bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Process Conditions on the Centrifugal Recovery of a Disabled Herpes Simplex Virus
Authors: Lotfian Pantea , Levy M. Susana , Coffin Robert. S. , Fearn T. , Ayazi‐Shamlou Parviz 
Published: February 2003

AbstractDespite continuous improvements in culturing and recovery techniques, high‐titer stocks of purified disabled herpes simplex virus type‐1 (HSV‐1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing‐out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV‐1 DIS. 80% virus recovery was achieved after 90 min at 26000 g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed‐angle centrifuge rotor operating at 26000 g. Plaque assays of recovered HSV‐1 DIS gave values on the order of 106 pfu/mL, compared to values typically above 109 pfu/mL obtained for the replication‐competent HSV‐1 viron.
Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells
Authors: Jensen Tor W. , Chen Yong , Miller William M. 
Published: February 2003

AbstractIncreases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)‐ and protamine sulfate (PS)‐aided retroviral transduction of NIH‐3T3 cells in a serum‐lot‐dependent manner. The increase in Pb‐aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS‐aided transduction was less effective on an absolute basis than Pb‐aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS‐aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb‐aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.
Improved Fermentation Processes for NS0 Cell Lines Expressing Human Antibodies and Glutamine Synthetase
Authors: Dempsey Jonathan , Ruddock Steve , Osborne Matthew , Ridley Alison , Sturt Simone , Field Ray 
Published: February 2003

AbstractTo meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed‐batch process was developed for GS‐NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine‐ and serum‐free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5‐fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin‐containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum‐and protein‐free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2‐fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10‐fold over the starting concentrations.
Metabolic Screening of Mammalian Cell Cultures Using Well‐Plates
Authors: Balcarcel R. Robert , Clark Lindsey M. 
Published: February 2003

AbstractFor use in a broad spectrum of cell culture applications, we have devised a novel method, termed High‐Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well‐plate technology and adaptations of well‐known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub‐milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T‐flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well‐plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.
Optimization of an Acoustic Cell Filter with a Novel Air‐Backflush System
Authors: Gorenflo Volker M. , Angepat Sumitra , Bowen Bruce D. , Piret James M. 
Published: February 2003

AbstractIncreasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air‐backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air‐backflush mode of operating a 10L acoustic separator was systematically tested at 107 cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h−1. The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 ± 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 107 cells/mL and 95% viability.
Strategies for Improved dCO2 Removal in Large‐Scale Fed‐Batch Cultures
Authors: Mostafa Sigma S. , Gu Xuejun (Sherry) 
Published: February 2003

AbstractCarbon dioxide buildup in large‐scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO2 (dCO2) level was 179 ± 9 mmHg at the pilot plant scale and 68 ± 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO2 levels between 40 and 120 mmHg throughout the 14‐day fed‐batch process. A cell‐free experiment was used to investigate the impact of the following parameters on dCO2 removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO2 was measured using a fiber optic based probe. dCO2 removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 μm pore size, 1 cm pipe i.d.). Open pipe provided 3‐ to 4‐fold better dCO2 removal for the same mass transfer coefficient ( kLa) value. A mathematical model and a bench‐scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed‐batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved kLa of dCO2 by 2‐fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO2 removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO2 level by 2.5‐fold (179 ± 9 vs 72 ± 9 mmHg). Titer at day 10 of culture improved by 1.5‐fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9–11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2‐fold higher titer compared to that of the historical control without compromising protein quality.
Heat Inactivation of Mammalian Cell Cultures for Biowaste Kill System Design
Authors: Gregoriades Niki , Luzardo Melissa , Lucquet Beatrice , Ryll Thomas 
Published: February 2003

AbstractA biowaste kill system was implemented to treat biological waste generated from a clinical manufacturing and R&D antibody facility. To confirm that design parameters of this continuous decontamination system are sufficient to inactivate mammalian cell culture waste, bench‐scale experiments were conducted. The biowaste kill system heat inactivates mammalian cell cultures before they are piped to a neutralization tank and subsequently released to the sewage system. Heat inactivation of cells is accomplished by exposing cells to 80 °C for 1 min. Small‐scale heat inactivation studies were performed on CHO, 293‐HEK, and hybridoma cells. Cells at 1 × 106 cells/mL or 1 × 107 cells/mL were exposed to 37, 60, 70, or 80 °C for 0, 30, 60, and 120 s. Viability based on trypan blue exclusion method and ability to proliferate was assessed after exposure to heat. Data suggest that exposure of cells to 80 °C for 60 s is sufficient to inactivate these cultures before they are released to the sewage system.
Fluid Mechanics, Cell Distribution, and Environment in Cell Cube Bioreactors
Authors: Auniņš John G. , Bader Brett , Caola Anthony , Griffiths Janet , Katz Maayan , Licari Peter , Ram Kripa , Ranucci Colette S. , Zhou Weichang 
Published: February 2003

AbstractCultivation of MRC‐5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine ( 1), is performed in the Cell Cube reactor, a laminar flow fixed‐bed bioreactor with an unusual diamond‐shaped, diverging‐converging flow geometry. These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation ( 2, 3). Early testing of the Cell Cube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase. Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained. Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment.
Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation
Authors: Joosten Christoph E. , Shuler Michael L. 
Published: February 2003

AbstractUsing a nonengineered Trichoplusia ni insect cell line, Tn‐4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N‐acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner‐flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post‐infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of β‐galactosidase and N‐acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular β‐galactosidase and N‐acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn‐4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.
Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 β1‐adrenoceptors versus Gly389 β1‐adrenoceptors in isolated human atrial myocardium
Authors: Sandilands A J , O'Shaughnessy K M , Brown M J 
Published: February 2003

We studied the biochemical and contractile responses of isolated human myocardial tissue expressing native receptor variants of the 389G>R β1‐adrenoceptor polymorphism.
Right atrial appendage was obtained from homozygous RR patients (n=37) and homozygous GG patients (n=17) undergoing elective cardiac surgery. The positive inotropic effect of noradrenaline in these tissues, mediated through β1‐adrenoceptors, was studied using electrically stimulated (1 Hz) atrial strips, as well as the effects of noradrenaline on cyclic AMP levels and cyclic AMP‐dependent protein kinase.
Tissue from RR homozygotes (n=14) showed significantly increased inotropic potency to noradrenaline (−log EC50, M=6.92±0.12) compared to GG homozygotes (n=8, −log EC50, M=6.36±0.11, P<0.005). This difference was not dependent on tissue basal force.
Tissue cyclic AMP levels (pmol mg−1) were also greater in RR homozygotes (basal 34.8±3.7 n=12, 300 nM noradrenaline 41.4±7.6 n=9, 30 μM noradrenaline 45.2±3.2 n=22, 0.2 mM isoprenaline 48.3±4.2 n=16) compared to GG homozygotes (basal 30.7±4.4 n=5, 300 nM noradrenaline 32.6±6.92 n=5, 30 μM noradrenaline 38.1±3.1 n=8, 0.2 mM isoprenaline 42.6±5.2 n=6, P=0.007). There were no differences between the variants in terms of cyclic AMP‐dependent protein kinase activity.
These data provide the first evidence that enhanced G‐protein coupling of the R389 β1‐adrenoceptor variant reported in rodent fibroblast expression systems is also present in native human receptors. The functional consequence of this is to significantly alter the inotropic potency of β1‐adrenoceptor activation depending on its genotype at the 389 position.

British Journal of Pharmacology (2003) 138, 386–392. doi:10.1038/sj.bjp.0705030
Effects of the AT1‐receptor antagonist eprosartan on the progression of left ventricular dysfunction in dogs with heart failure
Authors: Suzuki George , Mishima Takayuki , Tanhehco Elaine J , Sharov Victor G , Todor Anastassia , Rostogi Sharad , Gupta Ramesh C , Chaudhry Pervaiz A , Anagnostopoulos Petros V , Nass Omar , Goldstein Sidney , Sabbah Hani N 
Published: February 2003

We examined the effects of eprosartan, an AT1 receptor antagonist, on the progression of left ventricular (LV) dysfunction and remodelling in dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction, EF 30 to 40%).
Dogs were randomized to 3 months of oral therapy with low‐dose eprosartan (600 mg once daily, n=8), high‐dose eprosartan (1200 mg once daily, n=8), or placebo (n=8).
In the placebo group, LV end‐diastolic (EDV) and end‐systolic (ESV) volumes increased after 3 months (68±7 vs 82±9 ml, P<0.004, 43±1 vs 58±7 ml, P<0.003, respectively), and EF decreased (37±1 vs 29±1%, P<0.001). In dogs treated with low‐dose eprosartan, EF, EDV, and ESV remained unchanged over the course of therapy, whereas in dogs treated with high‐dose eprosartan, EF increased (38±1 vs 42±1%, P<0.004) and ESV decreased (41±1 vs 37±1 ml, P<0.006), Eprosartan also decreased interstitial fibrosis and cardiomyocyte hypertrophy.
We conclude that eprosartan prevents progressive LV dysfunction and attenuates progressive LV remodelling in dogs with moderate HF and may be useful in treating patients with chronic HF.

British Journal of Pharmacology (2003) 138, 301–309. doi:10.1038/sj.bjp.0705032
Functional characterization of α1‐adrenoceptor subtypes in vascular tissues using different experimental approaches:a comparative study
Authors: Gisbert Regina , Madrero Yolanda , Sabino Valentina , Noguera M Antonia , Ivorra M Dolores , D'Ocon Pilar 
Published: February 2003

The α1‐adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5‐methylurapidil (α1A selective) and BMY 7378 (α1D selective) have been used in different experimental procedures.
Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC50‐contraction=7.32±0.04; pEC50‐IPs=6.03±0.08) than in the tail artery (pEC50‐contraction=5.71±0.07; pEC50‐IPs=5.51±0.10). Although the maximum contraction was similar in both tissues (Emax‐tail=619.1±55.6 mg; Emax‐aorta‐698.2±40.8 mg), there were marked differences in the ability of these tissues to generate intracellular second messengers the tail artery being more efficient (Emax‐tail=1060±147%; Emax‐aorta=108.1±16.9%).
Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline‐induced maximal response in Ca2+‐containing or Ca2+‐free medium have been performed. A comparative analysis of the different procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed.
The CICa was the simplest method to characterize the predominant α1‐adrenoceptor subtype involved in the functional response of a tissue.
In aorta, where constitutively active α1D‐adrenoeptors are present, the use of different experimental procedures evidenced a complex equilibrium between α1D‐ and α1A‐adrenoceptor subtypes.
The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the different experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

British Journal of Pharmacology (2003) 138, 359–368. doi:10.1038/sj.bjp.0705033
Characterization of opioid receptors that modulate nociceptive neurotransmission in the trigeminocervical complex
Authors: Storer R J , Akerman S , Goadsby P J 
Published: February 2003

Opioid agonists have been used for many years to treat all forms of headache, including migraine. We sought to characterize opioid receptors involved in craniovascular nociceptive pathways by in vivo microiontophoresis of μ‐receptor agonists and antagonists onto neurons in the trigeminocervical complex of the cat.
Cats were anaesthetized with α‐chloralose 60 mg kg−1, i.p. and 20 mg kg−1, i.v. supplements after induction and surgical preparation using halothane. Units were identified in the trigeminocervical complex responding to supramaximal electrical stimulation of the superior sagittal sinus, and extracellular recordings of activity made.
Seven‐ or nine‐barrelled glass micropipettes incorporating tungsten recording electrodes in their centre barrels were used for microiontophoresis of test substances onto cell bodies.
Superior sagittal sinus (SSS)‐linked cells whose firing was evoked by microiontophoretic application of L‐glutamate (n=8 cells) were reversibly inhibited by microiontophoresis of H2N‐Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol (DAMGO) (n=12), a selective μ‐receptor agonist, in a dose dependent manner, but not by control ejection of sodium or chloride ions from a barrel containing saline.
The inhibition by DAMGO of SSS‐linked neurons activated with L‐glutamate could be antagonized by microiontophoresis of selective μ‐receptor antagonists D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) or D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP), or both, in all cells tested (n=4 and 6, respectively).
Local iontophoresis of DAMGO during stimulation of the superior sagittal sinus resulted in a reduction in SSS‐evoked activity. This effect was substantially reversed 10 min after cessation of iontophoresis. The effect of DAMGO was markedly inhibited by co‐iontophoresis of CTAP.
Thus, we found that μ‐receptors modulate nociceptive input to the trigeminocervical complex. Characterizing the sub‐types of opioid receptors that influence trigeminovascular nociceptive transmission is an important component to understanding the pharmacology of this synapse, which is pivotal in primary neurovascular headache.

British Journal of Pharmacology (2003) 138, 317–324. doi:10.1038/sj.bjp.0705034