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In JoVE (2)
- Indução de hipóxia e Testes em Cultura de Células
- Produção e Detecção de Espécies Reativas de Oxigênio (ROS) em Câncer
Other Publications (13)
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Blood
- Human Gene Therapy
- Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism
- Cancer Research
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Blood
- Blood
- The Journal of Biological Chemistry
- Molecular Therapy : the Journal of the American Society of Gene Therapy
- Clinical Immunology (Orlando, Fla.)
- Methods in Molecular Biology (Clifton, N.J.)
- The Journal of Biological Chemistry
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Articles by Patricia Yotnda in JoVE
JoVE General
Indução de hipóxia e Testes em Cultura de Células
Center for Cell and Gene Therapy, Baylor College of Medicine
Aqui propomos métodos simples para induzir hipóxia em culturas de células e testes simples para avaliar o estado hipóxico das culturas.
JoVE Clinical and Translational Medicine
Produção e Detecção de Espécies Reativas de Oxigênio (ROS) em Câncer
Center for Cell and Gene Therapy, Baylor College of Medicine
Aqui nós propor métodos simples para testar e avaliar a presença de espécies reativas de oxigênio nas células.
Other articles by Patricia Yotnda on PubMed
Bilamellar Cationic Liposomes Protect Adenovectors from Preexisting Humoral Immune Responses
Adenoviral vectors have been widely used for gene therapy, but they are limited both by the presence of a humoral immune response that dramatically decreases the level of transduction after reinjection and by their requirement for target cells to express appropriate receptors such as Coxsackie adenovirus receptor (CAR). To overcome both limits, we encapsulated adenovectors using bilamellar DOTAP:chol liposomes. Electron micrography (EM) showed that these liposomes efficiently encapsulated the vectors, allowing CAR-independent adenovector transduction of otherwise resistant cells. DOTAP:chol-encapsulated adenovectors encoding LacZ or alpha(1)-antitrypsin inhibitor (AAT) were also functionally resistant ex vivo and in vivo to the neutralizing effects of human anti-adenoviral antibodies, unlike other liposomal systems. Hence, bilamellar DOTAP:chol liposomes may be useful for applications using adenovectors in which the target cells lack adenoviral receptors or in which the recipient already has or develops a neutralizing antibody response that would otherwise inactivate readministered vector.
Transgenic Expression of CD40 Ligand Produces an in Vivo Antitumor Immune Response Against Both CD40(+) and CD40(-) Plasmacytoma Cells
Because tumor-specific antigens have been identified in multiple myeloma (MM), immunotherapy might provide an additional treatment modality for the disease. Expression of CD40 ligand (CD40L) proximate to the MM cells might serve this purpose, either by increasing their capacity to present self-antigens by activation through their CD40 receptor or by the recruitment of professional antigen-presenting cells (APCs) able to take up and present tumor-associated antigens. To distinguish between these possibilities and predict whether human CD40(-) myeloma might respond to this approach, we examined 3 murine plasmacytoma cell lines, 2 (MPC-11 and S107) expressing the CD40 molecule and 1 (X-24) lacking such expression. Syngeneic BALB/CBYJ mice were inoculated subcutaneously with tumor cells mixed with CL7.1 fibroblasts, retrovirally transduced to express either the mCD40L or the neo gene. For all 3 plasmacytoma cell lines, coinjection with CL7.1/mCD40L significantly reduced local tumor growth compared with controls. This effect was mediated by a systemic antitumor immune response, since mice immunized with tumor and CL7.1/mCD40L were resistant to subsequent challenge with tumor, and tumor growth inhibition was abolished when CD8(+) or CD4(+) lymphocytes were depleted. Because expression of CD40L gave equivalent protection from CD40(+) and CD40(-) tumors and transgenic-CD40L failed to up-regulate costimulatory molecules in either tumor, the protective effects of CD40L probably resulted from recruitment/activation of professional APCs rather than from CD40 activation of plasmacytoma cells. As further support of this concept, we found that mice were also well protected if CL7.1 and CD40L were injected together with apoptotic plasmacytoma cells from these tumors. Hence, transgenic CD40L expression may produce an antimyeloma immune response against either CD40(+) or CD40(-) tumors and may be of therapeutic value for both types of myeloma in humans.
Use of a Chimeric Adenovirus Vector Enhances BMP2 Production and Bone Formation
Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.
Reduced Inflammatory Reactions to the Inoculation of Helper-dependent Adenoviral Vectors in Traumatically Injured Rat Brain
Traumatic brain injury (TBI) causes delayed neuronal deficits that in principle could be prevented by timely intervention with therapeutic genes. However, appropriate vectors for gene transfer to the brain with TBI remain to be developed. First-generation adenoviruses (fgAd) are usually associated with inflammatory and toxic effects when inoculated into brains, despite their high efficiency of gene transfer to these tissues. In this study the authors attempted to determine whether a less immunogenic gene-transfer protocol can be established in the traumatically injured rat brain using helper-dependent adenoviruses (hdAd), a novel adenoviral construct with full deletion of viral coding sequences. Their results show that transgene expression from intrahippocampally inoculated hdAd is maintained for at least 2 months after TBI, in contrast to the much shorter duration of fgAd-mediated gene expression. There was only minimal secretion of proinflammatory IL-1beta and TNF-alpha after inoculation of hdAd. Furthermore, the hdAd-mediated gene expression was associated with less microglial proliferation, astrocytic activation, and macrophage infiltration than observed in fgAd-inoculated brains. There was no additional tissue loss after hdAd inoculation compared with PBS injection. Although both anti-adenoviral and neutralizing antibodies were found in serum after brain inoculation of hdAd, they did not appear to affect transgene expression. The results suggest that hdAd are less immunogenic vectors than conventional adenoviral vectors, and offer improved vehicles for long-term therapeutic transgene transfer to traumatically injured brains.
Induction of Therapeutic T-cell Responses to Subdominant Tumor-associated Viral Oncogene After Immunization with Replication-incompetent Polyepitope Adenovirus Vaccine
The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed in various EBV-associated malignancies have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and potential threat from their oncogenic potential. Here we have developed a replication- incompetent adenoviral vaccine that encodes multiple HLA class I-restricted CTL epitopes from LMP1 and LMP2 as a polyepitope. Immunization with this polyepitope vaccine consistently generated strong LMP-specific CTL responses in HLA A2/K(b) mice, which can be readily detected by both ex vivo and in vivo T-cell assays. Furthermore, a human CTL response to LMP antigens can be rapidly expanded after stimulation with this recombinant polyepitope vector. These expanded T cells displayed strong lysis of autologous target cells sensitized with LMP1 and/or LMP2 CTL epitopes. More importantly, this adenoviral vaccine was also successfully used to reverse the outgrowth of LMP1-expressing tumors in HLA A2/K(b) mice. These studies demonstrate that a replication-incompetent adenovirus polyepitope vaccine is an excellent tool for the induction of a protective CTL response directed toward multiple LMP CTL epitopes restricted through common HLA class I alleles prevalent in different ethnic groups where EBV-associated malignancies are endemic.
Liposomal Enhancement of the Antitumor Activity of Conditionally Replication-competent Adenoviral Plasmids
Many human tumors have a functional deficiency in p53. Numerous studies have taken advantage of this phenomenon to use a conditionally replication-competent adenovirus (Ad dl1520) that will grow in and lyse tumor cells while sparing normal tissues. However, success has been limited, in part due to difficulties in reaching a sufficiently high proportion of tumor cells. Preexisting or developing immune responses directed toward viral proteins further decrease the efficacy of the approach. We have developed a liposome-encapsulated conditionally replication-competent plasmid based on the dl1520 virus. Like the parent virus, this plasmid generates infectious particles following transfection of p53-defective, but not p53-wild-type tumor cells, but unlike the parent virus it is able to infect CAR-negative tumor cells. The antitumor efficacy of this infectious plasmid was demonstrated in mice with xenografted human tumors, in which it was active after both local and intravenous administration for subcutaneous tumors and following intravenous administration for disseminated malignancy. Activity was retained systemically, even in the presence of neutralizing antibody. Such liposomally encapsulated conditionally replication-competent plasmids may complement the use of conventional viral particles, particularly in settings in which liver uptake of adenoviral vector is undesirable or there are problematic inhibitory effects from humoral immune responses.
Targeted Delivery of Adenoviral Vectors by Cytotoxic T Cells
Effective targeting of vectors to tumor cells that have metastasized to multiple different tissue sites remains a major challenge for gene therapy. Tumor-specific cytotoxic T lymphocytes (CTLs) have been shown in animal models and in humans to be able to cross tissue barriers and traffic to tumor cells. However, their capacity to eliminate malignancy has been limited by tumor immune evasion strategies. We now use a model of Epstein-Barr virus-mediated malignancy to show that human CTLs themselves may be modified to release therapeutic vectors following engagement of their antigen-specific receptors and that these vectors will effectively transduce and destroy tumor targets. We generated EBV-specific CTLs that were transgenic for the adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand (CD40L) promoter. Following transduction with E1-deficient adenoviral vectors, these CTLs produced infectious virus when exposed to HLA-matched EBV-expressing targets, but not on exposure to major histocompatibility complex (MHC)-mismatched or otherwise irrelevant cells. This approach provides a means of delivering oncolytic/therapeutic vectors not only to locally accessible macroscopic tumors as is presently the case, but also to disseminated metastatic disease, while avoiding the risks associated with systemic administration of large doses of adenoviral vectors.
An Inducible Caspase 9 Safety Switch for T-cell Therapy
The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a "safety switch" that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies.
Assembly of the Kappa PreB Receptor Requires a V Kappa-like Protein Encoded by a Germline Transcript
By confining germline transcription as a byproduct of the mechanisms inherent to genetic rearrangements, the translation of respective mRNAs and their biological relevance might have been overlooked. Here we report the identification, cloning, and biochemical characterization of a human Vkappa-like protein that is encoded by a germline transcript. This surrogate protein assembles with the immunoglobulin mu heavy chain at the surface of B cell progenitors and precursors to form a kappa-like antigen receptor. These findings support the notion that germline transcription is not futile and stress the flexibility in eukaryotic gene usage and expression. In addition, the present study confirms the co-existence of surrogate lambda and kappa receptors that are proposed to work in concert to promote B lymphocyte maturation.
Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment
Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.
Fiber-modified Recombinant Adenoviral Constructs Encoding Hepatitis C Virus Proteins Induce Potent HCV-specific T Cell Response
Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) play an important role in HCV clearance. The frequency of HCV-specific T(CD8) in peripheral blood of HCV-infected donors is very low and HCV cannot be cultivated for reinfection of antigen presenting cells, making it difficult to detect T(CD8) of broad HCV specificities from peripheral blood mononuclear cells (PBMCs). We have developed a recombinant adenoviral system that efficiently reactivates and expands HCV-specific CTLs from PBMCs of HCV-infected donors. Replication-incompetent adenoviruses expressing individual HCV proteins (core and NS3) were produced and PBMCs from HCV-infected donors were transduced with these recombinant adeno-HCV constructs to stimulate HCV-specific CTL populations. T cells expanded from adeno-HCV stimulated cultures were potent producers of HCV-specific IFN-gamma and TNF-alpha and efficiently lysed target cells pulsed with HCV peptides. These constructs could stimulate T(CD8) directed towards multiple HCV peptides while preserving the determinant hierarchy. This approach therefore overcomes some of the shortcomings of the selective expansion of CTLs with peptide-based vaccine strategies. These findings provide an effective approach for the expansion of HCV-specific CTLs from PBMCs of HCV-infected patients and have potential for immunotherapeutic/vaccine development.
Hypoxic Tumors and Their Effect on Immune Cells and Cancer Therapy
The abnormal decrease or the lack of oxygen supply to cells and tissues is called hypoxia. This condition is commonly seen in various diseases such as rheumatoid arthritis and atherosclerosis, also in solid cancers. Pre-clinical and clinical studies have shown that hypoxic cancers are extremely aggressive, resistant to standard therapies (chemotherapy and radiotherapy), and thus very difficult to eradicate. Hypoxia affects both the tumor and the immune cells via various pathways. This review summarizes the most common effects of hypoxia on immune cells that play a key role in the anti-tumor response, the limitation of current therapies, and the potential solutions that were developed for hypoxic malignancies.
KCa1.1 Potassium Channels Regulate Key Proinflammatory and Invasive Properties of Fibroblast-like Synoviocytes in Rheumatoid Arthritis
Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.
