RESUMEN
The immunogenicity of recombinant adenoviruses (Ad) constitutes a major concern for their use in gene therapy. Antibody- and cell-mediated immune responses triggered by adenoviral vectors hamper long-term transgene expression and efficient viral readministration. We previously reported that interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha play an essential role in both the acute phase and antibody response against Ad, respectively. As TNF-alpha controls the immune response and the development of the immune system, we examined here the consequence of blockade of TNF-alpha activity through Ad-mediated gene delivery of a dimeric mouse TNFR1-IgG fusion protein on transgene expression from a second Ad. Ad encoding TNFR1-IgG (AdTNFR1-Ig) was injected intravenously along with Ad encoding beta-galactosidase or alpha1-antitrypsin transgene in wild-type (IL-6(+/+)) but also in IL-6-deficient mice (IL-6(-/-)) to analyze how TNF-alpha and IL-6 diminish liver gene transfer efficacy. Blockade of TNF-alpha leads to increased transgene expression in both wild-type and IL-6(-/-) mice due to a reduced inflammatory response and to diminished recruitment of macrophages and NK cells towards the liver. Antibody responses against adenoviral particles and expressed transgenes were only delayed in AdTNFR1-Ig-treated wild-type mice, but were markedly reduced in AdTNFR1-Ig-treated IL-6(-/-) mice. Finally, treatment of mice with etanercept, a clinically approved anti-TNF-alpha drug, confirmed the importance of controlling proinflammatory cytokines during gene therapy by adenoviral vectors.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae/genética , Terapia Genética/métodos , Interleucina-6/inmunología , Transducción Genética/métodos , Factor de Necrosis Tumoral alfa/inmunología , Adenoviridae/inmunología , Animales , Anticuerpos Antivirales/sangre , Autoanticuerpos/sangre , ADN Viral/genética , Etanercept , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Hepatitis/inmunología , Humanos , Inmunoglobulina G/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , ARN Mensajero/análisis , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Interleukin-6 (IL-6) plays a central role in the pathogenesis of several autoimmune and inflammatory diseases as well as B-cell lymphoproliferative disorders. This work describes the effects of the recombinant or adenovirally-delivered IL-6 superantagonist Sant7, anti-IL-6 and IL-6 receptor monoclonal antibodies in a severe murine model of human B-cell lymphoma induced in SCID mice by transplantation of an LCL-41 cell line variant (isotype-switched IgM>IgG). Survival of 60% of the animals treated with anti-gp130 was observed up to day 33, while about 20% of the animals survived with anti-gp80 and Sant7 treatment. No survival was observed with the anti-IL-6 monoclonal antibody treatment. No significant change in serum and peritoneal levels of human IL-6 (hIL-6) and soluble human IL-6 receptor (shIL-6R) was observed in the recombinant Sant7-treated group towards the control group. The anti-gp80 monoclonal antibody induced significant increase of both hIL-6R and hIL-6 in serum and peritoneum. The anti-gp130 monoclonal antibody treatment determined a reduction of the seric shIL-6R and a significant increase of the seric hIL-6. Anti-IL-6 monoclonal antibody administration resulted in a reduction of serum and in an increase of peritoneal hIL-6. Treatment with adenoviral Sant7 was associated with a reduction of circulating shIL-6R, hIgG and mSAP. However, only marginal anti-tumor efficacy of the adenoviral Sant7 was observed. Overall, the present data suggest a potential for anti-hIL-6 therapy in B-cell lymphomas. Less severe animal models might be useful to better evaluate Sant7 efficacy alone or in combination with other anti-IL-6 therapeutics.
Asunto(s)
Anticuerpos Monoclonales/química , Interleucina-6/análogos & derivados , Interleucina-6/inmunología , Linfoma de Células B/metabolismo , Receptores de Interleucina-6/inmunología , Adenoviridae/genética , Animales , Antígenos CD/biosíntesis , Línea Celular , Línea Celular Tumoral , Receptor gp130 de Citocinas , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoglobulina G/química , Inflamación , Interleucina-6/biosíntesis , Interleucina-6/sangre , Interleucina-6/metabolismo , Linfoma de Células B/inmunología , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones SCID , Trasplante de Neoplasias , Receptores de Interleucina-6/biosíntesis , Proteínas Recombinantes/química , Factores de TiempoRESUMEN
The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.
Asunto(s)
Adenoviridae/genética , Bacteriófagos/genética , Proteínas de la Cápside , Cápside/genética , Proteínas Portadoras/metabolismo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Células HeLa , Humanos , Integrinas/metabolismo , Microscopía Electrónica , Oligopéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción GenéticaRESUMEN
Using human lymphocytes from a group of 20 donors, we investigated (i) the X-ray-induced adaptive response (AR) after four different conditioning treatments of 1, 2, 4 and 6 cGy, (ii) chromosomal sensitivity to X-irradiation during G(2) and (iii) the G(2)/M checkpoint response. An AR was found in 11 of the 18 donors (approximately 60%). No correlation was found between the presence of AR and G(2) chromosomal radiosensitivity, or with donor sensitivity to the activation of the G(2)/M checkpoint by 30 cGy or a dose as low as 2 cGy. The AR was not related to any particular conditioning treatment, but was consistently present or absent in any one donor under all conditioning treatments used. As far as chromosomal breakage and induced mitotic delay in G(2) are concerned, large variability between individuals was observed, together with a close correlation in the same donor between mitotic delay induced by a low dose (2 cGy) and the frequency of aberrations.
Asunto(s)
Cromátides/efectos de la radiación , Aberraciones Cromosómicas , Fase G2/efectos de la radiación , Adulto , Anciano , Células Cultivadas , Cromosomas/efectos de la radiación , Daño del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mitosis/efectos de la radiación , Rayos XRESUMEN
BACKGROUND: The major concern for the use of adenoviral vectors for gene therapy is the viral-induced immune response that has been shown to be responsible for short-term transgene expression and inefficient viral readministration. In vivo studies and clinical trials with recombinant adenovirus have suggested a role for interleukin 6 (IL-6) in the inflammatory reaction that follows Ad-infection. IL-6 plays an important role in the acute-phase innate response, in the differentiation of B-cells and in the activation of the Th2 cell subsets. METHODS: To clarify the role of IL-6 in the immune response to Ad-vectors, we used IL-6 knock-out mice (IL-6 -/- ). E1/E3 deleted recombinant adenoviruses encoding reporter genes were administered to wild type or IL-6-/- mice; transgene expression kinetics and immune response were analyzed. RESULTS: Acute phase protein production was significantly diminished in IL-6 -/- mice after adenoviral injection. No significant difference between wild type and knock-out animals in the level or the nature of leucocyte recruitment in the liver was detectable. A minor decrease in the IgG response to Ad-recombinants was observed in knock-out mice. Gene transfer efficiency, both in terms of levels and duration of transgene expression, were comparable in IL-6+/+ and IL-6-/- mice. An increase in IL-1beta and tumor necrosis factor-alpha (TNF-alpha) levels was observed in the sera of IL-6 -/- mice as compared to wild type animals: this phenomenon represents a possible compensatory mechanism for the establishment of the immune phenotype observed in mutant mice. CONCLUSIONS: IL-6 plays a role in the acute phase response to adenoviral vectors. Nevertheless, possibly due to a compensatory mechanism exerted by other cytokines, the antibody and cellular responses to adenoviruses are very similar in wild type and IL-6 -/- mice.
Asunto(s)
Adenoviridae/genética , Formación de Anticuerpos/genética , Vectores Genéticos , Inflamación/inmunología , Interleucina-6/fisiología , Proteínas de Fase Aguda/genética , Animales , Humanos , Inmunidad Celular/genética , Inflamación/genética , Interleucina-6/genética , Ratones , Ratones NoqueadosRESUMEN
The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, and the related cytokine lymphotoxin alpha (LTalpha), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-alpha/LTalpha-/-), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-alpha/LTalpha-/- and TNF-alpha/LTalpha+/- mice versus TNF-alpha/LTalpha+/+ mice links antibody levels to TNF-alpha/LTalpha gene dosage. Due to the absence of neutralizing antibodies, the TNF-alpha/LTalpha knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-alpha in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Linfotoxina-alfa/fisiología , Mastadenovirus/genética , Mastadenovirus/inmunología , Factor de Necrosis Tumoral alfa/deficiencia , Reacción de Fase Aguda , Animales , Anticuerpos Antivirales/biosíntesis , Expresión Génica , Terapia Genética , Inmunidad Celular , Técnicas In Vitro , Hígado/inmunología , Hígado/virología , Activación de Linfocitos , Linfotoxina-alfa/genética , Mastadenovirus/patogenicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pruebas de Neutralización , Recombinación Genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The effect of X-irradiation (1 Gy) during G1 on the transition from G1 to S and on the length of G2 over cell cycles subsequent to irradiation was studied in human lymphocytes from six different donors. After irradiation a delay was observed in the onset of S phase, as was an extension of the G2 phase lasting throughout the three to four subsequent cell divisions. The extension of G2 and of the cell cycle as a whole is partly related to the presence of chromosome aberrations in the cell. This is demonstrated by: (i) the presence of a larger number of chromosome aberrations in M1 cells corresponding to sampling times longer after that of irradiation; (ii) the presence of a larger number of chromosome aberrations in cells with a longer G2. The most significant chromosome aberrations in this respect are isochromatid fragments. Lastly, we observed that irradiation during G1 activates another checkpoint governing the way mitosis proceeds. This takes the form of an extension of metaphase; in this case also, in some cells, activation of a possible checkpoint during preanaphase seems to be related to the presence of aberrations.
Asunto(s)
Ciclo Celular/efectos de la radiación , Fase G1/efectos de la radiación , Activación de Linfocitos/efectos de la radiación , Linfocitos/efectos de la radiación , Adulto , Anafase/efectos de los fármacos , Anafase/efectos de la radiación , Autorradiografía , Ciclo Celular/genética , Células Cultivadas , Aberraciones Cromosómicas/genética , Aberraciones Cromosómicas/fisiología , Colchicina/farmacología , Colchicina/toxicidad , Replicación del ADN/efectos de la radiación , Fase G2/efectos de la radiación , Humanos , Linfocitos/citología , Masculino , Metafase/efectos de los fármacos , Metafase/efectos de la radiación , Persona de Mediana Edad , Mitosis/efectos de la radiación , Mutagénesis/efectos de la radiación , Profase/efectos de los fármacos , Profase/efectos de la radiación , Fase S/efectos de la radiación , Telofase/efectos de los fármacos , Telofase/efectos de la radiación , Factores de TiempoRESUMEN
Experiments were carried out with human lymphocytes to test whether there was any relation between the changes that conditioning treatment can produce in cell progression or in mitotic delay induced by the challenge dose and the presence of an 'adaptive response' (AR). In experiments in which the cells were successively fixed after the challenge dose, the interaction between conditioning treatment and challenge was of the same sign for all the fixation times: therefore it is likely that modifications of the cytogenetic damage in primed cells is not a mere reflection of stage sensitivity. In experiments in which using 1 Gy as conditioning treatment we induced a drastic extension of G2, we did not observe any AR; therefore, even if conditioning treatment can induce modifications in the cell-cycle phases before and/or after challenge, there is probably no link between these modifications and the presence of an AR.
Asunto(s)
Adaptación Fisiológica/genética , Adaptación Fisiológica/efectos de la radiación , Linfocitos/efectos de la radiación , Mitosis/efectos de la radiación , Células Cultivadas , Daño del ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Fase G2/efectos de la radiación , Humanos , Linfocitos/citología , Masculino , Persona de Mediana Edad , Índice Mitótico/efectos de la radiación , Rayos XRESUMEN
Experiments were carried out with human lymphocytes to test the effect of low-dosage X-ray irradiation (2 cGy) on cell-cycle kinetics and on the mitotic delay induced by the conditioning pretreatment alone or by a subsequent high dose of X-ray. All the tests were performed using lymphocytes from two donors who had previously displayed considerable differences in the interaction between a low and a high dose of ionizing radiation. A dose of 2 cGy led to significant variations in mitotic indices (MI) which differed for the two donors in relation to variations in the times of irradiation and fixation after stimulation with PHA. Moreover in one of the two donors 4-6 h after challenge the pretreated cultures have a higher MI that the controls; on the other hand, conditioning treatment alone induces in the other donor an extension of both G2 and of the time taken by cells in S at the time of challenge to reach mitosis. These findings could in the future provide some insight into the problem of the variability of the adaptive response in human lymphocytes.