MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

Transforming growth factor beta (TGF-beta) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-beta production in response to Leishmania stimulation in healthy subjects. In this study, TGF-beta1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-beta1-producing cells were described as a distinct subpopulation of CD4(+) CD25(+) regulatory T cells. The suppressive properties of CD4(+) CD25(+) T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4(+) CD25(-) T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-beta1 did not reverse the suppressive activity of CD4(+) CD25(+) T cells activated by anti-CD3, it reversed the suppressive activity of CD4(+) CD25(+) T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-beta1 is involved in the suppressive activity of L. guyanensis-stimulated CD4(+) CD25(+) T cells from healthy controls.

Dexamethasone therapy and endogenous cortisol production in severe pediatric head injury.

A prospective randomised study was performed on 25 children aged 1.4 to 15.8 years with severe head injury (Glasgow Coma Scale less than or equal to 7) to determine the clinical effectiveness and the impact on endogenous cortisol production of high-dose steroid therapy. Thirteen patients (group 1) received dexamethasone 1 mg/kg/day during the first 3 days and 12 (group 2) not. All patients were treated with a standardized regimen. Urinary free cortisol was measured by radioimmunoassay, and the clinical data were recorded at hourly intervals. Outcome was assessed 6 months later using the Glasgow Outcome Scale. We found a higher frequency of bacterial pneumonias in the dexamethasone-treated patients (7/13 versus 2/12). Group 1 showed a suppression of endogenous cortisol production from day 1 to day 6. In group 2, mean free cortisol was up to 5-fold higher than under basal conditions. The results in group 2 showed that the endogenous steroid production reacts adequately to the stress of severe head injury. It probably is sufficient to elicit maximum glucocorticoid effects. There was no other statistically significant difference in the clinical and laboratory data between the two groups. We conclude that dexamethasone in high doses suppresses endogenous cortisol production up to 6 days and may increase the risk of bacterial infection without affecting the outcome or the clinical and laboratory data.

NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production?

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex that activates caspase 1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18. The NLRP3 inflammasome is activated by a wide range of danger signals that derive not only from microorganisms but also from metabolic dysregulation. It is unclear how these highly varied stress signals can be detected by a single inflammasome. In this Opinion article, we review the different signalling pathways that have been proposed to engage the NLRP3 inflammasome and suggest a model in which one of the crucial elements for NLRP3 activation is the generation of reactive oxygen species (ROS).

Economics of public governance with strategic production of information: four essays

SUMMARY This paper analyses the outcomes of the EEA and bilateral agreements vote at the level of the 3025 communities of the Swiss Confederation by simultaneously modelling the vote and the participation decisions. Regressions include economic and political factors. The economic variables are the aggregated shares of people employed in the losing, Winning and neutral sectors, according to BRUNETTI, JAGGI and WEDER (1998) classification, Which follows a Ricardo-Viner logic, and the average education levels, which follows a Heckscher-Ohlin approach. The political factors are those used in the recent literature. The results are extremely precise and consistent. Most of the variables have the predicted sign and are significant at the l % level. More than 80 % of the communities' vote variance is explained by the model, substantially reducing the residuals when compared to former studies. The political variables do have the expected signs and are significant as Well. Our results underline the importance of the interaction between electoral choice and participation decisions as well as the importance of simultaneously dealing with those issues. Eventually they reveal the electorate's high level of information and rationality. ZUSAMMENFASSUNG Unser Beitrag analysiert in einem Model, welches gleichzeitig die Stimm- ("ja" oder "nein") und Partizipationsentscheidung einbezieht, den Ausgang der Abstimmungen über den Beitritt zum EWR und über die bilateralen Verträge für die 3025 Gemeinden der Schweiz. Die Regressionsgleichungen beinhalten ökonomische und politische Variabeln. Die ökonomischen Variabeln beinhalten die Anteile an sektoriellen Arbeitsplatzen, die, wie in BRUNETTI, JAGGIl.1I1d WEDER (1998), in Gewinner, Verlierer und Neutrale aufgeteilt Wurden, gemäß dem Model von Ricardo-Viner, und das durchschnittliche Ausbildungsniveau, gemäß dem Model von Heckscher-Ohlin. Die politischen Variabeln sind die in der gegenwärtigen Literatur üblichen. Unsere Resultate sind bemerkenswert präzise und kohärent. Die meisten Variabeln haben das von der Theorie vorausgesagte Vorzeichen und sind hoch signifikant (l%). Mehr als 80% der Varianz der Stimmabgabe in den Gemeinden wird durch das Modell erklärt, was, im Vergleich mit früheren Arbeiten, die unerklärten Residuen Wesentlich verkleinert. Die politischen Variabeln haben auch die erwarteten Vorzeichen und sind signifikant. Unsere Resultate unterstreichen die Bedeutung der Interaktion zwischen der Stimm- und der Partizipationsentscheidung, und die Bedeutung diese gleichzeitig zu behandeln. Letztendlich, belegen sie den hohen lnformationsgrad und die hohe Rationalität der Stimmbürger. RESUME Le présent article analyse les résultats des votations sur l'EEE et sur les accords bilatéraux au niveau des 3025 communes de la Confédération en modélisant simultanément les décisions de vote ("oui" ou "non") et de participation. Les régressions incluent des déterminants économiques et politiques. Les déterminants économiques sont les parts d'emploi sectoriels agrégées en perdants, gagnants et neutres selon la classification de BRUNETTI, JAGGI ET WEDER (1998), suivant la logique du modèle Ricardo-Viner, et les niveaux de diplômes moyens, suivant celle du modèle Heckscher-Ohlin. Les déterminants politiques suivent de près ceux utilisés dans la littérature récente. Les résultats sont remarquablement précis et cohérents. La plupart des variables ont les signes prédits par les modèles et sont significatives a 1%. Plus de 80% de la variance du vote par commune sont expliqués par le modèle, faisant substantiellement reculer la part résiduelle par rapport aux travaux précédents. Les variables politiques ont aussi les signes attendus et sont aussi significatives. Nos résultats soulignent l'importance de l'interaction entre choix électoraux et décisions de participation et l'importance de les traiter simultanément. Enfin, ils mettent en lumière les niveaux élevés d'information et de rationalité de l'électorat.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro.

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFalpha and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

Control of brood male production in the Argentine ant Iridomyrmex humilis (Mayr)

The influence of various social factors on the production of males was investigated in the Argentine ant, Iridomyrmex humilis. In this polygynous species, the workers which are monomorphic are unable to lay reproductive eggs, so all the males are the progeny of the queens. Although male eggs appear to be laid by mated queens throughout the year, in large stock colonies males are reared periodically (every 3 or 4 months); males develop from brood taken from these colonies at any point in the cycle and given queenless or queenright (1 to 5 queens) units. This is in striking contrast to many other species of ants where it is generally assumed that male eggs are laid seasonnally. Comparative experiments suggest that several related factors influence the rearing of males as far as the pupal stage. Worker/larva ratio: The proportion of male larvae developing in standardized units in which the worker/larva ratio was varied from 0.25 to 25 demonstrated that low ratios inhibit male production. Queen influence: In standardized units where the worker/larva ratio was high the presence of queens did not inhibit the rearing of males suggesting that there is no queen inhibitory pheromone controlling male experimental production. Data suggest evidence that queens prevent male production by means of appropriation of food. Diet: Male larvae failed to pupate in experimental societies deprived of protein. Thus, the production of males appears to be controlled by the amount of food available to larvae. This depends on foraging activity, the quantity of brood in relation to the number of workers and the number of queens in the society.

Male production by workers in the polygynous ant Prolasius advenus

The ability of workers to produce male individuals is reported here for the first time in a species of the formicine ant genus Prolasius. We show that Prolasius advenus workers possess ovaries and demonstrate that they are able to produce adult males in queenless colonies. We also experimentally tested the influence of queen volatiles on the level of worker reproduction. Workers produced fewer eggs in treatments where they could perceive odors from queens. Some volatile compounds emitted by queens may thus have a signaling or inhibitory effect on worker reproduction. This effect of queen presence did not entirely stop worker reproduction, however, as adult males still emerged under these conditions. Worker-produced males were absent only in treatments with the physical presence of queens. Dissections of workers collected from queenright nests in the field revealed signs of egg-laying activity in more than half of individuals. Together, these results suggest that in nature P. advenus workers produce males at least in orphaned colonies or in situations where the physical presence of queens is limited.

Chronic potassium depletion increases adrenal progesterone production that is necessary for efficient renal retention of potassium.

Modern dietary habits are characterized by high-sodium and low-potassium intakes, each of which was correlated with a higher risk for hypertension. In this study, we examined whether long-term variations in the intake of sodium and potassium induce lasting changes in the plasma concentration of circulating steroids by developing a mathematical model of steroidogenesis in mice. One finding of this model was that mice increase their plasma progesterone levels specifically in response to potassium depletion. This prediction was confirmed by measurements in both male mice and men. Further investigation showed that progesterone regulates renal potassium handling both in males and females under potassium restriction, independent of its role in reproduction. The increase in progesterone production by male mice was time dependent and correlated with decreased urinary potassium content. The progesterone-dependent ability to efficiently retain potassium was because of an RU486 (a progesterone receptor antagonist)-sensitive stimulation of the colonic hydrogen, potassium-ATPase (known as the non-gastric or hydrogen, potassium-ATPase type 2) in the kidney. Thus, in males, a specific progesterone concentration profile induced by chronic potassium restriction regulates potassium balance.