Yield and proliferation rate of adipose-derived stromal cells as a function of age,body mass index and harvest site—increasing the yield by use of adherent and supernatant fractions?

Background aimsAdipose-derived stem cells are easily accessed and have a relatively high density compared with other mesenchymal stromal cells. Isolation protocols of adipose-derived stem cells (ASC) rely on the cell's ability to adhere to tissue culture plastic overnight. It was evaluated whether the floating ASC fractions are also of interest for cell-based therapies. In addition, the impact of age, body mass index (BMI) and harvest site was assessed.MethodsThe surface protein profile with the use of flow cytometry, the cell yield and the doubling time of passages 4, 5 and 6 of ASC from 30 donors were determined. Adherent and supernatant fractions were compared. The impact of age, BMI and harvest site on cell yield and doubling times was determined.ResultsBoth adherent and supernatant fractions showed high mean fluorescence intensities for CD13, CD29, CD44, CD73, CD90 and CD105 and comparatively low mean fluorescence intensities for CD11b, CD62L, intracellular adhesion molecule-1 and CD34. Doubling times of adherent and supernatant fractions did not differ significantly. Whereas the old age group had a significantly lower cell yield compared with the middle aged group, BMI and harvest site had no impact on cell yield. Finally, doubling times for passages 4, 5 and 6 were not influenced by the age and BMI of the donors, nor the tissue-harvesting site.ConclusionsThe floating ASC fraction is an equivalent second cell source just like the adherent ASC fraction. Donor age, BMI and harvest site do not influence cell yield and proliferation rate.     

Notes on Scleroderma,Puff balls and Geastrum of Azad Jammu and Kashmir,Pakistan

Six species of mushrooms allied to the Family Sclerodermataceae, Lycoperdaceae and Geastraceae have been described for the first time from Azad Jammu and Kashmir. These are Scleroderma aurantium, Calvatia verrucosia sp. nov., Lycoperdon pedicellaton sp. nov. L. sphaericon sp. nov., L. echinulaton sp. nov., and Geastrum heptaplex sp. nov.     

Effect of p53 haploinsufficiency on melphalan-induced genotoxic effects in mouse bone marrow and peripheral blood

Mice heterozygous for a p53 null mutation develop tumours induced by genotoxic carcinogens with a shorter latency than wild type mice and have been proposed as an alternate animal model for carcinogenicity testing. Some literature data suggest that p53+/- mice might also be more sensitive to the short-term effects of genotoxic agents and manifest a haploinsufficiency phenotype that could contribute to the higher tumour susceptibility. We have compared the induction of micronuclei in bone marrow and blood of p53+/- and p53+/+ isogenic mice after treatment with a single or multiple doses of melphalan (MLP), a crosslinking genotoxic carcinogen. We have also characterized the mechanism of micronucleus induction with CREST staining of kinetochore proteins to distinguish between chromosome break- and chromosome loss-induced micronuclei. Significant increases of micronucleated bone marrow polychromatic erythrocytes and blood reticulocytes were induced under all MLP exposure conditions. The frequency of micronucleated blood erythrocytes increased linearly with duration of exposure. Micronuclei were essentially a consequence of chromosome break events. After a single MLP dose, a significant reduction of the frequency of polychromatic erythrocytes in bone marrow of p53+/+ animals suggested the induction of cytotoxicity/cell cycle delay. This effect was not observed in p53+/- mice. We believe this finding to provide some evidence of a haploinsufficiency phenotype in the modulation of cell cycle/apoptotic pathways mediated by the p53 protein. In bone marrow of wild type mice, an increased effect of multiple MLP doses was detected over that of a single administration, whereas, in p53+/- mice, no differential effect was found of different exposure durations. Possibly, the probability of micronucleus formation increased under chronic exposure because of increased cell division in response to peripheral anemia and a reduction of p53 protein level had a small effect on cell cycle modulation and on such indirect mechanism of micronucleus induction. However, pairwise comparisons between the frequencies of cells with micronuclei in wild type and p53+/- mice under all exposure conditions did not show statistically significant differences, suggesting that the observed effects of p53 haploinsufficiency were weak and temporary and a higher/faster induction of irreversible chromosome damage could not account for the increased susceptibility of p53+/- mice to MLP-induced tumours.     

Head-to-nerve analysis of electromechanical impairments of diffuse axonal injury

Biomechanics and Modeling in Mechanobiology - The aim was to investigate mechanical and functional failure of diffuse axonal injury (DAI) in nerve bundles following frontal head impacts, by finite...     

Resequencing the susceptibility gene,ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Introduction

The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods

We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results

Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.

Conclusions

Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.     

Multiparameter telemetry as a sensitive screening method to detect vaccine reactogenicity in mice

Refined vaccines and adjuvants are urgently needed to advance immunization against global infectious challenges such as HIV, hepatitis C, tuberculosis and malaria. Large-scale screening efforts are ongoing to identify adjuvants with improved efficacy profiles. Reactogenicity often represents a major hurdle to the clinical use of new substances. Yet, irrespective of its importance, this parameter has remained difficult to screen for, owing to a lack of sensitive small animal models with a capacity for high throughput testing. Here we report that continuous telemetric measurements of heart rate, heart rate variability, body core temperature and locomotor activity in laboratory mice readily unmasked systemic side-effects of vaccination, which went undetected by conventional observational assessment and clinical scoring. Even minor aberrations in homeostasis were readily detected, ranging from sympathetic activation over transient pyrogenic effects to reduced physical activity and apathy. Results in real-time combined with the potential of scalability and partial automation in the industrial context suggest multiparameter telemetry in laboratory mice as a first-line screen for vaccine reactogenicity. This may accelerate vaccine discovery in general and may further the success of vaccines in combating infectious disease and cancer.     

Analysis of a genome-wide association study-linked locus (CCR6) in Asian rheumatoid arthritis

A genome-wide association study in Japan identified the C-C chemokine receptor type 6 gene (CCR6) as associated with rheumatoid arthritis (RA). This finding has not been validated in other Asian populations. A case-control study involving 996 subjects, comprising 440 controls and 556 RA patients, was done to determine their anticyclic citrullinated peptide (anti-CCP) antibody status and CCR6 polymorphism (rs3093024) genotype. Three hundred eighty-seven patients were anti-CCP positive and 153 anti-CCP negative. Logistic regression showed that allele A was likely to increase the risk of developing RA among females via a recessive model (odds ratio [OR]=1.55, 95% confidence interval [CI]=1.01, 2.39), whereas the risk effect appeared to be reduced among males via an additive model (OR=0.60, 95% CI=0.42, 0.85). Considering only subjects who are anti-CCP positive, allele A increased RA risk among females via a recessive model (OR=1.68, 95% CI=1.07, 2.64) but decreased the risk among males via an additive model (OR=0.59, 95% CI=0.39, 0.89). We showed that CCR6 polymorphism was a risk factor among females but a protective factor among males. Functional studies are warranted to unravel the pathophysiological relevance of the gene variant and other linked variants with RA.     

SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.     

Biodegradable thermoplastic composites based on polyvinyl alcohol and algae

Algae constitute a largely available, low value material from renewable resources of marine origin to be used for the production of eco-compatible composites. Fibers of the green alga Ulva armoricana from the French coast were positively evaluated for the production of composites with a hydrophilic, eco-compatible polymer, such as poly(vinyl alcohol) (PVA) as continuous matrix by casting of aqueous suspensions and compression molding. PVA, Ulva, and starch were also successfully processed by the melt in the presence of glycerol. Positive results were obtained for film-forming properties and mechanical characteristics also with limited amounts of PVA (40%) attesting for Ulva suitability to be introduced in composites (up to 30%). Degradation in soil of Ulva and an Ulva-based composites outlined a rapid mineralization of Ulva in the selected medium (over 80% in 100 days) while the composite samples underwent a mineralization rate affected by the different component propensity to degradation.