Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production.

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.

Bexarotene targets autophagy and is protective against thromboembolic stroke in aged mice with tauopathy.

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.

Deletion of Nuclear Factor kappa B p50 Subunit Decreases Inflammatory Response and Mildly Protects Neurons from Transient Forebrain Ischemia-induced Damage.

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.

ADAMTS-4 promotes neurodegeneration in a mouse model of amyotrophic lateral sclerosis.

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.

Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.

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.

Aging aggravates ischemic stroke-induced brain damage in mice with chronic peripheral infection.

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.

Blood graft composition after plerixafor injection in patients with NHL.

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.

Production of monocytic cells from bone marrow stem cells: therapeutic usage in Alzheimer's disease.

Accumulation of amyloid β (Aβ) is a major hallmark in Alzheimer's disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aβ 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.

Chondrogenesis and mineralization during in vitro culture of human mesenchymal stem cells on three-dimensional woven scaffolds.

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-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(ɛ-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.

Quantitative evaluation of spontaneously and surgically repaired rabbit articular cartilage using intra-articular ultrasound method in situ.

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 vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D-woven poly(epsilon-caprolactone) scaffolds.

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.

1H MR spectroscopic imaging of phospholipase-mediated membrane lipid release in apoptotic rat glioma in vivo.

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.

Poly-L-D-lactic acid scaffold in the repair of porcine knee cartilage lesions.

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.

Low spin-lock field T1 relaxation in the rotating frame as a sensitive MR imaging marker for gene therapy treatment response in rat glioma.

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).

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models.

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).

High-resolution magic angle spinning 1H NMR spectroscopy and reverse transcription-PCR analysis of apoptosis in a rat glioma.

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.

High-resolution magic-angle-spinning 1H NMR spectroscopy reveals different responses in choline-containing metabolites upon gene therapy-induced programmed cell death in rat brain glioma.

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.

Water diffusion in a rat glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death in vivo: correlation with cell density.

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.

Novel magnetic resonance imaging contrasts for monitoring response to gene therapy in rat glioma.

Magnetic resonance imaging relaxation times, T(1rho) and Carr-Purcell T(2) (CP-T(2)), were measured in a glioma herpes simplex virus-thymidine kinase gene therapy model. In treated tumors with >50% cell death by histology, T(1rho) and CP-T(2) measured with short spacing (tau(CP)) between centers of adiabatic refocusing pulses showed similar enhanced sensitivity to cytotoxic cell damage over CP-T(2) measured with long tau(CP) (long-tau(CP) T(2): 54.3 +/- 0.

Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo, ex vivo, and in vitro.

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%.

Assignment of 1H nuclear magnetic resonance visible polyunsaturated fatty acids in BT4C gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death.

Polyunsaturated fatty acids (PUFAs), as detected by (1)H nuclear magnetic resonance (NMR) spectroscopy, accumulate into BT4C glioma during ganciclovir-thymidine kinase gene therapy-induced programmed cell death (PCD). In this study, we have quantified the (1)H NMR visible lipids in vivo and characterized their biophysical and biochemical nature in these tumors during PCD both ex vivo and in vitro. Concentrations of (1)H NMR-detectable PUFAs increased 3-fold with pattern recognition identifying CH = CH and CH = CHCH(2)CH = CH as the most significant in monitoring the dynamics of PCD.

Early gene therapy-induced apoptotic response in BT4C gliomas by magnetic resonance relaxation contrast T1 in the rotating frame.

The design and evaluation of therapeutic gene transfection protocols and vectors are under extensive development. Magnetic resonance imaging (MRI) techniques can aid considerably in the development of experimental treatment approaches, as well as in determining treatment response by observing gross tissue morphology. However, through a unique set of contrast parameters, namely T1, T2, and diffusion, more information about tissue status can be obtained while delineating and classifying tumor characteristics in more detail.