Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MΦ and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MΦ showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.     

Arabidopsis type I metacaspases control cell death

Metacaspases are distant relatives of animal caspases found in protozoa, fungi, and plants. Limited experimental data exist defining their function(s), despite their discovery by homology modeling a decade ago. We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically control programmed cell death in Arabidopsis. AtMC1 is a positive regulator of cell death and requires conserved caspase-like putative catalytic residues for its function. AtMC2 negatively regulates cell death. This function is independent of the putative catalytic residues. Manipulation of the Arabidopsis type I metacaspase regulatory module can nearly eliminate the hypersensitive cell death response (HR) activated by plant intracellular immune receptors. This does not lead to enhanced pathogen proliferation, decoupling HR from restriction of pathogen growth.     

Combination of Trabectedin and Gemcitabine for Advanced Soft Tissue Sarcomas: Results of a Phase I Dose Escalating Trial of the German Interdisciplinary Sarcoma Group (GISG)

Background: Evaluation of the potential efficacy and safety of combination therapies for advanced soft tissue sarcomas (STS) has increased substantially after approval of trabectedin and pazopanib. Trabectedin’s introduction in Europe in 2007 depended mainly on its activity in so-called L-sarcomas (liposarcoma and leiomyosarcoma); combination of trabectedin with other chemotherapies used in STS seems of particular interest. Methods: We initiated within the German Interdisciplinary Sarcoma Group (GISG) a phase I dose escalating trial evaluating the combination of trabectedin and gemcitabine in patients with advanced and/or metastatic L-sarcomas (GISG-02; ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01426633","term_id":"NCT01426633"}}NCT01426633). Patients were treated with increasing doses of trabectedin and gemcitabine. The primary endpoint was to determine the maximum tolerated dose. Results: Five patients were included in the study. Two patients were treated on dose level 1 comprising trabectedin 0.9 mg/m² on day 1 and gemcitabine 700 mg/m² on days 1 + 8, every 3 weeks. Due to dose-limiting toxicity (DLT) in both patients (elevated transaminases and thrombocytopenia), an additional three patients were treated on dose level −1 with trabectedin 0.7 mg/m² plus gemcitabine 700 mg/m². Of these three patients, two demonstrated another DLT; therefore, the trial was stopped and none of the dose levels could be recommended for phase II testing. Conclusion: The GISG-02 phase I study was stopped with the conclusion that the combination of gemcitabine and trabectedin is generally not recommended for the treatment of patients with advanced and/or metastatic leiomyosarcoma or liposarcoma. Also, this phase I study strongly supports the necessity for careful evaluation of combination therapies.     

Pharmacokinetics and efficacy of intravenous and extradural tramadol in dogs

Tramadol is a synthetic opioid agonist used extensively in human and, to a lesser extent, veterinary medicine throughout the world. The clinical efficacy and pharmacokinetic profile of intravenous (IV) and extradural (ED) tramadol (2 mg/kg) and its o-desmethyl metabolite were studied in dogs undergoing tibial plateau levelling osteotomy (TPLO). Intra-operative cardiorespiratory variables were monitored and post-operative pain was assessed using the short form of the Glasgow Composite Pain Scale. A rapid (<5 min) and effective production of o-desmethyl tramadol was recorded. The pharmacokinetic profile was similar for tramadol and its metabolite irrespective of the route of administration. ED tramadol provided sufficient intra- and post-operative analgesia without significant clinical side-effects, but the post-operative analgesia was comparable to that following IV administration and the ED route could therefore not be considered a practical alternative to the IV route.     

An innovative stabilization/solidification treatment For contaminated soil remediation: demonstration project results

Background, aim, and scope

An innovative stabilization/solidification (S/S) process using high-performance additivated concrete technology was developed for remediating soil contaminated by metals from abandoned industrial sites. In order to verify the effectiveness of this new ex situ S/S procedure, an area highly contaminated by metallic pollutants (As, Cd, Hg, and Pb), due to the uncontrolled discharge of waste generated from artistic glass production on the island of Murano (Venice, Italy), was selected as a case study. The technique transforms the contaminated soil into an aggregate material suitable for reuse as on-site backfill. This paper reports the main results of the demonstration project performed in collaboration with the local environmental protection agency (ARPAV).

Materials and methods

An ex situ treatment for brownfield remediation, based on the transformation of contaminated soil into very dense, low porous, and mechanically resistant granular material, was set up and tested. Specific additives (water reducers and superplasticizers) to improve the stabilized material properties were developed and patented. A demonstration plant assembled on the study area to treat 6 m³ h^–1was then tested. After excavation, the contaminated soil was screened to remove coarse material. The fraction Ø?>?4 mm (coarse fraction), mainly composed of glass, brick, concrete, and stone debris, was directly reused on site after passing through a washing treatment section. The highly polluted fraction Ø?≤?4 mm (fine fraction) was treated in the S/S treatment division of the plant (European patent WO/2006/097272). The fine fraction was mixed with Portland cement and additives defined on the basis of the high performance concrete technique. the mixture was then granulated in a rolling-plate system. After 28 days curing in an onsite storage area to allow for cement hydration, the stabilized material was monitored before its in situ relocation. The chemical, mechanical, and ecotoxicological reliability and performance of the treatment was checked. Metal leachability was verified according to four leaching test methods: Italian Environmental Ministry Decree (1998), EN 12457 (2002) tout court, amended only with MgSO₄ and, lastly, with artificial sea water. The mechanical properties were measured according to BS (1990) and AASHTO (1999) to obtain the Aggregate Crushing Value and California Bearing Ratio, in that order. Moreover, leachate samples prepared with artificial seawater were assessed via the Crassostrea gigas embryotoxicity test and Vibrio fischeri bioluminescence inhibition test to discriminate the presence of potential ecotoxicological effects for the brackish and saltwater biota.

Results

Outcomes from all leachate samples highlighted the effectiveness of the remediation treatment, fully complying with the Italian legislation for non-hazardous material reuse under a physicochemical viewpoint. The stabilized granular material demonstrated high mechanical strength, low porosity, and leachability. Moreover, ecotoxicological surveys indicated the presence of low toxicity levels in leachate samples according to both toxicity tests.

Discussion

Remediated soil samples revealed a significant decrease in leachability of heavy metals as a consequence of the application of additivated cement that enhanced granular material properties, resulting in improved compactness due to the reduction in water content. The toxicity data confirmed this state-of-the-art technique, indicating that leachates could be deemed as minor acutely toxic.

Conclusions

The proposed S/S treatment proved to be able to remediate soil contaminated by heavy metals through trapping pollutants in pellet materials presenting adequate physicochemical, mechanical, and ecotoxicological properties in order to prevent leachability phenomena, their reclamation, and reuse being made easier by its granular form.

Recommendation and perspectives

This project foresees long-term monitoring activity over several years (until 2014) to consider treatment durability.     

Lipophilic (hydroxy)phenylacetates by solvent-free lipase-catalyzed esterification and transesterification in vacuo

Various long-chain alkyl (hydroxy)phenylacetates were prepared in high yield by lipase-catalyzed transesterification of the corresponding short-chain alkyl hydroxyphenylacetates and fatty alcohols in equimolar ratios. The reactions were performed in vacuo at moderate temperatures in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica (Novozym 435) was the most effective biocatalyst for the various transesterification reactions. Generally, Novozym 435-catalyzed transesterifications of short-chain alkyl (hydroxy)phenylacetates with long-chain alcohols led to higher conversions and enzyme activities than the corresponding esterifications. For example, the transesterification activity was up to 4-fold higher than the esterification activity for the formation of oleyl 4-hydroxy-3-methoxyphenylacetate using Novozym 435 as a biocatalyst. The relative transesterification activities were as follows: phenylacetate > 3-methoxyphenylacetate approximately 4-methoxyphenylacetate > 4-hydroxy-3-methoxyphenylacetate > 3-hydroxyphenylacetate approximately 4-hydroxyphenylacetate > 2-methoxyphenylacetate > 3,4-dihydroxyphenylacetate. With respect to the position of methoxy and hydroxy substituents, the transesterification activity of Novozym 435 decreased in the order meta approximately para > ortho. Compounds with inverse chemical structures, for example, tyrosyl oleate, were obtained by Novozym 435-catalyzed esterification and transesterification of fatty acids and their methyl esters, respectively, with 2-phenylethan-1-ols. In contrast to the transesterifications of short-chain alkyl (hydroxy)phenylacetates with fatty alcohols, higher conversions and enzyme activities were observed for the Novozym 435-catalyzed esterifications of (hydroxy)phenylethanols with long-chain fatty acids than the corresponding transesterifications with fatty acid methyl esters.     

Quantitative analysis of exudates from soil-living basidiomycetes in pure culture as a response to lead, cadmium and arsenic stress

Six different ectomycorrhizal fungi (Hebeloma velutipes, Piloderma byssinum, Paxillus involutus, Rhizopogon roseolus, Suillus bovinus and Suillus variegatus) and two saprotrophic fungi (Hypholoma fasciculare and Hypholoma capnoides) were exposed to metal stress induced by Pb, Cd and As. After pre-growth in a nutrient solution in Petri dishes, metal exposure was performed either in a nutrient rich solution or in a nutrient poor solution for seven days. The fungi were exposed to two different metal concentrations, low and high (Pb: 10 + 100 μM; Cd: 1 + 10 μM; As: 1 + 10 μM). Exudation of low molecular weight organic compounds (low molecular weight organic acids (LMWOA), amino acids and dissolved monosaccharides), as well as dissolved organic carbon was quantified as a potential response to the metal stress. The main LMWOA identified was oxalate. Oxalate exudation increased significantly in response to both low and high Pb and Cd concentrations, as well as low As exposure, relative to nutrient controls. Exposure to As and mixtures of metals (Pb + Cd, Pb + As) did not result in any significant increase in oxalate production compared to controls. The presence of a carbon source (glucose) in this study is likely to have been important for exudation of organic compounds. For the nutrient rich (+1 mM glucose) metal treatments exposure to Pb and Cd mainly increased exudation of oxalate and total amino acids. Production of dissolved monosaccharides, as well as DOC, did not increase significantly in response to metal exposure, irrespective of nutrient conditions. This may be explained by re-absorption of the organic compounds by the mycelium or by the fact that metals had no effect on exudation of these compounds.     

Cultivation of the Paracalanid Copepod,Bestiolina similis (Calanoida: Crustacea)

First feed production continues to be a major barrier to the cultivation of many fish species. Although copepod nauplii are a suitable food, consistency and high production have been difficult. Temporal changes in production in batch cultures of the calanoid copepod, Bestiolina similis, were investigated to develop management strategies for the use of copepod nauplii as a live food. Population abundances and female egg production rates were measured, and recruitment and mortality rates were calculated. Relative expression levels of a molecular biomarker for stress, heat shock protein hsp70, were determined using real‐time quantitative polymerase chain reaction. The population cycle included a period of rapid increase in abundances, followed by a steep decline and a period of stable but low population densities. Initially, egg production exceeded 25 eggs per female per day and low mortality rates prevailed. The population decline was preceded by upregulation of hsp70 and followed by an 80–90% decline in female fecundity and an increase in mortality rates. Egg production rates remained below four eggs per female per day even after new generations of females reached adulthood. The predictable population cycle provides opportunities to coordinate nauplius production rates with first feed needs of fish larvae.     

Biochemical markers of morphogenesis in long term horseradish (Armoracia lapathifolia Gilib.) tissue culture

Long term in vitro cultures of plant tissue spontaneously change at morphological, cellular and biochemical level. Horseradish tissues were cultivated in vitro for more than 16 years. Primary crown-gall tumours were induced with Agrobacterium tumefaciens on leaf explants excised from plantlets propagated in vitro. Subcultured on hormone-free MS medium, transformed tissue continued to grow in an unorganized way (TN tumour) or as teratogenous tumour (TM teratoma) forming malformed shoots which never rooted. The aim of this study was to establish the morphological and biochemical status of this long term cultures. The study was performed by verifying biochemical marker of transformation and morphogenesis, which had been investigated when the culture was established (peroxidase, PPX) as well as by introducing new markers which have not been analyzed so far in horseradish tissues (ascorbate peroxidase, APX; phenylalanine-ammonia lyase, PAL; and phenolics content). Morphological changes were observed only in teratoma tissue. Peroxidase activity and isoenzyme pattern indicated stability of horseradish tissue culture. The new biochemical markers were in a complete (PAL) and partial (APX) correlation with the PPX results and are therefore useful addition in biochemical characterisation of these tissues and can also be applied as markers of morphogenesis and tumorization.     

Polarized epithelium-sperm co-culture system reveals stimulatory factors for the secretion of mouse epididymal quiescin sulfhydryl oxidase 1

Spermatozoa acquire fertilization ability through post-translational modifications. These membrane surface alterations occur in various segments of the epididymis. Quiescin sulfhydryl oxidases, which catalyze thiol-oxidation reactions, are involved in disulfide bond formation, which is essential for sperm maturation, upon transition and migration in the epididymis. Using castration and azoospermia transgenic mouse models, in the present study, we showed that quiescin sulfhydryl oxidase 1 (QSOX1) protein expression and secretion are positively correlated with the presence of testosterone and sperm cells. A two-dimensional in vitro epithelium-sperm co-culture system provided further evidence in support of the notion that both testosterone and its dominant metabolite, 5α-dihydrotestosterone, promote epididymal QSOX1 secretion. We also demonstrated that immature caput spermatozoa, but not mature cauda sperm cells, exhibited great potential to stimulate QSOX1 secretion in vitro, suggesting that sperm maturation is a key regulatory factor for mouse epididymal QSOX1 secretion. Proteomic analysis identified 582 secretory proteins from the co-culture supernatant, of which 258 were sperm-specific and 154 were of epididymal epithelium-origin. Gene Ontology analysis indicated that these secreted proteins exhibit functions known to facilitate sperm membrane organization, cellular activity, and sperm-egg recognition. Taken together, our data demonstrated that testosterone and sperm maturation status are key regulators of mouse epididymal QSOX1 protein expression and secretion.