RESUMEN
CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antígenos CD28/inmunología , Antígenos CD5/inmunología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos , Antígenos CD28/antagonistas & inhibidores , Antígenos CD28/genética , Antígenos CD5/antagonistas & inhibidores , Antígenos CD5/genética , Extractos Celulares/farmacología , Línea Celular Tumoral , Proliferación Celular , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Femenino , Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Activación de Linfocitos/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Noqueados , Transducción de Señal , Linfocitos T Citotóxicos/inmunología , Receptor fas/genética , Receptor fas/inmunologíaRESUMEN
The engulfment and cell motility (ELMOs) family of proteins plays a crucial role in tumor cell migration and invasion. However, the function of ELMO3 is poorly defined. To elucidate its role in the development and progression of colorectal cancer (CRC), we examined the expression of ELMO3 in 45 cases of paired CRC tumor tissues and adjacent normal tissues. Furthermore, we assessed the effect of the knockdown of ELMO3 on cell proliferation, cell cycle, migration, invasion and F-actin polymerization in HCT116 cells. The result shows that the expression of ELMO3 in CRC tissues was significantly increased in comparison to the adjacent normal colorectal tissues. Moreover, this overexpression was associated with tumor size (p = 0.007), tumor differentiation (p = 0.001), depth of invasion (p = 0.009), lymph node metastasis (p = 0.003), distant metastasis (p = 0.013) and tumor, node, metastasis (TNM)-based classification (p = 0.000). In in vitro experiments, the silencing of ELMO3 inhibited cell proliferation, invasion, metastasis, and F-actin polymerization, and induced Gap 1 (G1) phase cell cycle arrest. Our study demonstrates that ELMO3 is involved in the processes of growth, invasion and metastasis of CRC, and could be used a potential molecular diagnostic tool or therapy target of CRC.
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Proteínas Adaptadoras Transductoras de Señales/genética , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/genética , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Actinas/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Femenino , Células HCT116 , Humanos , Metástasis Linfática/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: While the concept of angiogenesis blockade as a therapeutic intervention for cancer has been repeatedly demonstrated, the full promise of this approach has yet to be realized. Specifically, drugs such as VEGF-blocking antibodies or kinase inhibitors suffer from the drawbacks of resistance development, as well as off-target toxicities. Previous studies have demonstrated feasibility of specifically inducing immunity towards tumor endothelium without consequences of systemic autoimmunity in both animal models and clinical settings. METHOD: Placenta-derived endothelial cells were isolated and pretreated with interferon gamma to enhance immunogenicity. Syngeneic mice received subcutaneous administration of B16 melanoma, 4 T1 mammary carcinoma, and Lewis Lung Carcinoma (LLC), followed by administration of control saline, control placental endothelial cells, and interferon gamma primed endothelial cells (ValloVax™). Tumor volume was quantified. An LLC metastasis model was also established and treated under similar conditions. Furthermore, a safety analysis in non-tumor bearing mice bracketing the proposed clinical dose was conducted. RESULTS: ValloVax™ immunization led to significant reduction of tumor growth and metastasis as compared to administration of non-treated placental endothelial cells. Mitotic inactivation by formalin fixation or irradiation preserved tumor inhibitory activity. Twenty-eight day evaluation of healthy male and female mice immunized with ValloVax™ resulted in no abnormalities or organ toxicities. CONCLUSION: Given the established rationale behind the potential therapeutic benefit of inhibiting tumor angiogenesis as a treatment for cancer, immunization against a variety of endothelial cell antigens may produce the best clinical response, enhancing efficacy and reducing the likelihood of the development of treatment resistance. These data support the clinical evaluation of irradiated ValloVax™ as an anti-angiogenic cancer vaccine.
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Células Endoteliales/inmunología , Inmunización , Interferón gamma/uso terapéutico , Neoplasias/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Placenta/citología , Animales , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Proliferación Celular , Células Endoteliales/efectos de los fármacos , Femenino , Inmunidad , Interferón gamma/efectos adversos , Interferón gamma/farmacología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , Metástasis de la Neoplasia , Neoplasias/inmunología , EmbarazoRESUMEN
BACKGROUND: While substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival. METHOD: Vectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed. RESULT: Allogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10-16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4+CD25+FoxP3+ regulatory T cell differentiation in vitro. CONCLUSION: This study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.
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Antígeno B7-1/genética , Antígenos CD40/genética , Silenciador del Gen , Rechazo de Injerto/prevención & control , Trasplante de Corazón , Animales , Secuencia de Bases , Cartilla de ADN , Citometría de Flujo , Rechazo de Injerto/genética , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues in vivo presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and poly(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery in vitro and in vivo. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16-F10 cells was demonstrated in vitro. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy.
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Nanotubos de Carbono/química , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular , Humanos , Hígado/metabolismo , Ratones , Fosfatidiletanolaminas/química , Polietilenglicoles/química , Interferencia de ARN , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
CD5 is a member of the scavenger receptor cysteine-rich superfamily that is expressed on T cells and a subset of B cells (B1a) cell and can regulate the T cell receptor signaling pathway. Blocking CD5 function may have therapeutic potential in treatment of cancer by enhancing cytotoxic T lymphocyte recognition and ablation of tumour cells. The effect of administering an anti-CD5 antibody to block or reduce CD5 function as an immune checkpoint blockade to enhance T cell anti-tumour activation and function in vivo has not been explored. Here we challenged mice with poorly immunogenic 4T1 breast tumour cells and tested whether treatment with anti-CD5 monoclonal antibodies (MAb) in vivo could enhance non-malignant T cell anti-tumour immunity and reduce tumour growth. Treatment with anti-CD5 MAb resulted in an increased fraction of CD8+ T cells compared to CD4+ T cell in draining lymph nodes and the tumour microenvironment. In addition, it increased activation and effector function of T cells isolated from spleens, draining lymph nodes, and 4T1 tumours. Furthermore, tumour growth was delayed in mice treated with anti-CD5 MAb. These data suggest that use of anti-CD5 MAb as an immune checkpoint blockade can both enhance activation of T cells in response to poorly immunogenic antigens and reduce tumour growth in vivo. Exploration of anti-CD5 therapies in treatment of cancer, alone and in combination with other immune therapeutic drugs, is warranted.
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Neoplasias de la Mama , Linfocitos T CD8-positivos , Ratones , Animales , Humanos , Femenino , Inhibidores de Puntos de Control Inmunológico/farmacología , Linfocitos T Citotóxicos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Aloinjertos , Microambiente TumoralRESUMEN
The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αß(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVß2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quimerismo/efectos de los fármacos , Supresión Clonal/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Trasplante de Médula Ósea/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Supresión Clonal/efectos de los fármacos , Ciclofosfamida/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/inmunología , Inmunosupresores/uso terapéutico , Células Asesinas Naturales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Perforina/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Trasplante de Piel/inmunología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
BACKGROUND: We have previously demonstrated that immune modulation can be accomplished by administration of gene silenced dendritic cells (DC) using siRNA. In this study, we demonstrate the therapeutic utilization of shRNA-modified DC as an antigen-specific tolerogenic vaccine strategy for autoimmune arthritis. METHODS: A shRNA that specifically targets IL-12 p35 was designed and cloned into a plasmid vectors (IL-12 shRNA). Bone marrow-derived DC from DBA/1 mice were transfected with the IL-12 shRNA construct in vitro. Mice with collagen II (CII)-induced arthritis (CIA) were treated with the modified DCs expressing the shRNA. Recall response and disease progression were assessed. RESULTS: After gene silencing of IL-12 in DC, DC were shown to selectively inhibit T cell proliferation on recall responses and in an MLR. In murine CIA, we demonstrated that administration of IL-12 shRNA-expressing DC that were pulsed with CII inhibited progression of arthritis. The therapeutic effects were evidenced by decreased clinical scores, inhibition of inflammatory cell infiltration in the joint, and suppression of T cell and B cell responses to CII. CONCLUSION: We demonstrate a novel tolerance-inducing protocol for the treatment of autoimmune inflammatory joint disease in which the target antigen is known, utilizing DNA-directed RNA interference.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Células Dendríticas/inmunología , Silenciador del Gen , Interleucina-12/genética , Animales , Formación de Anticuerpos/inmunología , Artritis Experimental/patología , Colágeno Tipo II/inmunología , Memoria Inmunológica/inmunología , Inmunomodulación , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , ARN Interferente Pequeño/metabolismo , Linfocitos T/inmunología , TransfecciónRESUMEN
BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is a central mediator in the hepatic response to ischemia/reperfusion. Short hairpin RNA (shRNA) has been proven to be an effective means of harnessing the RNA interference pathway in mammalian cells. In the current study, we investigated whether silencing TNF-α gene with shRNA can prevent liver ischemic reperfusion injury (IRI). METHODS: Male BalB/c mice were randomized to TNF-α shRNA, scramble shRNA, or sham operation groups. TNF-α shRNA and scramble shRNA groups were injected 48 h before inducing IRI. IRI was induced via microaneurysm clamps applied to the left hepatic artery and portal vein. Six hours after reperfusion, IRI injury was examined by serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology, MPO, and MDA level, as well as by relative quantities of TNF-α mRNA. RESULTS: TNF-α expression induced by ischemia reperfusion in the liver was significantly suppressed after treatment with TNF-α shRNA compared with the group treated with scramble shRNA (P < 0.001). Mice treated with TNF-α shRNA showed lower peak values of AST and ALT than scramble shRNA treated mice (P < 0.001). On histopathologic slides, mice treated with TNF-α shRNA had significantly less ischemia/reperfusion injury based on Suzuki score than the scramble shRNA group, 3.57 ± 2.30 and 8.83 ± 0.98 respectively (P < 0.001), while the sham group was not significantly different from the TNF-alpha shRNA group, 0 ± 0 and 3.57 ± 2.30, respectively (P = 0.075). Liver tissue MDA levels were significantly lower in mice treated with TNF-α shRNA as compared with the group treated with scramble shRNA (P < 0.01). Immunohistochemical staining for MPO was significantly lower in mice treated with TNF-α shRNA compared with the group treated with shRNA (compared with treated with scramble shRNA group.) CONCLUSIONS: Liver IRI can be minimized through gene silencing of TNF-α. This may represent a novel therapy in the setting of transplantation and in other conditions associated with IRI of the liver.
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Terapia Genética/métodos , Hígado/fisiología , ARN Interferente Pequeño/farmacología , Daño por Reperfusión/genética , Daño por Reperfusión/terapia , Factor de Necrosis Tumoral alfa/genética , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Regulación hacia Abajo/genética , Hígado/patología , Masculino , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos BALB C , Peroxidasa/metabolismo , ARN Interferente Pequeño/genética , Daño por Reperfusión/patologíaRESUMEN
Translation of small interfering RNA (siRNA)-based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.
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Presentación de Antígeno/efectos de los fármacos , Antígenos CD40/antagonistas & inhibidores , Células Dendríticas/efectos de los fármacos , Terapia Genética/métodos , Terapia de Inmunosupresión/métodos , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Células de la Médula Ósea/citología , Antígenos CD40/biosíntesis , Antígenos CD40/genética , Antígenos CD40/inmunología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Células Dendríticas/inmunología , Sistemas de Liberación de Medicamentos , Inmunoconjugados/administración & dosificación , Lectinas Tipo C/inmunología , Liposomas , Activación de Linfocitos/efectos de los fármacos , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Receptores de Superficie Celular/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Induction of RNA interference with small interfering RNA (siRNA) has demonstrated therapeutic potential through the knockdown of target genes. We have previously reported that systemic administration of CD40 siRNA is capable of attenuating allergic symptoms but in an allergen-nonspecific fashion. However, siRNA-based allergen-specific therapy for allergy has not been developed. OBJECTIVE: We attempted to develop a new allergen-specific therapy for allergy using CD40-silenced and allergen-pulsed dendritic cells (DCs). METHODS: Bone marrow-derived DCs were silenced with CD40 siRNA and pulsed with ovalbumin (OVA). Mice had allergy after intraperitoneal sensitization with OVA and keyhole limpet hemocyanin, followed by intranasal challenge with the same allergens. The mice were treated with CD40-silenced and OVA-pulsed DCs (CD40-silenced OVA DCs) either before allergic sensitization or after establishing allergic rhinitis. RESULTS: Mice receiving CD40-silenced OVA DCs either before or after the establishment of allergic rhinitis showed remarkable reductions in allergic symptoms caused by OVA challenge, as well as anti-OVA IgE levels in sera. Additionally, CD40-silenced OVA DCs suppressed eosinophil infiltration at the nasal septum, OVA-specific T-cell responses, T-cell production of IL-4 and IL-5 after stimulation with OVA, and CD4(+)CD25(-) effector T-cell responses. Furthermore, CD40-silenced OVA DCs facilitated the generation of CD4(+)CD25(+) forkhead box protein 3-positive OVA-specific regulatory T cells, which inhibit allergic responses in vivo. However, CD40-silenced OVA DCs suppressed only OVA-specific allergy but did not inhibit keyhole limpet hemocyanin-induced allergy, suggesting that CD40-silenced OVA DCs induce allergen-specific tolerance. CONCLUSIONS: This study is the first to demonstrate a novel allergen-specific therapy for allergy through DC-mediated immune modulation after gene silencing of CD40.
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Antígenos CD40/antagonistas & inhibidores , Células Dendríticas/inmunología , Hipersensibilidad/terapia , Inmunoterapia/métodos , Interferencia de ARN/inmunología , Animales , Antígenos CD40/genética , Separación Celular , Citometría de Flujo , Ratones , Ovalbúmina/inmunología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized ß-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/ß-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.
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Sistemas CRISPR-Cas/genética , Neoplasias Pulmonares/genética , Placofilinas/metabolismo , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , beta Catenina/metabolismo , Humanos , Neoplasias Pulmonares/patologíaRESUMEN
Histone deacetylases (HDACs) are involved in tumor progression, and some have been successfully targeted for cancer therapy. The expression of histone deacetylase 4 (HDAC4), a class IIa HDAC, was upregulated in our previous microarray screen. However, the role of HDAC4 dysregulation and mechanisms underlying tumor growth and metastasis in nasopharyngeal carcinoma (NPC) remain elusive. Here, we first confirmed that the HDAC4 levels in primary and metastatic NPC tissues were significantly increased compared with those in normal nasopharyngeal epithelial tissues and found that high HDAC4 expression predicted a poor overall survival (OS) and progression-free survival (PFS). Functionally, HDAC4 accelerated cell cycle G1/S transition and induced the epithelial-to-mesenchymal transition to promote NPC cell proliferation, migration, and invasion in vitro, as well as tumor growth and lung metastasis in vivo. Intriguingly, knockdown of N-CoR abolished the effects of HDAC4 on the invasion and migration abilities of NPC cells. Mechanistically, HDAC3/4 binds to the E-cadherin promoter to repress E-cadherin transcription. We also showed that the HDAC4 inhibitor tasquinimod suppresses tumor growth in NPC. Thus, HDAC4 may be a potential diagnostic marker and therapeutic target in patients with NPC.
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Histona Desacetilasas/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/terapia , Proteínas Represoras/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Ratones , Ratones DesnudosRESUMEN
BACKGROUND: Ischemia/reperfusion injury is a major factor in graft quality and subsequent function in the transplantation setting. We hypothesize that the process of RNA interference may be used to "engineer" a graft to suppress expression of genes associated with inflammation, apoptosis, and complement, which are believed to cause ischemia/reperfusion injury. Such manipulation of pathological gene expression may be performed by treatment of the graft ex vivo with small interfering RNA (siRNA) as part of the preservation procedure. METHODS AND RESULTS: Heart grafts from BALB/c mice were preserved in UW solution (control) or UW solution containing siRNAs targeting tumor necrosis factor-alpha, C3, and Fas genes (siRNA solution) at 4 degrees C for 48 hours and subsequently transplanted into syngeneic recipients. Tumor necrosis factor-alpha, C3, and Fas genes were elevated by ischemia/reperfusion injury after 48 hours of preservation in UW solution. Preservation in siRNA solution knocked down gene expression at the level of messenger RNA and protein in the grafts after transplantation. All grafts preserved in siRNA solution showed strong contraction, whereas grafts preserved in control solution demonstrated no detectable contraction by high-frequency ultrasound scanning. siRNA solution-treated organs exhibited improved histology and diminished neutrophil and lymphocyte infiltration compared with control solution-treated organs. Furthermore, the treated heart grafts retained strong beating up to the end of the observation period (>100 days), whereas all control grafts lost function within 8 days. CONCLUSIONS: Incorporation of siRNA into organ storage solution is a feasible and effective method of attenuating ischemia/reperfusion injury, protecting cardiac function, and prolonging graft survival.
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Complemento C3/antagonistas & inhibidores , Trasplante de Corazón , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , ARN Interferente Pequeño/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Receptor fas/antagonistas & inhibidores , Animales , Complemento C3/genética , Silenciador del Gen , Supervivencia de Injerto , Masculino , Ratones , Ratones Endogámicos BALB C , Miocardio/patología , Factor de Necrosis Tumoral alfa/genética , Receptor fas/genéticaRESUMEN
BACKGROUND: The current paradigm for cord blood transplantation is that HLA matching and immune suppression are strictly required to prevent graft versus host disease (GVHD). Immunological arguments and historical examples have been made that the use of cord blood for non-hematopoietic activities such as growth factor production, stimulation of angiogenesis, and immune modulation may not require matching or immune suppression. METHODS: 114 patients suffering from non-hematopoietic degenerative conditions were treated with non-matched, allogeneic cord blood. Doses of 1-3 x 10(7) cord blood mononuclear cells per treatment, with 4-5 treatments both intrathecal and intravenously were performed. Adverse events and hematological, immunological, and biochemical parameters were analyzed for safety evaluation. RESULTS: No serious adverse effects were reported. Hematological, immunological, and biochemical parameters did not deviate from normal ranges as a result of therapy. CONCLUSION: The current hematology-based paradigm of need for matching and immune suppression needs to be revisited when cord blood is used for non-hematopoietic regenerative purposes in immune competent recipients.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedades Neurodegenerativas/terapia , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/sangre , Enfermedades Neurodegenerativas/inmunología , Trasplante Homólogo/efectos adversos , Adulto JovenRESUMEN
BACKGROUND: Apoptosis is an early event involved in cardiomyopathy associated with diabetes mellitus. Toll-like receptor (TLR) signaling triggers cell apoptosis through multiple mechanisms. Up-regulation of TLR4 expression has been shown in diabetic mice. This study aimed to delineate the role of TLR4 in myocardial apoptosis, and to block this process through gene silencing of TLR4 in the myocardia of diabetic mice. METHODS: Diabetes was induced in C57/BL6 mice by the injection of streptozotocin. Diabetic mice were treated with 50 µg of TLR4 siRNA or scrambled siRNA as control. Myocardial apoptosis was determined by TUNEL assay. RESULTS: After 7 days of hyperglycemia, the level of TLR4 mRNA in myocardial tissue was significantly elevated. Treatment of TLR4 siRNA knocked down gene expression as well as diminished its elevation in diabetic mice. Apoptosis was evident in cardiac tissues of diabetic mice as detected by a TUNEL assay. In contrast, treatment with TLR4 siRNA minimized apoptosis in myocardial tissues. Mechanistically, caspase-3 activation was significantly inhibited in mice that were treated with TLR4 siRNA, but not in mice treated with control siRNA. Additionally, gene silencing of TLR4 resulted in suppression of apoptotic cascades, such as Fas and caspase-3 gene expression. TLR4 deficiency resulted in inhibition of reactive oxygen species (ROS) production and NADPH oxidase activity, suggesting suppression of hyperglycemia-induced apoptosis by TLR4 is associated with attenuation of oxidative stress to the cardiomyocytes. CONCLUSIONS: In summary, we present novel evidence that TLR4 plays a critical role in cardiac apoptosis. This is the first demonstration of the prevention of cardiac apoptosis in diabetic mice through silencing of the TLR4 gene.
Asunto(s)
Apoptosis/genética , Silenciador del Gen , Hiperglucemia/prevención & control , Miocardio/patología , Receptor Toll-Like 4/genética , Animales , Caspasa 3/metabolismo , Inhibidores de Caspasas , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Hiperglucemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/enzimología , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Since the days of Medawar, the goal of therapeutic tolerogenesis has been a "Holy Grail" for immunologists. While knowledge of cellular and molecular mechanisms of this process has been increasing at an exponential rate, clinical progress has been minimal. To provide a mechanistic background of tolerogenesis, we overview common processes in the naturally occurring examples of: pregnancy, cancer, oral tolerance and anterior chamber associated immune deviation. The case is made that an easily accessible byproduct of plastic surgery, the adipose stromal vascular fraction, contains elements directly capable of promoting tolerogenesis such as T regulatory cells and inhibitory macrophages. The high content of mesenchymal and hematopoietic stem cells from this source provides the possibility of trophic/regenerative potential, which would augment tolerogenic processes by decreasing ongoing inflammation. We discuss the application of this autologous cell source in the context of rheumatoid arthritis, concluding with some practical examples of its applications.
Asunto(s)
Tejido Adiposo/patología , Artritis Reumatoide/inmunología , Trasplante de Células Madre Hematopoyéticas , Articulaciones/patología , Células del Estroma/patología , Tejido Adiposo/cirugía , Tejido Adiposo/trasplante , Anciano , Animales , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/patología , Artritis Reumatoide/fisiopatología , Artritis Reumatoide/terapia , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Autoanticuerpos/genética , Células Cultivadas , Femenino , Humanos , Articulaciones/metabolismo , Lipectomía , Células Madre Mesenquimatosas/inmunología , Actividad Motora , Péptidos Cíclicos/inmunología , Factor Reumatoide/biosíntesis , Factor Reumatoide/sangre , Factor Reumatoide/genética , Nicho de Células Madre , Células del Estroma/trasplante , Linfocitos T Reguladores/inmunologíaRESUMEN
Renal ischemia-reperfusion injury (IRI) can result in acute renal failure with mortality rates of 50% in severe cases. NK cells are important participants in early-stage innate immune responses. However, their role in renal tubular epithelial cell (TEC) injury in IRI is currently unknown. Our data indicate that NK cells can kill syngeneic TEC in vitro. Apoptotic death of TEC in vitro is associated with TEC expression of the NK cell ligand Rae-1, as well as NKG2D on NK cells. In vivo following IRI, there was increased expression of Rae-1 on TEC. FACS analyses of kidney cell preparations indicated a quantitative increase in NKG2D-bearing NK cells within the kidney following IRI. NK cell depletion in wild-type C57BL/6 mice was protective, while adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2(-/-)gamma(c)(-/-) mice with IRI. NK cell-mediated kidney injury was perforin (PFN)-dependent as PFN(-/-) NK cells had minimal capacity to kill TEC in vitro compared with NK cells from wild-type, FasL-deficient (gld), or IFN-gamma(-/-) mice. Taken together, these results demonstrate for the first time that NK cells can directly kill TEC and that NK cells contribute substantially to kidney IRI. NK cell killing may represent an important underrecognized mechanism of kidney injury in diverse forms of inflammation, including transplantation.
Asunto(s)
Apoptosis/inmunología , Células Epiteliales/inmunología , Enfermedades Renales/inmunología , Túbulos Renales/inmunología , Células Asesinas Naturales/inmunología , Daño por Reperfusión/inmunología , Traslado Adoptivo/métodos , Animales , Apoptosis/genética , Línea Celular , Células Epiteliales/patología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Enfermedades Renales/genética , Enfermedades Renales/patología , Trasplante de Riñón/inmunología , Túbulos Renales/patología , Células Asesinas Naturales/patología , Células Asesinas Naturales/trasplante , Ratones , Ratones Noqueados , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/inmunología , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
CD5, a member of the scavenger receptor cysteine-rich superfamily, is a marker for T cells and a subset of B cells (B1a). CD5 associates with T-cell and B-cell receptors and increased CD5 is an indication of B cell activation. In tumor-infiltrating lymphocytes (TILs) isolated from lung cancer patients, CD5 levels were negatively correlated with anti-tumor activity and tumor-mediated activation-induced T cell death, suggesting that CD5 could impair activation of anti-tumor T cells. We determined CD5 levels in T cell subsets in different organs in mice bearing syngeneic 4T1 breast tumor homografts and assessed the relationship between CD5 and increased T cell activation and effector function by flow cytometry. We report that T cell CD5 levels were higher in CD4+ T cells than in CD8+ T cells in 4T1 tumor-bearing mice, and that high CD5 levels on CD4+ T cells were maintained in peripheral organs (spleen and lymph nodes). However, both CD4+ and CD8+ T cells recruited to tumors had reduced CD5 compared to CD4+ and CD8+ T cells in peripheral organs. In addition, CD5high/CD4+ T cells and CD5high/CD8+ T cells from peripheral organs exhibited higher levels of activation and associated effector function compared to CD5low/CD4+ T cell and CD5low/CD8+ T cell from the same organs. Interestingly, CD8+ T cells among TILs and downregulated CD5 were activated to a higher level, with concomitantly increased effector function markers, than CD8+/CD5high TILs. Thus, differential CD5 levels among T cells in tumors and lymphoid organs can be associated with different levels of T cell activation and effector function, suggesting that CD5 may be a therapeutic target for immunotherapeutic activation in cancer therapy.
Asunto(s)
Antígenos CD5/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias Pulmonares/inmunología , Activación de Linfocitos/inmunología , Animales , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Subgrupos de Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Intranasally administered dendritic cells (DCs) migrate into blood and thymus to induce immune responses. Regulatory dendritic cells (DCs) are also useful agents for allergy control. However, to the best of our knowledge, the effects of intranasal administration of regulatory DCs on allergy have not been reported until now. Therefore, we examined the effects of intranasal route of administration of CD40-silenced DCs on allergic responses and compared these with the effects of other administration routes, based on our previous findings on the inhibitory effects of CD40-silenced DCs on allergic responses. METHODS: Mice with allergic rhinitis were treated intranasally, subcutaneously, intraperitoneally, or intravenously with CD40-silenced ovalbumin (OVA)-pulsed DCs that were transfected with CD40 siRNAs and pulsed with OVA antigen. The effects of these DCs on allergic reactions and symptoms were estimated. RESULTS: Intranasal, subcutaneous, intraperitoneal, or intravenous administration of OVA-pulsed CD40-silenced DCs inhibited allergic responses and symptoms in mice. Furthermore, intranasal administration of OVA-pulsed CD40-silenced DCs significantly reduced allergic symptoms and the number of eosinophils in the nasal mucosa compared with subcutaneous, intraperitoneal, or intravenous administration of these DCs. Intranasal administration of OVA-pulsed CD40-silenced DCs resulted in significantly up-regulated IL-10, IL-35, and Foxp3 expression, and enhanced the percentage of CD11c+CD40- and CD4+CD25+ cells within the cervical lymph nodes compared to subcutaneous, intraperitoneal, or intravenous routes of administration. CONCLUSIONS: We believe that this is the first report to demonstrate that regulatory DCs infiltrate into the cervical lymph nodes after intranasal administration of these cells and that intranasal administration of regulatory DCs is more effective for the induction of tolerance in the nasal mucosa than subcutaneous, intraperitoneal, or intravenous administration.