BackgroundThymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut.ObjectiveIn human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions.MethodsWe evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo.ResultsWe showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli.ConclusionsWe have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform.

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a "universal" marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, singleparticle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca 2+ overload caused by Ca 2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca 2+ entry through plasma membrane. This results in Ca 2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei, the etiological agent of melioidosis. The crystal structure of OppA(Bp), reported here at 2.1 Å resolution, was the basis for a computational analysis that identified three potential epitopes. In parallel, antigen proteolysis and immunocapturing allowed us to identify three additional peptides. All six potential epitopes were synthesized as free peptides and tested for their immunoreactivity against sera from healthy seronegative, healthy seropositive, and recovered melioidosis patients. Three synthetic peptides allowed the different patient groups to be distinguished, underlining the potential of this approach. Extension of the computational analysis, including energy-based decomposition methods, allowed rationalizing results of the predictive analyses and the immunocapture epitope mapping. Our results illustrate a structure-based epitope discovery process, whose application may expand our perspectives in the diagnostic and vaccine design fields.