Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways

WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the β-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced β-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the β-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.     

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.     

Trophic niche partitioning of cryptic species of long-eared bats in Switzerland: implications for conservation

Dietary niche partitioning is postulated to play a major role for the stable coexistence of species within a community, particularly among cryptic species. Molecular markers have recently revealed the existence of a new cryptic species of long-eared bat, Plecotus macrobullaris, in the European Alps. We studied trophic niches as well as seasonal and regional variations of diet in eight colonies of the three Plecotus species occurring in Switzerland. Faeces were collected monthly from individuals returning to roost after foraging. Twenty-one arthropod categories were recognized from the faeces. All three species fed predominantly on Lepidoptera, which made up 41%, 87% and 88% (means across colonies) of the diet composition of P. auritus, P. macrobullaris and P. austriacus, respectively. The occurrence of numerous fragments of both diurnal and flightless insects in the diet of P. auritus (but rarely in the diet of the other two species) indicates that this species mostly gleans prey from substrates. P. austriacus and P. macrobullaris are more typical aerial feeders. The latter two species have narrow trophic niches, whilst P. auritus has a much broader diet. Comparison of intraspecific and interspecific niche overlaps in P. auritus and P. macrobullaris in sympatry suggests dietary niche partitioning between these two species. In contrast, the high similarity of the trophic niches of P. austriacus and P. macrobullaris, associated with a typical parapatric distribution, indicates competitive exclusion. The best conservation measures are preservation and restoration of habitats offering a high abundance of moths, the major prey of the three Plecotus species.     

DNA-methylation profiling distinguishes malignant melanomas from benign nevi

DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma. We determined technical feasibility for high-throughput DNA-methylation array-based profiling using formalin-fixed paraffin-embedded tissues for selection of candidate DNA-methylation differences between melanomas and nevi. Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray. Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β-value differences of ≥ 0.2. Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of > 0.95. Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons. This first report of a DNA-methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.     

Changes in waist circumference and the incidence of diabetes in middle-aged men and women

Background

Waist circumference (WC) is positively associated with diabetes, but the association with changes in WC (DWC) is less clear. We investigated the association between DWC and the subsequent risk of diabetes in middle-aged men and women, and evaluated the influence from concurrent changes in body mass index (DBMI).

Methodology/Principal Findings

Data on 15,577 men and 20,066 women from the Danish Diet, Cancer and Health study were analyzed. Anthropometry was assessed in 1993–97 and 1999–02. Information on diabetes was obtained from The Danish National Diabetes Register. Hazard ratios (HR) were calculated from Cox'' proportional hazard models with individuals considered at risk from 1999–02 until December 31 2006. During 5.4 years of follow-up, 1,027 and 876 new cases of diabetes occurred among men and women, respectively. WC was positively associated with diabetes in both sexes also with adjustment for covariates and BMI. DWC was positively associated with diabetes in women, but not in men (HR per 5 cm change = 1.09 (1.04∶1.15) in women, and 1.00 (0.94, 1.07) in men with adjustment for covariates, baseline WC, BMI and DBMI). Associations with DWC were not notably different in sub-groups stratified according to baseline WC or DBMI, or when individuals with diseases or diabetes occurring within the first years of follow-up were excluded.

Conclusions/Significance

While this study confirmed that WC is positively associated with the risk of diabetes in middle-aged men and women, it surprisingly showed that changes in WC were not associated with the subsequent risk of diabetes in men, and only weakly positively associated with the risk of diabetes in women. Accordingly, these findings suggest that a reduction in WC may be a weak or insufficient or target for prevention of diabetes in middle-aged men and women.     

Increased sensitivity to broadly neutralizing antibodies of end-stage disease R5 HIV-1 correlates with evolution in Env glycosylation and charge

Background

Induction of broadly neutralizing antibodies, such as the monoclonal antibodies IgGb12, 2F5 and 2G12, is the objective of most antibody-based HIV-1 vaccine undertakings. However, despite the relative conserved nature of epitopes targeted by these antibodies, mechanisms underlying the sensitivity of circulating HIV-1 variants to broadly neutralizing antibodies are not fully understood. Here we have studied sensitivity to broadly neutralizing antibodies of HIV-1 variants that emerge during disease progression in relation to molecular alterations in the viral envelope glycoproteins (Env), using a panel of primary R5 HIV-1 isolates sequentially obtained before and after AIDS onset.

Principal Findings

HIV-1 R5 isolates obtained at end-stage disease, after AIDS onset, were found to be more sensitive to neutralization by TriMab, an equimolar mix of the IgGb12, 2F5 and 2G12 antibodies, than R5 isolates from the chronic phase. The increased sensitivity correlated with low CD4⁺ T cell count at time of virus isolation and augmented viral infectivity. Subsequent sequence analysis of multiple env clones derived from the R5 HIV-1 isolates revealed that, concomitant with increased TriMab neutralization sensitivity, end-stage R5 variants displayed envelope glycoproteins (Envs) with reduced numbers of potential N-linked glycosylation sites (PNGS), in addition to increased positive surface charge. These molecular changes in Env also correlated to sensitivity to neutralization by the individual 2G12 monoclonal antibody (mAb). Furthermore, results from molecular modeling suggested that the PNGS lost at end-stage disease locate in the proximity to the 2G12 epitope.

Conclusions

Our study suggests that R5 HIV-1 variants with increased sensitivity to broadly neutralizing antibodies, including the 2G12 mAb, may emerge in an opportunistic manner during severe immunodeficiency as a consequence of adaptive molecular Env changes, including loss of glycosylation and gain of positive charge.