RESUMEN
Infection is one of the main causes of orthopedic implants failure, with antibiotic-resistant bacteria playing a crucial role in this outcome. In this work, antimicrobial nanogels were developed to be applied in situ as implant coating to prevent orthopedic-device-related infections. To that regard, a broad-spectrum antimicrobial peptide, Dhvar5, was grafted onto chitosan via thiol-norbornene "photoclick" chemistry. Dhvar5-chitosan nanogels (Dhvar5-NG) were then produced using a microfluidic system. Dhvar5-NG (1010 nanogels (NG)/mL) with a Dhvar5 concentration of 6 µg/mL reduced the burden of the most critical bacteria in orthopedic infections - methicillin-resistant Staphylococcus aureus (MRSA) - after 24 h in medium supplemented with human plasma proteins. Transmission electron microscopy showed that Dhvar5-NG killed bacteria by membrane disruption and cytoplasm release. No signs of cytotoxicity against a pre-osteoblast cell line were verified upon incubation with Dhvar5-NG. To further explore therapeutic alternatives, the potential synergistic effect of Dhvar5-NG with antibiotics was evaluated against MRSA. Dhvar5-NG at a sub-minimal inhibitory concentration (109 NG/mL) demonstrated synergistic effect with oxacillin (4-fold reduction: from 2 to 0.5 µg/mL) and piperacillin (2-fold reduction: from 2 to 1 µg/mL). This work supports the use of Dhvar5-NG as adjuvant of antibiotics to the prevention of orthopedic devices-related infections.
RESUMEN
BACKGROUND: To evaluate the prevalence of oral cancer in Brazil according to the clinical stage, anatomical location, alcoholism and smoking. MATERIAL AND METHODS: Data referring to 31,217 cases of oral cancer, from 2000 to 2010, were obtained from the Integrator Module of the Hospital Registry of Cancer. Inconsistent data ("non-classified" cases) was eliminated and 21,160 cases were analyzed. The frequency distribution according to clinical stage, anatomical location, alcoholism and smoking was analyzed descriptively and through a binary logistic regression model (α<0.05). The clinical stage (dependent variable) was dichotomized in early stage (I and II) or advanced stage (III and IV). The year of diagnosis, anatomical location and deleterious habits (alcoholism and smoking) were considered independent variables. RESULTS: The most frequent characteristics were: oropharynx location (n=3856, 18.41%), clinical stage IV (n=11924, 56.09%) and combined use of alcohol and tobacco (n=19226; 61.59%). The year 2009 (p<0.01, PR = 1.162, CI-95%=1.053-1.283) and location at the base of tongue (p<0.01, PR = 2.485, CI-95% = 2.182-2.807) presented a higher prevalence ratio for advanced stage oral cancer. The combined use of alcohol and tobacco showed a higher prevalence rate for the advanced clinical stage of cancer (p<0.01, PR =1.449, CI-95%=1.382-1.520) if compared to individuals without habits, or just alcoholics. CONCLUSIONS: Higher prevalence of advanced stage of oral cancer is related to the localization at the base of the tongue and to the concomitant use of alcohol and tobacco. Therefore, it can be suggested that all these characteristics lead to a worse prognosis of oral cancer.
Asunto(s)
Alcoholismo/complicaciones , Neoplasias de la Boca/epidemiología , Neoplasias de la Boca/etiología , Fumar/efectos adversos , Brasil , Estudios Transversales , Humanos , Neoplasias de la Boca/patología , Estadificación de Neoplasias , PrevalenciaRESUMEN
PURPOSE: This Phase I, multicenter, randomized study (ClinicalTrials.gov NCT01220128) evaluated the safety and immunogenicity of recombinant Wilms' tumor 1 (WT1) protein combined with the immunostimulant AS15 (WT1-immunotherapeutic) as neoadjuvant therapy administered concurrently with standard treatments in WT1-positive breast cancer patients. METHODS: Patients were treated in 4 cohorts according to neoadjuvant treatment (A: post-menopausal, hormone receptor [HR]-positive patients receiving aromatase inhibitors; B: patients receiving chemotherapy; C: HER2-overexpressing patients on trastuzumab-chemotherapy combination; D: HR-positive/HER2-negative patients on chemotherapy). Patients (cohorts A-C) were randomized (2:1) to receive 6 or 8 doses of WT1-immunotherapeutic or placebo together with standard neoadjuvant treatment in a double-blind manner; cohort D patients received WT1-immunotherapeutic in an open manner. Safety was assessed throughout the study. WT1-specific antibodies were assessed pre- and post-vaccination. RESULTS: Sixty-two patients were randomized; 60 received ≥ one dose of WT1-immunotherapeutic. Two severe toxicities were reported: diarrhea (cohort C; also reported as a grade 3 serious adverse event) and decreased left ventricular ejection fraction (cohort B; also reported as a grade 2 adverse event). Post-dose 4 of WT1-immunotherapeutic, 10/10 patients from cohort A, 0/8 patients from cohort B, 6/11 patients from cohort C, and 2/3 patients from cohort D were humoral responders. The sponsor elected to close the trial prematurely. CONCLUSIONS: Concurrent administration of WT1-immunotherapeutic and standard neoadjuvant therapy was well tolerated and induced WT1-specific antibodies in patients receiving neoadjuvant aromatase inhibitors. In patients on neoadjuvant chemotherapy or trastuzumab-chemotherapy combination, the humoral response was impaired or blunted, likely due to either co-administration of corticosteroids and/or the chemotherapies themselves.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer , Proteínas Recombinantes/administración & dosificación , Proteínas WT1/administración & dosificación , Anticuerpos/inmunología , Antígenos de Neoplasias/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/patología , Terapia Combinada , Femenino , Humanos , Inmunoterapia , Terapia Neoadyuvante , Estadificación de Neoplasias , Proteínas Recombinantes/inmunología , Resultado del Tratamiento , Proteínas WT1/inmunologíaRESUMEN
Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In this study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response was evaluated in mice infected with Leishmania braziliensis. Balb/c mice were infected with L. braziliensis and subsequently treated with three sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA-3, Foxp3 and T-bet) were analysed by quantitative PCR. The parasite load in the treated group was significantly lower than in the untreated group (P<.0001); in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (P=.012 and P=.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (P=.0001). 5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA.
Asunto(s)
Ácido Aminolevulínico/uso terapéutico , Leishmania braziliensis/parasitología , Leishmaniasis Cutánea/terapia , Fotoquimioterapia , Fármacos Fotosensibilizantes/uso terapéutico , Animales , Citocinas/biosíntesis , Interferón gamma , Leishmania braziliensis/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , ARN Mensajero , Factores de Transcripción/biosíntesisRESUMEN
PURPOSE: The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. PATIENTS AND METHODS: Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. RESULTS: 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. CONCLUSIONS: The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500â µg PRAME immunotherapeutic dose. TRIAL REGISTRATION NUMBER: NCT01149343, Results.
RESUMEN
Human cardiac stem/progenitor cells (hCPCs) have been shown to be capable to regenerate contractile myocardium. However, because of their relative low abundance in the heart, in vitro expansion of hCPC is mandatory to achieve necessary quantities for allogeneic or autologous cardiac regeneration therapy applications (10(6)-10(9) cells/patient). Up to now, cell number requirements of ongoing phase I/IIa trials have been fulfilled with production in static monolayer cultures. However, this manufacturing process poses critical limitations when moving to the following clinical phases where hundreds of patients will be enrolled. For this, increased process yield is required, while guaranteeing the quality of the cell-based products. In this work, we developed and validated a robust, scalable, and good manufacturing practice (GMP)-compatible bioprocess for the expansion of high-quality hCPC. We applied platforms extensively used by the biopharmaceutical industry, such as microcarrier technology and stirred systems, and assessed culture conditions' impact on hCPC's quality and potency, as required by regulatory agencies. Complementary analytical assays including gene expression microarrays and mass spectrometry-based approaches were explored to compare transcriptome, proteome, surface markers, and secretion profiles of hCPC cultured in static monolayers and in stirred microcarrier-based systems. Our results show that stirred microcarrier-based culture systems enabled achieving more than 3-fold increase in hCPC expansion, when compared with traditional static monolayers, while retaining cell's phenotype and similar "omics" profiles. These findings demonstrate that this change in the production process does not affect cell's identity and quality, with potential to be translated into a transversal production platform for clinical development of stem-cell therapies.
Asunto(s)
Miocardio/enzimología , Proteómica/métodos , Células Madre/citología , Trasplante Homólogo , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Humanos , Espectrometría de Masas , Microesferas , Fenotipo , Proteoma/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Virus-like particles (VLPs) are a particular subset of subunit vaccines which are currently explored as safer alternatives to live attenuated or inactivated vaccines. VLPs derived from retrovirus (retroVLPs) are commonly used as scaffolds for vaccine candidates due to their ability to incorporate heterologous envelope proteins. Pseudotyping retroVLPs is however not a selective process therefore, host cellular proteins such as tetraspanins are also included in the membrane. The contribution of these host-proteins to retrovirus immunogenicity remains unclear. In this work, human cells silenced and not silenced for tetraspanin CD81 were used to produce CD81(-) or CD81(+) retroVLPs. We first analyzed mice immune response against human CD81. Despite effective silencing of CD81 in retroVLP producing cells, both humoral and cellular immune responses showed persistent anti-CD81 immunogenicity, suggesting cross reactivity to related antigens. We thus compared the incorporation of related tetraspanins in retroVLPs and showed that decreased CD81 incorporation in CD81(-) retro-VLPs is compensated by an increased incorporation of CD9 and CD63 tetraspanins. These results highlight the dynamic nature of host-derived proteins incorporation in retroVLPs membrane, which should be considered when retrovirus-based biopharmaceuticals are produced in xenogeneic cells.
Asunto(s)
Reacciones Cruzadas , Retroviridae , Tetraspanina 28/inmunología , Tetraspaninas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Femenino , Silenciador del Gen , Células HEK293 , Humanos , Inmunidad Celular , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Tetraspanina 28/genética , Tetraspanina 29/genética , Tetraspanina 29/inmunología , Tetraspanina 30/genética , Tetraspanina 30/inmunología , Tetraspaninas/genéticaRESUMEN
Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.
Asunto(s)
Adenovirus Caninos/genética , Sistema Nervioso Central/metabolismo , Vectores Genéticos , Transducción Genética , Adenovirus Humanos/genética , Animales , Transporte Axonal , Diferenciación Celular , Supervivencia Celular , Clonación Molecular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Transgenes , Tropismo ViralRESUMEN
Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Ácido Láctico/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Retroviridae/crecimiento & desarrollo , Carga Viral , Cultivo de Virus/métodos , Línea Celular , Regulación hacia Abajo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Serina-Treonina Quinasas/genética , Piruvato Deshidrogenasa Quinasa Acetil-TransferidoraRESUMEN
This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.
Asunto(s)
Clonación Molecular/métodos , Pruebas Genéticas/métodos , Retroviridae/metabolismo , Línea Celular , Terapia Genética/métodos , Vectores Genéticos , Humanos , Retroviridae/genética , Transducción GenéticaRESUMEN
The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.
Asunto(s)
Adenovirus Caninos/crecimiento & desarrollo , Medio de Cultivo Libre de Suero , Cultivo de Virus/métodos , Animales , Reactores Biológicos , Proliferación Celular , Perros , Células de Riñón Canino Madin DarbyRESUMEN
Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Benzodiazepinonas/administración & dosificación , Benzodiazepinonas/farmacología , Caspasa 3/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Replicación del ADN , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Pirazoles/administración & dosificación , Pirazoles/farmacología , Timocitos/metabolismoRESUMEN
Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.
Asunto(s)
Adenovirus Caninos/genética , Replicación Viral , Adenovirus Caninos/fisiología , Animales , Autofagia , Supervivencia Celular , Replicación del ADN , Perros , Terapia Genética , Vectores Genéticos , Genoma Viral , Células de Riñón Canino Madin Darby , Transducción GenéticaRESUMEN
The objective of this study was to evaluate the histopathological grade of malignancy in a series of lower lip squamous cell carcinomas (LLSCCs) using three histopathological grading systems (invasive front grading system, World Health Organization (WHO) grading system, and histological risk assessment), and to correlate this with clinical parameters (tumour size/extent, regional lymph node metastasis, and clinical stage). Haematoxylin-eosin-stained histological sections obtained from 59 cases of LLSCC were analyzed by light microscopy. Grading of the invasive tumour front showed a significant association between low grade of malignancy and the absence of regional lymph node metastasis (P=0.030) and initial clinical stage (P=0.043). No significant associations were observed between the clinical parameters analyzed and the WHO system (P>0.05). Using the risk assessment, a highly significant association was observed between the risk score and regional lymph node metastasis (P=0.004) and clinical stage (P=0.002). In addition, the lymphocytic infiltrate was significantly associated with regional lymph node metastasis (P=0.017) and clinical stage (P=0.040). The results of the present study suggest that, among the histopathological grading systems evaluated, the histological risk assessment is the best option to predict the biological behaviour of LLSCCs.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de los Labios/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Pronóstico , Medición de RiesgoRESUMEN
Biopharmaceuticals derived from enveloped virus comprise an expanding market of vaccines, oncolytic vectors and gene therapy products. Thus, increased attention is given to the development of robust high-titer cell hosts for their manufacture. However, the knowledge on the physiological constraints modulating virus production is still scarce and the use of integrated strategies to improve hosts productivity and upstream bioprocess an under-explored territory. In this work, we conducted a functional genomics study, including the transcriptional profiling and central carbon metabolism analysis, following the metabolic changes in the transition 'parental-to-producer' of two human cell lines producing recombinant retrovirus. Results were gathered into three comprehensive metabolic maps, providing a broad and integrated overview of gene expression changes for both cell lines. Eight pathways were identified to be recruited in the virus production state: amino acid catabolism, carbohydrate catabolism and integration of the energy metabolism, nucleotide metabolism, glutathione metabolism, pentose phosphate pathway, polyamines biosynthesis and lipid metabolism. Their ability to modulate viral titers was experimentally challenged, leading to improved specific productivities of recombinant retrovirus up to 6-fold. Within recruited pathways in the virus production state, we sought for metabolic engineering gene targets in the low producing phenotypes. A mining strategy was used alternative to the traditional approach 'high vs. low producer' clonal comparison. Instead, 'high vs. low producer' from different genetic backgrounds (i.e. cell origins) were compared. Several genes were identified as limiting in the low-production phenotype, including two enzymes from cholesterol biosynthesis, two enzymes from glutathione biosynthesis and the regulatory machinery of polyamines biosynthesis. This is thus a frontier work, bridging fundamentals to technological research and contributing to enlarge our understanding of enveloped virus production dynamics in mammalian cell hosts.
Asunto(s)
Ingeniería Celular , Virus de la Leucemia del Gibón/metabolismo , Virus de la Leucemia Murina/metabolismo , Infecciones por Retroviridae/metabolismo , Animales , Células HEK293 , Humanos , Virus de la Leucemia del Gibón/genética , Virus de la Leucemia Murina/genética , Ratones , Infecciones por Retroviridae/genéticaRESUMEN
Canine adenovirus type 2 (CAV-2) vectors overcome many of the clinical immunogenic concerns related to vectors derived from human adenoviruses (AdVs). In addition, CAV-2 vectors preferentially transduce neurons with an efficient traffic via axons to afferent regions when injected into the brain. To meet the need for preclinical and possibly clinical uses, scalable and robust production processes are required. CAV-2 vectors are currently produced in E1-transcomplementing dog kidney (DK) cells, which might raise obstacles in regulatory approval for clinical grade material production. In this study, a GMP-compliant bioprocess was developed. An MDCK-E1 cell line, developed by our group, was grown in scalable stirred tank bioreactors, using serum-free medium, and used to produce CAV-2 vectors that were afterwards purified using column chromatographic steps. Vectors produced in MDCK-E1 cells were identical to those produced in DK cells as assessed by SDS-PAGE and dynamic light scatering measurements (diameter and Zeta potential). Productivities of â¼10(9) infectious particles (IP) ml(-1) and 2 × 10(3) IP per cell were possible. A downstream process using technologies transferable to process scales was developed, yielding 63% global recovery. The total particles to IP ratio in the purified product (<20:1) was within the limits specified by the regulatory authorities for AdV vectors. These results constitute a step toward a scalable process for CAV-2 vector production compliant with clinical material specifications.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/aislamiento & purificación , Adenoviridae/aislamiento & purificación , Animales , Perros , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Células de Riñón Canino Madin DarbyRESUMEN
The manufacture of enveloped virus, particularly retroviral (RV) and lentiviral (LV) vectors, faces the challenge of low titers that are aggravated under serum deprivation culture conditions. Also, the scarce knowledge on the biochemical pathways related with virus production is still limiting the design of rational strategies for improved production yields. This work describes the adaptation to serum deprivation of two human RV packaging cell lines, 293 FLEX and Te Fly and its effects on lipid biosynthetic pathways and infectious vector production. Total lipid content as well as cellular cholesterol were quantified and lipid biosynthesis was assessed by (13)C-NMR spectroscopy; changes in gene expression of lipid biosynthetic enzymes were also evaluated. The effects of adaptation to serum deprivation in lipid biosynthesis were cell line specific and directly correlated with infectious virus titers: 293 FLEX cells faced severe lipid starvation-up to 50% reduction in total lipid content-along with a 68-fold reduction in infectious vector titers; contrarily, Te Fly cells were able to maintain identical levels of total lipid content by rising de novo lipid biosynthesis, particularly for cholesterol-50-fold increase-with the consequent recovery of infectious vector productivities. Gene expression analysis of lipid biosynthetic enzymes further confirmed cholesterol production pathway to be prominently up-regulated under serum deprivation conditions for Te Fly cells, providing a genotype-phenotype validation for enhanced cholesterol synthesis. These results highlight lipid metabolism dynamics and the ability to activate lipid biosynthesis under serum deprivation as an important feature for high retroviral titers. Mechanisms underlying virus production and its relationship with lipid biosynthesis, with special focus on cholesterol, are discussed as potential targets for cellular metabolic engineering.
Asunto(s)
Proliferación Celular , Medio de Cultivo Libre de Suero/química , Vectores Genéticos , Metabolismo de los Lípidos , Retroviridae/crecimiento & desarrollo , Vías Biosintéticas/genética , Línea Celular , Citosol/química , Perfilación de la Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Carga ViralRESUMEN
Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the immunogenicity of RV-based vector and enhancing in vivo half-life. Such tools can also be applied to lentiviral or other enveloped viral vectors.
Asunto(s)
Productos Biológicos/química , Regulación hacia Abajo , Vectores Genéticos , Retroviridae/química , Retroviridae/genética , Tetraspanina 28/análisis , Animales , Productos Biológicos/administración & dosificación , Productos Biológicos/aislamiento & purificación , Línea Celular , Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen , Humanos , Ratones , Retroviridae/crecimiento & desarrollo , Retroviridae/aislamiento & purificaciónRESUMEN
Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.
Asunto(s)
Baculoviridae/genética , Técnicas de Cultivo de Célula , Vectores Genéticos , Lentivirus/genética , Lentivirus/aislamiento & purificación , Animales , Línea Celular , Etanolaminas , Organismos Modificados Genéticamente , Ratas , Transducción Genética , TransfecciónRESUMEN
The presence of chromosomal aberrations induced in circulating lymphocytes from breast cancer patients during chemotherapy was analyzed. Ten breast cancer patients undergoing neoadjuvant chemotherapy and ten healthy women (controls) were evaluated. Metaphases were obtained from cultures of peripheral lymphocytes stimulated with phytohemaglutinin and metaphase blockage was achieved with colchicine. One hundred metaphases were analyzed for chromosomal aberrations and 1,000 cells for the mitotic index. No significant differences were observed regarding the frequency of chromosomal aberrations, number of cells with chromosomal aberrations and mitotic index between the controls and patients before chemotherapy. However, after the first chemotherapy cycle, the numbers of chromosomal aberrations and cells with them was greater. After the third cycle, the mitotic index was lower, but the fifth cycle produced an increase in relation to the third and fourth cycles. The results suggest that chemotherapy raises the number of chromosomal aberrations and favors persistence of stable chromosomal abnormalities.