Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30–100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-γ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3+CD4+CD25+ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.

The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S phase transition of the cell cycle in normal lymphocytes. Surprisingly, Skp2 enhances c-Myc-induced S phase transition and activates c-Myc target genes in a Myc-dependent manner. Further, Myc-induced transcription was shown to be Skp2 dependent, suggesting interdependence between c-Myc and Skp2 in activation of transcription. Moreover, Myc-dependent association of Skp2, ubiquitylated proteins, and subunits of the proteasome to a c-Myc target promoter was demonstrated in vivo. The results suggest that Skp2 is a transcriptional cofactor for c-Myc and indicates a close relationship between transcription activation and transcription factor ubiquitination.

Exosomes from APC are nano-vesicles that can induce antigen-specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated. We could detect exosome-induced antigen-specific CD8 + T cell responses in peripheral blood from humans. Exosomes from monocyte-derived DC (MDDC) were loaded with a mix of 23 immunogenic peptides from EBV, CMV and influenza virus, and added to autologous peripheral CD8 + T cells. IFN-c-producing cells were detected by enzyme-linked immunospot assay (ELISPOT). MDDC-exosomes induced IFN-c production in CD8 + T cells without addition of DC. The response was exosome dose dependent, and dependent on exosomal MHC class I. Furthermore, we detected an enhanced T cell stimulatory capacity by exosomes from lipopolysaccharide-matured MDDC compared to exosomes from immature MDDC. Exosomes could also induce TNF-a production. These results show, for the first time, that exosomes can directly stimulate human peripheral CD8 + T cells in an antigen-specific manner and that ELISPOT is a suitable method for detecting exosome-induced peripheral T cell responses. This system may provide a useful tool when developing exosomes as therapeutic agents.

No abstract

We investigated the effect of spherical gold nanoparticles on immature dendritic cells (DCs). Conventionally produced nanoparticles had a maturating effect on the DCs--a result of lipopolysaccharide (LPS) contamination. By modification of the production process, low-LPS particles were obtained, which had practically no effect on phenotypic maturation or cytokine production of the DCs. Our findings emphasize the importance of high purity in the production of nanoparticles, since possible contaminants may interfere with the assessment of biological/medical effects. They also highlight that nanoparticles can function as carriers of immune modulating contaminants.