Characterization of extracellular vesicles produced by single human embryos at early stages of development

Background: Extracellular vesicles (EVs) are recognized as potent vehicles for intercellular communication. To date, there is little information available regarding the role of EVs during the early stages of human embryonic development. The aim of this study was to develop techniques for the recovery of EVs secreted by a single human embryo in an in vitro culture system. The EVs were characterized according to size, concentration and electrical surface properties (zeta potential), in order to understand the role of EVs production in human embryos for determination of their quality at early stages of development. Methods: Human embryos were produced by in vitro fertilization (IVF) for 24 h in fertilization medium, cultured individually for 48 h (3 days) in cleavage medium and additionally 48 h in blastocyst medium (day 5). Conditioned media, at days 3 and 5 post-IVF, was collected and EVs were isolated using a series of centrifugations and size-exclusion chromatography. The size, concentration and zeta potential of EVs were characterized using a nanoparticle tracking analysis. Results: Using this method of isolation, we were able to collect and characterize EVs produced by a single human embryo. Analysis confirmed the presence of EVs at early stages of development, with the concentration of EVs being higher in early blastocysts (day 5), as compared to 4-8 cell-stage embryos (day 3). Moreover, already at day 3, we were able to discriminate between embryos that were properly developing and those that were later visually determined as degrading at day 5. The data indicates that embryos following normal development at day 3 but degrading at later stages (day 5) were producing significantly higher number of EVs (with size range of 100-160 nm) compared with those developing properly at day 3 and later progressing to early blastocysts at day 5. Summary/conclusion: In conclusion, we have developed a sensitive protocol for the isolation of EVs from human embryos cultured individually. We have demonstrated that human embryos secrete EVs in varying amounts and sizes during the early stages of their development. Further investigations are needed to establish EV characteristics of early human embryo as a quality marker for human clinical embryology

Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T-cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection

Background This randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. Methods Twelve HIV-1+ patients on suppressive cART with baseline CD4 T-cell counts >400 cells/mm3 blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n = 3); (2) vaccine alone (n = 4); or (3) IL-2, GM-CSF and rhGH (n = 5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. Results Median baseline CD4 T-cell count was 757 cells/mm3 (interquartile range [IQR] 567–886 cells/mm3), median age 48 years (IQR 42–51 years), and plasma HIV-1-RNA <50 copies/ml for all subjects. Patients who received vaccine plus IL-2, GM-CSF and rhGH (group 1) showed the most marked changes. Assessing mean changes from baseline to week 48 revealed significantly elevated numbers of CD4 T cells (p = 0.0083) and improved CD4/CD8 T-cell ratios (p = 0.0033). This was accompanied by a significant reduction in expression of CD38 on CD4 T cells (p = 0.0194), significantly increased IFN-γ and IL-2 production in response to Gag (p = 0.0122) and elevated IFN-γ production in response to Tat (p = 0.041) at week 48 compared to baseline. Subjects in all treatment groups showed significantly reduced PD-1 expression at week 48 compared to baseline, with some reductions in proviral DNA. Conclusions Multifarious immunotherapeutic approaches in the context of fully suppressive cART further reduce immune activation, and improve both CD4 T-lymphocyte counts and HIV-1-specific T-cell responses (NCT01130376)

Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies

Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration

MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription–polymerase chain reaction (RT–PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites