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The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.
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Reactores Biológicos , Vectores Genéticos , Cultivo de Virus , Terapia GenéticaRESUMEN
Stroke is a highly debilitating, often fatal disorder for which current therapies are suitable for only a minor fraction of patients. Discovery of novel, effective therapies is hampered by the fact that advanced age, primary age-related tauopathy or comorbidities typical to several types of dementing diseases are usually not taken into account in preclinical studies, which predominantly use young, healthy rodents. Here we investigated for the first time the neuroprotective potential of bexarotene, an FDA-approved agent, in a co-morbidity model of stroke that combines high age and tauopathy with thromboembolic cerebral ischemia. Following thromboembolic stroke bexarotene enhanced autophagy in the ischemic brain concomitantly with a reduction in lesion volume and amelioration of behavioral deficits in aged transgenic mice expressing the human P301L-Tau mutation. In in vitro studies bexarotene increased the expression of autophagy markers and reduced autophagic flux in neuronal cells expressing P301L-Tau. Bexarotene also restored mitochondrial respiration deficits in P301L-Tau neurons. These newly described actions of bexarotene add to the growing amount of compelling data showing that bexarotene is a potent neuroprotective agent, and identify a novel autophagy-modulating effect of bexarotene.
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Autofagia/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Accidente Cerebrovascular/prevención & control , Tauopatías/tratamiento farmacológico , Tetrahidronaftalenos/farmacología , Tromboembolia/prevención & control , Envejecimiento , Animales , Bexaroteno , Ratones , Ratones Transgénicos , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Tauopatías/metabolismo , Tauopatías/patología , Tromboembolia/metabolismo , Tromboembolia/patologíaRESUMEN
Transient forebrain ischemia induces delayed death of the hippocampal pyramidal neurons, particularly in the CA2 and medial CA1 area. Early pharmacological inhibition of inflammatory response can ameliorate neuronal death, but it also inhibits processes leading to tissue regeneration. Therefore, research efforts are now directed to modulation of post-ischemic inflammation, with the aim to promote beneficial effects of inflammation and limit adverse effects. Transcription factor NF-κB plays a key role in the inflammation and cell survival/apoptosis pathways. In the brain, NF-κB is predominantly found in the form of a heterodimer of p65 (RelA) and p50 subunit, where p65 has a transactivation domain while p50 is chiefly involved in DNA binding. In this study, we subjected middle-aged Nfkb1 knockout mice (lacking p50 subunit) and wild-type controls of both sexs to 17 min of transient forebrain ischemia and assessed mouse performance in a panel of behavioral tests after two weeks of post-operative recovery. We found that ischemia failed to induce clear memory and motor deficits, but affected spontaneous locomotion in genotype- and sex-specific way. We also show that both the lack of the NF-κB p50 subunit and female sex independently protected CA2 hippocampal neurons from ischemia-induced cell death. Additionally, the NF-κB p50 subunit deficiency significantly reduced ischemia-induced microgliosis, astrogliosis, and neurogenesis. Lower levels of hippocampal microgliosis significantly correlated with faster spatial learning. We conclude that NF-κB regulates the outcome of transient forebrain ischemia in middle-aged subjects in a sex-specific way, having an impact not only on neuronal death but also specific inflammatory responses and neurogenesis.
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BACKGROUND: A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteoglycanases are specialized in the degradation of chondroitin sulfate proteoglycans and participate in mechanisms mediating neuroplasticity. Despite the beneficial effect of ADAMTS-4 on neurorepair after spinal cord injury, the functions of ADAMTS proteoglycanases in other CNS disease states have not been studied. Therefore, we investigated the expression, effects and associated mechanisms of ADAMTS-4 during amyotrophic lateral sclerosis (ALS) in the SOD1(G93A) mouse model. RESULTS: ADAMTS-4 expression and activity were reduced in the spinal cord of SOD1(G93A) mice at disease end-stage when compared to WT littermates. To counteract the loss of ADAMTS-4, SOD1(G93A) and WT mice were treated with saline or a recombinant ADAMTS-4 before symptom onset. Administration of ADAMTS-4 worsened the prognosis of SOD1(G93A) mice by accelerating clinical signs of neuromuscular dysfunctions. The worsened prognosis of ADAMTS-4-treated SOD1(G93A) mice was accompanied by increased degradation of perineuronal nets enwrapping motoneurons and increased motoneuron degeneration in the lumbar spinal cord. Motoneurons of ADAMTS-4-treated SOD1(G93A) mice were more vulnerable to degeneration most likely due to the loss of their extracellular matrix envelopes. The decrease of neurotrophic factor production induced by ADAMTS-4 in vitro and in vivo may also contribute to a hostile environment for motoneuron especially when devoid of a net. CONCLUSIONS: This study suggests that the reduction of ADAMTS-4 activity during the progression of ALS pathology may be an adaptive change to mitigate its neurodegenerative impact in CNS tissues. Therapies compensating the compromized ADAMTS-4 activity are likely not promising approaches for treating ALS.
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Proteínas ADAM/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Procolágeno N-Endopeptidasa/metabolismo , Médula Espinal/metabolismo , Proteína ADAMTS4 , Esclerosis Amiotrófica Lateral/patología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones Transgénicos , Superóxido Dismutasa/metabolismoRESUMEN
Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.
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Adenoviridae/fisiología , Cultivo de Virus , Reactores Biológicos , Proliferación Celular , Medios de Cultivo , Vectores Genéticos , Células HEK293 , Humanos , Replicación ViralRESUMEN
Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all of which alter peripheral inflammatory processes with concomitant impact on stroke outcome. The majority of the stroke patients are elderly, but the impact of interactions between aging and inflammation on stroke remains unknown. We thus investigated the influence of age on the outcome of stroke in animals predisposed to systemic chronic infection. Th1-polarized chronic systemic infection was induced in 18-22 month and 4-month-old C57BL/6j mice by administration of Trichuris muris (gut parasite). One month after infection, mice underwent permanent middle cerebral artery occlusion and infarct size, brain gliosis, and brain and plasma cytokine profiles were analyzed. Chronic infection increased the infarct size in aged but not in young mice at 24 h. Aged, ischemic mice showed altered plasma and brain cytokine responses, while the lesion size correlated with plasma prestroke levels of RANTES. Moreover, the old, infected mice exhibited significantly increased neutrophil recruitment and upregulation of both plasma interleukin-17α and tumor necrosis factor-α levels. Neither age nor infection status alone or in combination altered the ischemia-induced brain microgliosis. Our results show that chronic peripheral infection in aged animals renders the brain more vulnerable to ischemic insults, possibly by increasing the invasion of neutrophils and altering the inflammation status in the blood and brain. Understanding the interactions between age and infections is crucial for developing a better therapeutic regimen for ischemic stroke and when modeling it as a disease of the elderly.
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Envejecimiento/fisiología , Lesiones Encefálicas/etiología , Isquemia Encefálica/patología , Isquemia Encefálica/parasitología , Tricuriasis/patología , Trichuris/crecimiento & desarrollo , Animales , Lesiones Encefálicas/inmunología , Lesiones Encefálicas/parasitología , Lesiones Encefálicas/patología , Isquemia Encefálica/inmunología , Quimiocina CCL2/metabolismo , Enfermedad Crónica , Citocinas/sangre , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos/metabolismo , Infarto de la Arteria Cerebral Media/parasitología , Infarto de la Arteria Cerebral Media/patología , Inflamación/inmunología , Inflamación/parasitología , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/patología , Distribución Aleatoria , Tricuriasis/inmunología , Regulación hacia ArribaRESUMEN
Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.
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Microglía/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Superóxidos/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/patología , Animales , Células del Asta Anterior/enzimología , Astrocitos/enzimología , Línea Celular , Activación Enzimática , Inducción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Muscimol/análogos & derivados , Muscimol/farmacología , NADPH Oxidasa 1 , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/genética , Transporte de Proteínas , Superóxido Dismutasa , Superóxido Dismutasa-1 , Factor de Necrosis Tumoral alfa/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: Plerixafor is used to mobilize CD34(+) hematopoietic stem cells from bone marrow to circulation. Limited data are available in regard to graft cellular content collected after plerixafor. OBJECTIVES: The aim of this study was to assess effects of plerixafor added to chemomobilization on graft CD34(+) cell subclasses, lymphocyte subsets, engraftment, and post-transplant course in non-Hodgkin lymphoma (NHL) patients. METHODS: Thirty-four patients with NHL were included. All patients received chemotherapy plus G-CSF to mobilize stem cells. Nineteen patients received plerixafor pre-emptively owing to poor mobilization or poor collection yields. The rest of the patients constituted the control group. Flow cytometric analyzes were performed from cryopreserved graft samples. Also, data on post-transplant engraftment and outcome were collected. RESULTS: The proportion of primitive stem cells (CD34(+) CD133(+) CD38(-) ) was significantly higher after the plerixafor injection when compared to the first collection in the control group. The amount of T cells (CD3(+) ), helper (CD3(+) CD4(+) ) T subsets, and suppressor (CD3(+) CD8(+) ) T subsets in the graft was all significantly higher in the plerixafor group. Also, the amount of NK cells (CD3(-) CD16/56(+) ) was higher. Engraftment after high-dose therapy was comparable between the groups, but leukocyte and platelet count at 6 months were higher in patients receiving plerixafor-mobilized grafts. CONCLUSION: Plerixafor, when used pre-emptively in addition to chemomobilization, seems to mobilize more primitive CD34(+) stem cells, T lymphocytes, and NK cells. Whether these differences are associated with immune reconstitution, long-term engraftment, or patient outcomes needs to be evaluated in larger patient groups with longer follow-up.
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Fármacos Anti-VIH/administración & dosificación , Antígenos CD34 , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas , Compuestos Heterocíclicos/administración & dosificación , Linfoma no Hodgkin/sangre , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Bencilaminas , Ciclamas , Femenino , Citometría de Flujo , Supervivencia de Injerto/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Humanos , Células Asesinas Naturales/patología , Recuento de Leucocitos , Linfoma no Hodgkin/patología , Linfoma no Hodgkin/terapia , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica , Linfocitos T/patología , Trasplante AutólogoRESUMEN
Accumulation of amyloid ß (Aß) is a major hallmark in Alzheimer's disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aß burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell-based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM-derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aß uptake assay in vitro and Aß degradation assay of natively formed Aß deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC-derived cells for therapeutic purposes. HSC-derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aß reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.
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Enfermedad de Alzheimer/terapia , Células Madre Hematopoyéticas/fisiología , Monocitos/fisiología , Monocitos/trasplante , Péptidos beta-Amiloides/metabolismo , Animales , Separación Celular , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacosRESUMEN
Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(É-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(É-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.
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Condrogénesis/fisiología , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Células Cultivadas , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana EdadRESUMEN
During the last decade, a major effort has been devoted to developing surgical methods for repairing localized articular cartilage lesions. Despite some promising results no ultimate breakthrough in surgical cartilage repair has been achieved. Improvements in repair techniques would benefit from more sensitive and quantitative methods for long-term follow-up of cartilage healing. In this study, the potential of a new ultrasound technique for detecting the compositional and structural changes in articular cartilage after surgery, using recombinant human type II collagen gel and spontaneous repair was, investigated. Rabbit knee joints containing intact (n = 13) and surgically (n = 8) or spontaneously (n = 5) repaired tissue were imaged in situ at 6 months after the operation using a clinical intravascular high-frequency (40 MHz) ultrasound device. Based on the ultrasound raw data, ultrasound reflection coefficient (R), integrated ultrasound reflection coefficient (IRC), apparent integrated backscattering coefficient (AIB) and ultrasound roughness index (URI) were determined for each sample. URI was significantly higher in both repair groups than in intact cartilage (p < 0.05). The reflection parameters (R and IRC) were significantly lower in surgically repaired cartilage (p < 0.05) than in intact cartilage. Furthermore, AIB was significantly higher in surgically repaired cartilage than in intact tissue (p < 0.05). To conclude, the integrity of the rabbit articular cartilage repair could be quantitatively evaluated with the nondestructive ultrasound approach. In addition, clinically valuable qualitative information on the changes in cartilage integration, structure and composition could be extracted from the ultrasound images. In the present study, the structure and properties of repaired tissue were inferior to native tissue at 6 months after the operation. The applied ultrasound device and probes are FDA approved and, thus, applicable for the quantitative in vivo evaluation of human articular cartilage.
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Cartílago Articular/lesiones , Cartílago Articular/cirugía , Traumatismos de la Rodilla/diagnóstico por imagen , Traumatismos de la Rodilla/cirugía , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/cirugía , Ultrasonografía Intervencional/métodos , Animales , Cartílago Articular/diagnóstico por imagen , Conejos , Resultado del TratamientoRESUMEN
Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were combined with adult human mesenchymal stem cells (hMSC) to engineer mechanically functional cartilage constructs in vitro. The specific objectives were to: (i) produce PCL scaffolds with cartilage-like mechanical properties, (ii) demonstrate that hMSCs formed cartilage after 21 days of culture on PCL scaffolds, and (iii) study effects of scaffold structure (loosely vs. tightly woven), culture vessel (static dish vs. oscillating bioreactor), and medium composition (chondrogenic additives with or without serum). Aggregate moduli of 21-day constructs approached normal articular cartilage for tightly woven PCL cultured in bioreactors, were lower for tightly woven PCL cultured statically, and lowest for loosely woven PCL cultured statically (p<0.05). Construct DNA, total collagen, and glycosaminoglycans (GAG) increased in a manner dependent on time, culture vessel, and medium composition. Chondrogenesis was verified histologically by rounded cells within a hyaline-like matrix that immunostained for collagen type II but not type I. Bioreactors yielded constructs with higher collagen content (p<0.05) and more homogenous matrix than static controls. Chondrogenic additives yielded constructs with higher GAG (p<0.05) and earlier expression of collagen II mRNA if serum was not present in medium. These results show feasibility of functional cartilage tissue engineering from hMSC and 3D-woven PCL scaffolds.
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Cartílago/metabolismo , Células Madre Mesenquimatosas/fisiología , Poliésteres/química , Andamios del Tejido , Adulto , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Cartílago/citología , Células Cultivadas , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Humanos , Artropatías/patología , Artropatías/terapia , Masculino , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Poliésteres/metabolismo , Estrés Mecánico , Ingeniería de Tejidos/métodosRESUMEN
The purpose of the current study was to determine regional spatiotemporal differences and to gain insight on the mechanisms responsible for lipid accumulation during apoptotic cell death using in vivo MR spectroscopic imaging in combination with histology and biochemical membrane lipid analyses. Rats bearing BT4C gliomas were treated with ganciclovir (GCV) for 14 days, and combined in vivo quantitative MR spectroscopic imaging (MRSI) of gliomas with histology and a biochemical analysis of major cell membrane constituents. By using 1H MRSI in vivo in combination with histology, we were able to demonstrate previously unattainable regional lipid concentration differences in tumors during GCV-induced apoptosis, with 5-microL tissue volume resolution. Our results also show that, during treatment, phospholipase A2 (PLA2) expression is significantly elevated by 37+/-13% (P<0.05) and tumor cell membranes loose a significant proportion of unsaturated fatty acyl moieties (56+/-6 mmol/kg, P<0.05). These changes are reflected in both histology and significant MR-visible lipid accumulation, demonstrating that phospholipid hydrolysis in tissue undergoing apoptosis can be imaged with MRSI. Our work demonstrates the versatility of 1H MRSI in studying apoptosis in vivo, which is likely to pave way for the use of MRSI in both experimental and clinical anticancer trials.
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Apoptosis , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Espectroscopía de Resonancia Magnética/métodos , Lípidos de la Membrana/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Ganciclovir/farmacología , Glioma/tratamiento farmacológico , Etiquetado Corte-Fin in Situ , Fosfolipasas A2/metabolismo , RatasRESUMEN
Articular cartilage injuries cause a major clinical problem because of the negligible repair capacity of cartilage. Autologous chondrocyte transplantation is a surgical method developed to repair cartilage lesions. In the operation, cartilage defect is covered with a periosteal patch and the suspension of cultured autologous chondrocytes is injected into the lesion site. The method can form good repair tissue, but new techniques are needed to make the operation easier and to increase the postoperative biomechanical properties of the repair tissue. In this study, we investigated poly-L,D-lactic acid (PLDLA) scaffolds alone or seeded with autologous chondrocytes in the repair of circular 6-mm cartilage lesions in immature porcine knee joints. Spontaneous repair was used as a reference. Histologic evaluation of the repair tissue showed that spontaneous repair exhibited higher scores than either PLDLA scaffold group (with or without seeded chondrocytes). The scaffold material was most often seen embedded in the subchondral bone underneath the defect area, probably because of the hardness of the PLDLA material. However, some of the cell-seeded and nonseeded scaffolds contained cartilaginous tissue, suggesting that invasion of mesenchymal cells inside nonseeded scaffolds had occurred. Hyaluronan deposited in the scaffold had possibly acted as a chemoattractant for the cell recruitment. In conclusion, the PLDLA scaffold material used in this study was obviously mechanically too hard to be used for cartilage repair in immature animals.
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Condrocitos/patología , Condrocitos/trasplante , Fracturas del Cartílago/patología , Fracturas del Cartílago/cirugía , Regeneración Tisular Dirigida/métodos , Ácido Láctico/química , Polímeros/química , Ingeniería de Tejidos/métodos , Animales , Células Cultivadas , Traumatismos de la Rodilla/patología , Traumatismos de la Rodilla/cirugía , Ácido Láctico/uso terapéutico , Poliésteres , Polímeros/uso terapéutico , Porcinos , Resultado del TratamientoRESUMEN
PURPOSE: To prospectively assess the effectiveness of T1 relaxation in the rotating frame (T1 rho) dispersion and the low spin-lock radiofrequency field (B(1)) T1 rho magnetic resonance (MR) imaging relaxation time in noninvasive monitoring of gene therapy response in BT4C glioma in rats. MATERIALS AND METHODS: All animal studies were approved by the ethical committee of the National Laboratory Animal Center. Rats with BT4C gliomas (n=9) were treated with herpes simplex virus thymidine kinase gene therapy and were compared with untreated rats (n=5). Absolute T1 rho at a B(1) range of 2.0 x 10(-6) to 1.4 x 10(-4) T, T1, T2, and apparent diffusion constant were measured at 4.7 T during treatment. Statistical significance was tested by using repeated-measures analysis of variance. RESULTS: A significant (P<.05) lengthening of T1 rho was observed beginning on the 4th day of treatment, and T1 rho values increased to be approximately 80% higher than values observed before treatment. These changes preceded T1 and T2 changes and resembled those of water diffusion. The T1 rho was associated with a treatment-induced decrease in cell density; this was the only measured MR imaging property that provided significant (P<.05) Pearson correlation with cell density in the tumor border. T1 rho relaxation dispersion, however, did not offer additional benefits over those offered in one B(1) experiment in the early phase of treatment. CONCLUSION: T1 rho with low B(1) is an excellent MR imaging marker of early gene therapy response in gliomas. The low B(1) approach is not limited by specific absorption rate restrictions; this finding suggests that spin-lock methods could be applicable in clinical settings. (
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Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioma/diagnóstico , Glioma/terapia , Interpretación de Imagen Asistida por Computador/métodos , Imagen por Resonancia Magnética/métodos , Animales , Línea Celular Tumoral , Glioma/genética , Aumento de la Imagen/métodos , Pronóstico , Ratas , Reproducibilidad de los Resultados , Rotación , Sensibilidad y Especificidad , Marcadores de Spin , Resultado del TratamientoRESUMEN
Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication-competent SFV vector VA7-EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7-EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV-positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T-cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti-SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.
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Adenocarcinoma/terapia , Neoplasias Encefálicas/terapia , Glioma/terapia , Neoplasias Pulmonares/terapia , Viroterapia Oncolítica , Virus de los Bosques Semliki/genética , Adenocarcinoma/virología , Animales , Neoplasias Encefálicas/virología , Línea Celular Tumoral , Vectores Genéticos , Glioma/virología , Humanos , Neoplasias Pulmonares/virología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ratas , Tasa de Supervivencia , Transfección , Trasplante Heterólogo , Replicación ViralRESUMEN
The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS 1H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.
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Apoptosis , Glioma/patología , Espectroscopía de Resonancia Magnética/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Glioma/genética , Metabolismo de los Lípidos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Protones , ARN Neoplásico/análisis , Ratas , Ratas Endogámicas , Factores de Tiempo , Receptor fasRESUMEN
Changes in the concentrations of choline-containing metabolites (CCM) have been implicated in both cell proliferation and death processes. In this study, high-resolution magic-angle-spinning (HRMAS) 1H NMR spectroscopy was used to study metabolite changes in the CCM chemical shift region in rat glioma ex vivo during apoptosis induced by thymidine kinase-ganciclovir gene therapy. Cell density and apoptotic activity in the tumours were quantified by histological methods. HRMAS 1H NMR was able to resolve peaks from choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), taurine (Tau) and myo-inositol (myo-Ins), all of which contribute to the in vivo 1H NMR peak centred at 3.23 ppm. The early phase of apoptosis (treatment day 4), with a approximately 2.8-fold increase in the number of apoptotic nuclei (at constant cell density of 1.8 +/- 0.1 x 10(5) cells/mm3) was associated with increases in resonance intensity from GPC and PC, while Cho and Tau remained unchanged. Later stage apoptosis, accompanied by synchronous cell death (cell density declined to 0.7 +/- 0.02 x 10(5) cells/mm3), resulted in a significant decline in Tau relative to untreated tumours, while the contents of CCMs and myo-Ins detectable by 1H HRMAS were unchanged. These observations demonstrate that, while the in vivo 1H NMR peak at 3.23 ppm is indicative of cellular processes involved in apoptosis, the biochemical changes monitored by this resonance involve a number of different and chemically distinct metabolites.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Colina/análisis , Terapia Genética/métodos , Glioma/metabolismo , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Femenino , Glioma/diagnóstico , Glioma/genética , Glioma/terapia , Interpretación de Imagen Asistida por Computador/métodos , Protones , Ratas , Marcadores de Spin , Resultado del TratamientoRESUMEN
PURPOSE: To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)-thymidine kinase gene therapy-induced programmed cell death (PCD) in vivo. MATERIALS AND METHODS: The trace of the diffusion tensor (Dav = 1/3TraceD), T2, and spin density were determined by MRI and the apparent diffusion coefficient (ADC) of water by diffusion nuclear MR (NMR) spectroscopy using largely varying b values and diffusion times (tD) at 4.7 T. Cell count and apoptotic cells were quantified by histological means. RESULTS: Decline in cell count was strongly associated with increase in both Dav and T2. Spin density ratio between tumor and contralateral parietal cortex increased with a very similar time course as Dav and T2, indicating net water gain into the eradicating tumor. Diffusion spectroscopy showed a nonmonoexponential signal decay at all tD values ranging from 14-192 msec. During PCD, the ADC of the component yielding fast diffusion coefficient (D1), as acquired with tD > or = 47 msec, increased with kinetics similar to those of Dav (tD = 4.8 msec). The fractional size of D1 increased by 10% to 15% throughout the entire tD range. Apparent water residence time of the slow diffusion component, D2, shortened from a value of 38.3 +/- 1.7 msec on day 0 to 33.4 +/- 0.5 msec by day 8. CONCLUSION: The present results show that reduced cell density and increased water content, leading to altered water microenvironment, are associated with increased water diffusion coefficient in eradicating gliomas as a result of PCD.