RESUMO
Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.
Assuntos
Âmnio/citologia , Exossomos/metabolismo , MicroRNAs/metabolismo , Apoptose , Carcinogênese/patologia , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Exossomos/ultraestrutura , Feminino , Humanos , Neoplasias Ovarianas/patologia , Tetraspanina 30/metabolismoRESUMO
The impact of high temperatures on mortality is well known, but not all deaths that occur during heat waves can be explained by this effect. We evaluated whether an additional mechanism caused by periods of sustained heat without nightly cooling influenced mortality during the European heat wave in 2003 and whether this mechanism is different for varying causes of death. We obtained daily counts of total and cause-specific mortality for Essen, Germany, for the years 2000-2006. We used time-series regression methods to separate a possible additional effect of sustained heat from the temperature effect and included air pollution, influenza epidemics, long-term and seasonal trends, days of week and bank holidays as covariates. The maximum daily relative risk of all-cause mortality during the heat wave was 1.28 (95% CI 1.06-1.53). The maximum relative risks of cardiovascular and neoplastic mortality were 1.25 (95% CI 0.95-1.65) and 1.35 (95% CI 1.00-1.82), respectively. The effect on respiratory mortality was delayed; the maximum relative risk was 1.66 (95% CI 1.19-2.23) 6 days after the heat wave. We found that periods with sustained heat especially affected respiratory mortality, whereas for cardiovascular and neoplastic mortality no distinct influence could be shown.
Assuntos
Transtornos de Estresse por Calor/mortalidade , Temperatura Alta/efeitos adversos , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/epidemiologia , Alemanha/epidemiologia , Transtornos de Estresse por Calor/complicações , Humanos , Umidade , Neoplasias/complicações , Neoplasias/epidemiologia , Ozônio/análise , Material Particulado/análise , Doenças Respiratórias/complicações , Doenças Respiratórias/epidemiologia , Fatores de RiscoRESUMO
During the 2003 heat wave an increase in mortality was observed in several European countries. Evidence suggests that the heat wave effect on mortality varies based upon underlying disease. In this study we examined the effects of the 2003 heat wave on all-cause and cause-specific mortality (neoplasms, cardiovascular and respiratory diseases) in a large west German city. Daily weather data for Essen was obtained from the German meteorological service. Death certificates for all deaths in Essen from 2002 to 2003 were coded according to the World Health Organization (WHO) guidelines. Mean numbers of daily deaths during and after the heat wave were compared with the average mortality in summer months (reference period). Poisson generalized additive models, adjusted for weekday and season, were fitted for overall and cause-specific mortality for the entire study period. During the 2003 heat wave (August 6-12), daily mortality increased by 15% (neoplasms), 30% (cardiovascular), and 61% (respiratory), with a decrease in the week after the heat wave of 17% for neoplasms and a sustained rise for respiratory mortality (77%). Regression analysis showed an association between heat and overall mortality in 2003 and greatest associations for respiratory mortality. Even the comparatively short heat wave in Essen in the year 2003 was associated with a rise in overall and cause-specific mortality. Different mechanisms appear to influence cause-specific mortality, with strongest associations for respiratory mortality. Harvesting might play a role in mortality due to neoplasms.
Assuntos
Causas de Morte , Transtornos de Estresse por Calor/mortalidade , Temperatura Alta/efeitos adversos , Atestado de Óbito , Alemanha/epidemiologia , Humanos , Análise de Regressão , Estações do Ano , Organização Mundial da SaúdeRESUMO
BACKGROUND: Human cell lines are the most innovative choice of host cell for production of biopharmaceuticals since they allow for authentic posttranslational modification of therapeutic proteins. We present a new method for generating high and stable protein expressing cell lines based on human amniocytes without the requirement of antibiotic selection. RESULTS: Primary amniocytes from routine amniocentesis samples can be efficiently transformed with adenoviral functions resulting in stable human cell lines. Cotransfection of the primary human amniocytes with a plasmid expressing adenoviral E1 functions plus a second plasmid containing a gene of interest resulted in permanent cell lines expressing up to 30 pg/cell/day of a fully glycosylated and sialylated protein. Expression of the gene of interest is very stable for more than 90 passages and, importantly, was achieved in the absence of any antibiotic selection. CONCLUSION: We describe an improved method for developing high protein expressing stable human cell lines. These cell lines are of non-tumor origin, they are immortalized by a function not oncogenic in human and they are from an ethically accepted and easily accessible cell source. Since the cell can be easily adapted to growth in serum-free and chemically defined medium they fulfill the requirements of biopharmaceutical production processes.
Assuntos
Líquido Amniótico/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular/citologia , Linhagem Celular/fisiologia , Melhoramento Genético/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Antibacterianos , Proliferação de Células , Humanos , Transfecção/métodosRESUMO
Intravascular injection of adenoviral vectors may result in a toxic and potentially lethal reaction, the mechanism of which is poorly understood. We noted that mice demonstrated a transient change in behavior that was characterized by inactivity and lethargy within minutes after intravenous injection of relatively low doses of adenoviral vectors (including high-capacity gutless vectors). Moreover, immediately after vector injection a significant drop in blood pressure was measured that most probably was caused by the systemic activation of endothelial cells as monitored by detection of phosphorylated Akt/PKB kinase, activated endothelial nitric oxide synthase (eNOS), and nitrotyrosine. The activation of the endothelium was the result of the interaction of viral particles with Kupffer cells, which are resident macrophages of the liver representing the first line of defense of the innate immune system. Surprisingly, the uptake of vector particles by Kupffer cells not only resulted in their strong activation, but also in their nearly complete disappearance from the liver. Our results suggest that the toxicity of intravenously injected adenoviral vectors may be directly linked to the activation and destruction of Kupffer cells.
Assuntos
Adenoviridae/genética , Células Endoteliais/metabolismo , Células de Kupffer/metabolismo , Tirosina/análogos & derivados , Animais , Pressão Sanguínea , Eicosanoides/metabolismo , Endotélio/metabolismo , Endotélio Vascular/metabolismo , Ativação Enzimática , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos , Imuno-Histoquímica , Injeções Intravenosas , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fosforilação , Radioimunoensaio , Baço/metabolismo , Fatores de Tempo , Tirosina/metabolismoRESUMO
Tissue macrophages, in particular hepatic Kupffer cells (KCs), contribute to early inflammatory responses following adenoviral vector administration. This study evaluates the effect of selective and transient (3 days) depletion of KCs by a single injection of clodronate liposomes on the in vivo performance of high-capacity adenoviral (HC-Ad) vectors. In KC-depleted C57BL/6 and C3H mice increased and stabilized hAAT levels were observed following intravenous injection of HC-Ad vectors expressing human alpha-1 anti-trypsin (hAAT) either from the hAAT promoter or from the human cytomegalovirus promoter. Comparable increases in hAAT levels were obtained in mice preinjected with a transcriptionally silent HC-Ad vector. Interestingly, in the majority of animals of both strains depletion of KCs was sufficient to prevent the generation of anti-hAAT antibodies, resulting in prolonged transgene expression. Thus, short-term and selective depletion of hepatic macrophages at the same time significantly increased hepatic transgene expression and reduced the humoral immune response to the transgenic protein.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Células de Kupffer/imunologia , Fígado/metabolismo , Transgenes , Animais , Citomegalovirus/genética , Ensaio de Imunoadsorção Enzimática , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , alfa 1-Antitripsina/genéticaRESUMO
High-capacity adenoviral (HC-Ad) vectors expressing B-domain-deleted human or canine factor VIII from different liver-specific promoters were evaluated for gene therapy of hemophilia A. Intravenous administration of these vectors into hemophilic FVIII-deficient immunodeficient SCID mice (FVIIIKO-SCID) at a dose of 5 x 10(9) infectious units (IU) resulted in efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 15 000 mU/mL), correcting the bleeding diathesis. Injection of only 5 x 10(7) IU still resulted in therapeutic FVIII levels. In immunocompetent hemophilic FVIII-deficient mice (FVIIIKO), FVIII expression levels peaked at 75 000 mU/mL but declined thereafter because of neutralizing anti-FVIII antibodies and a cellular immune response. Vector administration did not result in thrombocytopenia, anemia, or elevation of the proinflammatory cytokine interleukin-6 (IL-6) and caused no or only transient elevations in serum transaminases. Following transient in vivo depletion of macrophages before gene transfer, significantly higher and stable FVIII expression levels were observed. Injection of only 5 x 10(6) HC-Ad vectors after macrophage depletion resulted in long-term therapeutic FVIII levels in the FVIIIKO and FVIIIKO-SCID mice. Intravenous injection of an HC-Ad vector into a hemophilia A dog at a dose of 4.3 x 10(9) IU/kg led to transient therapeutic canine FVIII levels that partially corrected whole-blood clotting time. Inhibitory antibodies to canine FVIII could not be detected, and there were no signs of hepatotoxicity or of hematologic abnormalities. These results contribute to a better understanding of the safety and efficacy of HC-Ad vectors and suggest that the therapeutic window of HC-Ad vectors could be improved by minimizing the interaction between HC-Ad vectors and the innate immune system.
Assuntos
Adenovírus Humanos/genética , Fator VIII/análise , Terapia Genética , Vetores Genéticos/uso terapêutico , Hemofilia A/terapia , Animais , Anticorpos Heterófilos/biossíntese , Anticorpos Heterófilos/imunologia , Apolipoproteína C-II , Apolipoproteínas C/genética , Apolipoproteínas E/genética , Ácido Clodrônico/farmacologia , DNA Recombinante/análise , DNA Recombinante/genética , Doenças do Cão/genética , Doenças do Cão/terapia , Cães , Fator VIII/genética , Fator VIII/imunologia , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Hemofilia A/sangue , Hemofilia A/genética , Hemofilia A/veterinária , Hemorragia/prevenção & controle , Injeções Intravenosas , Fígado/metabolismo , Testes de Função Hepática , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Animais , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Especificidade da EspécieRESUMO
In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.