Development of a 3D human in vitro skin co‐culture model for detecting irritants in real‐time

Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant‐induced inflammatory reaction and cytotoxicy. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi®)] at sub‐toxic concentrations and studied interleukin‐1 alpha (IL‐1α) release from human keratinocytes and activation of NF‐κB in human fibroblasts. NF‐κB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi®. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF‐κB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi® was required for NF‐κB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF‐κB. A technical advantage of using transfected cells in 3D was an ability to detect NF‐κB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL‐1α was measured in keratinocyte‐conditioned medium after incubation with SDS or Formi®, which correlated with fibroblast NF‐κB activity. In summary, this work has value for developing 3D tissue engineered co‐culture models for the in vitro testing of irritant chemicals at sub‐toxic concentrations, as an alternative to in vivo models. Biotechnol. Bioeng. 2010;106: 794–803. © 2010 Wiley Periodicals, Inc.     

Seroprevalence of Toxoplasma gondii in pregnant women and cats in Grenada, West Indies

Prevalence of antibodies against Toxoplasma gondii was studied in 534 pregnant women and 40 domestic cats in Grenada, West Indies. Antibodies (IgG) for T. gondii were sought in human sera by an enzyme-linked immunosorbent assay and in cat sera by using the modified agglutination test (MAT). Antibodies were found in 57 % of pregnant women. Seroprevalence increased with age; 51% of 15- to 19-yr-old women (100 total) had antibodies versus 60% of 20- to 24-yr-old women (127 total). Antibodies to T. gondii (MAT, 1:25 serum dilution) were found in 35% of cats; titers were 1:25 in 7 cats, 1:50 in 4 cats, and 1:500 in 3 cats. Epidemiological data suggested that the ingestion of food or water contaminated with oocysts was an important mode of transmission of T. gondii to women.     

Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents

In this study, we asked the question of whether cells in 3D culture cope more effectively with cytotoxic agents than cells in 2D. The sensitivities of human skin cells (keratinocytes, dermal fibroblasts and endothelial cells) to oxidative stress (hydrogen peroxide) and to a potentially toxic heavy metal (silver) when cultured under 2D and 3D conditions were investigated. The results show a marked resistance of cells to a given dose of hydrogen peroxide or silver nitrate causing a 50% loss of viability in 3D cultures, when compared to the same cells grown in 2D. There was also an improvement in the ability of cells to withstand both stresses when cells were in co-culture rather than in mono-culture. Foetal calf serum was found to have a mild protective effect in 2D culture but this was not extended to findings in 3D culture. This study suggests that dermatotoxicity testing using 3D co-cultures might be more likely to reflect true physiological responses to xenobiotic materials than existing models that rely on 2D mono-cultures.     

RecFOR Is Not Required for Pneumococcal Transformation but Together with XerS for Resolution of Chromosome Dimers Frequently Formed in the Process

Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA ^- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci. These observations presumably apply to most naturally transformable species.     

Near-Infrared Laser Adjuvant for Influenza Vaccine

Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.     

Obesity-Induced Insulin Resistance in Human Skeletal Muscle Is Characterised by Defective Activation of p42/p44 MAP Kinase

Insulin resistance (IR), an impaired cellular, tissue and whole body response to insulin, is a major pathophysiological defect of type 2 diabetes mellitus. Although IR is closely associated with obesity, the identity of the molecular defect(s) underlying obesity-induced IR in skeletal muscle remains controversial; reduced post-receptor signalling of the insulin receptor substrate 1 (IRS1) adaptor protein and downstream effectors such as protein kinase B (PKB) have previously been implicated. We examined expression and/or activation of a number of components of the insulin-signalling cascade in skeletal muscle of 22 healthy young men (with body mass index (BMI) range, 20–37 kg/m²). Whole body insulin sensitivity (M value) and body composition was determined by the hyperinsulinaemic (40 mU. min⁻¹.m⁻².), euglycaemic clamp and by dual energy X-ray absorptiometry (DEXA) respectively. Skeletal muscle (vastus lateralis) biopsies were taken before and after one hour of hyperinsulinaemia and the muscle insulin signalling proteins examined by western blot and immunoprecipitation assay. There was a strong inverse relationship between M-value and BMI. The most striking abnormality was significantly reduced insulin-induced activation of p42/44 MAP kinase, measured by specific assay, in the volunteers with poor insulin sensitivity. However, there was no relationship between individuals'' BMI or M-value and protein expression/phosphorylation of IRS1, PKB, or p42/44 MAP kinase protein, under basal or hyperinsulinaemic conditions. In the few individuals with poor insulin sensitivity but preserved p42/44 MAP kinase activation, other signalling defects were evident. These findings implicate defective p42/44 MAP kinase signalling as a potential contributor to obesity-related IR in a non-diabetic population, although clearly multiple signalling defects underlie obesity associated IR.     

Syntaxin Binding Protein 1 Is Not Required for Allergic Inflammation via IgE-Mediated Mast Cell Activation

Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor) proteins and SM (Sec1/Munc18) family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1), a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs) from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W^sh mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.     

Natural born killers: an invasive amphipod is predatory throughout its life-history

Introduced predators can have profound impacts on prey populations, with subsequent ramifications throughout entire ecosystems. However, studies of predator–prey interaction strengths in community and food-web analyses focus on adults or use average body sizes. This ignores ontogenetic changes, or lack thereof, in predatory capabilities over the life-histories of predators. Additionally, large individual predators might not be physically capable of consuming very small prey individuals. Both situations are important to resolve, as native prey may or may not therefore experience ontogenetic or size refuges from invasive predators. Here, we find that the freshwater amphipod invader, Gammarus pulex, is predatory throughout its development from juvenile through to adult. All size classes collected in the field had a common prey, nymphs of the mayfly Baetis rhodani, in their guts. In an experiment with predator, prey and experimental arenas scaled for body size, G. pulex juveniles and adults consumed B. rhodani in all size-matched categories. In a second experiment, the largest G. pulex individuals were able to prey on the smallest B. rhodani. Thus, the prey do not benefit from any ontogenetic or size refuge from the predator. This corroborates with the known negative population abundance relationships between this invasive predator and its native prey species. Understanding and predicting invasive predator impacts will be best served when interactions among all life-history stages of predator and prey are considered.     

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ∼3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ∼100 to ∼900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.     

Simultaneous chiral separation of methylamphetamine and common precursors using gas chromatography/mass spectrometry

Methylamphetamine, ephedrine, and pseudoephedrine were derivatized using trifluoroacetic anhydride and enantiomers of each were analyzed using gas chromatography coupled to mass spectrometry (GC/MS) fitted with a γ‐cyclodextrin (Chiraldex™ G‐PN) chiral column. A temperature‐programmed method was developed and optimized and the results compared with those obtained using a previously published isothermal GC method applied to GC/MS analysis. Trifluoroacetylated 3‐(trifluoromethyl)phenethylamine hydrochloride was used as an internal standard, and mass fragmentation patterns are proposed for all derivatives analyzed. Qualitative validation of the optimized chromatographic conditions was completed in accordance with the guidelines published by the United Nations Office on Drugs and Crime (UNODC). Under conditions of repeatability and reproducibility, the method gave relative retention times with a relative standard deviation of less than 0.02% for all six analytes of interest. This surpasses the UNODC's acceptance criteria of 2% for validation of qualitative precision. Ephedrine and pseudoephedrine are common precursors in the clandestine manufacture of methylamphetamine. Seizures of illicit methylamphetamine therefore often contain mixtures of these optically active compounds. The simultaneous enantioseparation of these compounds to produce a profile would provide valuable information to law enforcement agencies regarding the provenance of a methylamphetamine seizure. Chirality, 2011. © 2011 Wiley‐Liss, Inc.