Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.     

Conserved expression of mouse Six1 in the pre-placodal region (PPR) and identification of an enhancer for the rostral PPR

All cranial sensory organs and sensory neurons of vertebrates develop from cranial placodes. In chick, amphibians and zebrafish, all placodes originate from a common precursor domain, the pre-placodal region (PPR), marked by the expression of Six1/4 and Eya1/2. However, the PPR has never been described in mammals and the mechanism involved in the formation of PPR is poorly defined. Here, we report the expression of Six1 in the horseshoe-shaped mouse ectoderm surrounding the anterior neural plate in a pattern broadly similar to that of non-mammalian vertebrates. To elucidate the identity of Six1-positive mouse ectoderm, we searched for enhancers responsible for Six1 expression by in vivo enhancer assays. One conserved non-coding sequence, Six1-14, showed specific enhancer activity in the rostral PPR of chick and Xenopus and in the mouse ectoderm. These results strongly suggest the presence of PPR in mouse and that it is conserved in vertebrates. Moreover, we show the importance of the homeodomain protein-binding sites of Six1-14, the Six1 rostral PPR enhancer, for enhancer activity, and that Dlx5, Msx1 and Pax7 are candidate binding factors that regulate the level and area of Six1 expression, and thereby the location of the PPR. Our findings provide critical information and tools to elucidate the molecular mechanism of early sensory development and have implications for the development of sensory precursor/stem cells.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Schistosoma mansoni: Tyrosine,a putative in vivo substrate of phenol oxidase

l-Tyrosine, l-[3,4]dihydroxyphenylalanine (l-DOPA), and dopamine are known to be in vitro substrates for Schistosoma mansoni phenol oxidase. Since all three compounds are present in the female schistosome, it is not clear which one serves as the substrate for phenol oxidase in intact S. mansoni. However, the concentration of l-tyrosine in the female schistosome (252 ng/mg worm) is 4-fold higher than the K_m of phenol oxidase for this amino acid while the concentrations of l-DOPA and dopamine (0.954 and 0.790 ng/mg worm, respectively) are 100- and 500-fold lower than the K_m of these substrates. Tri-l-tyrosine methyl ester is oxidized at less than 3% of the rate of l-tyrosine methyl ester. A tyrosine:lysine peptide and chymotrypsinogen are not oxidized. Female S. mansoni do not incorporate l-tyrosine into proteins to a significantly greater extent than l-leucine. The results suggest that free l-tyrosine is the substrate for S. mansoni phenol oxidase in vivo.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca²⁺ signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca²⁺ transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca²⁺ transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca²⁺ release from the intracellular Ca²⁺ stores. Thus, our in vivo data suggest that microglial Ca²⁺ signals occur mostly under pathological conditions and identify a Ca²⁺ store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.     

Schistosoma mansoni: Role in vivo of complement in primary infection of mice

The role of complement in the control of the primary Schistosoma mansoni infection in mice was investigated in vivo. The number of recovered adult schistosomes 6–7 weeks postinfection was used as a parasitological criterion of immunity. No significant difference in the worm burden was observed between C5-sufficient and C5-deficient mice. In contrast, when cobra venom factor (CVF) was injected into normal or C5-deficient mice 24 hr before challenge, a significant increase of the worm burden was noticed in comparison to the untreated mice. These results indicated that, although C5 and probably the late complement components are not essential for the control of the primary infection, the alternative pathway and some of its components are involved. In fact, the injection of C3 2 hr before infection of CVF-treated mice completely restored the immunity. A role for C3, in association with effector cells, in the nonspecific immunity occurring in the first hours after a primary S. mansoni infection is suggested.     

In vitro and in vivo interaction of moxidectin with BCRP/ABCG2

The study characterizes the interaction between BCRP/ABCG2 and moxidectin by means of cellular transport, and pharmacokinetic studies in Bcrp1 (−/−) and wild-type mice. Milbemycin moxidectin ([³H]-moxidectin) was tested for its ability to be transported across MCDK-II epithelial monolayer cultures transfected with BCRP. In a second approach, accumulation assays by BCRP-expressing Xenopus laevis oocytes were carried out. Finally, pharmacokinetic studies were performed in order to establish the role of the transporter in milk secretion and tissue distribution. The efflux was negligible in polarized cells but moxidectin was efficiently transported in BCRP-expressing X. laevis oocytes. The transport was blocked by an acridone derivative, a novel BCRP inhibitor. Moxidectin secretion into breast milk was decreased in Bcrp1-knockout mice and the milk to plasma ratio was 2-fold higher in wild-type mice after i.v. administration. Drug accumulation in intestinal content, bile, and intestine was higher in wild-type mice but the plasma concentration was not different.Moxidectin is identified as a BCRP substrate since its Bcrp1-mediated secretion into breast milk and the involvement of Bcrp1 in intestinal and bile secretion has been demonstrated. This interaction has pharmacokinetic and toxicological consequences. The most important toxicological consequences of the interaction between BCRP and moxidectin may be related with the presence of drug residues in milk.     

Increased per cell IFN-γ productivity indicates recent in vivo activation of T cells

Immunization with vaccinia virus causes long-term immunity. Efforts have been made to characterize the T cells responsible for this protection. Recently, T cell subsets were described that not only co-express multiple cytokines, but also show increased per cell cytokine productivity. These highly productive cells are often considered to be the most protective. We used ELISPOT assays to measure per cell IFN-γ productivity of vaccinia-specific T cells in childhood immunized adults immediately before and at different time points after vaccinia re-vaccination. Apart from an increase in frequency, we found a marked increase of IFN-γ productivity following vaccinia re-vaccination. However, these changes were short-lived as both parameters quickly returned to baseline values within 22 days after re-vaccination. Therefore, increased per cell IFN-γ productivity seems to be a sign of recent in vivo T cell activation rather than a stable marker of a distinct T cell subset responsible for long-term immune protection.     

Discovery of a crystalline sulforaphane analog with good solid-state stability and engagement of the Nrf2 pathway in vitro and in vivo

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23?°C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.     

Echinostoma revolutum: Labeling of miracidia with radioselenium in vivo and assay for host finding

Radioactivity was incorporated into Echinostoma revolutum worms and eggs when ⁷⁵Semethionine was administered intraperitoneally to mice infected with the fluke parasites. The levels of incorporation of radioactivity increased in proportion to the amounts of radioselenium used. During the period 3–10 days p.i. the maximum egg-bound radioactivity was, in general, achieved 3 days after the administration of the radioisotope, but substantial radiolabeling was obtained at all isotope levels until Day 10. The radioactivity of the miracidium constituted 29–34% of that of the egg. The radiolabeling procedure did not interfere with the biological characteristics (behavioral activity, infectivity) of the radiolabeled miracidia. Thus, the use of such labeled miracidia for host-finding studies seems acceptable. A radioisotope tracer system for assaying E. revolutum miracidial host finding was described. This system employs exposure of the first intermediate host snail, Biomphalaria alexandrina, to radiolabeled miracidia. A linear proportionality was found to exist between the number of penetrating miracidia and the amount of snail-bound radioactivity. Thus, snail-bound radioactivity retained after exposure to radiolabeled miracidia can be used to measure miracidial host-finding capacity under various experimental conditions.