Dopamine inhibits luteinizing hormone synthesis and release in the juvenile European eel: a neuroendocrine lock for the onset of puberty

In various adult teleost fishes, LH ovulatory peak is under a dual neurohormonal control that is stimulatory by GnRH and inhibitory by dopamine (DA). We investigated whether DA could also be involved in the inhibitory control of LH at earlier steps of gametogenesis by studying the model of the European eel, Anguilla anguilla, which remains at a prepubertal stage until the oceanic reproductive migration. According to a protocol previously developed in the striped bass, eels received sustained treatments with GnRH agonist (GnRHa), DA-receptor antagonist (pimozide), and testosterone (T) either alone or in combination. Only the triple treatment with T, GnRHa, and pimozide could trigger dramatic increases in LH synthesis and release as well as in plasma vitellogenin levels and a stimulation of ovarian vitellogenesis. Thus, in the prepubertal eel, removal of DA inhibition is required for triggering GnRH-stimulated LH synthesis and release as well as ovarian development. To locate the anatomical support for DA inhibition, the distribution of tyrosine hydroxylase (TH) in the brain and pituitary was studied by immunocytochemistry. Numerous TH-immunoreactive cell bodies were observed in the preoptic anteroventral nucleus, with a dense tract of immunoreactive fibers reaching the pituitary proximal pars distalis, where the gonadotrophs are located. This pathway corresponds to that mediating the inhibition of LH and ovulation in adult teleosts. To our knowledge, this is the first demonstration of a pivotal role for DA in the control of LH and puberty in a juvenile teleost. These data support the view that DA inhibition on LH secretion is an ancient evolutionary component in the neuroendocrine regulation of reproduction that may have been partially maintained throughout vertebrate evolution.     

Glycosylation study of the major genetic variants of human alpha1-acid glycoprotein and of their pharmacokinetics in the rat

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants, the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. AAG is also an extensively glycosylated protein which possesses five N-linked glycans exhibiting substantial heterogeneity in their structures. The first objective of this study was to investigate the glycosylation of the two major gene products of AAG, i.e. the A variant and a mixture of the F1 and S variants (F1*S). To this end, we combined a chromatographic method for the fractionation of the AAG variants with a lectin-binding assay to characterise the glycosylation of purified glycoproteins. Secondly, because the oligosaccharides can influence the disposition of AAG, a kinetic study of the AAG variants was carried out in the rat. After intravenous administration of whole human AAG, the separation and quantification of the AAG variants in plasma was performed by application of specific methods by isoelectric focusing and immunonephelometry. The binding studies carried out on a panel of lectins showed significant differences in the lectin-binding characteristics of the separated F1*S and A variants, accounting for differences in the degree of branching of their glycan chains and substitution with sialic acid and fucose. The plasma concentration-time profiles of the F1*S and A variants were biphasic, and only small differences were observed between the variants for their initial and terminal half-lives, clearance and distribution volume. This indicates that the structural differences between the two AAG gene products do not affect their pharmacokinetics in the rat. Specific drug transport roles have been previously demonstrated for the F1*S and A variants, calling for further investigations into their effects on the disposition of drugs they bind in plasma. The present study shows that such investigations are possible without being complicated by kinetic differences between these variants.     

Endothelium Dependent Vasomotion and In Vitro Markers of Endothelial Repair in Patients with Severe Sepsis: An Observational Study

Background

Outcome in sepsis is mainly defined by the degree of organ failure, for which endothelial dysfunction at the macro- and microvascular level is an important determinant. In this study we evaluated endothelial function in patients with severe sepsis using cellular endothelial markers and in vivo assessment of reactive hyperaemia.

Materials and Methods

Patients with severe sepsis (n = 30) and 15 age- and gender- matched healthy volunteers were included in this study. Using flow cytometry, CD34+/KDR+ endothelial progenitor cells (EPC), CD31+ T-cells, and CD31+/CD42b- endothelial microparticles (EMP) were enumerated. Migratory capacity of cultured circulating angiogenic cells (CAC) was assessed in vitro. Endothelial function was determined using peripheral arterial tonometry at the fingertip.

Results

In patients with severe sepsis, a lower number of EPC, CD31+ T-cells and a decreased migratory capacity of CAC coincided with a blunted reactive hyperaemia response compared to healthy subjects. The number of EMP, on the other hand, did not differ. The presence of organ failure at admission (SOFA score) was inversely related with the number of CD31+ T-cells. Furthermore, the number of EPC at admission was decreased in patients with progressive organ failure within the first week.

Conclusion

In patients with severe sepsis, in vivo measured endothelial dysfunction coincides with lower numbers and reduced function of circulating cells implicated in endothelial repair. Our results suggest that cellular markers of endothelial repair might be valuable in the assessment and evolution of organ dysfunction.     

Both the hippocampus and striatum are involved in consolidation of motor sequence memory

Functional magnetic resonance imaging (fMRI) was used to investigate the cerebral correlates of motor sequence memory consolidation. Participants were scanned while training on an implicit oculomotor sequence learning task and during a single testing session taking place 30 min, 5 hr, or 24 hr later. During training, responses observed in hippocampus and striatum were linearly related to the gain in performance observed overnight, but not over the day. Responses in both structures were significantly larger at 24 hr than at 30 min or 5 hr. Additionally, the competitive interaction observed between these structures during training became cooperative overnight. These results stress the importance of both hippocampus and striatum in procedural memory consolidation. Responses in these areas during training seem to condition the overnight memory processing that is associated with a change in their functional interactions. These results show that both structures interact during motor sequence consolidation to optimize subsequent behavior.     

Disulfide Bond-mediated multimerization of Ask1 and its reduction by thioredoxin-1 regulate H(2)O(2)-induced c-Jun NH(2)-terminal kinase activation and apoptosis

Apoptosis signal-regulated kinase-1 (Ask1) lies upstream of a major redox-sensitive pathway leading to the activation of Jun NH(2)-terminal kinase (JNK) and the induction of apoptosis. We found that cell exposure to H(2)O(2) caused the rapid oxidation of Ask1, leading to its multimerization through the formation of interchain disulfide bonds. Oxidized Ask1 was fully reduced within minutes after induction by H(2)O(2). During this reduction, the thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) became covalently associated with Ask1. Overexpression of Trx1 accelerated the reduction of Ask1, and a redox-inactive mutant of Trx1 (C35S) remained trapped with Ask1, blocking its reduction. Preventing the oxidation of Ask1 by either overexpressing Trx1 or using an Ask1 mutant in which the sensitive cysteines were mutated (Ask1-DeltaCys) impaired the activation of JNK and the induction of apoptosis while having little effect on Ask1 activation. These results indicate that Ask1 oxidation is required at a step subsequent to activation for signaling downstream of Ask1 after H(2)O(2) treatment.     

Modulation of Macrophage Activation State Protects Tissue from Necrosis during Critical Limb Ischemia in Thrombospondin-1-Deficient Mice

Background

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Methods and Findings

Using a genetic model of tsp-1 ^−/− mice subjected to femoral artery excision, we report that tsp-1 ^−/− mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1 ^−/− and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1 ^−/− mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1 ^−/− mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1 ^−/− mice, thereby demonstrating that macrophages mediated tissue protection in these mice.

Conclusion

This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.     

Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.     

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

Background

Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.

Results

Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.

Conclusion

We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.