Does smoking, age or gender affect the protein phenotype of extracellular vesicles in plasma?

AbstractExtracellular vesicles (EVs) are involved in several diseases, which have formed the basis for the potential use of EV analyses in a clinical setting. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers.With this extensive study of 161 healthy individuals it was elucidated that certain markers of plasma EVs are influenced by demographic variations such as gender, age and smoking status. When the purpose is to use EVs as a diagnostic tool, it should be emphasized how important it is to choose the correct demographic group when comparing marker levels of plasma EVs

Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells

BACKGROUND: Wnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain. RESULTS: By combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin(−) Sca-1(+) c-Kit(+) cells. Surprisingly, ND lacking Wnt3a contributed to Lin(−) Sca-1(+) c-Kit(+) cell expansion, an effect that was not mediated through β-catenin. CONCLUSIONS: The data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12951-016-0218-5) contains supplementary material, which is available to authorized users

New Enhancing MRI Lesions Associate with IL-17, Neutrophil Degranulation and Integrin Microparticles: Multi-Omics Combined with Frequent MRI in Multiple Sclerosis

Background: Blood–barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood–barrier disruption as the initial event of the evolution of new lesions. Methods: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs. Results: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1β. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1β levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution. Conclusions: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1β clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation.Background: Blood-barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood-barrier disruption as the initial event of the evolution of new lesions.Methods: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs.Results: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1β. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1β levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution.Conclusions: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1β clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors

Cancer treatment: the combination of vaccination with other therapies

Harnessing of the immune system by the development of ‘therapeutic’ vaccines, for the battle against cancer has been the focus of tremendous research efforts over the past two decades. As an illustration of the impressive amounts of data gathered over the past years, numerous antigens expressed on the surface of cancer cells, have been characterized. To this end, recent years research has focussed on characterization of antigens that play an important role for the growth and survival of cancer cells. Anti-apoptotic molecules like survivin that enhance the survival of cancer cells and facilitate their escape from cytotoxic therapies represent prime vaccination candidates. The characterization of a high number of tumor antigens allow the concurrent or serial immunological targeting of different proteins associated with such cancer traits. Moreover, while vaccination in itself is a promising new approach to fight cancer, the combination with additional therapy could create a number of synergistic effects. Herein we discuss the possibilities and prospects of vaccination when combined with other treatments. In this regard, cell death upon drug exposure may be immunogenic or non-immunogenic depending on the specific chemotherapeutics. Also, chemotherapy represents one of several options available for clearance of CD4+ Foxp3+ regulatory T cells. Moreover, therapies based on monoclonal antibodies may have synergistic potential in combination with vaccination, both when used for targeting of tumor cells and endothelial cells. The efficacy of therapeutic vaccination against cancer will over the next few years be studied in settings taking advantage of strategies in which vaccination is combined with other treatment modalities. These combinations should be based on current knowledge not only regarding the biology of the cancer cell per se, but also considering how treatment may influence the malignant cell population as well as the immune system

Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction

This work was funded by National Institutes of Health (NIH; http://www.nih.gov) Grants R01EY024140 and R21EY022466 (to M.C.C.) and R01EY019494 (to M.H.E.). Our research is also funded in part by NIH Core Grant P30EY021725 (to Robert E. Anderson, OUHSC) and an unrestricted grant from Research to Prevent Blindness Inc. (http://www.rpbusa.org) to the Dean A. McGee Eye Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank Bolanle Adebayo (Cameron University, Lawton OK), Craig Land (Oklahoma State University, Stillwater OK), Nathan Jia (Oklahoma Christian University, Edmond OK), Kobbe Wiafe (Oklahoma School of Science and Mathematics, Oklahoma City OK), and Amanda Roehrkasse and Madhu Parkunan (OUHSC) for intellectual discussions and technical assistance. The authors also acknowledge thank Nanette Wheatley, Dr. Feng Li, and Mark Dittmar (OUHSC Live Animal Imaging Core, P30EY021725) for their invaluable technical assistance.This work was presented in part at the 2014 Association for Research in Vision and Ophthalmology Annual Conference in Orlando FL.The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.Yeshttp://www.plosone.org/static/editorial#pee