Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) gamma. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFN gamma than DCs stimulated with the cytokine cocktail: tumor necrosis factor-alpha, IL-1 beta, IL-6, and prostaglandin E-2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFN gamma were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFN gamma, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFN gamma secretion. Thus, MoDCs generated ill SFM efficiently stimulate T-cell IFN gamma production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Visualisation of macropinosome maturation by the recruitment of sorting nexins

We report that phosphoinositol-binding sorting nexin 5 ( SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Circannual rhythms of maturation in the female rainbow trout and their entrainment by changes in photoperiod

The importance of endogenous rhythms in the photoperiodic control of the annual reproduction cycle in female rainbow trout was investigated. The effect of photoperiod regimes on the different stages of maturation was assessed by recording the timing of ovulation and from quantifying associated changes in serum oestradiol-17,testosterone and total calcium. Maintained under constant 6L:18D and constant temperature for up to four years, rainbow trout exhibited an endogenous rhythm of maturation with a periodicity of approximately one year. This rhythm of maturation appears to be driven by an autonomous circannual oscillator or clock which can be dissociated from the neuroendocrine mechanisms controlling gonadal maturation. Under conditions of constant 18L:6D or LL the periodicity of the maturation rhythm was 5.5-6 months; it is suggested that this periodicity may be caused by a splitting or uncoupling of at least two circannual clocks involved in the control of maturation. Abrupt changes in the length of the photoperiod act as a zeitgeber to entrain the endogenous rhythm of maturation. Whether the timing of maturation is advanced or delayed depends primarily on the direction of the change in photoperiod and its timing in relation to the phase of the rhythm, with the magnitude of the alteration in photoperiod having only a supplementary effect. The effect of specific changes in photoperiod on the entrainment of the maturation cycle can be described in terms of a phase-response curve. Photic information is transduced, probably by the pineal gland, into a daily rhythm of melatonin; exposure of rainbow trout to skeleton and resonance photoperiod regimes indicated that daylength measurement is effected by endogenous circadian clock(s) rather than by hour-glass mechanisms. A gating mechanism is closely associated with the circannual clock which determines the timing of onset of maturation in virgin female rainbow trout, only allowing fish that have attained a threshold stage of development to undergo gonadal maturation. Collectively the results support the hypothesis that the female rainbow trout exhibits an endogenous circannual rhythm of maturation which can be entrained by changes in photoperiod.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P <0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P <0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P <0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P <0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P <0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome. © Journal compilation © 2008 The Physiological Society.

Seawater acidification affects the physiological energetics and spawning capacity of the Manila clam Ruditapes philippinarum during gonadal maturation

Ocean acidification is predicted to have widespread implications for marine bivalve mollusks. While our understanding of its impact on their physiological and behavioral responses is increasing, little is known about their reproductive responses under future scenarios of anthropogenic climate change. In this study, we examined the physiological energetics of the Manila clam Ruditapes philippinarum exposed to CO2-induced seawater acidification during gonadal maturation. Three recirculating systems filled with 600 L of seawater were manipulated to three pH levels (8.0, 7.7, and 7.4) corresponding to control and projected pH levels for 2100 and 2300. In each system, temperature was gradually increased ca. 0.3 °C per day from 10 to 20 °C for 30 days and maintained at 20 °C for the following 40 days. Irrespective of seawater pH levels, clearance rate (CR), respiration rate (RR), ammonia excretion rate (ER), and scope for growth (SFG) increased after a 30-day stepwise warming protocol. When seawater pH was reduced, CR, ratio of oxygen to nitrogen, and SFG significantly decreased concurrently, whereas ammonia ER increased. RR was virtually unaffected under acidified conditions. Neither temperature nor acidification showed a significant effect on food absorption efficiency. Our findings indicate that energy is allocated away from reproduction under reduced seawater pH, potentially resulting in an impaired or suppressed reproductive function. This interpretation is based on the fact that spawning was induced in only 56% of the clams grown at pH 7.4. Seawater acidification can therefore potentially impair the physiological energetics and spawning capacity of R. philippinarum.

A key role for PTP1B in dendritic cell maturation, migration and T cell activation

Acknowledgements We thank the Iain Fraser Flow Cytometry Centre and the Medical Research Facility of the University of Aberdeen. We are grateful to Drs West, Zaru, and Davidson (University of Dundee) for the scientific discussion and technical assistance. Wethank Derek Mitchell (University of Dundee) for aiding with the quantification of focal contacts. Funding This work was supported by Saving Sight in Grampian and the Development Trust of the UoA (both to J.V.F.). Work on this project was partly funded by project grants from British Heart Foundation and European Foundation for the Study of Diabetes/Lilly diabetes programme grant (to M.D.).

A key role for PTP1B in dendritic cell maturation, migration and T cell activation

Acknowledgements We thank the Iain Fraser Flow Cytometry Centre and the Medical Research Facility of the University of Aberdeen. We are grateful to Drs West, Zaru, and Davidson (University of Dundee) for the scientific discussion and technical assistance. Wethank Derek Mitchell (University of Dundee) for aiding with the quantification of focal contacts. Funding This work was supported by Saving Sight in Grampian and the Development Trust of the UoA (both to J.V.F.). Work on this project was partly funded by project grants from British Heart Foundation and European Foundation for the Study of Diabetes/Lilly diabetes programme grant (to M.D.).

Étude de l'expression génique des molécules reliées à l'apoptose durant la maturation des cellules dendritiques : rôle pro-apoptotique de fas

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.