Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia

Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two a-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.

A calcium-dependent interaction between calmodulin and the calponin homology domain of human IQGAP1

IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered.

Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells

Umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB-ECFC with human fetal-mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p <.001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC-conditioned media, leading to 1.8-2.2× higher ALP levels and a 1.4-1.5× increase in calcium deposition (p <.01) in a dose-dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF-ßs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB-ECFC. Subcutaneous implantation of UCB-ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2-fold increase in host neovascularization compared to hfMSC-only implants (p = .001). We conclude that this study shows that UCB-ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB-ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower.

Structure-Phenotype Correlations of Human CYP21A2 Mutations in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by adrenal cortex. The predominant causes of the disorder are mutations in the CYP21A2 gene that encodes a Cytochrome P450 21-hydroxylase enzyme, which is central to steroidogenesis. The severity of the disease depends upon the extent of impaired enzymatic activity and can be classified under severe Classical form or the mild Non-Classical form, Molecular characterisation of CYP21A2 mutations can be used to predict clinical phenotype and disease severity based upon changes it brings in 21-hydroxylase enzyme structure. A humanized model of CYP21A2 has been used to map and investigate the structural role of all known disease-causing mutations. A structural explanation of clinical manifestation allows us to put forward criteria that might allow the prediction of clinical severity of the disease.

Independent and Interdependent Immunoregulatory Effects of IL-27, IFN-β, and IL-10 in the Suppression of Human Th17 Cells and Murine Experimental Autoimmune Encephalomyelitis

IFN-ß, IL-27, and IL-10 have been shown to exert a range of similar immunoregulatory effects in murine and human experimental systems, particularly in Th1- and Th17-mediated models of autoimmune inflammatory disease. In this study we sought to translate some of our previous findings in murine systems to human in vitro models and delineate the interdependence of these different cytokines in their immunoregulatory effects. We demonstrate that human IL-27 upregulates IL-10 in T cell-activated PBMC cultures and that IFN-ß drives IL-27 production in activated monocytes. IFN-ß-driven IL-27 is responsible for the upregulation of IL-10, but not IL-17 suppression, by IFN-ß in human PBMCs. Surprisingly, IL-10 is not required for the suppression of IL-17 by either IL-27 or IFN-ß in this model or in de novo differentiating Th17 cells, nor is IL-27 signaling required for the suppression of experimental autoimmune encephalomyelitis (EAE) by IFN-ß in vivo. Furthermore, and even more surprisingly, IL-10 is not required for the suppression of Th17-biased EAE by IL-27, in sharp contrast to Th1-biased EAE. In conclusion, IFN-ß and IL-27 both induce human IL-10, both suppress human Th17 responses, and both suppress murine EAE. However, IL-27 signaling is not required for the therapeutic effect of IFN-ß in EAE. Suppression of Th17-biased EAE by IL-27 is IL-10-independent, in contrast to its mechanism of action in Th1-biased EAE. Taken together, these findings delineate a complex set of interdependent and independent immunoregulatory mechanisms of IFN-ß, IL-27, and IL-10 in human experimental models and in murine Th1- and Th17-driven autoimmunity.

Determining mycotoxins and mycotoxigenic fungi in food and feed:Developing biomarkers of human exposure to mycotoxins

Exposure assessment is a critical part of epidemiological studies into the effect of mycotoxins on human health. Whilst exposure assessment can be made by estimating the quantity of ingested toxins from food analysis and questionnaire data, the use of biological markers (biomarkers) of exposure can provide a more accurate measure of individual level of exposure in reflecting the internal dose. Biomarkers of exposure can include the excreted toxin or its metabolites, as well as the products of interaction between the toxin and macromolecules such as protein and DNA. Samples in which biomarkers may be analysed include urine, blood, other body fluids and tissues, with urine and blood being the most accessible for human studies. Here we describe the development of biomarkers of exposure for the assessment of three important mycotoxins; aflatoxin, fumonisin and deoxynivalenol. A number of different biomarkers and methods have been developed that can be applied to human population studies, and these approaches are reviewed in the context of their application to molecular epidemiology research.

Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses

In animal models, variations in early maternal care are associated with differences in hypothalamic-pituitary-adrenal(HPA) stress response in the offspring, mediated via changes in the epigenetic regulation of glucocorticoid receptor (GR) gene (Nr3c1) expression.

Therapeutic Effects of Human Mesenchymal Stem Cells in Ex Vivo Human Lungs Injured with Live Bacteria

Rationale: Mesenchymal stem cells secrete paracrine factors that can regulate lung permeability and decrease inflammation, making it a potentially attractive therapy for acute lung injury. However, concerns exist whether mesenchymal stem cells' immunomodulatory properties may have detrimental effects if targeted toward infectious causes of lung injury. Objectives: Therefore, we tested the effect of mesenchymal stem cells on lung fluid balance, acute inflammation, and bacterial clearance. Methods: We developed an Escherichia coli pneumonia model in our ex vivo perfused human lung to test the therapeutic effects of mesenchymal stem cells on bacterial-induced acute lung injury. Measurements and Main Results: Clinical-grade human mesenchymal stem cells restored alveolar fluid clearance to a normal level, decreased inflammation, and were associated with increased bacterial killing and reduced bacteremia, in part through increased alveolar macrophage phagocytosis and secretion of antimicrobial factors. Keratinocyte growth factor, a soluble factor secreted by mesenchymal stem cells, duplicated most of the antimicrobial effects. In subsequent in vitro studies, we discovered that human monocytes expressed the keratinocyte growth factor receptor, and that keratinocyte growth factor decreased apoptosis of human monocytes through AKT phosphorylation, an effect that increased bacterial clearance. Inhibition of keratinocyte growth factor by a neutralizing antibody reduced the antimicrobial effects of mesenchymal stem cells in the ex vivo perfused human lung and monocytes grown in vitro injured with E. coli bacteria. Conclusions: In E. coli-injured human lungs, mesenchymal stem cells restored alveolar fluid clearance, reduced inflammation, and exerted antimicrobial activity, in part through keratinocyte growth factor secretion.

Antibacterial Effect of Human Mesenchymal Stem Cells is Mediated in Part from Secretion of the Antimicrobial Peptide LL-37

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

The effect of human-body shadowing on indoor UWB TOA-based ranging systems

Because of its superior time resolution, ultra-wide bandwidth (UWB) transmission can be a highly accurate technique for ranging in indoor localization systems. Nevertheless, the presence of obstructions may deteriorate the ranging performance of the system. Indoor environments are often densely populated with people. However, t h e effect of the human body presence has been scarcely investigated so far within the UWB ranging context. In this work, we investigate this effect by conducting UWB measurements and analyzing the ranging performance of the system. Two measurement campaigns were performed in the 3-5.5 GHz band, in an anechoic chamber and a laboratory environment. The range estimates were obtained by employing the threshold-based first peak detection technique. Analysis results revealed that the human body significantly attenuates the direct-path signal component. On the other hand, in this study it does not introduce a significant range error since the length difference between the diffracted paths around the body and the direct-path is less than the spatial resolution of the measurement setup. © 2012 IEEE.

Ex Vivo Expansion of Human Outgrowth Endothelial Cells Leads to IL-8-Mediated Replicative Senescence and Impaired Vasoreparative Function

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue-engineering requires efficient ex-vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex-vivo expansion, which is associated with enlarged cell size, ß-galactosidase activity, DNA damage, tumour suppressor pathway activation and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in-vitro and in-vivo. Integrated transcriptome-proteome analysis identified inflammatory signalling pathways as major mechanistic components of the OEC senescence programme. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex-vivo lifespan, delayed replicative senescence and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. The current study also suggests that modulation of the senescence-associated secretory phenotype (SASP) could be used to optimise OEC therapy.

Effect of in vitro non-enzymatic glycosylation of human skin collagen on susceptibility to collagenase digestion

The effect of glycosylation on susceptibility of skin collagen to collagenase digestion was studied in a skin sample obtained at autopsy from the interscapular region of a 24 year old white male who had died of an acute illness and who had no history of diabetes. Homogeneous suspensions of insoluble collagen were prepared, and were incubated in 50 mmol l-1 dextrose at pH 7.35 and 37 degrees C for 7 days. Non-enzymatic glycosylation measured by the weak acid hydrolysis/thiobarbituric acid method increased from 13.1 +/- 1.0 (n = 5) to 45.2 +/- 5.5 (n = 8) nmol fructose per 10 mg collagen (P less than 0.001). Digestion of collagen using clostridial collagenase was monitored by measuring (a) hydroxyproline content and (b) absorption at 206 nm of the supernatant after centrifugation to remove substrate. The rate of digestion was similar in glycosylated and control collagen. We conclude that the ketoamine link formed in non-enzymatic glycosylation does not increase the resistance of collagen to enzymatic digestion. The possibility remains that subsequent rearrangement of this link could be important in this respect.