A thin‐walled polydimethylsiloxane bioreactor for high‐density hepatocyte sandwich culture

In vitro drug testing requires long‐term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24‐well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long‐term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days. Biotechnol. Bioeng. 2013; 110: 1663–1673. © 2012 Wiley Periodicals, Inc.     

Structure-based design of novel dihydroisoquinoline BACE-1 inhibitors that do not engage the catalytic aspartates

The structure–activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.     

Promoters maintain their relative activity levels under different growth conditions

Most genes change expression levels across conditions, but it is unclear which of these changes represents specific regulation and what determines their quantitative degree. Here, we accurately measured activities of ～900 S. cerevisiae and ～1800 E. coli promoters using fluorescent reporters. We show that in both organisms 60–90% of promoters change their expression between conditions by a constant global scaling factor that depends only on the conditions and not on the promoter's identity. Quantifying such global effects allows precise characterization of specific regulation—promoters deviating from the global scale line. These are organized into few functionally related groups that also adhere to scale lines and preserve their relative activities across conditions. Thus, only several scaling factors suffice to accurately describe genome‐wide expression profiles across conditions. We present a parameter‐free passive resource allocation model that quantitatively accounts for the global scaling factors. It suggests that many changes in expression across conditions result from global effects and not specific regulation, and provides means for quantitative interpretation of expression profiles.     

Deficiency of C5L2 Increases Macrophage Infiltration and Alters Adipose Tissue Function in Mice

Background

Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT) of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP), and C5a, involved in innate immunity.

Aim

We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl) and C5L2 knock-out (C5L2^−/−) mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO) conditions.

Results

In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold) both over time and with DIO. By contrast, in C5L2^−/−, there was no change in C5aR in WAT. C5L2^−/− mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro-) vs M2 (anti-inflammatory) macrophage proportion was unchanged but C5L2^−/− adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2^−/− mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2^−/− mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance.

Conclusion

Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.     

Short-Term Effect of Pollen and Spore Exposure on Allergy Morbidity in the Brussels-Capital Region

Belgium is among the European countries that are the most affected by allergic rhinitis. Pollen grains and fungal spores represent important triggers of symptoms. However, few studies have investigated their real link with disease morbidity over several years. Based on aeroallergen counts and health insurance datasets, the relationship between daily changes in pollen, fungal spore concentrations and daily changes in reimbursable systemic antihistamine sales has been investigated between 2005 and 2011 in the Brussels-Capital Region. A Generalized Linear Model was used and adjusted for air pollution, meteorological conditions, flu, seasonal component and day of the week. We observed an augmentation in drug sales despite no significant increase in allergen levels in the long term. The relative risk of buying allergy medications associated with an interquartile augmentation in pollen distributions increased significantly for Poaceae, Betula, Carpinus, Fraxinus and Quercus. Poaceae affected the widest age group and led to the highest increase of risk which reached 1.13 (95% CI [1.11–1.14]) among the 19- to 39-year-old men. Betula showed the second most consistent relationship across age groups. Clear identification of the provoking agents may improve disease management by customizing prevention programmes. This work also opens several research perspectives related to impact of climate modification or subpopulation sensitivity.     

Inhibitory Effect of Nasal Intermittent Positive Pressure Ventilation on Gastroesophageal Reflux

Non-invasive intermittent positive pressure ventilation can lead to esophageal insufflations and in turn to gastric distension. The fact that the latter induces transient relaxation of the lower esophageal sphincter implies that it may increase gastroesophageal refluxes. We previously reported that nasal Pressure Support Ventilation (nPSV), contrary to nasal Neurally-Adjusted Ventilatory Assist (nNAVA), triggers active inspiratory laryngeal closure. This suggests that esophageal insufflations are more frequent in nPSV than in nNAVA. The objectives of the present study were to test the hypotheses that: i) gastroesophageal refluxes are increased during nPSV compared to both control condition and nNAVA; ii) esophageal insufflations occur more frequently during nPSV than nNAVA. Polysomnographic recordings and esophageal multichannel intraluminal impedance pHmetry were performed in nine chronically instrumented newborn lambs to study gastroesophageal refluxes, esophageal insufflations, states of alertness, laryngeal closure and respiration. Recordings were repeated without sedation in control condition, nPSV (15/4 cmH₂O) and nNAVA (~ 15/4 cmH₂O). The number of gastroesophageal refluxes recorded over six hours, expressed as median (interquartile range), decreased during both nPSV (1 (0, 3)) and nNAVA [1 (0, 3)] compared to control condition (5 (3, 10)), (p < 0.05). Meanwhile, the esophageal insufflation index did not differ between nPSV (40 (11, 61) h^-1) and nNAVA (10 (9, 56) h^-1) (p = 0.8). In conclusion, nPSV and nNAVA similarly inhibit gastroesophageal refluxes in healthy newborn lambs at pressures that do not lead to gastric distension. In addition, the occurrence of esophageal insufflations is not significantly different between nPSV and nNAVA. The strong inhibitory effect of nIPPV on gastroesophageal refluxes appears identical to that reported with nasal continuous positive airway pressure.     

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy

Background

The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions.

Methodology/Principal Findings

Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8⁺ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8⁺ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings.

Conclusions/Significance

Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8⁺ T cell-mediated cytotoxicity.     

GbEXPATR,a species‐specific expansin,enhances cotton fibre elongation through cell wall restructuring

Cotton provides us the most important natural fibre. High fibre quality is the major goal of cotton breeding, and introducing genes conferring longer, finer and stronger fibre from Gossypium barbadense to Gossypium hirsutum is an important breeding strategy. We previously analysed the G. barbadense fibre development mechanism by gene expression profiling and found two homoeologous fibre‐specific α‐expansins from G. barbadense, GbEXPA2 and GbEXPATR. GbEXPA2 (from the D_T genome) is a classical α‐expansin, while its homoeolog, GbEXPATR (A_T genome), encodes a truncated protein lacking the normal C‐terminal polysaccharide‐binding domain of other α‐expansins and is specifically expressed in G. barbadense. Silencing EXPA in G. hirsutum induced shorter fibres with thicker cell walls. GbEXPA2 overexpression in G. hirsutum had no effect on mature fibre length, but produced fibres with a slightly thicker wall and increased crystalline cellulose content. Interestingly, GbEXPATR overexpression resulted in longer, finer and stronger fibres coupled with significantly thinner cell walls. The longer and thinner fibre was associated with lower expression of a number of secondary wall‐associated genes, especially chitinase‐like genes, and walls with lower cellulose levels but higher noncellulosic polysaccharides which advocated that a delay in the transition to secondary wall synthesis might be responsible for better fibre. In conclusion, we propose that α‐expansins play a critical role in fibre development by loosening the cell wall; furthermore, a truncated form, GbEXPATR, has a more dramatic effect through reorganizing secondary wall synthesis and metabolism and should be a candidate gene for developing G. hirsutum cultivars with superior fibre quality.