Name: stem-cell based engineered immunity against hiv infection in the humanized mouse model
Uploaded: 2016-07-02T13:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

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The authors have nothing to disclose.

Com CAR e baseado em HSC ganhando impulso imunidade projetado para os estudos clínicos, é importante ter um modelo animal adequado para examinar de perto a diferenciação e função destas células manipuladas. Neste protocolo, descrevemos os métodos para a construção e teste de camundongos humanizados com células-tronco geneticamente modificados projetados contra o HIV. É importante ter a transdução eficiente de células estaminais, antes do transplante. No entanto, devido à capacidade de células T a proliferar no reconhecimento de células alvo, os baixos níveis de modificação de células-tronco eram suficientes para gerar uma resposta robusta contra a replicação do HIV 8. No entanto, para alcançar alto nível de modificação de células-tronco, recomendamos a utilização de células CD34 + transduzidas na mistura de proteína gelatinosa com timo autólogo, em vez de fígado e pedaços timo para a cirurgia de ratos que tinham sido descritos em outros lugares 13-15. mistura proteica gelatinosa é um solubilizEd membrana basal do tecido rico em proteínas da matriz extracelular. Sob condições fisiológicas normais, a mistura gelatinosa polimerizar proteína para produzir uma matriz reconstituído, biologicamente activa e estável, que iria permitir a ligação eficaz e diferenciação das células estaminais 20. reconstituição célula humana pode ser verificada por sangramento retro-orbital e citometria de fluxo 6 - 8 semanas pós-cirurgia. Para assegurar que o vector não é tóxico para a sobrevivência de células-tronco e a renovação, é recomendado para titular o vector em células CD34 + antes da experiência, conforme descrito em 1.2.6. A capacidade do modelo de ratinho NSG-BLT para suportar a infecção das mucosas, viremia consistente e respostas imunes celulares a torna um modelo muito útil para estudar a patologia imunológica HIV e terapias baseadas em células para o tratamento de uma infecção por HIV. Mais importante ainda, as células T geradas a partir dos ratinhos NSG-BLT são seleccionados no tecido tímico autólogo, permitindo que o investigador para estudar o destinodo gene modificado células estaminais após a selecção tímica 8,21. Com o método descrito, temos sido capazes de obter 40% -90% de reconstituição imune celular humana de forma consistente. Os baixos níveis de reconstituição de células humanas pode resultar de vários fatores, incluindo as habilidades da pessoa que executa a cirurgia e a qualidade dos tecidos para transplante. Para atingir um alto nível de reconstituição de células humanas, é importante assegurar os transplantes estão correctamente colocados por baixo da cápsula do rim. Além disso, é altamente recomendável para examinar cada implante timo preparado para a cirurgia em microscópio de luz e descartar quaisquer peças questionáveis. Embora o modelo humanizado rato BLT é uma ferramenta promissora para o estudo da imunidade projetados contra o HIV (revisto em 1,15,22), que tem suas próprias limitações. Ou seja, este modelo não imitam perfeitamente um sistema imunológico periférico humano. Estudos têm demonstrado desenvolvimento prejudicado de hiper-mutante, comutação de classe IgG Antibody 1,23. Além disso, utilizando camundongos imuno-deficientes é tecnicamente desafiador e mantendo estes ratos saudável requer recursos consideráveis ​​e treinamento. infecções oportunistas sutis podem se manifestar como diferenças significativas entre as amostras e, potencialmente, ter consequências negativas para a experimentação. Por isso, é importante ter instalações bem preparadas e pessoal devidamente treinado para manter a integridade do futuro 1,24 dados. Com estas limitações em mente, o modelo do rato NSG-BLT humanizado ainda fornece uma ferramenta importante para o estudo da imunidade engenharia baseada em células-tronco, como é demonstrado por estes exemplos 4,8. Com a tendência de desenvolvimento de receptores de antigénios quiméricos baseados em HIV ampla anticorpos 10 e modificação do domínio de sinalização para o carro mais eficiente neutralização 25, este modelo e do protocolo pode ser utilizado para caracterizar e investigar a funcionalidade do gene CEL modificadols com uma nova geração de carros. Além disso, este modelo pode potencialmente acomodar estudos sobre a terapia com base imune (tal como o bloqueio do receptor inibitório) em conjunto com a imunidade de engenharia.

Apesar do sucesso da terapia anti-retroviral combinada, a infecção por HIV é ainda uma doença ao longo da vida. A resposta imune celular contra o HIV desempenha um papel muito importante no controlo da replicação do HIV. Os recentes avanços na manipulação de células-tronco tem permitido para o rápido desenvolvimento da terapia genética abordagens para HIV tratamento 1-3. Como resultado, é importante dispor de um modelo animal apropriado que permita estudo in vivo da eficácia de terapias à base de células contra o HIV. Trabalhando com HIV em modelos animais é complicada pelo facto de o vírus infecta apenas células humanas. Para contornar essa limitação, os cientistas recorreram ao uso de modelos de doenças como o vírus Simian Immunodeficiency (SIV) em macacos Rhesus 4,5. Infelizmente, há grandes limitações neste modelo devido às diferenças inerentes entre as espécies e as diferenças entre SIV e HIV. Além disso, apenas as instalações altamente especializados são capable de apoiar o trabalho com primatas não-humanos e cada macaque requer um grande investimento. Assim, existe uma necessidade premente de um modelo que utiliza o sistema imunitário humano, que seja susceptível a infecção por HIV / patogénese, e é financeiramente menos proibitivo. O não-obesos diabéticos (NOD) -severe imunodeficientes combinados (SCID) de cadeia knockout gamma -Common (c γ - / -) (ou NSG) Blood / Fígado / Thymus (BLT) modelo de rato humanizado é cada vez mais provado ser uma ferramenta importante para estudar a infecção pelo HIV. Por implantação de células-tronco hematopoiéticas (HSCs) e timo fetal, os ratinhos são capazes de recapitular o desenvolvimento e um sistema imune humano 1-3. Um tipo de terapia genética baseada em células estaminais envolve &#39;reorientação das células T periféricas de alvo do VIH através da reprogramação de células estaminais hematopoiéticas (HSCs) de se diferenciar em células T específicas para o antigénio. Nós temos mostrado previamente que HSCs de engenharia com um clonado anti-HIV re específica molecular das células TCeptor (TCR) contra o epitopo SL9 (aminoácidos 77-85; SLYNTVATL) do HIV-1 Gag pode redireccionar células estaminais em células T maduras formando que suprimem a replicação do HIV no modelo de ratinho humanizado BLT NSG-6. A advertência do uso de um TCR clonado molecular é de que seja limitado a um subtipo específico de antigénio de leucócitos humanos (HLA) que vai limitar a aplicação desta terapia. receptores de antigénios quiméricos (CAR), por outro lado, pode ser universalmente aplicado para todos os subtipos de HLA. Os estudos iniciais foram realizados utilizando um CARRO construído com os domínios extracelular e transmembranar de CD4 humano fundidos com o domínio intracelular de ζ CD3 sinalização (denominado a CD4ζCAR). CD4ζCAR expressa em células T CD8 podem reconhecer do envelope de HIV e desencadear uma resposta de células T citotóxicas que é semelhante à que mediada por um receptor de células T 7. Nós demonstramos recentemente que hsc humanas podem ser modificados com CD4ζCAR, que pode então diferenciam-se em múltiplos Li hematopoiéticasneages, incluindo células T funcionais capazes de suprimir a replicação do HIV no modelo de ratinho humanizado 8. Com o rápido avanço em terapias de receptores de antigénios quiméricos para o cancro 9, e a caracterização permanente de anticorpos neutralizantes amplos 10-12 potentes contra o HIV que permitem a construção de veículos de anticorpo de cadeia simples, é perceptível que muitas novas construções de candidatos, além de CD4ζCAR , irão ser gerados e testados para a terapia genética baseada em células estaminais de doenças do HIV e outras doenças. Além disso, o modelo de rato NSG-BLT humanizado contendo estes carros antígeno-específicas também pode fornecer uma ferramenta útil para examinar de perto as respostas das células T humanas in vivo. Importante, a nossa protocolo difere dos métodos descritos anteriormente para a construção de murganhos humanizado de BLT 13-15 em que a mistura proteica em HSCs gelatinosa é usado em lugar de troncos de fígado fetal 16. Este protocolo descreve: 1) construção de humanicamundongos BLT zed projetados com CD4ζCAR; e 2) a caracterização da diferenciação das células geneticamente modificadas; e 3) a caracterização da funcionalidade das células geneticamente modificadas.

<table><tbody><tr><td>CD34 microbead kit</td><td>Miltenyi</td><td>130-046-702</td><td>For sorting human CD34+ progenitor cells</td></tr><tr><td>Bambanker</td><td>Wako</td><td>302-14681</td><td>For freezing cells</td></tr><tr><td>QIAamp Viral RNA kit&amp;nbsp;</td><td>Qiagen</td><td>52904</td><td>For measuring viral load in the serum</td></tr><tr><td>MACSQuant Flow Cytometer</td><td>Miltenyi</td><td></td><td>For flow analysis</td></tr><tr><td>BD LSRFortessa&amp;trade;</td><td>BD biosciences</td><td></td><td>For flow analysis</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H6254-500MG&amp;nbsp;</td><td>For tissue digestion</td></tr><tr><td>Deoxyribonuclease I&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Worthington</td><td>LS002006&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>Collagenase</td><td>Life technology</td><td>17104-019&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>CFX Real time PCR detection system</td><td>Biorad</td><td></td><td>For measuring viral load and gene expression</td></tr><tr><td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td><td>The Jackson Laboratory</td><td>5557</td><td>For constructing the humanized mice</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Thermo Fisher Scientific</td><td>10378016</td><td>For culturing cells</td></tr><tr><td>Piperacillin/tazobactam</td><td>Pfizer</td><td>Zosyn</td><td>Anti-fungal</td></tr><tr><td>Amphotericin B (Fungizone antimycotic)</td><td>Thermo Fisher Scientific</td><td>15290-018</td><td>Anti-fungal</td></tr><tr><td>Autoclip Wound Clips, 9 mm - 1,000 units</td><td>Becton Dickinson</td><td>427631&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Medline</td><td>DYNJP2707&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sutures, 4-0, vicryl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>23000J304H&amp;nbsp;&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Alcohol prep pads&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>3583006818</td><td>For surgery</td></tr><tr><td>Gloves, surgical, 6 1/2</td><td>Owens and Minor</td><td>4075711102</td><td>For surgery</td></tr><tr><td>Yssel&amp;rsquo;s Serum-Free T-Cell Medium</td><td>Gemini Bio-products</td><td>400-102</td><td>For CD34+ cell transduction</td></tr><tr><td>Human Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9511</td><td>For CD34+ cell transduction</td></tr></tbody></table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

Com o rápido desenvolvimento de terapias baseadas em genes de células estaminais contra o HIV, não é requisito para pressionar um modelo animal para estudar a diferenciação hematopoiética e a função imunológica das células geneticamente modificadas. A medula humanizado / fígado / timo (BLT) modelo do rato permite a total reconstituição de um sistema imune humano na periferia, que inclui células T, células B, células NK e monócitos. O implante de timo humano também permite a selecção tímica de células T em tecido tímico autólogo. Em adição ao estudo da infecção por HIV, o modelo fica como uma ferramenta poderosa para estudar diferenciação, desenvolvimento e funcionalidade de células derivadas de células estaminais hematopoiéticas (HSCs). Aqui destacamos a construção de humanizada não-obesos diabéticos (NOD) -severe imunodeficientes combinados (SCID) de cadeia knockout gamma -Common (c γ - / -) ratos -Bone de medula / Fígado / Thymus (NSG-BLT) com HSCs transduzidas com o receptor do antigénio quimérico de CD4 (CD4CAR)vetor lentivírus. Nós mostramos que o CD4CAR HSCs podem diferenciar com sucesso em múltiplas linhagens e têm actividade anti-HIV. O objetivo do estudo é demonstrar a utilização do modelo de rato NSG-BLT como um modelo in vivo para a imunidade projetados contra o HIV. Vale a pena notar que, devido lentivírus e tecido humano é usado, as experiências e cirurgias devem ser realizadas em uma cabine de segurança biológica Classe II em um Nível de Biossegurança 2 (BSL2) com precauções especiais (BSL2 +) instalações.

A Figura 1 mostra um esboço da construção de ratinhos humanizados BLT com células estaminais modificado. 10 semanas após a cirurgia de implante, os ratinhos foram sacrificados para avaliar a diferenciação e desenvolvimento de células do gene modificado. Como mostrado na Figura 2, vários tecidos linfóides (sangue, baço, timo e medula óssea) foram colhidas a partir de um rato que foi modificado com CD4ζCAR. O CD4ζCAR utilizados no presente protocolo contém receptor de antigénio quimérico CD4 e GFP que pode ser detectada por um anticorpo anti-CD4 e a expressão de GFP 8. As células foram isoladas e coradas com anticorpo contra o CD45 humano, assim como anticorpos anti-CD4 e analisadas por citometria de fluxo. GFP e as células duplamente positivas CD4 foi detectada, indicando a presença de células CD4CAR + em vários tecidos linfóides. Para investigar a diferenciação das células do gene modificado,<html> os esplenócitos foram corados com anticorpos contra CD45 humano (linfócitos), CD3 (células T), CD19 (células B), CD14 (monócitos e macrófagos) e CD337 (células NK). Como mostrado na Figura 3, as células estaminais hematopoiéticas CD4ζCAR diferenciar em linhagens múltiplas. Para investigar se as células CD4CAR modificados são funcionais, nós coincubated esplenócitos com células-alvo que as células CD4CAR reconheceria (células T1 infectada pelo HIV ou células T1 não infectados como controle). As células foram co-incubadas durante a noite e o inibidor de transporte de proteína adicionou-se durante um adicional de 6 horas. Em seguida, as células foram fixadas e permeabilizadas para corar para a expressão intracelular de citocinas, tais como IFNy e TNFa. Como mostrado na Figura 4, as células que expressam CAR produziu maior quantidade de IFNy e TNFa com células infectadas T1. carga / 54048 / 54048fig1.jpg &quot;/&gt; Figura 1: Esquema de construção de camundongos humanizados BLT com células-tronco modificadas FT:. Timo fetal. FL:. Fígado fetal <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54048/54048fig2.jpg" /> Figura 2:. Células modificadas do receptor de antigénio quimérico de CD4 pode ser detectado em vários tecidos linfóides do rato com CD4CAR modificado HSCs foram sacrificados 10 semanas após a cirurgia e vários tecidos linfóides foram colhidos e as células foram coradas com anticorpos CD4 CD45 anti-humano e anti-humanos e analisadas por citometria de fluxo. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig2large.jpg" target="_blank">por favor clique aqui para ver uma versão maior desta figura.</a> Figura 3:.. Células modificadas do receptor antígeno quimérico CD4 podem se diferenciar em várias linhagens esplenócitos de ratinhos CD4CAR modificado foram colhidas e coradas com anticorpos contra CD45 humana, CD3, CD19, CD14 e CD337 e analisadas por citometria de fluxo <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig3large.jpg" target="_blank">Por favor clique aqui para ver uma maior versão desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54048/54048fig4.jpg" /> Figura 4:. Ex ensaio in vivo de citocinas de células CD4 + T CAR esplenócitos de HIV camundongos infectados CD4CAR foram estimuladas com HIV células T1 infectado ou não infectado e sua produção intracelular de citocinas é mostrado. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig4large.jpg" target="_blank">Por favor clique aqui para vie</a>wa versão maior desta figura.

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Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

De células-tronco Baseado Imunidade Engineered contra infecção por HIV no modelo humanizado mouse

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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10.2K visualizações.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

Vídeo: Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Utilizando o rato como um BLT humanizado baseado Stem Modelo Gene Therapy célula tumoral

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Imunologia e Infecção

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Testando imunoterápicos contra o câncer em um modelo humanizado de camundongo com tumores humanos

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Pesquisa do câncer

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Humanizado NOD/SCID/IL2rγnula (hu-NSG) Mouse modelo para estudos de latência e a replicação do HIV

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Estabelecimento do modelo humanizado duplo do rato de TK-NOG para a patogénese HIV-associada do fígado

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Infecção por HIV crônica, aguda e reativada em modelos de camundongos imunodeficientes humanizados

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Camundongos NOG humanizados para exposição intravaginal ao HIV e tratamento da infecção pelo HIV

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

Tratamento Antirretroviral Combinacional Oral em Camundongos Humanizados Infectados pelo HIV-1

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Source: Lanis, J. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Isolando células-tronco hematopoiéticas do sangue do cordão umbilical para gerar camundongos do sistema imunológico humano

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

JoVE Encyclopedia of Experiments

Imunologia

Immunotherapy

Model Development and Disease Study

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Source: Dagur, R. S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58645">Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Desenvolvendo um modelo de camundongo humanizado com células hepáticas humanas e células imunológicas

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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De células-tronco Baseado Imunidade Engineered contra infecção por HIV no modelo humanizado mouse

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