Antinuclear autoantibodies are more prevalent in COPD in association with low body mass index but not with smoking history.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with a higher prevalence of antinuclear autoantibodies (ANAs). However, a significant subgroup of patients is ANA negative. It remains to be determined which patient groups carry autoantibodies. METHODS: The association of smoking behaviour, disease status, gender, age and body mass index (BMI) with the presence of autoantibodies in the serum was determined in 124 patients with COPD and 108 non-COPD control subjects. In addition, the role of B cells in autoantibody generation in COPD was investigated by sequencing the antibody repertoire of B cells in the lungs of patients with COPD and of ex-smoking and never-smoking control subjects. RESULTS: Patients with COPD had a significantly higher risk of being serum positive for ANAs (OR 3.12, 95% CI 1.68 to 5.76, p<0.001). ANAs were not significantly associated with age, smoking status, gender or pack-years of smoking. Within the COPD population, subjects with BMI <22 kg/m2 had a significantly higher risk of ANAs (OR 4.93, 95% CI 1.50 to 16.50, p=0.009) than those with normal or high BMI. The antibody repertoire of B cells in the lungs of patients with COPD had a high frequency of positively charged CDR3 residues, a feature which is associated with self-reactive antibodies. CONCLUSION: The results show that COPD is a heterogeneous disease with respect to the prevalence of ANAs. ANAs are primarily associated with the presence of COPD and with low BMI, but not with smoking and forced expiratory volume in 1 s.

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing.

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors.

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.

Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances.

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO(2)) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD(+)). Oxidative stress, as a resultof which the NAP(P)(+)/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO(2) to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO(2) levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.

Error analysis of metabolic-rate measurements in mammalian-cell culture by carbon and nitrogen balances.

The analysis of metabolic fluxes of large stoichiometric systems is sensitive to measurement errors in metabolic uptake and production rates. It is therefore desirable to independently test the consistency of measurement data, which is possible if at least two elemental balances can be closed. For mammalian-cell culture, closing the C balance has been hampered by problems in measuring the carbon-dioxide production rate. Here, it is shown for various sets of measurement data that the C balance can be closed by applying a method to correct for the bicarbonate buffer in the culture medium. The measurement data are subsequently subject to measurement-error analysis on the basis of the C and N balances. It is shown at 90% reliability that no gross measurement errors are present, neither in the measured production- and consumption rates, nor in the estimated in- and outgoing metabolic rates of te subnetwork, that contains the glycolysis, the pentose-phosphate, and the glutaminolysis pathways.

Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells.

The effect of added ammonia on the intracellular fluxes in hybridoma cells was investigated by metabolic-flux balancing techniques. It was found that, in ammonia-stressed hybridoma cells, the glutamate-dehydrogenase flux is in the reverse direction compared to control cells. This demonstrates that hybridoma cells are able to prevent the accumulation of ammonia by converting ammonia and alpha-ketoglutarate into glutamate. The additional glutamate that is produced by this flux, as compared to the control culture, is converted by the reactions catalyzed by alanine aminotransferase (45% of the extra glutamate) and aspartate aminotransferase (37%), and a small amount is used for the biosynthesis of proline (6%). The remaining 12% of the extra glutamate is secreted into the culture medium. The data suggest that glutamate dehydrogenase is a potential target for metabolic engineering to prevent ammonia accumulation in high-cell-density culture.

Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells.

The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. In order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints: (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using 13CO2 and 13C-lactate measurements of 1 - 13C-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions "maximize ATP" and "maximize NADH" are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas, are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors.

Metabolic flux analysis of hybridoma cells in different culture media using mass balances.

The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O(2), CO(2), NH(4), MAB, and the intracellular amino acid pools have been determined for two different steady-states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO(2) or the NAD(P)H mass balance into account is shown to be in agreement with the measured O(2) and CO(2) metabolic rates.The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day(-1) are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that (i)The majority of the consumed glucose (>90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO(2).(iii)The flux from glutamate to alpha-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 x 10(-12) to 13 x 10(-12) mol . cell(-1) . day(-1)) compared to other fluxes in both culture media.

Determination of the respiration quotient in mammalian cell culture in bicarbonate buffered media.

The determination of the respiration quotient (RQ = CER/OUR) has not been used so far as a tool for understanding animal cell metabolism. This is due to problems in measuring the carbon dioxide evolution rate (CER) rather than the oxygen uptake rate (OUR). The determination of the CER is complicated by the use of bicarbonate in the medium. Using liquid and gas balances we have derived an equation for continuous culture to quantify the amount of CO(2) that comes from the bicarbonate in the feed. Under cell-free conditions, values predicted by this equation agree within 4% with the experimental results. In continuous culture using hybridoma cells, the CO(2) from the feed, as determined by an IR-gas analyzer, was found to represent a significant amount of the total measured CO(2) in the off-gas (50% in a suboptimal, and 30% in high-growth medium). Furthermore, the problem of CO(2) loss from the medium during medium preparation and storage was solved using both a theoretical and an experimental approach. RQ values in continuous culture were evaluated for two different growth media. Small but significant differences in RQ were measured, which were matched by differences in specific antibody rates and other metabolic quotients. In a medium with Primatone RL, an enzymatic hydrolysate of animal cell tissue that causes a more than twofold increase in cell density, the RQ was found to be 1.05, whereas in medium without Primatone RL (but containing amino acids equivalent in composition and concentration to Primatone RL) the RQ was found to be 0.97. We suggest the RQ to be a useful parameter for estimating the physiological state of cells. Its determination could be a suitable tool for both the on-line control of animal cell cultivations and the understanding of cell metabolism.