Resistin,Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens

Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present in the form of ulcerative colitis (UC) or as Crohn’s disease (CD). Biomarkers are needed for reliable diagnosis and disease monitoring in IBD, especially in pediatric patients. Plasma samples from a pediatric IBD cohort were interrogated using an aptamer-based screen of 1322 proteins. The elevated biomarkers identified using the aptamer screen were further validated by ELISA using an independent cohort of 76 pediatric plasma samples, drawn from 30 CD, 30 UC, and 16 healthy controls. Of the 1322 proteins screened in plasma from IBD patients, 129 proteins were significantly elevated when compared with healthy controls. Of these 15 proteins had a fold change greater than 2 and 28 proteins had a fold change >1.5. Neutrophil and extracellular vesicle signatures were detected among the elevated plasma biomarkers. When seven of these proteins were validated by ELISA, resistin was the only protein that was significantly higher in both UC and CD (p < 0.01), with receiver operating characteristic area under the curve value of 0.82 and 0.77, respectively, and the only protein that exhibited high sensitivity and specificity for both CD and UC. The next most discriminatory plasma proteins were elastase and lactoferrin, particularly for UC, with receiver operating characteristic area under the curve values of 0.74 and 0.69, respectively. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients using two independent diagnostic platforms and two independent patient cohorts.     

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

Background

Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.

Aim

Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).

Methods and Results

Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.

Conclusion

Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.     

Exosomes derived from MSC pre-treated with oridonin alleviates myocardial IR injury by suppressing apoptosis via regulating autophagy activation

This study aimed to investigate the molecular mechanisms underlying the role of bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes in ischaemia/reperfusion (IR)-induced damage, and the role of oridonin in the treatment of IR. Exosomes were isolated from BMMSCs. Western blot analysis was done to examine the expression of proteins including CD63, CD8, apoptotic-linked gene product 2 interacting protein X (AliX), Beclin-1, ATG13, B-cell lymphoma-2 (Bcl-2), apoptotic peptidase activating factor 1 (Apaf1) and Bcl2-associated X (Bax) in different treatment groups. Accordingly, the expression of CD63, CD81 and AliX was higher in BMMSCs-EXOs and IR + BMMSCs-EXOs + ORI groups compared with that in the BMMSCs group. And BMMSCs-derived exosomes inhibited the progression of IR-induced myocardial damage, while this protective effect was boosted by the pre-treatment with oridonin. Moreover, Beclin-1, ATG13 and Bcl-2 were significantly down-regulated while Apaf1 and Bax were significantly up-regulated in IR rats. And the presence of BMMSCs-derived exosomes partly alleviated IR-induced dysregulation of these proteins, while the oridonin pre-treatment boosted the effect of these BMMSCs-derived exosomes. The inhibited proliferation and promoted apoptosis of H9c2 cells induced by hypoxia/reperfusion (HR) were mitigated by the administration of BMMSCs-derived exosomes. Meanwhile, HR also induced down-regulation of Beclin-1, ATG13 and Bcl-2 expression and up-regulation of Apaf1 and Bax, which were mitigated by the administration of BMMSCs-derived exosomes. And oridonin pre-treatment boosted the effect of BMMSCs-derived exosomes. In conclusion, our results validated that BMMSCs-derived exosomes suppressed the IR-induced damages by participating in the autophagy process, while the pre-treatment with oridonin could boost the protective effect of BMMSCs-derived exosomes.     

Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes

Exosomes are 40–100 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep? density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40–100 nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2–8, EPHB1–4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling.     

Proteome analysis of circulating exosomes in health and breast cancer

Microvesicles were isolated from blood plasma and total blood of healthy females and breast cancer patients by filtration and ultracentrifugation. According to flow cytometry, different subpopulations of exosomes were represented in blood of healthy donors and cancer patients at different levels with median fluorescence intensity (MFI) values in both groups arranged in the following order: CD24/СD9 > СD9/СD81 > CD9/CD63 = CD24/CD63. Concentration of exosomes in blood plasma of healthy females estimated by nanoparticle tracking analysis (NTA) did not exceed (3.71 ± 1.15) × 10⁷ particles/mL of blood and did not differ from that in plasma of breast cancer patients, which averaged (3.99 ± 1.03) × 10⁷ particles/mL of blood. Concentration of total exosomes in blood (including exosomes from plasma and blood cell surface-bound exosomes) did not depend on the presence/absence of a tumor; the values were (7.66 ± 0.7) × 10⁷ particles/mL of healthy blood and (9.4 ± 1.24) × 10⁷ particles/mL of blood from cancer patients. Comparative analysis of exosomes using 2-D electrophoresis with subsequent analysis of 2-D proteomic maps revealed proteins missing in blood or differentially expressed in healthy females and breast cancer women. The data presented provide the possibility for identification of exosomal proteomic markers and isolation of tumor-specific exosomes, which contributes to the development of breast cancer diagnostics.     

Treatment of human neuroblastoma cell line SH-SY5Y with HSP27 siRNA tagged-exosomes decreased differentiation rate into mature neurons

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2′-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.     

Mesenchymal stroma/stem‐like cells of GARP knockdown inhibits cell proliferation and invasion of mouse colon cancer cells (MC38) through exosomes

Mesenchymal stroma/stem‐like cells (MSCs) have antitumour activity, and MSC‐derived exosomes play a role in the growth, metastasis and invasion of tumour cells. Additionally, glycoprotein A repetition predominant (GARP) promotes oncogenesis in breast cancer. Therefore, GARP is speculated to be a target gene for cancer therapy. We aimed to explore the therapy role of MSC‐derived exosomes targeting GARP in mouse colon cancer cell MC38. We successfully established a GARP knockdown system using three kinds of siRNA‐GARP in MSC cells. Exosomes were isolated from MSC and siGARP‐MSC cells, and verified by the exosome surface protein markers CD9, CD63 and CD81. GARP expression was significantly decreased in siGARP‐MSC exosomes compared with that of MSC exosomes. We found that siGARP‐MSC exosomes inhibited cell proliferation, migration and invasion of MC38 cells, using CCK‐8, colony formation, wound‐healing and Transwell invasion assays. Furthermore, siGARP‐MSC exosomes impeded IL‐6 secretion and partly inactivated JAK1/STAT3 pathway, measured using ELISA and RT‐qPCR. In conclusion, MSC‐derived exosomes targeting GARP are a potential strategy for cancer therapy.     

Genetic Association of Peptidoglycan Recognition Protein Variants with Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a common disease, includes Crohn''s disease (CD) and ulcerative colitis (UC), and is determined by altered gut bacterial populations and aberrant host immune response. Peptidoglycan recognition proteins (PGLYRP) are innate immunity bactericidal proteins expressed in the intestine. In mice, PGLYRPs modulate bacterial populations in the gut and sensitivity to experimentally induced UC. The role of PGLYRPs in humans with CD and/or UC has not been previously investigated. Here we tested the hypothesis that genetic variants in PGLYRP1, PGLYRP2, PGLYRP3 and PGLYRP4 genes associate with CD and/or UC and with gender and/or age of onset of disease in the patient population. We sequenced all PGLYRP exons in 372 CD patients, 77 UC patients, 265 population controls, 210 familial CD controls, and 24 familial UC controls, identified all polymorphisms in these populations, and analyzed the variants for significant association with CD and UC. We identified 16 polymorphisms in the four PGLYRP genes that significantly associated with CD, UC, and/or subgroups of patient populations. Of the 16, 5 significantly associated with both CD and UC, 6 with CD, and 5 with UC. 12 significant variants result in amino acid substitutions and based on structural modeling several of these missense variants may have structural and/or functional consequences for PGLYRP proteins. Our data demonstrate that genetic variants in PGLYRP genes associate with CD and UC and may provide a novel insight into the mechanism of pathogenesis of IBD.     

ExoCarta: A compendium of exosomal proteins and RNA

Exosomes, membrane microvesicles (40–100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary‐conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type‐specific proteins being incorporated in the exosomes, some of these so‐called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type‐specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type‐derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web ( http://exocarta.ludwig.edu.au ). We believe that this community resource will be of great biological importance for any future exosome analyses.     

Differences in Visceral Fat and Fat Bacterial Colonization between Ulcerative Colitis and Crohn’s Disease. An In Vivo and In Vitro Study

Crohn’s disease (CD) is notably characterized by the expansion of visceral fat with small adipocytes expressing a high proportion of anti-inflammatory genes. Conversely, visceral fat depots in ulcerative colitis (UC) patients have never been characterized. Our study aims were a) to compare adipocyte morphology and gene expression profile and bacterial translocation in omental (OM) and mesenteric (MES) adipose tissue of patients with UC and CD, and b) to investigate the effect of bacterial infection on adipocyte proliferation in vitro. Specimens of OM and MES were collected from 11 UC and 11 CD patients, processed and examined by light microscopy. Gene expression profiles were evaluated in adipocytes isolated from visceral adipose tissue using microarray and RTqPCR validations. Bacteria within adipose tissue were immuno-detected by confocal scanning laser microscopy. Adipocytes were incubated with Enterococcus faecalis and cells counted after 24h. Morphology and molecular profile of OM and MES revealed that UC adipose tissue is less inflamed than CD adipose tissue. Genes linked to inflammation, bacterial response, chemotaxis and angiogenesis were down-regulated in adipocytes from UC compared to CD, whereas genes related to metallothioneins, apoptosis pathways and growth factor binding were up-regulated. A dense perinuclear positivity for Enterococcus faecalis was detected in visceral adipocytes from CD, whereas positivity was weak in UC. In vitro bacterial infection was associated with a five-fold increase in the proliferation rate of OM preadipocytes. Compared to UC, visceral adipose tissue from CD is more inflamed and more colonized by intestinal bacteria, which increase adipocyte proliferation. The influence of bacteria stored within adipocytes on the clinical course of IBD warrants further investigations.     

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV‐1 infection

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV‐1‐infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re‐routed to non‐viral EVs in a Nef‐dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.     

Detection of Tumor Cell-Specific mRNA and Protein in Exosome-Like Microvesicles from Blood and Saliva

The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell–specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.     

Exosomes with immune modulatory features are present in human breast milk

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-gamma 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.     

不同血清量标本外泌体提取及微小RNA的检测结果比较

本文旨在探讨Qiagen exoRNeasy Serum/Plasma试剂盒提取血清标本中外泌体所需的最适血清量。采用Qiagen exoRNeasy Serum/Plasma 试剂盒分别对250、500、1 000 μL血清中的外泌体进行抽提,使用透射电子显微镜检测分离的外泌体大小和形态,蛋白质免疫印迹法检测外泌体蛋白标记CD63和TSG101的表达,实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测外泌体中微小RNA-122(microRNA-122,miR-122)的表达。结果显示,透射电子显微镜下可见血清外泌体呈圆形或椭圆形,直径30～150 nm,有完整的膜结构。蛋白免疫印迹法检测外泌体CD63和TSG101阳性。实时荧光定量PCR检测慢性乙型肝炎患者250、500、1 000 μL血清外泌体中miR-122表达量,与正常人相比,分别上调22.44、21.48、20.69倍(P＝0.42)。结果提示,在临床血清样本体积有限的情况下,采用 Qiagen exoRNeasy Serum/Plasma 试剂盒提取血清中外泌体,减少血清量至250 μL也可达到所需实验目的。     

Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)*

Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca²⁺ efflux pump, plasma membrane Ca²⁺ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4–64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.     

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm ± 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of “normal” early pregnancy.     

Identifying candidate genes for discrimination of ulcerative colitis and Crohn’s disease

In this study we aimed to screen effective biomarkers for differential diagnosis of ulcerative colitis (UC) and Crohn’s disease (CD). By using the gene expression profile dataset GSE24287 including 47 ileal CD, 27 UC and 25 non-inflammatory bowel diseases control downloaded from Gene Expression Omnibus database, we identified the differentially expressed genes (DEGs) between UC patients and controls as well as between CD patients and controls (|log₂FC(fold change)| > 1 and p < 0.05). Then Gene Ontology (GO) functional enrichment analyses were performed for these DEGs in two groups, followed by the construction of weight PPI (protein–protein interaction) networks. Subnets enriched for the PPIs and differentially expressed genes were constructed based on the weight PPI networks. The overlapping genes between the genes in the top 10 subnets with smallest p value and the DEGs were selected as the candidate genes of disease. A total of 75 DEGs were identified in UC group and 87 ones in CD group. There were 69 and 57 specific DEGs in CD group and UC group, respectively. The DEGs in CD group were mainly enriched in “inflammatory response” and “defense response”, while the most significantly enriched GO terms in UC group were “anion transport” and “chemotaxis”. FOS and SOCS3 were identified as candidate genes for CD and other three genes HELB, ZBTB16 and FAM107A were candidate genes for UC. In conclusion, there were distinct genetic alterations between UC and CD. The candidate genes identified in current study may be used as biomarkers for differential diagnosis of CD and UC.