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The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.
Assuntos
Ciclotídeos , Infecções por HIV , Neoplasias , Ratos , Animais , Ciclotídeos/farmacologia , Linhagem Celular , Receptores CXCR4RESUMO
We have previously reported that overexpression of Programmed Death -1 Homolog (PD-1H) in human monocytes leads to activation and spontaneous secretion of multiple pro inflammatory cytokines. Here we evaluate changes in monocytes gene expression after enforced PD-1H expression by gene array. The results show that there are significant alterations in 51 potential candidate genes that relate to immune response, cell adhesion and metabolism. Genes corresponding to pro-inflammatory cytokines showed the highest upregulation, 7, 3.2, 3.0, 5.8, 4.4 and 3.1 fold upregulation of TNF-α, IL-1 ß, IFN-α, γ, λ and IL-27 relative to vector control. The data are in agreement with cytometric bead array analysis showing induction of proinflammatory cytokines, IL-6, IL-1ß and TNF-α by PD-1H. Other genes related to inflammation, include transglutaminase 2 (TG2), NF-κB (p65 and p50) and toll like receptors (TLR) 3 and 4 were upregulated 5, 4.5 and 2.5 fold, respectively. Gene set enrichment analysis (GSEA) also revealed that signaling pathways related to inflammatory response, such as NFκB, AT1R, PYK2, MAPK, RELA, TNFR1, MTOR and proteasomal degradation, were significantly upregulated in response to PD-1H overexpression. We validated the results utilizing a standard inflammatory sepsis model in humanized BLT mice, finding that PD-1H expression was highly correlated with proinflammatory cytokine production. We therefore conclude that PD-1H functions to enhance monocyte activation and the induction of a pro-inflammatory gene expression profile.
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A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV) infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Vacinas de DNA/administração & dosagem , Infecção por Zika virus/patologia , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Transgênicos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Zika virus/patogenicidade , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
IL-10 is a crucial anti-inflammatory cytokine which can also exert a seemingly divergent immunostimulatory effects under certain conditions. We found high levels of the cytokine in a xenogeneic GVHD model where NOD-scid IL2rγcnull (NSG) mice were transplanted with human PBMCs in presence of IL-2. Presence of exogenous IL-10 altered the kinetics of IL-2 induced human T cell reconstitution in vivo, showing an initial delay, followed by rapid expansion. Further, compared to IL-2 alone, treatment with IL-2 in combination with IL-10 increased survival in most animals and completely protected â¼20% of mice from GVHD. Additionally, IL-2 induced expansion of both CD4+ and CD8+ xenoreactive T cells whereas a combination of IL-2 and IL-10 resulted in selective expansion of CD4+ T cells only. TCR Vß repertoire analysis of CD4+ T cells showed that in contrast to IL-2 alone, simultaneous presence of both cytokines drastically reduced the Vß repertoire of the expanded CD4+ T cells. Highly restricted Vß usage was also observed when the cytokine combination was tested in an allogeneic GVHD model where NOD-scid IL2rγcnull mice expressing HLA-DR4 (NSG-DR4) were transplanted with purified CD4+ T cells from HLA-DR4 negative donors. Taken together, our results demonstrate that IL-10 can profoundly modulate the subset composition and repertoire of responding T cells during GVHD.
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Because endogenous interferon type I (IFN-I) produced by HIV-1 infection might complicate the analysis of therapeutically administered IFN-I, we tested different humanized mouse models for induction of IFN-I during HIV-1 infection. While HIV-1 induced high levels of IFN-α in BLT mice, IFN-I was undetectable following infection in the Hu-PBL mouse model, in which only T cells expand. We therefore tested the effect of treatment with Pegylated IFN-2 (pegasys), in Hu-PBL mice. Pegasys prevented CD4 T cell depletion and reduced the viral load for 10 days, but the effect waned thereafter. We next expressed IFN-I subsets (IFN-α2, -α6, -α8, -α14, and -ß) in Hu-PBL mice by hydrodynamic injection of plasmids encoding them and 2 days later infected the mice with HIV-1. CD4 T cell depletion was prevented in all subtypes of IFN-I-expressing mice by day 10. However, at day 40 post-infection, protection was seen in IFN-ß- and IFN-α14-expressing mice, but not the others. The viral load followed an inverse pattern and was highest in control mice and lowest in IFN-ß- and IFN-α14-expressing mice until day 40 after infection. These results show that gene therapy with plasmids encoding IFN-ß and -α14, but not the commonly used -α2, confers long-term suppression of HIV-1 replication.
Assuntos
Transferência Adotiva , Terapia Genética/métodos , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Interferon-alfa/genética , Interferon beta/genética , Leucócitos Mononucleares/transplante , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Humanos , Interferon-alfa/biossíntese , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Interferon beta/biossíntese , Interferon beta/imunologia , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos NOD , Camundongos SCID , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Carga Viral , Replicação ViralRESUMO
RNA interference (RNAi) provides a powerful tool to silence specific gene expression and has been widely used to suppress host factors such as CCR5 and/or viral genes involved in HIV-1 replication. Newer nuclease-based gene-editing technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, also provide powerful tools to ablate specific genes. Because of differences in co-receptor usage and the high mutability of the HIV-1 genome, a combination of host factors and viral genes needs to be suppressed for effective prevention and treatment of HIV-1 infection. Whereas the continued presence of small interfering/short hairpin RNA (si/shRNA) mediators is needed for RNAi to be effective, the continued expression of nucleases in the gene-editing systems is undesirable. Thus, RNAi provides the only practical way for expression of multiple silencers in infected and uninfected cells, which is needed for effective prevention/treatment of infection. There have been several advances in the RNAi field in terms of si/shRNA design, targeted delivery to HIV-1 susceptible cells, and testing for efficacy in preclinical humanized mouse models. Here, we comprehensively review the latest advances in RNAi technology towards prevention and treatment of HIV-1.
Assuntos
Síndrome da Imunodeficiência Adquirida/prevenção & controle , Síndrome da Imunodeficiência Adquirida/terapia , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , HIV-1 , Interferência de RNA , Terapêutica com RNAi/métodos , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency. RESULTS: Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes. CONCLUSIONS: By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , RNA/química , Proteínas Associadas a CRISPR/genética , Linhagem Celular , Desoxirribonuclease I/genética , Deleção de Genes , Humanos , Células Jurkat , MutaçãoRESUMO
Attempts at eliciting neutralizing antibodies against human immunodeficiency virus (HIV)-1 have generally failed. Computationally designed epitope-scaffold platforms allow transplantation of structural epitopes to scaffold proteins. Human rhinovirus (HRV) allows such engrafting of HIV-1 epitopes on the surface scaffold proteins. However, since HRV infects only humans and great apes, the efficacy of chimeric HRV-based live viral vaccines is difficult to assess in animal models. Here, we used human ICAM-1 transgenic (hICAM-1 Tg) mice that support productive HRV infection to assess the efficacy of chimeric HRV expressing the HIV-1 membrane proximal external region (MPER) epitope, 4E10. Intranasal immunization with chimeric HRV in transgenic mice effectively induced antibodies that recognized 4E10 peptide as well as HIV-1 Env trimer. Importantly, the immunized mouse sera were able to neutralize HIV strains including those belonging to clades B and C. Moreover, intranasal immunization could bypass pre-existing immunity to HRV. Thus, chimeric HRV appears to provide a viable vaccine vehicle for HIV-1 immunization in humans.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/imunologia , Rhinovirus/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/química , Apresentação de Antígeno/imunologia , Modelos Animais de Doenças , Epitopos/química , Epitopos/genética , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Imunização , Molécula 1 de Adesão Intercelular/genética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Ligação Proteica/imunologia , Conformação ProteicaRESUMO
Although patients with GVHD have elevated serum levels of IL10, whether its role is protective or pathogenic remains unclear. Here, we used a humanized mouse model to study the role of IL-10 in GVHD. When human PBMCs were engrafted in NOD-scid IL2rγc(null) mice expressing human IL-10, the T cells underwent massive expansion resulting in lethality by day 21, whereas control mice survived for at least 40 days. Histopathology of the liver showed extensive mononuclear cell infiltration in IL-10 expressing but not in control mice. Corresponding to their aggressiveness, the T cells in the IL-10 group exhibited predominantly an effector memory phenotype (CD45RO(+)CD27(-)) while in control mice, the T cells were of transitional memory phenotype (CD45RO(+)CD27(+)). Further, IL-10 receptor blocking antibody was able to protect the animals from GVHD. Since our results demonstrate a direct pathogenic role for IL-10, blockade of IL-10 signaling may provide a therapeutic option for GVHD.
Assuntos
Doença Enxerto-Hospedeiro/fisiopatologia , Interleucina-10/metabolismo , Linfócitos T/citologia , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos AnimaisRESUMO
Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc(-/-) mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Replicação Viral/genética , Animais , Contagem de Linfócito CD4 , Linhagem Celular , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Infecções por HIV/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Camundongos , Receptores CCR5/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Transdução GenéticaRESUMO
Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.
Assuntos
Células Dendríticas/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/efeitos dos fármacos , Antígenos Virais/genética , Antígenos Virais/imunologia , Células Dendríticas/citologia , Células Dendríticas/virologia , Engenharia Genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Imunização , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/síntese química , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/virologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/virologiaRESUMO
Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.
Assuntos
Antígenos B7/metabolismo , Infecções por HIV/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Infecções por HIV/imunologia , Humanos , Macrófagos/imunologia , Monócitos/imunologia , Carga Viral/imunologiaRESUMO
CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4(+) T cells. Both activated and resting primary CD4(+) T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4(+) T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN-transduced resting CD4(+) T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy.
RESUMO
Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called "Berlin patient" who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.
Assuntos
Terapia Genética/tendências , Infecções por HIV/genética , Infecções por HIV/terapia , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Terapia Genética/métodos , HumanosRESUMO
Hypersecretion of cytokines by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here, we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient nonobese diabetic (NOD)/SCID/IL2Rγ(-/-) mice, and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, compared with immunocompetent WT mice, NOD/SCID/IL2Rγ(-/-) mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ(-/-) mice transplanted with human hematopoietic stem cells [humanized bone marrow liver thymic mice (BLT) mice] showed high serum levels of HMGB1, as well as multiple human but not murine proinflammatory cytokines, and died uniformly, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short acetylcholine receptor (AchR)-binding peptide [rabies virus glycoprotein (RVG)-9R] effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis, and rescued humanized mice from CLP-induced mortality. siRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that CLP in humanized mice provides a model to study human sepsis, HMGB1 siRNA might provide a treatment strategy for human sepsis, and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used to suppress a variety of human inflammatory diseases.
Assuntos
Células Dendríticas/citologia , Proteína HMGB1/metabolismo , Macrófagos/citologia , Sepse/metabolismo , Animais , Citocinas/metabolismo , Inativação Gênica , Técnicas de Transferência de Genes , Humanos , Sistema Imunitário , Inflamação , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Peptídeos/química , RNA Interferente Pequeno/metabolismoRESUMO
Regulatory T cells are essential to maintain immune homeostasis and prevent autoimmunity. Therapy with in vitro expanded human nT(Regs) is being tested to prevent graft versus host disease, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate appropriately and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model for studying the in vivo reconstitutive capability of human nT(Regs) is a major impediment for investigating the behavior of adoptively transferred nT(Regs)in vivo. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nT(Regs) in NOD-SCID IL2rγc(null) mice. We also demonstrate that these in vivo reconstituted T(Regs) traffic to different organs of the body and retain suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted T(Regs) are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel 'hu-T(Reg) mouse' model offers a pre-clinical platform to study the in vivo function and stability of human nT(Regs) and their ability to modulate autoimmune diseases and GVHD.
Assuntos
Expressão Gênica , Interleucina-2/genética , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplante , Transferência Adotiva , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Interleucina-2/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Fenótipo , Plasmídeos/administração & dosagem , Plasmídeos/genética , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Humanized mice have recently emerged as powerful translational animal models for studying human hematopoiesis, immune interactions, and diseases of the human immune system. Several important advances in the humanized mouse technology have been reported over the last few years, thereby resulting in improved engraftment, high levels of human chimerism, and sustained human hematopoiesis. This chapter describes the detailed procedures for generating various humanized mouse models including hu-PBL, hu-HSC, and BLT models and discusses considerations for choosing the appropriate model system.
Assuntos
Modelos Animais de Doenças , Integrinas/metabolismo , Leucócitos/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Animais , Feminino , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , Integrinas/genética , Masculino , CamundongosRESUMO
siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.
Assuntos
Pareamento Incorreto de Bases/genética , RNA Interferente Pequeno/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Sequência Conservada , Células HEK293 , Humanos , Conformação de Ácido Nucleico , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/metabolismo , TermodinâmicaRESUMO
Targeting the HIV entry and assembly pathways holds promise for development of novel anti-HIV gene therapy vectors. We characterized discrete dominant negative (DN) Gag and Envelope mutants for their anti-HIV-1 activity. We show here that capsid mutants (Q155N and Y164A) are more potent inhibitors of WT HIV than the matrix mutant 1GA. Both the Envelope mutants tested, V513E and R515A, were equally effective and a combination of Gag and Envelope DN genes significantly enhanced potency. Interestingly, the DN mutants acted at multiple steps in the virus life cycle rather than solely disrupting virus release or infection. Inhibition mediated by R515A could be partially attributed to the Envelope cytoplasmic tail, as deletion of R515A tail partially abrogated its DN effect. Finally, the Y164A/R515A double mutant expressed in a lentiviral vector was effective at inhibiting HIV replication in CD34+ hematopoietic stem cell-derived macrophages, demonstrating the therapeutic potential of our approach.