RESUMEN

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1ß, IL-8, LIF and TGFß2.

RESUMEN

Lentiviral vectors are an important tool for gene transfer research and gene therapy purposes. However, the low stability of these vectors affects their production, storage, and efficacy in preclinical and clinical settings. In the present work the mechanism underlying the thermosensitivity of lentiviral vectors was evaluated. For lentiviral vectors pseudotyped with amphotropic and RDpro envelopes, the capacity to perform reverse transcription was lost rapidly at 37 degrees C, in high correlation with the loss of infectivity. The vector with RDpro envelope presented a higher level of stability than that with amphotropic envelope for both the reverse transcription process and viral infectivity. Reverse transcriptase enzyme inactivation and viral template RNA degradation were not implicated in the loss of the viral capacity to perform reverse transcription. Furthermore, early entry steps in the infection process do not determine the rate of viral inactivation, as the amount of viral RNA and p24 protein entering the cells decreased slowly for both vectors. Taken together, it can be concluded that the reverse transcription process is thermolabile and thus determines the rate of lentiviral inactivation. Strategies to stabilize the reverse transcription process should be pursued to improve the applicability of lentiviral vectors in gene therapy.

Asunto(s)

Vectores Genéticos/genética , Lentivirus/genética , Transcripción Reversa/genética , Temperatura , Animales , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Activación Enzimática , Genoma Viral/genética , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , ARN Viral/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus

RESUMEN

BACKGROUND: The loss of gene transfer capacity in retroviral vectors constitutes a major disadvantage in the development of retroviral vectors for gene therapy applications. In the present work the loss of a vector's capacity to perform reverse transcription was studied as a possible explanation for the low stability of retroviral vectors from the production stage to the target cell gene transfer event. METHODS: Inactivation studies were performed with murine leukemia virus vectors at 37 degrees C and several residual activities were tested, including viral infectivity, reverse transcription capacity, reverse transcriptase (RT) activities and viral RNA stability. RESULTS: The results indicate a high correlation between loss of infectivity and the capacity of the virus to perform the initial steps of reverse transcription. To further understand the thermosensitivity of the reverse transcription process, the two enzyme activities of RT were investigated. The results indicate that, although the inactivation rate of the DNA polymerase is faster than that of RNase H, the decline of these two enzyme activities is significantly slower than that of reverse transcription. Also, viral RNA stability is not implicated in the loss of the virus capacity to perform reverse transcription as the rate of viral RNA degradation was very slow. Furthermore, it was observed that the amount of viral RNA that entered the cells decreased slowly due to viral inactivation at 37 degrees C. CONCLUSIONS: The reverse transcription process is thermolabile and this sensitivity determines the rate of retroviral inactivation. Strategies targeting stabilization of the reverse transcription complex should be pursued to improve the applicability of retroviral vectors in gene therapy studies.

Asunto(s)

Técnicas de Transferencia de Gen , Vectores Genéticos/fisiología , Virus de la Leucemia Murina/fisiología , Transcripción Reversa , Inactivación de Virus , Línea Celular , Terapia Genética , Vectores Genéticos/genética , Genoma Viral , Humanos , Virus de la Leucemia Murina/enzimología , Virus de la Leucemia Murina/genética , Estabilidad del ARN , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H/genética

RESUMEN

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and alpha-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.

Asunto(s)

Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/virología , Fructosa/metabolismo , Vectores Genéticos , Glucosa/metabolismo , Retroviridae/crecimiento & desarrollo , Cultivo de Virus , Adenosina Trifosfato/análisis , Alanina/análisis , Recuento de Células , Medios de Cultivo/química , Ácidos Grasos/biosíntesis , Ácido Glutámico/biosíntesis , Glutamina/biosíntesis , Humanos , Ácido Láctico/análisis , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Fosfocreatina/análisis

RESUMEN

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Asunto(s)

Membrana Celular/metabolismo , Vectores Genéticos , Retroviridae , Animales , Calorimetría , Línea Celular , Membrana Celular/química , Membrana Celular/virología , Espectroscopía de Resonancia por Spin del Electrón , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Conformación Proteica , Retroviridae/genética , Retroviridae/metabolismo , Temperatura , Transducción Genética , Inactivación de Virus

RESUMEN

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Asunto(s)

Carbohidratos/farmacología , Medios de Cultivo/química , Terapia Genética , Vectores Genéticos/fisiología , Retroviridae/fisiología , Ensamble de Virus/efectos de los fármacos , Recuento de Células , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fructosa/farmacología , Galactosa/farmacología , Glucosa/farmacología , Células HCT116 , Semivida , Humanos , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/fisiología , Sorbitol/farmacología , Transgenes

RESUMEN

The increasing numbers of pre-clinical and clinical trials where recombinant adenoviral vectors are used for gene therapy and vaccination require the development of cost-effective and reproducible large scale purification strategies of the biologically active particles. Alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation method, such as fixed bed chromatography strategies, have been developed, but the yields of final recovery remain too low due mainly to the capture and concentration steps taking place before and between the chromatographic stages. In this study, a rapid and efficient scale-able purification protocol allowing to obtain concentrated, pure and bioactive adenoviral vectors was developed. This allows efficient levels of binding to the column media and vector purification without centrifugation or filtration steps. Expanded bed chromatography followed by hollow fiber concentration allows the capture of viral particles directly from cellular extracts with high efficiency and vector purification is achieved in less than one working day with a minimal amount of sample handling, thus presenting an improvement over existing processes. The overall process yield reached 32%, representing an eight-fold improvement over results reported previously, while the purity is comparable to that obtained with the CsCl method.

Asunto(s)

Adenoviridae/genética , Cromatografía por Intercambio Iónico/métodos , Vectores Genéticos/aislamiento & purificación , Adenoviridae/crecimiento & desarrollo , Adenoviridae/aislamiento & purificación , Western Blotting , Línea Celular , ADN Viral/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Reacción en Cadena de la Polimerasa , Ultrafiltración , Ensayo de Placa Viral , Proteínas Virales/análisis

RESUMEN

Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 x 10(2) particles/microl) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA.

Asunto(s)

Virus de la Leucemia Murina/fisiología , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vectores Genéticos/genética , Virus de la Leucemia Murina/genética , Sensibilidad y Especificidad

RESUMEN

The optimization of production and purification processes is usually approached by engineers from a strictly biotechnological point of view. The present paper envisages the definition and application of an optimization model that takes into account the impact of both biological and technological issues upon the optimization protocols and strategies. For this purpose, the optimization of three analogous but different systems comprising animal cell growth and bioparticle production is presented. These systems were: human immunodeficiency 1 (HIV-1) and porcine parvovirus (PPV) virus-like particles (VLPs) produced in insect cells and retrovirus produced in mammalian cells. For the systematization of the optimization process four levels of optimization were defined-product, technology, design and integration. In this paper, the limits of each of the optimization levels defined are discussed by applying the concept to the systems described. This analysis leads to decisions regarding the production of VLPs and retrovirus as well as on the points relevant for further process development. Finally, the definition of the objective function or performance index, the possible strategies and tools for bioprocess optimization are described. Although developed from the three described processes, this approach can, based on the recent literature evidence reviewed here, be applied more universally for the process development of complex biopharmaceuticals.

Asunto(s)

Reactores Biológicos , Biotecnología/métodos , Microbiología Industrial/métodos , Modelos Biológicos , Retroviridae/aislamiento & purificación , Virión/aislamiento & purificación , Animales , Almacenaje de Medicamentos/métodos , VIH-1/aislamiento & purificación , Humanos , Insectos , Parvovirus Porcino/aislamiento & purificación , Control de Calidad , Porcinos , Análisis de Sistemas , Integración de Sistemas

RESUMEN

Improved, human-based packaging cell lines allow the production of high-titer, RCR-free retroviral vectors. The utility of these cell lines for the production of clinical grade vectors critically depends on the definition of optimal conditions for scaled-up cultures. In this work, a clone derived from the TE Fly GALV packaging cell (Duisit et al. Hum. Gene Ther. 1999, 10, 189) that produces high titers of a lacZ containing retroviral vector with a Gibbon Ape Leukemia Virus envelope glycoprotein was used. This clone can produce (2-5) x 10(6) PFU cm(-3) in small scale cultures and has been evaluated for growth and vector production in different reactor systems. The performances of fixed bed reactors [CellCube (Costar) and Celligen (New Brunswick)] and stirred tank reactors [microcarriers and clump cultures] were compared. The cells showed a higher apparent growth rate in the fixed bed reactor systems than in the suspension systems, probably as a result of the fact that aggregation and/or formation of clumps led to a reduced viability and reduced growth of cells in the interior of the clumps. As a consequence, the final cell density and number were in average 3- to 7-fold higher in the fixed bed systems in comparison to the suspension culture systems. The average titers obtained ranged from 0.5 to 2.1 x 10(7) PFU cm(-3) for the fixed bed and microcarrier systems, while the clump cultures produced only (2-5) x 10(5) PFU cm(-3). The differences in titers reflect cell densities as well as specific viral vector production rates, with the immobilization and microcarrier systems exhibiting an at least 10-fold higher production rate in comparison to the clump cultures. A partial optimization of the culture conditions in the Celligen fixed bed reactor, consisting of a 9-fold reduction of the seeding cell density, led to a 5-fold increased vector production rate accompanied by an average titer of 3 x 10(7) PFU cm(-3) (maximum titer (4-5) x 10(7) PFU cm(-3)) in the fixed bed reactor. The performance evaluation results using mathematical models indicated that the fixed bed bioreactor has a higher potential for retroviral vector production because of both the higher reactor productivity and the lower sensitivity of productivity in relation to the changes in final retrovirus titer in the range of 3 x 10(6) to 15 x 10(6) PFU cm(-3).

Asunto(s)

Reactores Biológicos , Vectores Genéticos/biosíntesis , Retroviridae/genética , Línea Celular

RESUMEN

Here we describe the cloning, localization, and characterization of a novel mammalian endo-apyrase (LALP1) in human and mouse. The predicted human LALP1 gene encodes a 604-amino acid protein, whereas the mouse Lalp1 gene encodes a 606-amino acid protein. The human and mouse genes have 88% amino acid sequence identity. These genes share considerable homologies with hLALP70, a recently discovered mammalian lysosomal endo-apyrase. The human LALP1 gene resides on chromosome 10q23-q24 and contains 12 exons and 11 introns covering a genomic region of approximately 46 kilobase pairs. The subcellular localization and enzymatic activity of LALP1 indicated that LALP1 is indeed an endo-apyrase with substrate preference for nucleoside triphosphates UTP, GTP, and CTP.

Asunto(s)

Apirasa/química , Apirasa/genética , Cromosomas Humanos Par 10 , Pirofosfatasas/química , Pirofosfatasas/genética , Secuencia de Aminoácidos , Animales , Apirasa/metabolismo , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Citidina Trifosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Masculino , Mamíferos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Pirofosfatasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transfección , Uridina Trifosfato/metabolismo

RESUMEN

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.

Asunto(s)

Reactores Biológicos , Terapia Genética , Retroviridae/fisiología , Replicación Viral , Línea Celular , Cromatografía en Gel , Retroviridae/genética , Ensamble de Virus

RESUMEN

Although retroviruses are a promising tool for gene therapy, there are two major problems limiting the establishment of viable industrial processes: retrovirus stability and low final yield in the supernatant. This fact emphasizes the need for an effective process optimization, not only at a genetic level but also at a bioprocess engineering level. In part 1 of this paper a mathematical model was developed to optimize the bioreaction yield by determining the best retrovirus harvest strategy in perfusion cultures. PA317 cells producing recombinant retroviruses were used to develop and test this model. Cell culture was performed in stirred tanks using porous supports. The parameters of the proposed model were experimentally determined for batch and perfusion cultures at 32 and 37 degrees C both with and without additives to enhance production; the model was then validated. This model allowed the determination of the optimal values of all operational variables included: batch and perfusion duration and perfusion rate. The highest productivity (2682 virus cm(-)(3) h(-)(1)) was obtained under the following conditions: batch at 37 degrees C for 53 h followed by perfusion at 32 degrees C for 23 h with a perfusion rate of 0.107 h(-)(1). This value was 3.5-fold higher than the best result obtained in batch cultures for the same conditions of titer and quality. A sensitivity analysis of the parameters showed that the parameters that affect most the final productivity depend on the bioreaction phase: cell growth in batch culture and production and product degradation in perfusion culture. In part 2 of this paper, this model is extended to the first step of downstream processing, and the addition of further steps to the process is discussed in order to achieve global process optimization.

Asunto(s)

Reactores Biológicos , Biotecnología/métodos , Modelos Biológicos , Retroviridae/crecimiento & desarrollo , Animales , Butiratos/farmacología , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Células Cultivadas/virología , Dexametasona/farmacología , Terapia Genética/métodos , Semivida , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/crecimiento & desarrollo , Reproducibilidad de los Resultados , Retroviridae/genética , Sensibilidad y Especificidad , Temperatura

RESUMEN

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing.

Asunto(s)

Endopeptidasas/metabolismo , VIH-1/patogenicidad , Spodoptera/enzimología , Adenosina Trifosfato/análisis , Albúminas/metabolismo , Animales , Caseínas/metabolismo , Células Cultivadas , Endopeptidasas/análisis , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Productos del Gen gag/metabolismo , VIH-1/metabolismo , Espectroscopía de Resonancia Magnética , Precursores de Proteínas/metabolismo , Factores de Tiempo , Transfección

RESUMEN

Urofacial (Ochoa) syndrome is an autosomal recessive disease characterized by distorted facial expression and urinary abnormalities. Previously, we mapped the UFS gene to chromosome 10q23-q24 and narrowed the interval to one YAC clone of 1410 kb. Here, we have constructed a BAC/PAC contig of the 1-Mb region using STS content mapping with 42 BAC/PAC-end sequences, 9 previously reported and 16 newly identified microsatellite markers, and 14 EST markers. A total of 26 polymorphic microsatellite markers were genotyped for 31 UFS patients from Colombia and 2 patients from the United States. Haplotype analyses suggest that the UFS gene is located within two overlapping BAC clones, a region of <360 kb of DNA sequence. We tested 42 EST markers previously mapped to the D10S1709-D10S603 interval against the BAC/PAC contig and identified 11 ESTs located in the 1-Mb region. Four of the 11 ESTs mapped to the 360-kb UFS critical region. Shotgun sequencing of the two BAC clones and BLASTN search of the EST databases revealed 3 other ESTs contained in the UFS critical region. These results will facilitate the cloning and identification of the UFS gene.

Asunto(s)

Cromosomas Humanos Par 10/genética , ADN/genética , Expresión Facial , Vejiga Urinaria Neurogénica/genética , Bacteriófago P1/genética , Cromosomas Bacterianos/genética , Clonación Molecular , Mapeo Contig , Salud de la Familia , Femenino , Marcadores Genéticos , Haplotipos , Humanos , Masculino , Linaje , Mapeo Físico de Cromosoma , Polimorfismo Genético , Síndrome

RESUMEN

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Asialoglicoproteínas/inmunología , Hemaglutininas Virales/inmunología , Orosomucoide/análogos & derivados , Polilisina/inmunología , Simplexvirus/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunación/métodos , Proteínas del Envoltorio Viral/inmunología , Animales , Receptor de Asialoglicoproteína , Asialoglicoproteínas/metabolismo , Antígenos CD4/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Hígado/citología , Hígado/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Orosomucoide/inmunología , Orosomucoide/metabolismo , Plásmidos , Polilisina/metabolismo , Receptores de Superficie Celular/metabolismo , Transfección , Proteínas del Envoltorio Viral/biosíntesis

RESUMEN

Type I and type II interferons (IFNs) are known to exert antitumor effects on a variety of tissues and cell types. We have previously shown that the type I IFN IFN alpha induces the expression of the cyclin-dependent kinase inhibitor p21WAF1 and inhibits the cell cycle of the human prostate adenocarcinoma cell line, DU145, that carries mutations in the tumor suppressor gene products p53 and pRB. We now show that the type II IFN IFN gamma similarly induces the expression of p21WAF1 and inhibits the cell cycle of DU145 cells. In addition, we show that while both IFNs exert antiproliferative activity, only IFN gamma induced phenotypic changes in these cells that accompanied the antiproliferative effect. For example, IFN gamma, but not IFN alpha, caused a significant reduction in epidermal growth factor receptor expression as well as an increase in the adhesion molecules intercellular adhesion molecule-1 and integrin alpha3. These phenotypic changes in DU145 cells are suggestive of the acquisition of a non-tumorigenic state. Consistent with these findings, IFN gamma showed a significantly lower invasive ability in in vitro assays using invasion chambers. Thus, IFN gamma inhibits both the cell cycle and the metastatic potential of DU145 cells independent of the p53 and RB status, and our data describe a mechanism for mediating the antitumor capabilities of IFN gamma that bypasses tumor suppressor genes like p53.

Asunto(s)

Ciclo Celular/efectos de los fármacos , Ciclinas/biosíntesis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inducción Enzimática/efectos de los fármacos , Humanos , Interferón-alfa/farmacología , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

We show, in this study, that type I IFN induction of the cyclin-dependent kinase (cdk) inhibitor p21WAF1 in the human Burkitt lymphoma B cell-line Daudi and ensuing cell cycle arrest correlate with the terminal differentiation of these cells, and is ultimately followed by apoptosis and cell death. The expression of p21WAF1 paralleled the onset of G1 arrest and the reduction of surface IgM expression which was used as a marker of the differentiation response, and the IFN treated cells acquired a typical plasma cell-like morphology. The type II IFN IFNgamma, which does not inhibit the growth of Daudi cells, did not induce the expression of p21WAF1, nor affect the expression of surface IgM. The induction of p21WAF1 which paralleled the inhibition of the phosphorylation of the retinoblastoma protein, pRB, was preceded by the strong reduction in c-myc levels. We propose that the coupled down-regulation of c-myc and induction of p21WAF1 may be crucial to the induction of differentiation and G1 arrest in Daudi cells by type I IFN. Growth arrest and differentiation was followed by apoptosis and cell death, and was accompanied by the induction of the activity of the apoptotic ICE-family protease CPP32. G1 arrest and differentiation followed by apoptotic cell death are characteristics of terminal differentiation. Thus, our data suggest that the induction of p21WAF1 and G1 arrest mediated by type I IFN in Daudi cells is part of terminal differentiation response in these cells, highlighting a role for type I IFN as B cell terminal differentiation factors.

Asunto(s)

Apoptosis/efectos de los fármacos , Linfocitos B/patología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/biosíntesis , Fase G1/efectos de los fármacos , Interferón Tipo I/farmacología , Apoptosis/genética , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Linfoma de Burkitt , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/fisiología , Fase G1/genética , Humanos , Células Tumorales Cultivadas

RESUMEN

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.

Asunto(s)

Spodoptera/virología , Virión , Cultivo de Virus/métodos , Animales , Baculoviridae , Western Blotting , División Celular , Línea Celular , Productos del Gen gag/biosíntesis , Consumo de Oxígeno , Spodoptera/citología , Virión/crecimiento & desarrollo , Virión/aislamiento & purificación

RESUMEN

The effect of medium viscosity upon cell growth and aggregate characteristics of baby hamster kidney (BHK) cells cultivated in stirred tanks was evaluated. Two thickening agents were tested, 9300 MW dextran and a low-viscosity sodium (carboxymethyl)-cellulose; both were used in two different sets of experiments: (i) 250 cm3 Wheaton spinner flasks with a ball impeller operated at 45 rpm; (ii) 500 cm3 Corning spinner flasks with a paddle impeller, operated at constant power dissipation (88 cm2 s-3). Aggregate diameter and the fraction of cells in aggregates increased with the increase in viscosity. Power laws were applied to the experimental results. A dependence of aggregate size upon power dissipation of the order of -0.19 and kinematic viscosity of 0.34 and 0.49 for the constant agitation and constant power dissipation tests were obtained, respectively. A model based upon the entire universal-equilibrium range (i.e., the entire spectrum of isotropic eddies) was used to predict theoretical relationships between the variables studied. The model leads to a power dependence of -0.25 for the energy dissipated in the entire universal-equilibrium range and between 0.25 and 0.5 for the kinematic viscosity in the viscous dissipation subrange, depending on the energy correlation used; it also gives a good explanation for the dependence of aggregate size on the hydrodynamics of the vessel.

Asunto(s)

Biotecnología/métodos , Riñón/citología , Viscosidad , Animales , Carboximetilcelulosa de Sodio , Agregación Celular/fisiología , División Celular/fisiología , Células Cultivadas , Cricetinae , Medios de Cultivo , Dextranos