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The temperature dependence of the in-plane magnetic penetration depth, lambda(ab)(T), has been measured in a c-axis oriented polycrystalline CaC(6) bulk sample using a high-resolution mutual inductance technique. A clear exponential behavior of lambda(ab)(T) has been observed at low temperatures, strongly suggesting isotropic s-wave pairing. Data fit using the standard BCS theory yields lambda(ab)(0) = (720 +/- 80) A and delta(0) = (1.79 +/- 0.08) meV. The ratio 2delta(0)/k(B)T(c) = (3.6 +/- 0.2) gives indication for a weakly coupled superconductor.

The temperature dependence of the in-plane magnetic penetration depth, $\lambda_{ab}(T)$, has been measured in a c-axis oriented polycrystalline CaC$_{6}$ bulk sample using a high-resolution mutual inductance technique. A clear exponential behavior of $\lambda_{ab}(T)$ has been observed at low temperatures, strongly suggesting isotropic s-wave pairing. Data fit using the standard BCS theory yields $\lambda_{ab}(0)=(720\pm 80)$ Angstroem and $\Delta(0)=(1.79\pm 0.08)$ meV. The ratio $2\Delta(0)/k_{_B}T_{c}=(3.6\pm 0.2)$ gives indication for a conventional weakly coupled superconductor., Comment: To appear in Phys. Rev. Lett

In this study we describe the changes in medical practices and nosocomial infection rates in obstetrics observed through a surveillance network in the South East of France.The maternity units which belong to this network participated in voluntary surveillance using the network's methodology. The criteria for the diagnosis of nosocomial infections were in accordance with the methods described by the Centers for Disease Control and Prevention.101240 pregnancies including 18503 caesareans (18.3%) were included in the network from 1997 to 2000. During the study period, nosocomial infection rates following caesarean section and vaginal delivery decreased respectively from 7.8% to 4.3% (p0.001) and from 2.2% to 0.9% (p0.001). This evolution was compared with the evolution of collected risk factors.In spite of the methodology limits, general value of surveillance and infection control programs in maternity units has been confirmed by the results of this surveillance network. During the study period, both obstetrics-related risk factors for nosocomial infection and observed hospital-acquired infection rates were dramatically reduced, what prove an improvement of quality of care in maternity units.

Background Hereditary neuropathy with liability to pressure palsy (HNPP) first described in 1947, has been showed to be due to a 1.5Mb deletion, which includes the peripheral myelin protein 22 (PMP22) gene, on chromosome 17p11.2. HNPP is more common than previously thought. Objective We describe the clinical and molecular features in a three generation family where the index case became acutely disabled following surgery for cervical spondylosis. Method A total of 14 (including the index case) were examined. Eleven had clinical evidence of disease. The disability of this group ranged from asymptomatic (1), mild (4), moderate (4), severe (1) to death (1). The findings on examination ranged from a single nerve involvement to a confluent mononeuropathy multiplex. The patient who died had marked proximal and distal weakness. The immediate cause of death is unknown. On history two individuals (now deceased) were said to be affected. A further 2 subjects who were not available for examination but who provided blood for molecular analysis were said to be normal. A PCR based strategy for the determination of the PMP22 gene dose was undertaken in 15 subjects. Results Four of these individuals (2 on history and 2 on examination) who were clinically normal had no deletions. All the clinically affected individuals all exhibited the appropriate deletion. One was clinically normal but carried the deletion. Sample from a 16th individual, who was not examined, was insufficient. Conclusion This study is the first report of the existence of HNPP in South Africa. Cases are probably being missed. The correct diagnosis is important, as with appropriate measures and patient education, disability can be significantly reduced. Resume Introduction Decrite en 1947, la neuropathie hereditaire avec hypersensibilite a la pression (NHHP) est une neuropathie hereditaire sensitivomotrice a transmission autosomique dominante. Cette affection est liee a un defaut de synthese d\'une proteine de la myeline : la proteine PMP22 (peripheral myelin protein 22) en rapport dans pres de 90% des cas a une deletion de 1,5 megabases dans la region 17p11.2 incluant le gene PMP22. L\'HNPP parait plus frequent qu\'on ne le pensait. Objectif Nous decrivons les aspects cliniques et moleculaires d\'une famille sud africaine d\'origine indienne, sur trois generations, decouverte lors d\'une decompensation aigue suite a une intervention pour une myelopathie cervicarthrosique. Materiel et methodes 14 cas ont ete etudies. 11 patients avaient des signes cliniques evidents. Le groupe a ete classe selon les aspects suivants : asymptomatique, discret, modere, severe et deces. Les constatations etaient en rapport avec une atteinte tronculaire nerveuse, unique ou multiple. . Une enquete familiale a permis de realiser des examens cliniques et biologiques . Resultats Le PCR a ete pratique chez 15 patients. Quatre des patients asymptomatiques n\'avaient pas de deletion. Tous les patients symptomatiques avaient une deletion. Un patient asymptomatique etait porteur d\'une deletion.. Le patient decede de cause inconnue presentait une faiblesse proximale et distale tres marquee Conclusion Il s\'agit du premier cas rapporte en Republique Sud-Africaine sans que cela ne prejuge du nombre de cas qui est vraisemblablement sous-estime. Le diagnostic de HPPN ne doit pas etre omis compte tenu des eventuelles consequences fonctionnelles. (Af. J. of Neurological Sciences: 2003 22(1))

Although the emotional and motivational characteristics of dreaming have figured prominently in folk and psychoanalytic conceptions of dream production, emotions have rarely been systematically studied, and motivation, never. Because emotions during sleep lack the somatic components of waking emotions, and they change as the sleeper awakens, their properties are difficult to assess. Recent evidence of limbic system activation during REM sleep suggests a basis in brain architecture for the interaction of motivational and cognitive properties in dreaming. Motivational and emotional content in REM and NREM laboratory mentation reports from 25 participants were compared. Motivational and emotional content was significantly greater in REM than NREM sleep, even after controlling for the greater word count of REM reports.

The Australian grain industry relies heavily on phosphine to meet domestic and international market demand for high-quality grain, free of insects. Phosphine usage has increased markedly over the past 10 years, because of market reluctance to accept chemical residues and resistance in target pests to grain protectants. The threat that insects may also develop resistance to phosphine led to resistance-monitoring projects being initiated across all cereal-growing regions of Australia. The rationale was that the industry needed to be proactive in developing strategies to combat resistance when it evolved, and required early warning of the development of resistance and a scientific assessment of its likely impact. With industry support, these projects have now amalgamated to form a national phosphine resistance monitoring and management programme. Insect population samples are collected from farms, grain merchants, mills and central storages, and tested for resistance. If the resistance is classified as significant, then action is taken to eradicate or control the strain and, where feasible, to prevent its further distribution. In addition, research is undertaken to fully characterize the resistance and to develop control options such as changes to fumigation concentrations and exposure periods. Although the three collaborating laboratories are widely spaced geographically, they maintain close links through data sharing on an Internet-accessible database. They share a common procedures manual, and the groups independently confirm diagnoses of significant resistance made in other laboratories. They also hold regular national workshops to benchmark their suite of bioassays and other techniques and report at least annually to the industry through various forums. The Australian approach is unique in that it has drawn together primary producers, bulk handlers, chemical companies, industry funding organizations and government research institutions from across the country to combat the national threat of phosphine resistance.

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.

As has been shown for many kinds of organisms, barriers to interspecific hybridization may differ in strength between reciprocal crosses, resulting in a bias in the probability that one or the other species may be the maternal or paternal parent of hybrids. The fern Dryopteris Xtriploidea, the "backcross" hybrid between the diploid D. intermedia and the tetraploid D. carthusiana, occurs in large numbers in nature, providing an opportunity to investigate whether such a bias exists. Differences in the chloroplast genome distinguishing the two parental species were discovered in the sequence of the trnL region following amplification by polymerase chain reaction (PCR), including a Mse I restriction site. This allowed rapid identification of the donor of the chloroplast genome, and therefore the maternal parent of each hybrid, assuming chloroplast DNA to be maternally inherited in Dryopteris. Analysis was carried out on 127 hybrids, shown to be of independent origin using allozymes, occurring at three localities in Virginia and West Virginia. When samples from all localities were pooled, 91 possessed the D. carthusiana trnL genotype and 36 possessed the D. intermedia genotype, a ratio that is significantly different (P0.001) from the null hypothesis of no gender bias. The strength of the bias differed significantly among the three sites, however, with bias at the West Virginia site much stronger (5.6:1 carthusiana:intermedia; P0.001) than at either Virginia site (1.55:1 and 1.43:1 carthusiana:intermedia, respectively; P0.05 in both cases). The cause of the strong bias in the West Virginia sample is unknown, as is the cause of the population differences. Causes of bias could include differences between the parental species related to their ploidy difference, including sizes of gametes and/or gametangia, sperm motility, breeding system (D. intermedia is outcrossing while D. carthusiana is selfing), or the nature and strength of interspecific isolating mechanisms.

The question is addressed as to whether observed parameter(s) characterizing single bubble burst (bubble jet height and speed) can be used to predict cell damage in sparged animal cell cultures. Bubble burst profiles are examined in the presence of realistic concentrations of fetal calf serum (FCS) or Pluronic F-68 using a high-speed video technique. The damage to TBC3 hybridoma cells from bubble sparging, characterized as a first-order decline, is reduced by even very small concentrations of both FCS and Pluronic F-68, but neither single bubble burst parameters nor surface properties give usable correlations with death rate constants.copy 1999 John WileySons, Inc.

Recent studies have suggested that Bcl-2 can affect cell cycle re-entry by inhibiting the transition from G0/G1 to S phase. In this study, we have taken a novel route to the study of the relationship between Bcl-2 expression and cell cycle progression. Continuous cultures of pEF (control) and Bcl-2 transfected murine hybridoma cells were operated at a range of dilution rates from 0.8 day-1 down to 0.2 day-1. The specific growth rate of the pEF cell line was the same as the dilution rate down to a value of 0.6 day-1. However, as the dilution rate was reduced stepwise to 0.2 day-1, the growth rate levelled-off at approximately 0.55 day-1 and this coincided with a fall in culture viability. By contrast, the specific growth rate of the Bcl-2 transfected cell line followed the dilution rate down to a value of 0.3 day-1 with high levels of cell survival. At high dilution rates, the cell cycle distributions were very similar for both cell lines. However, the distributions diverged as the dilution rate was reduced and, at a rate of 0.2 day-1, the percentage of G1 cells in the Bcl-2 culture was 80%, compared to only 56% in the pEF cell population. This corresponded with a greater extension in the duration of the G1 phase in the Bcl-2 cells, which was 1.7 days at the lowest dilution rate tested, compared to only 0.6 day for the pEF cell line. The durations of the G2/M and S phases remained constant throughout the culture. The maximum doubling time was 1.2 days in the pEF culture compared to 2.3 days in the Bcl-2 culture. Analysis of amino acids, ammonia and lactate concentrations indicated that the observed effects on cell cycle dynamics were probably not due to differences in the culture environment. It is suggested that the expression of Bcl-2 can effect G1 to S phase transition in continuously cycling cells, but this is only apparent at suboptimal growth rates.

To investigate early onset pneumonia in a neurosurgical intensive care unit, we studied a cohort of patients over a 13-month period and compared neurotrauma (T) with non-neurotrauma (NT) patients. Data were abstracted from the infection surveillance database. Five hundred and sixty-five adults were hospitalized in the neurosurgical intensive care unit. 57.9% had trauma and 129 patients developed 152 episodes of pneumonia. Incidence rates, restricted to the 129 first episodes of pneumonia, were 20.1 versus 15.7/1000 patient days and 34.2 versus 27.9/1000 ventilation days, in the T and NT groups respectively. In both groups, the distribution of risk stratified by hospital days was bimodal, being highest during the first three days. However, the risk was higher for T patients (at day 3, 20/1000 ventilation days versus 10.2/1000 ventilation days). The daily risk peaked again at days 5 and 6, and thereafter remained low. Pneumonia occurring within the first three days, or early onset pneumonia (EOP), was associated with trauma (P = 0.036) and, in the NT group only, with a Glasgow coma scale score lower than 9 (P = 0.062). EOP was caused by Staphylococcus aureus (33%), Haemophilus spp. (23%), other Gram-positive cocci (22%), and other Gram-negative bacilli (GNB) (19%); whereas after the third day GNB other than Haemophilus spp. accounted for 45.4% of isolates (P = 0.11). This large series confirms the high incidence of EOP in neurosurgical intensive care units, particularly among trauma patients, in relation to risk factors different from those seen in other intensive care patients. Further studies are needed to elaborate specific preventive measures during early care.

It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by inhibiting their apoptotic response can give improved process performance in terms of cell number and viability in intensive cell culture. Two cell perfusion systems, one using a spin filter and the other an ultrasonic filter, are compared using two cell lines. One cell line is transfected with the bcl-2 gene (TB:C3 bcl-2) which encodes the ‘anti-apoptotic’ human bcl-2 protein and the other cell line (TB:C3 pEF) with a negative transfection vector. Both reactor systems give similar retention performance for both cell lines. Bcl-2 transfected cells reach higher cell densities than the control cell line, and the percentage of apoptotic cells is clearly lower than with pEF cells. The maximum cell numbers of the bcl-2 cell line are 1.2110 7 ml 1 in the ultrasonic filter culture and 1.58 10 7 ml 1 in the spin filter culture, respectively. Using the pEF cell line the maximum cell number reaches 6.0 10 6 ml 1 with ultrasonic retention and 5.9 10 6 ml 1 in the spin filter. The use of ultrasound in this cell retention system has no apparent influence on cell growth, productivity or viability. Selective retention of viable cells is detectable but the effect of removing non-viable cells is negligible. © 1998 Elsevier Science B.V. All rights reserved.

Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John WileySons, Inc.

The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m−3 there was no apparent damage. At 320 W m−3 cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m−3 in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate. © 1995 John Wiley & Sons, Inc.

Insect cell growth can be significantly improved by close attention to the conditions used in the inoculum stages. Initial cell concentration, spent medium carry over and inoculum phase withdrawal significantly influenced the growth kinetics of Spodoptera frugiperda (Sf9) cells. The percentage of cells infected with wild and recombinant baculovirus AcNPV and (in the later case) the beta-galactosidase yield in fresh medium was appreciably affected by the stage of the growth curve that cells were in when infected and by the multiplicity of infection (MOI). However, the cell density at the time of infection and the medium condition showed little direct influence on infectivity. There may, however, be an indirect influence in that these factors determine the relative distribution of cells in the cell cycle. The infectivity is then in turn affected by the relative frequency of cells in the G1, S and G2/M phases. Insect cell specific oxygen uptake rates (1.3-3.4 x 10(-17) mol per cell per s) were essentially similar to or less than those measured for hybridoma cells. However, when Sf9 cells were infected with baculovirus, the specific oxygen uptake rate increased by up to 40%.

The effects of initial serum concentration, pH and stirrer speed on cell aggregation in CHO suspension cell culture were investigated. The results showed that cell aggregation was appreciably affected by the stage of the growth curve and cell density. The number of clumps increased during exponential and stationary phases. Increased serum concentration resulted in the formation of larger aggregate. However initial pH over a range of 6.8–7.6 showed no influence on either maxiumum cell number or cell aggregation. The effect of agitation on cell aggregation could not be distinguished from its effects on cell growth.

The death of cells undergoing apoptosis is characterised by chromatin cleavage at the internucleosomal sites which results in extensive fragmentation of DNA. Several methods are used to assay apoptosis but their main disadvantage is their inability to discriminate and quantify viable, apoptotic and necrotic cells in a heterogenous population. In this work we have demonstrated the usefulness of flow cytometry to measure several parameters in cultured hybridoma cells undergoing apoptosis induced by either camptothecin (a topoisomerase inhibitor) treatment or following glutamine limitation. Overall, the results indicated that flow cytometry can be applied to study the induction and kinetics of apoptosis. They also suggested that the detailed morphology and biochemical characteristics of cells undergoing apoptosis vary not only between cell lines, but also between different triggers of apoptosis.

Cell lines derived from heamopoetic lineages are widely used as hosts for the production of biologicals. It has recently been demonstrated that these cell lines undergo high levels of the active death programme commonly referred to as apoptosis. The effect of over-expression of the apoptosis supressor gene Bcl-2 on the properties of a human transformed B-cell line (61E) under various conditions was compared to the control cell line (SV2g). In stationary batch cultures, there was a slight increase in the maximum cell density which was most marked at the sub-optimal FCS level of 5%. More importantly, there was a clear increase in the duration of the culture. When cells were cultured in agitated suspension culture without prior adaptation, the 61E cells grew significantly better suggesting that the Bcl-2 gene protected the cells from death by apoptosis induced by the hydrodynamic environment. The induction of apoptosis following growth arrest is a major barrier to the development of culture strategies that optimise specific productivity by reducing the growth rate. Results presented here suggest that suppression of apotosis by enhanced Bcl-2 expression under these conditions allows the maintenance of cell culture in a growth-arrested state for much longer than would otherwise be possible.

A novel wedged AFO footwear combination (NAFOFC) to improve gait stability was assessed in 10 DMD boys. Primary outcome measure was gait analysis. At visit 1 the boys were cast for the NAFOFC, assessments included: muscle strength, Hammersmith Assessment of Motor Ability (HAMA) and contractures, the ’Activity Scale for Kids’ (ASK); and issued an activity logger (activPal) to use daily for six weeks. At visit 2 NAFOFCs were fitted, the activPAL was re-issued for 6 weeks, and gait was videoed. At 12 weeks (visit 3), boys completed a gait analysis, the ASK and a user satisfaction questionnaire. Preliminary results have been collected for 8 boys: four were household walkers and four community walkers. Timed 10 m walks ranged from 7 to 20 s. HAMA scores ranged from 8 to 35. Boys with HAMA 5% were not able to use the NAFOFC successfully. Six out of eight boys completed the data collection: independence (ASK scores) improved for four boys. Only one boy showed improvements in activPAL data: an increase in hours/day spent standing of 6%, and stepping 2%. Five boys found the NAFOFC to be comfortable, four were happy to use them, but only three found them useful. Boys reported NAFOFC to be too heavy, cosmetically unacceptable or walking felt less stable: these boys were more affected by weakness, contractures, and walked less. For NAFOFC to be acceptable and increase participation boys must be community walkers or be independent household walkers who are struggling to maintain their level of mobility. NAFOFC in DMD might improve ambulation in some boys if used at the correct stage of disease progression.

The levels of necrosis and apoptosis were quantified in CHO, SF9 insect cells, and murine plasmacytoma and hybridoma cells by acridine orange staining and fluorescence microscopy. This technique enabled the visualisation of a classic morphological feature of apoptotic cells, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was also employed to show an additional characteristic of the process, the endonuclease mediated fragmentation of DNA into multiples of 180 base pairs. Necrosis was found to be the dominant mechanism of cell death in the cultures of CHO and SF9. However, significant levels of apoptosis were found in the plasmacytoma and hybridoma cell lines. Around 81% of dead hybridoma cells were apoptotic by day 5 of a batch culture. By day 4 of the plasmacytoma batch culture the level of apoptosis was approximately 60% of dead cells.

Experimental results are presented on factors influencing cell growth, wild-type and recombinant virus production and recombinant protein yield in insect cells. While the growth of Spodoptera frugiperda (Sf9) cells was mildly inhibited by intense agitation, infection with wild-type baculovirus was unaffected by such conditions. Specific oxygen uptake rate (OUR) during the growth phase was similar to or slightly lower than that of mouse hybridomas. The specific OUR increased temporarily following infection (by 57% for wild-type and by 20% for a recombinant baculovirus), but returned to its former level one day after infection. The growth phase in which cells are infected as well as the amount of virus added (MOI) influence both rate of infection and final infectivity. Increasing the MOI from 1 to 50 only increased the yield of recombinant protein by ~ 13%. A gradual shift to higher DNA content following virus infection was observed by flow cytometry (FC) and this can be used as an early indicator of infectivity. The use of FC was also demonstrated for monitoring the expression of recombinant proteins.

The relationship between growth and interferon-gamma (IFN-gamma) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. The Ks and mu max-values were found to be 0.771% (v/v) serum and 0.031 h-1 respectively. The average specific IFN-gamma production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN-gamma production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.

Flow cytometry has been used to study the mechanisms of damage to mammalian cells by hydrodynamic forces. Cell damage resulted from the stresses created both by bubble entrainment and by bubble bursting caused by vortex formation in highly agitated culture. Damage to the antigen molecules on the cell surface together with increasing leakage and passive transport were observed. Pluronic F-68 reduced the efflux of fluorescein out of cells suggesting the possibility of plugging damaged parts of the membrane or change in membrane molecular organisation. Surface-associated immunoglobulin molecules were also protected by Pluronic F-68. The loss of plasma membrane integrity was followed by the destruction of cytoplasmic matrix. The nuclei were last to be destroyed. The fraction of metabolically active cells was drastically decreased by the intense hydrodynamic forces. However the mitochondrial transmembrane potential, which is linked to the ATP requirements of cells was well as the mean cell size of the live cell population, remained unaffected by the stressful hydrodynamic conditions. F-Actin content peaked during the early exponential phase and declined during the mid and late exponential and death phases of batch culture. The increase of actin during exponential phase was faster in stationary culture than in agitated culture. The increase was associated with the proliferative rate. Furthermore, the response to hydrodynamic forces was not related either to cell size difference or to the stage in the cell cycle.

The comparative effects of Pluronic on cell growth of hybridomas were studied. The addition of 0.05% Pluronic decreased the steady-state cell number in continuous culture by about 12% compared to a non-supplemented culture. In short-term experiments, results demonstrated a gradual decrease in cell number with increasing concentration of Pluronic. Such growth inhibition was found to be a result of lowering the rate of DNA synthesis.

TB/C3 mouse hybridoma cells have been grown at 2 controlled dO2 conditions by headspace and sparged oxygenation. Also a variety of sparging rates and sparger sizes and positions have been employed. Headspace oxygenation at dO2 levels from 5% to 100% of saturation give essentially the same performance as controls. Sparging is generally damaging to cells, the extent of damage decreasing with reduced sparging rate until at below about 0.02 vvm results equivalent to the unsparged conditions are obtained. Damage is clearly linked with bubble-cell interactions at the air-medium interface where bubbles bursting in clusters and of a size less than 5 mm appear to be the most lethal. When the interaction of air sparging with the agitator flow leads to an increase in the number of smaller bubbles and cluster bursts, cell damage is further increased. Pluronic F-68 reduces damage very significantly. Biological aspects are briefly discussed in the light of various biological tests. The practical implications of this work for large scale, free suspension cell culture are outlined.

A selection of mouse hybridoma cell lines showed a variation of approximately two orders of magnitude in intracellular monoclonal antibody contents. The different levels directly influenced apparent specific monoclonal antibody productivity during the death phase but not during the growth phase of a batch culture. The pattern of changes in specific productivity during culture remained basically similar even though at different levels for all cell lines tested. Arresting the cells in the G1 phase using thymidine increased the specific productivity, cell volume and intracellular antibody content but at the same time led to decreased viability. In continuous culture DNA synthesis decreased with decreasing dilution rate though without an accompanying change in cell cycle and cell size distributions. The data shows both the decrease in viability and intracellular antibody content to be important factors which influence the negative association between specific antibody productivity and growth rate. In high cell density perfusion culture, when the cell cycle was prolonged by slow growth, viability was low and dead, but not lysed, cells were retained in the system, the specific antibody productivity was nearly two fold higher than that obtained in either batch or continuous cultures. The results imply that the prolongation of G1 phase and the increase in death rate of cells storing a large amount of antibody together cause an apparent increase in specific antibody productivity.

Introduction Les peptides natriuretiques favorisent la lipolyse et pourraient jouer un role dans les maladies metaboliques. Nous avons etudie les associations de plusieurs polymorphismes et des haplotypes des genes NPPA et NPPB avec le diabete de type 2 et l’hyperglycemie moderee a jeun dans l’etude DESIR. Materiels et methodes 5212 sujets ont ete inclus dans cette etude de suivi. Quatre polymorphismes (tag SNPs, 1 en NPPA, rs5065 et 3 en NPPB, rs198381/ rs198389/rs198388) ont ete selectionnes sur HapMap et ont ete genotypes (Taqman). Le BNP plasmatique a ete mesuree dans un echantillon de 369 sujets (IRMA). Resultats Le genotype CC du SNP rs198381 est associe a des valeurs plus elevees de la glycemie a jeun (TT : 5.35 ± 0,84 mM, n = 3 886, TC : 5,38 ± 0,81, n = 1 180, and CC : 5, 57 ± 1,05, n = 106, p = 0,005) de l’insulinemie (TT : 47, 1 ± 31,2 pM, TC : 46,5 ± 27,8 and CC : 54,2 ± 33,5, p = 0,001), et du HOMA-IR (TT : 1,66 ± 1,30, TC : 1,65 ± 1,16 and CC : 2,05 ± 1,55, p = 0,0001). Il y a eu 566 nouveaux cas de DT2/IFG au cours des 9 ans de suivi. Les alleles rares des polymorphismes rs198381, rs198388 et rs5065 sont associes a une augmentation de la prevalence (OR = 1,31, p = 0,0004 ; OR = 1,18, p = 0,004 et OR = 1,29, p = 0,0003, respectivement) et de l’incidence (OR = 1,22, p = 0,02 ; OR = 1,18, p = 0,04 et OR = 1,20, p = 0,03, respectivement) de DT2/IFG. Six haplotypes decrivent 98 % de la variation genetique. Compares a TTGT (l’haplotype le plus frequent, 55 %), les haplotypes CTAC, TCAC et TTAC sont associes a un risque plus eleve de DT2/IFG (OR = 1,86, p = 0,00001 ; OR = 1,66, p = 0,01 et OR = 2,22, p = 0,0001, respectivement). Les alleles rares des SNPs rs5065 et rs198381 sont associes a des concentrations plus elevees de BNP (rs5065, TT : 3,1 pg/ml, TC : 3,3 et CC : 7,6, p = 0,009 ; rs198381, TT : 3,1 pg/ml, TC : 3,4 et CC : 5,2, p = 0,03). L’haplotype CCAC est associe a un taux plus eleve de BNP ( p = 0,008). Nous avons replique les associations entre ces variations genetiques et le DT2 dans deux grandes populations diabetiques de type 2 : DIABHYCAR (n = 3137) et D2NG (n = 2712). Conclusion Les variantes des genes des peptides natriuretiques sont associees au risque de DT2. Cette association pourrait s’expliquer par un effet sur les concentrations plasmatiques de BNP et l’insulino-resistance.

Cell cycle, cell size and rhodamine 123 fluorescence in cell populations of two batch cultures were analysed and quantified with a fluorescence-activated cell sorter (FACS). Two cultures derived from either exponential or stationary phase innocula were investigated in order to demonstrate the dependency of the subsequent cell growth on innoculum condition. The results demonstrated that the level of activity of cells in the innoculum culture could have a significant effect on cellular activity during the initial phase of the inoculated culture, as it advances through its growth cycle. Positive correlation was found between the cell size and mitochondrial activity (as measured by rhodamine 123 uptake) with S and G2 fractions as the cell progressed through the cell cycle. The enumeration of the fractions of cell cycle phases has helped in prediction of the changes in cell numbers following perturbation of the culture condition.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of glow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating “stationary” phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.

Epstein-Barr virus (EBV), a human herpesvirus endemic in all human populations, is the causal agent of infectious monoucleosis (IM), and is also linked to certain kinds of cancer. In vitro , EBV forms tightly latent infections of host cell lines: this lack of a simple permissive tissue culture system has made it difficult to obtain large quantities of virus for study. The B95–8 cell line, established by infecting Marmoset B lymphocytes with EBV from an IM patient, is unusual in supporting a small proportion of cells which spontaneously release virus: enhancement of this population by adding the tumour promoter TPA, followed by a labour intensive, low yield ultracentrifugation step, is the standard method of preparing virus, and B95–8 the most studied source of EBV. Here we report the combined use of Sap A, a non-tumour-promoting analogue of TPA, large-scale mammalian cell culture and contained downstream processing by crossflow filtration, in a new strategy for EBV production. It has yielded virus preparations of unprecedented size, quality and infectious titre, making possible previously uncomtemplatable experiments and facilitating production of potential EBV vaccines. KEYWORDS : Cell culture; Epstein-Barr Virus; Induction; Membrane Filtration; Vaccine

Several different metabolic events in murine hybridoma cells are affected by both nutrient depletion and waste metabolites. The incorporation of [3H] thymidine into DNA and [35S] methionine into protein and the reduction of MTT salt by active mitochondria are highly affected by the depletion of both glucose and glutamine. Serum depletion results in a 20% inhibition of thymidine incorporation; MTT reduction is also inhibited by 25%, whereas methionine incorporation is unaffected. Lactic acid and ammonia additions result in 79%, 75% and 57% inhibition of thymidine and methionine incorporation and MTT reduction respectively. KEYWORDS : Hybridoma; cell culture; DNA synthesis; protein synthesis; metabolic activity.

Dispersion and mass-transfer characteristics and fluidization parameters influencing the performance of a small pilot-plant immobilized enzyme reactor are evaluated. The suitability of a dispersed plug-flow model to predict the conversions obtained in the enzymatic reaction (starch → glucose) catalyzed by amyloglucosidase immobilized to solid and porous carriers is assessed. The performance of a fluidized-bed reactor is compared on the basis of a normalized residence time with that of a fixed bed and found to be superior.

The response of 3 murine hybridomas to increasing speeds of agitation from 100 to 450 rpm with propellers and Rushton turbines in surface-aerated bioreactors of 1.4 l has been determined with pO 2 being kept above a minimum level of 20%. Cell growth and viability, antibody production, glucose consumption, lactate production and metabolic activity have been measured and found to be unaffected over this range of speeds. Scanning electron micrographs (SEM) showed no detectable cell damage even at the highest intensity of agitation. However, once the culture was continuously sparged with air the net cell growth rate and the maximum cell number fell markedly, that fall increasing with increasing agitator speed. To generalise these findings and compare them with literature reports, the implications of this speed range on the turbulence parameters have been discussed. The implications for scale-up are also considered.

One hundred and fifteen male weanling rats of the Sprague–Dawley strain were placed on a basal ration designed to deplete their tissue reserves of vitamin E and selenium. They were then placed on a diet capable of precipitating acute hepatic necrosis within a few days. At 0, 12, 24, 36, 48, 60, 72, 78, 84, 90, 96, 114, 120, 132, 144, and 160 hours, groups of rats, including any which showed obvious signs of liver damage, were anesthetized with pentobarbital and blood samples were taken from the aorta via a syringe. Determinations of arginase activity of the plasma showed that greatly elevated values occurred only at the time of development of the acute liver damage. It is concluded that the disappearance of arginine from the blood of rats in which acute liver necrosis has developed is due to the liberation of arginase from the dead and dying liver cells thus destroying the amino acid.

Due to the nature of animal cell suspension culture, physical conditions during growth such as pH, pO2, pCO2, agitation and aeration are inter-related and can exhibit individual or combined effects on cells. Evaluation of their influence on hybridoma growth and monoclonal antibody production can only be achieved by the use of well-instrumented stirred tank reactors. To determine the effect of the various physical conditions, mouse hybridomas, excreting antibody specific for human IgG, have been cultured in a 2 litre mechanically agitated round bottomed glass vessel, equipped with facilities to monitor and control these parameters during cell growth. Batch culture has been carried out at 36 degrees C in medium supplemented with 5% foetal calf serum (FCS). Clones have also been grown successfully with 2% FCS additions and in serum free medium, after careful adaptation periods. At least two clones have been assessed for comparative purposes.

Fermentation involves a highly complex series of interactions between biochemical and physical processes [1], many of the components of which are not readily measured on-line. Therefore, the rapid and reliable estimation of derived variables is becoming increasingly important.

Sparger aeration, with or without mechanical agitation is the simplest method of providing an oxygen supply. Although earlier workers have demonstrated the sensitivity of mammalian cells to air bubbles, recent successful applications of the airlift principle to hybridoma culture indicate that under some conditions, the cells can withstand these effects. Therefore, this work has as its objective the elucidation of the relationship between gas-liquid interfaces and the survival of mammalian cells. Simple, 0.5 litre bubble columns with sintered discs are used batch-wise to study the effects of sparging on hybridomas and other mammalian cells in suspension culture. The effects of bubble diameters, superficial gas velocities and the non-ionic surfactant, Pluriol PE 6800, are investigated in cultures grown in RPM1 1640 with 5% foetal calf serum and 6 ppm silicone antifoam. From these studies it has become apparent that cell viability and survival in the presence of bubbles depend on: Cell type--Some cell lines are shown to be particularly sensitive to the presence of bubbles, although no gross morphological differences are detected by electron microscopy. Bubble sizes--At a superficial gas velocity of 0.42 X 10(-4)m/s (5 cc/min gas flow rate), small bubbles are shown to be more detrimental to the cells than the larger ones. Bubble frequency/superficial gas velocities--Increasing superficial gas velocities (0.42 X 10(-4) to 8.5 X 10(-4) m/s) result in decreasing cell viability. Where the presence of bubbles is detrimental to cell growth, the addition of the non-ionic surfactant, Pluriol PE 6800, has a concentration dependent protective effect. Surface tension, viscosity and bubble diameter data for typical medium will be presented. A novel application of microscopy to which video film systems can be applied for direct visualisation of the cells in the bubble column has been developed. From these studies, it is indicative that both the geometry of the system and the cell type are important in mammalian cell culture scale-up strategy.