Microsatellite markers developed for a Swedish population of sand lizard (<Emphasis Type="Italic">Lacerta agilis</Emphasis>)

Populations of sand lizards (Lacerta agilis) are declining throughout its north-western range. Here we characterize fifteen new microsatellite markers developed specifically for parentage analysis in a small Swedish population of sand lizards. These loci were screened in the Asketunnan population and a much larger and genetically diverse Hungarian population, with heterozygosities ranging from (0.217–0.875) and (0.400–0.974), respectively. All loci were in Hardy-Weinberg Equilibrium in the Swedish population but eight loci had significant heterozygote deficiencies in the Hungarian population. Two loci were significantly linked in both populations. These microsatellite loci are likely to be applicable in research on other sand lizard populations throughout Europe.     

Single and Multicomponent Gas Phase Diffusion in a Porous Media: Modeling and Laboratory Measurements

Subsurface vapor migration of volatile chemicals may impact ambient and indoor air quality, increasing the importance to investigate the fate and transport of these chemicals. This project involved both modeling and experimental work to study the vapor phase transport behavior of single, binary, and tertiary component systems present in the gas phase. The experimental phase resulted in the development of a diffusion cell for measuring vapor phase transport. Three organic compounds (toluene, cumene, and isooctane) common to petroleum-based products were selected. The objective of this research project was to evaluate how the rate of a component diffusing alone in a stagnant gas mixture compares to the rate of the same component when diffusing in the presence of multiple diffusing species. The equipment was first validated by measuring the unobstructed gas phase diffusion fluxes for each organic compound. The diffusion coefficients were then calculated from the experimentally measured diffusive fluxes using Fick's Law at 20 and 25°C and compared to the respective literature values. The experimental/literature (E/L) ratio was calculated for toluene, cumene, and isooctane. The range of the average E/L ratio for the single component data sets is 0.93 to 1.05. The validation data provided the baseline for extending the research to multicomponent data. The multi-component systems research was characterized as either binary systems or a three-component system. The binary systems were either isooctane/tolu-ene or isooctane/cumene. The three-component system consisted of a mixture of all three compounds. For both temperatures and all compounds the flux rate decreased for any single component due to the dilution effect by incorporation into a mixture. Applying Fick's Law to calculate the effective diffusion coefficient for each compound that corresponded to the resulting concentration gradient by the mixture, an enhancement in the diffusive flux of each individual species was observed. This enhancement can be explained by a compositional coupling of each component to all others which results in a total vapor phase mass flux comprised of both diffusive and pseudo-advective mass transport. This pseudo-advective component is attributed to simultaneous diffusion of other species in the presence of the one of interest. Since this research project incorporated a mixture of toluene and cumene present in a background carrier solvent of isooctane, by calculating the ratios Dexp(3-component)/ Dexp(2-component) and Dexp(2-compo-nent)/Dexp(single component), an estimate is obtained of the enhancement effect due to the advective component of simultaneously diffusing chemicals. The diffusivity ratios for the three-component system compared to the dual component system ranged from 0.8 to 3.7. The diffusivity ratios for individual compounds were for 1.5-3.7 cumene, 0.8-1.2 for toluene, and 1.0-1.2 for isooctane. The diffusivity ratios for the dual component system to the single component systems ranged from 0.8 to 4.0. The range of diffusivity ratios for individual compounds were for 2.0-4.0 for cumene, 0.8-1.6 for toluene, and 1.1-1.4 for isooctane. A ratio greater than 1.0 indicated an enhancement effect on the molecular diffusion rate due to the presence of one or more additional diffusing chemical species present. The majority of fate and transport models are based on single component behavior modeled by Fick's Law using the pure gas phase diffusion coefficient. The enhancement of the individual diffusive flux in a multicomponent mixture observed in this study and accounted for by pseudo-advec-tive mass transport results in an under-prediction of the actual multicomponent diffusive fluxes. It is recommended that a more rigorous diffusion equation such as the Stefan-Maxwell equation be considered for incorporation into vapor phase transport models when modeling multicomponent/ contaminant systems.     

Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.     

Transcriptome-based identification of new anti-anti-inflammatory and vasodilating properties of the n-3 fatty acid docosahexaenoic acid in vascular endothelial cell under proinflammatory conditions

ScopeHigh intakes of n-3 fatty acids exert anti-inflammatory effects and cardiovascular protection, but the underlying molecular basis is incompletely defined. By genome-wide analysis we searched for novel effects of docosahexaenoic acid (DHA) on gene expression and pathways in human vascular endothelium under pro-inflammatory conditions.ConclusionsEndothelial exposure to DHA regulates novel genes and related pathways. Such unbiased identification should increase our understanding of mechanisms by which n-3 fatty acids affect human diseases.     

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.     

An observational cohort study of the kynurenine to tryptophan ratio in sepsis: association with impaired immune and microvascular function

Both endothelial and immune dysfunction contribute to the high mortality rate in human sepsis, but the underlying mechanisms are unclear. In response to infection, interferon-γ activates indoleamine 2,3-dioxygenase (IDO) which metabolizes the essential amino acid tryptophan to the toxic metabolite kynurenine. IDO can be expressed in endothelial cells, hepatocytes and mononuclear leukocytes, all of which contribute to sepsis pathophysiology. Increased IDO activity (measured by the kynurenine to tryptophan [KT] ratio in plasma) causes T-cell apoptosis, vasodilation and nitric oxide synthase inhibition. We hypothesized that IDO activity in sepsis would be related to plasma interferon-γ, interleukin-10, T cell lymphopenia and impairment of microvascular reactivity, a measure of endothelial nitric oxide bioavailability. In an observational cohort study of 80 sepsis patients (50 severe and 30 non-severe) and 40 hospital controls, we determined the relationship between IDO activity (plasma KT ratio) and selected plasma cytokines, sepsis severity, nitric oxide-dependent microvascular reactivity and lymphocyte subsets in sepsis. Plasma amino acids were measured by high performance liquid chromatography and microvascular reactivity by peripheral arterial tonometry. The plasma KT ratio was increased in sepsis (median 141 [IQR 64–235]) compared to controls (36 [28–52]); p<0.0001), and correlated with plasma interferon-γ and interleukin-10, and inversely with total lymphocyte count, CD8+ and CD4+ T-lymphocytes, systolic blood pressure and microvascular reactivity. In response to treatment of severe sepsis, the median KT ratio decreased from 162 [IQR 100–286] on day 0 to 89 [65–139] by day 7; p = 0.0006) and this decrease in KT ratio correlated with a decrease in the Sequential Organ Failure Assessment score (p<0.0001). IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis and may link these two fundamental processes in sepsis pathophysiology.     

Fibroblast growth factor 2: good or bad guy in the joint?

Fibroblast growth factor 2 (FGF2) is a highly abundant growth factor found within the pericellular matrix of articular chondrocytes, but studies investigating its role have been conflicting. The paper reported by Yan and colleagues in the previous issue of Arthritis Research & Therapy proposes that differences in responses to FGF2 are most likely due to changes in the balance between the two major articular cartilage FGF receptors, FGFR1 and FGFR3. They show that the catabolic and anti-anabolic effects of FGF2 are mediated primarily through FGFR1 whereas the beneficial effects are through FGFR3. This balance is dynamic and is altered in disease and following growth factor stimulation in vitro.     

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

Introduction

We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.

Methods

Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.

Results

1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).

Conclusions

Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.