RESUMEN
In classical Hodgkin lymphoma (cHL), responsiveness to immune-checkpoint blockade (ICB) is associated with specific tumor microenvironment (TME) and peripheral blood features. The role of ICB in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is not established. To gain insights into its potential in NLPHL, we compared TME and peripheral blood signatures between HLs using an integrative multiomic analysis. A discovery/validation approach in 121 NLPHL and 114 cHL patients highlighted >2-fold enrichment in programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) gene expression for NLPHL versus cHL. Multiplex imaging showed marked increase in intra-tumoral protein expression of PD-1+ (and/or TIGIT+) CD4+ T-cells and PD-1+CD8+ T-cells in NLPHL compared to cHL. This included T-cells that rosetted with lymphocyte predominant (LP) and Hodgkin Reed-Sternberg (HRS) cells. In NLPHL, intra-tumoral PD-1+CD4+ T-cells frequently expressed TCF-1, a marker of heightened T-cell response to ICB. The peripheral blood signatures between HLs were also distinct, with higher levels of PD-1+TIGIT+ in TH1, TH2, and regulatory CD4+ T-cells in NLPHL versus cHL. Circulating PD-1+CD4+ had high levels of TCF-1. Notably, in both lymphomas, highly expanded populations of clonal TIGIT+PD-1+CD4+ and TIGIT+PD-1+CD8+ T-cells in the blood were also present in the TME, indicating that immune-checkpoint expressing T-cells circulated between intra-tumoral and blood compartments. In in vitro assays, ICB was capable of reducing rosette formation around LP and HRS cells, suggesting that disruption of rosetting may be a mechanism of action of ICB in HL. Overall, results indicate that further evaluation of ICB is warranted in NLPHL.
RESUMEN
Immune evasion, due to abnormal expression of programmed-death ligands 1 and 2 (PD-L1/PD-L2), predicts poor outcomes with chemoimmunotherapy in diffuse large B-cell lymphoma (DLBCL). Immune checkpoint inhibition (ICI) has limited efficacy at relapse but may sensitise relapsed lymphoma to subsequent chemotherapy. ICI delivery to immunologically intact patients may thus be the optimal use of this therapy. In the phase II AvR-CHOP study, 28 patients with treatment-naive stage II-IV DLBCL received sequential avelumab and rituximab priming ("AvRp;" avelumab 10 mg/kg and rituximab 375 mg/m2 2-weekly for 2 cycles), R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone for 6 cycles) and avelumab consolidation (10 mg/kg 2-weekly for 6 cycles). Grade 3/4 immune-related adverse events occurred in 11%, meeting the primary endpoint of a grade ≥3 irAE rate of <30%. R-CHOP delivery was not compromised but one patient ceased avelumab. Overall response rates (ORR) after AvRp and R-CHOP were 57% (18% CR) and 89% (all CR). High ORR to AvRp was observed in primary mediastinal B-cell lymphoma (67%; 4/6) and molecularly-defined EBV-positive DLBCL (100%; 3/3). Progression during AvRp was associated with chemorefractory disease. Two-year failure-free and overall survival were 82% and 89%. An immune priming strategy with AvRp, R-CHOP and avelumab consolidation shows acceptable toxicity with encouraging efficacy.
Asunto(s)
Linfoma de Células B Grandes Difuso , Recurrencia Local de Neoplasia , Humanos , Rituximab , Vincristina , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Ciclofosfamida , Prednisona , Doxorrubicina , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Resultado del TratamientoRESUMEN
Here, we present a novel case of a patient with chronic lymphocytic leukemia (CLL) who received CTLA-4 and then PD-1 immune-checkpoint blockade (ICB) as treatment for concomitant metastatic melanoma. Whereas the metastatic melanoma was responsive to ICB, the CLL rapidly progressed (but responded to ICB cessation and ibrutinib). There were no new genetic mutational drivers to explain the altered clinical course. PD-1/PD-L1/PD-L2 and CTLA-4/CD80/CD86 expression was not increased in CLL B cells, CD8+ or CD4+ T-cell subsets, or monocytes. The patient's CLL B cells demonstrated strikingly prolonged in vitro survival during PD-1 blockade, which was not observed in samples taken before or after ICB, or with other patients. To our knowledge, a discordant clinical course to ICB coupled with these biological features has not been reported in a patient with dual malignancies.
Asunto(s)
Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Leucemia Linfocítica Crónica de Células B , Melanoma , Receptor de Muerte Celular Programada 1 , Neoplasias Cutáneas , Humanos , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Progresión de la Enfermedad , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Melanoma/tratamiento farmacológico , Melanoma/etiología , Melanoma/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Antígeno B7-H1 , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Antineoplásicos/inmunología , Antineoplásicos/uso terapéuticoRESUMEN
Targeted antibody therapies improve outcomes for chronic lymphocytic leukemia (CLL) patients. However, resistance often develops. We have previously shown that resistance to therapeutic antibodies, by monocyte derived macrophages (referred to as nurse like cells, NLCs), from CLL patients is characterized by suppression of antibody dependent phagocytosis (ADP). The mechanism(s) contributing to the muted ADP responses remain unresolved. In this regard, an innate immune checkpoint was recently described that uses the CD47:SIRPα axis to suppress phagocytic responses by macrophages. In this study we examine whether the SIRPα axis regulates ADP responses to the anti-CD20 antibody, obinutuzumab, by NLCs. Using siRNA depletion strategies we show that SIRPα is a suppressor of ADP responses. Moreover, we show that this innate immune checkpoint contributes to the resistance phenotype in NLCs derived from CLL patients. Finally, we show that SIRPα suppression is mediated via the phosphatase, Shp1, which in turn suppresses SYK-dependent activation of ADP. Thus, we identify a druggable pathway that could be exploited to enhance sensitivity to existing therapeutic antibodies used in CLL. This is the first study to show that activation of the CD47:SIRPα innate immune checkpoint contributes to ADP resistance in NLCs from CLL patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Antígenos de Diferenciación , Antineoplásicos Inmunológicos , Leucemia Linfocítica Crónica de Células B , Macrófagos , Fagocitosis , Receptores Inmunológicos , Humanos , Anticuerpos Monoclonales Humanizados/farmacología , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antígeno CD47/genética , Células Cultivadas , Inmunidad Innata/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal , Quinasa Syk/metabolismoRESUMEN
There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.
Asunto(s)
Adipocitos/citología , Adipogénesis , Tejido Adiposo/citología , Técnicas de Cultivo de Célula/instrumentación , Células Madre Mesenquimatosas/citología , Adipocitos/patología , Tejido Adiposo/patología , Técnicas de Cultivo de Célula/economía , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo/economía , Técnicas de Cocultivo/instrumentación , Humanos , Neoplasias Renales/patología , Macrófagos/citología , Macrófagos/patología , Células Madre Mesenquimatosas/patología , Esferoides Celulares/citología , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
Fcγ receptor (FcγR) signalling in monocyte derived macrophages from chronic lymphocytic leukaemia (CLL) patients is poorly understood. This signalling pathway is the key determinant of the ability of the macrophages to respond to therapeutic antibodies in current clinical use for CLL. Muted FcγR signalling activity accompanies disease progression and results in resistance to therapeutic antibodies. The molecular mechanisms controlling FcγR signalling and resistance are unknown. Here, we demonstrate that the class I phosphoinositide 3-kinase (PI3K) catalytic subunit p110δ is essential for CLL-derived macrophages to respond to therapeutic antibodies. Inhibition of p110δ in the macrophages reduces FcγR-mediated antibody immune responses. Surprisingly, our studies indicated that FcγR downstream signalling is independent of SYK and BTK activity. Thus, we show that FcγR antibody responses occur via a previously unidentified p110δ-dependent pathway, which is independent of the previously described SYK/BTK activation pathway. These data provide novel insights into the effectors of antibody responses. Our data also provide mechanistic insights into therapy resistance in CLL.
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Formación de Anticuerpos/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Macrófagos/inmunología , Fosfatidilinositol 3-Quinasa/inmunología , Línea Celular , Línea Celular Tumoral , Humanos , Células Jurkat , Recuento de Leucocitos/métodos , Fagocitosis/inmunología , Proteínas Tirosina Quinasas/inmunología , Receptores de IgG/inmunología , Transducción de Señal/inmunología , Quinasa Syk/inmunologíaAsunto(s)
Biomarcadores de Tumor , Proteína Ligando Fas/sangre , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/mortalidad , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Masculino , Pronóstico , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
How do children and youth come to understand what it means to be a member of a particular race, gender, and other social groups? How do they come to hold beliefs about the groups that they do and do not belong to? Both news stories and fictional narratives that we are tuned into as a culture tell stories about what it means to be a member of a particular social group. In this review article, we relate the latest scientific knowledge on news and entertainment media representations of race, gender and other social categories and what they tell us about how these messages are taken in and processed by developing minds. We include research on identity development, social learning about members of other groups, and both positive and negative behavioral outcomes to cultural messages about race, gender, and other social categories. We offer recommendations for stakeholders to understand the role of the media in educating youth about race, gender and other social categories.
Asunto(s)
Desarrollo Infantil , Relaciones Interpersonales , Grupos Minoritarios/psicología , Medios de Comunicación Sociales , Niño , Medios de Comunicación , Humanos , Medios de Comunicación de MasasRESUMEN
Osteosarcoma (OS) accounts for 56% of malignant bone cancers in children and adolescents. Patients with localized disease rarely develop metastasis; however, pulmonary metastasis occurs in approximately 50% of patients and leads to a 5-year survival rate of only 10-20%. Therefore, identifying the genes and pathways involved in metastasis, as new therapeutic targets, is crucial to improve long-term survival of OS patients. Novel markers that define metastatic OS were identified using comparative transcriptomic analyses of two highly metastatic (C1 and C6) and two poorly metastatic clonal variants (C4 and C5) isolated from the metastatic OS cell line, KHOS. Using this approach, we determined that the metastatic phenotype correlated with overexpression of thioredoxin reductase 2 (TXNRD2) or vascular endothelial growth factor (VEGF). Validation in patient biopsies confirmed TXNRD2 and VEGF targets were highly expressed in 29-42% of metastatic OS patient biopsies, with no detectable expression in non-malignant bone or samples from OS patients with localised disease. Auranofin (AF) was used to selectively target and inhibit thioredoxin reductase (TrxR). At low doses, AF was able to inhibit TrxR activity without a significant effect on cell viability whereas at higher doses, AF could induce ROS-dependent apoptosis. AF treatment, in vivo, significantly reduced the development of pulmonary metastasis and we provide evidence that this effect may be due to an AF-dependent increase in cellular ROS. Thus, TXNRD2 may represent a novel druggable target that could be deployed to reduce the development of fatal pulmonary metastases in patients with OS.
Asunto(s)
Auranofina/farmacología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Animales , Antirreumáticos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Relación Dosis-Respuesta a Droga , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteosarcoma/genética , Osteosarcoma/patología , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxina Reductasa 2/genética , Factor A de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL. EXPERIMENTAL DESIGN: The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5(+)/CD19(+) CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents. RESULTS: Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L-related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells. CONCLUSION: This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols.
Asunto(s)
Selectina L/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Médula Ósea/metabolismo , Médula Ósea/patología , Células Cultivadas , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Selectina L/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
RNA interference (RNAi) may provide a therapeutic solution to many pulmonary epithelium diseases. However, the main barrier to the clinical use of RNAi remains the lack of efficient delivery vectors. Research has mainly concentrated on the intranasal route of delivery of short interfering RNA (siRNA) effector molecules for the treatment of respiratory diseases. However, this may be complicated in a diseased state due to the increased fluid production and tissue remodeling. Therefore, we investigated our hydration of a freeze-dried matrix (HFDM) formulated liposomes for systemic delivery to the lung epithelium. Here, we show that 45 ± 2% of epithelial murine lung cells receive siRNA delivery upon intravenous (IV) liposomal administration. Furthermore, we demonstrate that liposomal siRNA delivery resulted in targeted gene and protein knockdown throughout the lung, including lung epithelium. Taken together, this is the first description of lung epithelial delivery via cationic liposomes, and provides a proof of concept for the use of IV liposomal RNAi delivery to specifically knockdown targeted genes in the respiratory system. This approach may provide an attractive alternate therapeutic delivery strategy for the treatment of lung epithelium diseases.Molecular Therapy - Nucleic Acids (2013) 2, e96; doi:10.1038/mtna.2013.22; published online 4 June 2013.
RESUMEN
Chronic lymphocytic leukemia (CLL) is predominantly a disease of accumulation rather than rapid proliferation. To date, no cell lines exist, as CLL cells undergo rapid apoptosis when cultured in vitro, suggesting that a favorable in vivo microenvironment is required. To identify survival signals we cultured primary CLL peripheral blood mononuclear cells (PBMCs) at high density, which has previously been shown to dramatically improve survival. Using antibody arrays we measured the level of 42 cytokines in culture supernatants and showed that inerleukin-6 (IL-6), IL-8, CXCL2 and CCL2 were highly up-regulated in culture. This is the first report to describe a role for CCL2 and CXCL2 in CLL cell survival. Importantly, CXCL2, IL-6 and IL-8 were significantly up-regulated in primary patient plasma. The addition of either CXCL2 or CCL2 enhanced CLL cell survival, while antibodies blocking these chemokines reduced survival. Co-culture of CLL cells and PBMC accessory cells separated by transwells provided a similar degree of survival protection compared to normal culture, whereas CLL cells cultured alone died rapidly. Interestingly, CCL2 and CXCL2 appeared to be produced by CLL cells but only when co-cultured with accessory cells. Thus, we speculate that accessory cells release soluble factors that promote the production of these pro-survival chemokines from CLL cells and physical interactions are not required. Our data support the concept that the CLL microenvironment is critical, and suggests that soluble factors are more important than physical interactions.
Asunto(s)
Quimiocina CCL2/metabolismo , Quimiocina CXCL2/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Comunicación Celular , Supervivencia Celular , Quimiocina CCL2/sangre , Quimiocina CXCL2/sangre , Femenino , Humanos , Interleucina-6/sangre , Interleucina-6/metabolismo , Interleucina-8/sangre , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.
Asunto(s)
Resinas Acrílicas/química , Materiales Biocompatibles/química , Preparaciones de Acción Retardada/química , Técnicas de Transferencia de Gen , Oligodesoxirribonucleótidos/metabolismo , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/farmacología , Catálisis , Cationes/química , Cationes/metabolismo , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada/metabolismo , Preparaciones de Acción Retardada/farmacología , Femenino , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Imitación Molecular , Nanopartículas , Oligodesoxirribonucleótidos/farmacología , Tamaño de la Partícula , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.
Asunto(s)
Alginatos/química , Portadores de Fármacos/química , Oligonucleótidos Antisentido/administración & dosificación , Polietilenglicoles/química , ARN Interferente Pequeño/administración & dosificación , Vagina/metabolismo , Administración Intravaginal , Animales , Western Blotting , Composición de Medicamentos , Epitelio/metabolismo , Femenino , Liofilización , Técnicas de Silenciamiento del Gen/métodos , Ácido Glucurónico/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Ácidos Hexurónicos/química , Humanos , Lamina Tipo A/genética , Lípidos/química , Liposomas , Ratones , Ratones Endogámicos C57BL , Tamaño de la PartículaRESUMEN
Although small interfering RNA (siRNA) treatment holds great promise for the treatment of cancers, the field has been held back by the availability of suitable delivery vehicles. For cervical cancer the E6 and E7 oncogenes are ideal siRNA targets for treatment. The purpose of the present study was to explore the potential of dendrosomes for the delivery of siRNA targeting E6 and E7 proteins of cervical cancer cells in vitro. Optimization of dendrimer generation and nitrogen-to-phosphate (N/P) ratio was carried out using dendrimer-fluorescein isothiocyanate oligo complexes. The optimized N/P ratios were used in formulating complexes between dendrimers and siRNA targeting green fluorescence protein (siGFP). Although formulation 4D100 (dendrimer-siRNA complex) displayed the highest GFP knockdown, it was also found to be highly toxic to cells. In the final formulation 4D100 was encapsulated into dendrosomes so as to mask these toxic effects. The optimized dendrosomal formulation (DF), DF3 was found to possess a siGFP-entrapment efficiency of 49.76% +/- 1.62%, vesicle size of 154 +/- 1.73 nm, and zeta potential of +3.21 +/- 0.07 mV. The GFP knockdown efficiency of DF3 (dendrosome) was found to be almost identical to that of 4D100, but the former was completely nontoxic to the cells. DF3 containing siRNA against E6 and E7 was found to knock down the target genes considerably, as compared with the other formulations tested. Our results imply that dendrosomes hold potential for the delivery of siRNA and that a suitable targeting strategy could be useful for applications in vivo. FROM THE CLINICAL EDITOR: siRNA treatment holds great promise for the treatment of cancers, but overall, the availability of suitable delivery vehicles remains a major issue. The purpose of this study was to explore the potential of dendrosomes for the delivery of siRNA targeting specific proteins in cervical cancer cells in vitro. The results suggest that dendrosomes hold potential for the delivery of siRNA and a suitable targeting strategy could be useful for applications in vivo.
Asunto(s)
Dendrímeros/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Western Blotting , Muerte Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Fluoresceína-5-Isotiocianato , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Tamaño de la Partícula , Neoplasias del Cuello Uterino/patologíaRESUMEN
Recent studies on Hodgkin's lymphoma (HL) have indicated that patients with active disease display functional impairment of Ag-specific CD8+ T cells due to expansion of regulatory T cells at sites of disease and in the peripheral blood. Adoptive cellular immunotherapy based on EBV-specific CD8+ T cells has been explored with limited success to date. It has been proposed that improved targeting of these CD8+ T cells toward viral Ags that are expressed in HL may enhance future therapeutic vaccine strategies. In this study, we have developed a novel replication-deficient adenoviral Ag presentation system that is designed to encode glycine alanine repeat-deleted EBV nuclear Ag 1 covalently linked to multiple CD8+ T cell epitopes from latent membrane proteins 1 and 2. A single stimulation of CD8+ T cells from healthy virus carriers, and patients with HL with this adenoviral construct in combination with IL-2, was sufficient to reverse the functional T cell impairment and restored both IFN-gamma production and cytolytic function. More importantly, these activated CD8+ T cells responded to tumor cells expressing membrane proteins and recognized novel EBNA1 epitopes. Flow cytometric analysis revealed that a large proportion of T cells expanded from patients with HL were CD62L(high) and CD27(high), and CCR7(low), consistent with early to mid effector T cells. These findings provide an important platform for translation of Ag-specific adoptive immunotherapy for the treatment of EBV-associated malignancies such as HL and nasopharyngeal carcinoma.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Enfermedad de Hodgkin/terapia , Inmunoterapia Adoptiva , Proteínas de la Matriz Viral/inmunología , Adenoviridae/genética , Secuencia de Aminoácidos , Infecciones por Virus de Epstein-Barr/complicaciones , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Femenino , Vectores Genéticos , Herpesvirus Humano 4/inmunología , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/virología , Humanos , Técnicas In Vitro , Activación de Linfocitos/inmunología , Masculino , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/inmunología , Infecciones Tumorales por Virus/complicaciones , Proteínas de la Matriz Viral/genéticaRESUMEN
The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.
Asunto(s)
Química Orgánica/métodos , Epítopos , Vacunas Estreptocócicas/síntesis química , Streptococcus pyogenes/inmunología , Vacunas Sintéticas/química , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Secuencia de Aminoácidos , Animales , Toxina del Cólera/farmacología , Cisteína/química , Evaluación Preclínica de Medicamentos/métodos , Femenino , Sueros Inmunes , Inmunoglobulina G/análisis , Lipoproteínas/síntesis química , Lipoproteínas/inmunología , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Vacunas Estreptocócicas/inmunología , Vacunas Estreptocócicas/farmacología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacologíaRESUMEN
When applied to facial recognition, the levels of processing paradigm has yielded consistent results: faces processed in deep conditions are recognized better than faces processed under shallow conditions. However, there are multiple explanations for this occurrence. The own-race advantage in facial recognition, the tendency to recognize faces from one's own race better than faces from another race, is also consistently shown but not clearly explained. This study was designed to test the hypothesis that the levels of processing findings in facial recognition are a result of interest and attention, not differences in processing. This hypothesis was tested for both own and other faces with 105 Caucasian general psychology students. Levels of processing was manipulated as a between-subjects variable; students were asked to answer one of four types of study questions, e.g., "deep" or "shallow" processing questions, while viewing the study faces. Students' recognition of a subset of previously presented Caucasian and African-American faces from a test-set with an equal number of distractor faces was tested. They indicated their interest in and attention to the task. The typical levels of processing effect was observed with better recognition performance in the deep conditions than in the shallow conditions for both own- and other-race faces. The typical own-race advantage was also observed regardless of level of processing condition. For both own- and other-race faces, level of processing explained a significant portion of the recognition variance above and beyond what was explained by interest in and attention to the task.