L. plantarum,L. salivarius,and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner

Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103⁺ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-γ, IL5, IL10, and IL17 production by CD4⁺ and CD8⁺ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.     

Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for approximately 45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A-->G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far.     

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.     

Chronotype and environmental light exposure in a student population

ABSTRACT

In humans and most other species, changes in the intensity and duration of light provide a critical set of signals for the synchronisation of the circadian system to the astronomical day. The timing of activity within the 24 h day defines an individual’s chronotype, i.e. morning, intermediate or evening type. The aim of this study was to investigate the associations between environmental light exposure, due to geographical location, on the chronotype of university students. Over 6 000 university students from cities in the Northern Hemisphere (Oxford, Munich and Groningen) and Southern Hemisphere (Perth, Melbourne and Auckland) completed the Munich ChronoType Questionnaire. In parallel, light measures (daily irradiance, timing of sunrise and sunset) were compiled from satellite or ground stations at each of these locations. Our data shows that later mid-sleep point on free days (corrected for oversleep on weekends MFS_sc) is associated with (i) residing further from the equator, (ii) a later sunset, (iii) spending more time outside and (iv) waking from sleep significantly after sunrise. However, surprisingly, MSF_sc did not correlate with daily light intensity at the different geographical locations. Although these findings appear to contradict earlier studies suggesting that in the wider population increased light exposure is associated with an earlier chronotype, our findings are derived exclusively from a student population aged between 17 and 26 years. We therefore suggest that the age and occupation of our population increase the likelihood that these individuals will experience relatively little light exposure in the morning whilst encountering more light exposure later in the day, when light has a delaying effect upon the circadian system.     

Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of Azorhizobium caulinodans

Both the wild type and an isogenic hydrogenase-negative mutant of Azorhizobium caulinodans growing ex planta on N₂ as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O₂ tension. Production or consumption of O₂, H₂, and CO₂ was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O₂ tension: the growth yield decreased, and the specific O₂ consumption and CO₂ production increased. A similar dependency on the dissolved O₂ tension was found for the mutant with 2.5% H₂ in the influent gas. The H₂/N₂ ratio (moles of H₂ evolved per mole of N₂ consumed via nitrogenase) of the mutant, growing with or without 2.5% H₂, increased with increasing dissolved O₂ tensions. This increase in the H₂/N₂ ratio was small but significant. The dependencies of the ATP/N₂ ratio (moles of ATP consumed per mole of N₂ fixed) and the ATP/2e^- ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O₂ tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection.     

Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA-virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA-virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA-virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.     

Thirty-four years of pollen monitoring: an evaluation of the temporal variation of pollen seasons in Belgium

For the first time in Belgium, fluctuations in airborne pollen quantities over a 34 years period have been analyzed. Seven pollen types have been selected comprising the most clinically relevant in Belgium nowadays (birch, alder, hazel and grasses) and others that are known to be allergenic in other European countries and frequently found in Belgium (plane, ash and mugwort). Pollen monitoring was performed with a seven-day recording volumetric spore trap placed in Brussels. We measured increasing airborne pollen for four trees, namely alder, hazel, ash and plane. Although the total pollen index for birch has not increased significantly, an increasing trend in the annual amount of days above the concentration threshold of 80 pollen grains/m³ was clearly observed. Concerning temporal variations, the pollen season has tended to end earlier for birch, ash and plane and the peak concentration of the pollen of plane has been appearing earlier in the year. In the investigated period, the pollen seasons of grasses and mugwort have tended to become less severe. Furthermore, we reported a temporal shift of the grass pollen season, beginning and ending earlier, together with an advance of the annual peak date.     

Acute and phase-shifting effects of ocular and extraocular light in human circadian physiology

Light can influence physiology and performance of humans in two distinct ways. It can acutely change the level of physiological and behavioral parameters, and it can induce a phase shift in the circadian oscillators underlying variations in these levels. Until recently, both effects were thought to require retinal light perception. This view was challenged by Campbell and Murphy, who showed significant phase shifts in core body temperature and melatonin using an extraocular stimulus. Their study employed popliteal skin illumination and exclusively considered phase-shifting effects. In this paper, the authors explore both acute effects and phase-shifting effects of ocular as well as extraocular light. Twelve healthy males participated in a within-subject design and received all of three light conditions--(1) dim ocular light/no light to the knee, (2) dim ocular light/bright extraocular light to the knee, and (3) bright ocular light/no light to the knee--on separate nights in random order. The protocol consisted of an adaptation night followed by a 26-h period of sustained wakefulness, during which a 4-h light pulse was presented at a time when maximal phase delays were expected. The authors found neither immediate nor phase-shifting effects of extraocular light exposure on melatonin, core body temperature (CBT), or sleepiness. Ocular bright-light exposure reduced the nocturnal circadian drop in CBT, suppressed melatonin, and reduced sleepiness significantly. In addition, the 4-h ocular light pulse delayed the CBT rhythm by -55 min compared to the drift of the CBT rhythm in dim light. The melatonin rhythm shifted by -113 min, which differed significantly from the drift in the melatonin rhythm in the dim-light condition (-26 min). The failure to find immediate or phase-shifting effects in response to extraocular light in a within-subjects design in which effects of ocular bright light are confirmed strengthens the doubts raised by other labs of the impact of extraocular light on the human circadian system.     

Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation

Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.