The epithelial barrier and immunoglobulin A system in allergy.

Airway and intestinal epithelial layers represent first-line physical barriers, playing a key role in mucosal immunity. Barrier dysfunction, characterized by alterations such as disruption of cell-cell apical junctions and aberrant epithelial responses, probably constitutes early and key events for chronic immune responses to environmental antigens in the skin and in the gut. For instance, barrier dysfunction drives Th2 responses in atopic disorders or eosinophilic esophagitis. Such epithelial impairment is also a salient feature of allergic asthma and growing evidence indicates that barrier alterations probably play a driving role in this disease. IgA has been identified as the most abundant immunoglobulin in mucosa, where it acts as an active barrier through immune exclusion of inhaled or ingested antigens or pathogens. Historically, it has been thought to represent the serum factor underlying reaginic activity before IgE was discovered. Despite several studies about regulation and major functions of IgA at mucosal surfaces, its role in allergy remains largely unclear. This review aims at summarizing findings about epithelial functions and IgA biology that are relevant to allergy, and to integrate the emerging concepts and the recent developments in mucosal immunology, and how these could translate to clinical observations in allergy.

Loss of EZH2-like or SU(VAR)3-9-like proteins causes simultaneous perturbations in H3K27 and H3K9 tri-methylation and associated developmental defects in the fungus Podospora anserina.

BACKGROUND: Selective gene silencing is key to development. It is generally accepted that H3K27me3-enriched heterochromatin maintains transcriptional repression established during early development and regulates cell fate. Conversely, H3K9me3-enriched heterochromatin prevents differentiation but constitutes protection against transposable elements. We exploited the fungus Podospora anserina, a valuable alternative to higher eukaryote models, to question the biological relevance and functional interplay of these two distinct heterochromatin conformations. RESULTS: We established genome-wide patterns of H3K27me3 and H3K9me3 modifications, and found these marks mutually exclusive within gene-rich regions but not within repeats. We generated the corresponding histone methyltransferase null mutants and showed an interdependence of H3K9me3 and H3K27me3 marks. Indeed, removal of the PaKmt6 EZH2-like enzyme resulted not only in loss of H3K27me3 but also in significant H3K9me3 reduction. Similarly, removal of PaKmt1 SU(VAR)3-9-like enzyme caused loss of H3K9me3 and substantial decrease of H3K27me3. Removal of the H3K9me binding protein PaHP1 provided further support to the notion that each type of heterochromatin requires the presence of the other. We also established that P. anserina developmental programs require H3K27me3-mediated silencing, since loss of the PaKmt6 EZH2-like enzyme caused severe defects in most aspects of the life cycle including growth, differentiation processes and sexual reproduction, whereas loss of the PaKmt1 SU(VAR)3-9-like enzyme resulted only in marginal defects, similar to loss of PaHP1. CONCLUSIONS: Our findings support a conserved function of the PRC2 complex in fungal development. However, we uncovered an intriguing evolutionary fluidity in the repressive histone deposition machinery, which challenges canonical definitions of constitutive and facultative heterochromatin.

Dynamics of polymorphic nanostructures: from growth to collapse.

The deposition of preformed clusters on surfaces offers new possibilities to build complex artificial nanostructures, the shape of which depends on the cluster size. We describe routes for generating unusual polymorphic nanoislands, which constitute unique platforms for exploring instabilities. As coverage increases, the constraints accumulated in such nanostructures induce spectacular flattening collapse processes, which are not observed when the constraints are imposed by the substrate.

Instability driven fragmentation of nanoscale fractal islands.

Formation and evolution of fragmentation instabilities in fractal islands, obtained by deposition of silver clusters on graphite, are studied. The fragmentation dynamics and subsequent relaxation to the equilibrium shapes are controlled by the deposition conditions and cluster composition. Sharing common features with other materials' breakup phenomena, the fragmentation instability is governed by the length-to-width ratio of the fractal arms.

One step screening of retroviral producer clones by real time quantitative PCR.

BACKGROUND: Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. METHODS: We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. RESULTS: We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. CONCLUSION: We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.