo l&#39;anestesia e la sutura e il sec...

I vaccini sono tipicamente iniettati per via sottocutanea o intramuscolare stimolazione delle risposte immunitarie. Il successo di questa procedura richiede drenaggio efficace di vaccino ai linfonodi dove le cellule presentanti l&#39;antigene possono interagire con i linfociti per la generazione di risposte T e B-cellule. Inoltre, la forza e il tipo di risposte immunitarie indotte dipendono anche dalla densità o frequenza di tali interazioni, nonché il microambiente stesso, specialmente il contenuto di citochine. Poiché solo una piccola frazione di un vaccino iniettato in un tessuto periferico raggiunge un linfonodo, vaccinazioni di topi e nell&#39;uomo mediante iniezione diretta del vaccino con linfonodi, il sito sono stati generati risposte immunitarie, sono state eseguite. Nell&#39;uomo, la procedura è guidata da ultrasuoni, una procedura utilizzata anche per la somministrazione di agenti di imaging per la visualizzazione e la diagnosi del sistema linfatico. Nei topi, la procedura è invasiva. Qui, una piccola (5-10 mm) incision è realizzato nella regione inguinale di animali anestetizzati 1, il linfonodo è localizzato e immobilizzato con una pinza, e un volume di 10-20 ml di vaccino viene iniettato sotto controllo visivo; 10 microlitri è usato per prime iniezioni e in topi giovani con piccoli linfonodi, considerando che 20 microlitri può essere iniettato nei linfonodi di età superiore o topi già innescate, che hanno i linfonodi più grandi. L&#39;incisione può essere chiuso con un punto singolo con suture chirurgiche. Con questo metodo, i topi sono vaccinati con il plasmide DNA 2,3, RNA messaggero 4, 1,3,5,6 peptide, proteina 7-10, particelle 11, 12 batteri così come adiuvanti 7,13, e forte miglioramento immunitario Sono state osservate risposte contro tutti i tipi di vaccini. Il metodo endolinfatica della vaccinazione è particolarmente indicata nelle situazioni in cui la vaccinazione convenzionale produce immunità insufficiente o in cui la quantità di vaccino disponibile è limitato o very costoso. Nell&#39;uomo, il metodo endolinfatica di immunizzazione è stato applicato a pazienti allergici 14,15 o di pazienti con cancro 16-21. Sebbene l&#39;idea corrente è che il metodo endolinfatica è più invasivo rispetto ad altri metodi iniettabili come le iniezioni intramuscolari e sottocutanee, la percezione del dolore non è superiore dopo una puntura venosa 15. Si prevede che la vaccinazione endolinfatica diventerà alternativa o complementare ad altri metodi di vaccini profilattici e terapeutici particolarmente. Questo articolo descrive in dettaglio come viene eseguita la procedura di vaccinazione endolinfatica nei topi. Tutte le procedure descritte, sono state approvate dall&#39;Agenzia veterinario cantonale di Zurigo ed eseguiti secondo le linee guida federali svizzere e direttive sulla protezione degli animali utilizzati a fini scientifici.

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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

I vaccini sono tipicamente iniettati per via sottocutanea o intramuscolare per la stimolazione delle risposte immunitarie. Il successo di questo richiede drenaggio efficiente di vaccino ai linfonodi dove le cellule presentanti l&#39;antigene possono interagire con i linfociti per la generazione delle risposte immunitarie voluto. La forza e il tipo di risposte immunitarie indotte dipendono anche dalla densità o frequenza di interazioni nonché il microambiente, in particolare il contenuto di citochine. Poiché solo una minima parte di vaccini iniettati perifericamente raggiunge linfonodi, le vaccinazioni di topi e gli esseri umani sono stati eseguiti mediante iniezione diretta di vaccino nei linfonodi inguinali, cioè l&#39;iniezione endolinfatica. Nell&#39;uomo, la procedura è guidata da ultrasuoni. Nei topi, una piccola (5-10 mm) incisione viene fatta nella regione inguinale di animali anestetizzati, il linfonodo è localizzato e immobilizzato con una pinza, e un volume di 10-20 ml di vaccino viene iniettato sotto controllo visivo.L&#39;incisione viene chiusa con un solo punto usando suture chirurgiche. I topi sono stati vaccinati con il plasmide DNA, RNA, peptidi, proteine, particelle e batteri e coadiuvanti, e si è osservata una forte miglioramento delle risposte immunitarie contro tutti i tipi di vaccini. Il metodo endolinfatica della vaccinazione è particolarmente indicata nelle situazioni in cui la vaccinazione convenzionale produce immunità insufficiente o in cui la quantità di vaccino disponibile è limitato.

La procedura di iniezioni intralinfatiche nei topi, nonostante la natura chirurgica, è semplice e relativamente veloce. Una persona esperta in grado di eseguire la procedura in 3-4 min. L&#39;incisione che è chiuso con un punto guarisce in genere entro due giorni (Figura 1) Endolinfatica vaccinazione o immunizzazione è stato eseguito con mRNA, DNA plasmidico, peptidi, proteine, virus e batteri. Figura 2 illustra la produzione anticorpale dopo immunizzazio...

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

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccinazione profilattica e terapeutica spesso non riesce a stimolare forti risposte immunitarie dovuto settimana drenaggio del vaccino ai linfonodi e conseguente scarsa coinvolgimento delle cellule immunitarie. Mediante iniezione diretta di vaccino da linfonodi, cosiddetta iniezione endolinfatica, l&#39;efficacia del vaccino può essere fortemente migliorata e dosi di vaccino può essere ridotta.

Endolinfatica immunoterapia e di vaccinazione nei topi

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 Views.  University Hospital Zurich.  Vaccinazione profilattica e terapeutica spesso non riesce a stimolare forti risposte immunitarie dovuto settimana drenaggio del vaccino ai linfonodi e conseguente scarsa coinvolgimento delle cellule immunitarie. Mediante iniezione diretta di vaccino da linfonodi, cosiddetta iniezione endolinfatica, l'efficacia del vaccino può essere fortemente migliorata e dosi di vaccino può essere ridotta. 

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.

Metastasi sperimentale e CTL adottivi Immunoterapia modello di trasferimento del mouse

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

Ricerca

Immunologia e infezioni

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.

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

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.

Preparazione del Tumor Antigen-caricati mature cellule dendritiche per l&#39;immunoterapia

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.

High-throughput screening per largo spettro chimico inibitori di RNA virus

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.

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

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.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

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.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel 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.

Trasferimento adottivo stereotactic di citotossici cellule immunitarie nei modelli murini di xenotrapianti Multiforme di Glioblastoma umano ortotopico

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.

Ottimizzazione del interferone-gamma ELISpot analisi di risposte a cellula T misura nel modello cavia dopo la vaccinazione

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.

Analisi degli analoghi dell'acido iofenostico nella piccola mangusta indiana (Herpestes Auropunctatus) Sera per l'uso come marcatore biologico di vaccinazione della rabbia orale

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