Sera Of Healthy Individuals Research Articles

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

Mitochondrial damage and the resultant oxidative stress are associated with neurodegenerative diseases, ageing, and cancer. However, the triggers of mitochondrial damage remain unclear. We previously reported that cell-free chromatin particles (cfChPs) released from the billions of cells that die in the body every day can readily enter healthy cells and damage their DNA. Here, we show that cfChPs isolated from the sera of healthy individuals, when applied to NIH3T3 mouse fibroblast cells, cause physical damage to mitochondrial DNA (mtDNA). cfChPs also induce ultrastructural changes, increase mitochondrial mass, alter mitochondrial shape, upregulate mitochondrial outer membrane protein translocase of the outer membrane 20, and change mitochondrial membrane potential. Furthermore, a marked increase was observed in mitochondrial superoxide (ROS) production, as detected by MitoSOX Red, and intracellular superoxide dismutase-1 activation. ROS production was also activated when a conditioned medium containing cfChPs released from hypoxia-induced dying NIH3T3 cells was applied to healthy NIH3T3 cells. ROS activation was significantly reduced when the conditioned medium was pre-treated with three different cfChP-deactivating agents: anti-histone antibody-complexed nanoparticles, DNase I, and the novel pro-oxidant combination of the nutraceuticals resveratrol and copper. Given that 1 × 109–1 × 1012 cells die in the body every day, we hypothesise that cfChPs from dying cells are the major physiological triggers for mtDNA damage and ROS production. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions linked to oxidative stress.

Specific Recognition of β-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients.

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH2)3SH (glycan G29SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH2)3SH (glycan G32SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.

Use of MicroRNAs as biomarkers in the early diagnosis of prostate cancer.

Introduction:Prostate cancer (PCa) is a common type of cancer in western countries and prominent cause of mortality in men. The aim of the study was to evaluate miRNAs as biomarkers in PCa in healthy individuals and prostate cancer with patients, and examined the effect of miRNA levels on tumour mass.Material and methods:Twenty prostate cases, age (mean and range) 61,4±12.1 (45-73), and twenty healthy men, age 59,3±11.2 (44-70) were included to the study. Seven miRNAs including two internal controls (Let7c, miR125b, miR141, miR145, miR 155, miR181 ve miR192) were evaluated in two groups.Results:The mean and range of prostate spesific antigen (PSA) in cancer cases and healthy individuals were 6.79±2.84 ng/ml (2.25-14.7) and 3.8±2.2 ng/ml (1.3-7.8) respectively. The level of miR141 was significantly lower in PCa cases than healthy individuals (p=0,004), and miR155 was significantly higher (p=0,005) in PCa cases. Both miRNAs were explored sensitive and specific in the ROC analysis. Tumour mass were found to be associated with the level of miR-125b and miR-145.Conclusion:validation studies are required in wider patient groups in the subject of tumor effect and miRNA biomarkers in prostate cancer.

Serum Th17 and TNF-α distinguish between patients with occult hepatitis B infection, chronic hepatitis B infection and healthy individuals.

Chronic hepatitis B (CHB) is classified into five phases based on virus-host interactions: immune tolerance, immune clearance, inactive carrier state, reactive phase and occult hepatitis B infection (OBI). OBI is an uncommon asymptomatic phase of CHB that can be reactivated when the immune system is compromised, occasionally giving rise to severe liver disease. Host immune factors play essential roles in all phases of the CHB infection. Cytokines may alter infection course, influencing the propensity for and the progression of CHB and thus warrant study. Three clinical groups were studied: 48 healthy individuals (HI), 28 patients with persistent positive anti-HBc serological markers and negative HBsAg over time, who were diagnosed as OBI and 12 patients with active CHB. OBI patients were defined by three independent detections of the hepatitis B virus genome through nested PCR and real-time PCR. Quantitative measurement of 20 Th1, Th2 and Th17 human cytokines was performed in the sera of HI, OBI and CHB patients. Levels of IFN-γ, TNF-β, IL-28A, IL-4, IL-5, IL-13, IL-1β, IL-6, IL-21, IL-22, IL-23, GM-CSF and MIP-3α were similar between groups. IL-2, IL-12p70, IL-10, IL-17F and TGF-β1 were similar in HI and OBI, but higher in CHB. TNF-α and the IL-17A:IL-17F ratio were significantly different between the three groups. TNF-α was progressively higher in HI, OBI and CHB (P = 0.004), while the IL-17A:IL-17F ratio was 1.1 in HI, 3.4 in OBI and 0.4 in CHB. Detection and levels of these pro-inflammatory cytokines in OBI patients suggest that they are undergoing a silent hepatic inflammatory process.

Antibodies to Heat Shock Proteins 90\u03b1 and 90\u03b2 in Psoriasis

One of many hypotheses of psoriasis pathogenesis supposes an overexpression of heat shock proteins (Hsps) in different skin layers and systemic immunologic response to them. Hsp90 is one of the most abundant chaperone in eukaryotic cells. The number of studies concerning the role of Hsp90 and anti-Hsp90 antibodies in etiopathogenesis of various diseases is also constantly expanding. Still, there are not many reports concerning potential involvement of this Hsp family or anti-Hsp90 immunization in pathomechanism of psoriasis. The aim of the study was the estimation of anti-Hsp90α and anti-Hsp90β IgG antibodies in the sera of the psoriatic patients at different phases of disease activity in comparison to the sera of healthy individuals. The study material consisted of sera from psoriasis patients (n = 80) in active phase and in the remission phase and healthy individuals (n = 80). Concentrations of anti-Hsp90α and anti-Hsp90β IgG antibodies were determined using ELISA technique. In the patients with psoriasis (both in the active phase of the disease and in the remission phase) concentrations of anti-Hsp90α antibodies were significantly higher than in healthy individuals and they correlated positively with psoriasis area severity index values. The mean concentrations of anti-Hsp90β antibodies in the psoriatic patients and healthy controls were comparable. The obtained results indicate an existence of increased immunological response to Hsp90α in psoriasis. It may suggest the role of the extracellular form of this chaperone and/or anti-Hsp90α antibodies in etiopathogenesis of this dermatosis. The inhibition of Hsp90α may represent a novel therapeutic approach to treat psoriasis.

Fabrication and application of an amperometric lysine biosensor based on covalently immobilized lysine oxidase nanoparticles onto Au electrode

The fabrication of an amperometric lysine biosensor is described in this study, wherein nanoparticles (NPs) of lysine oxidase (LOx) are covalently immobilized onto gold electrode (AuE). The LOxNPs were prepared by desolvation method and characterized by UV Vis spectroscopy, Fourier transform infra red (FTIR) spectroscopy and transmission electron microscopy (TEM). The LOxNPs/AuE modified working electrode was studied by scanning electron microscope (SEM) and cyclic voltammetric (CV) techniques. The electrode exhibited optimum current within 3.5 s at applied potential, 0.8 V, pH 6.5 and temperature, 35 °C. The sensor displayed a linear relationship between lysine concentration and current in the range 10–800 µM with a limit of detection of 10 µM. Within assay and between batch coefficients of variation were 0.0751% and 0.0637% respectively. The analytical recoveries of added lysine at 10 µM and 20 µM in sera were 98.39% and 98.23% respectively. There was a good correlation between level of lysine in sera and milk samples (R2 = 0.999 and R2 = 0.98 respectively) as determined by the standard spectrophotometric method and the present method. The biosensor measured lysine levels in milk, pharmaceutical tablet and sera of healthy individuals and cancer patients. The biosensor showed slight interference by common interferents found in serum.

IgM ANCA in healthy individuals and in patients with ANCA-associated vasculitis

Low levels of IgM auto-antibodies have been reported in health and disease. IgM anti-neutrophil cytoplasmic antibodies (ANCA) have been reported in patients with ANCA-associated vasculitis (AAV). We sought to investigate if healthy individuals may have IgM ANCA in their sera. The first aim of the study was to determine whether IgM ANCA was present in healthy individuals and in patients with ANCA-associated vasculitis. The second aim was to determine what happens to IgM ANCA levels over time. The third aim was to determine whether bacterial infections affected IgM ANCA levels in non-AAV patients. Sera from healthy individuals and patients with AAV were tested for IgM ANCA by immunofluorescence on fixed neutrophils, immunoprecipitation, Western blot and ELISA. Peripheral blood mononuclear cells were isolated and tested by ELISpot for circulating IgM ANCA B cells. To determine whether infection affected IgM ANCA levels, we studied non-AAV patients with bacterial endocarditis or Staphylococcus aureus bacteraemia and measured IgM ANCA levels over time. IgM ANCA is detectable in both healthy individuals and patients with AAV and the titres decreased with increasing age. Circulating IgM ANCA B cells were identified by ELISpot. In the presence of infection, we could not find a significant change in IgM ANCA levels. We report the presence of low-level specific IgM ANCA in the sera of healthy individuals and in patients with ANCA-associated vasculitis. Bacterial infection did not affect the level of IgM ANCA in this small study.

SAT-006 IgM ANCA in healthy individuals and in patients with ANCA-associated vasculitis

Low levels of IgM auto-antibodies have been reported in health and disease. IgM anti-neutrophil cytoplasmic antibodies (ANCA) have been reported in patients with ANCA-associated vasculitis (AAV). We sought to investigate if healthy individuals may have IgM ANCA in their sera. The first aim of the study was to determine whether IgM ANCA was present in healthy individuals and in patients with ANCA-associated vasculitis. The second aim was to determine whether bacterial infections affected IgM ANCA levels in non-AAV patients. Sera from healthy individuals and patients with AAV were tested for IgM ANCA by immunofluorescence on fixed neutrophils, immunoprecipitation, western blot and ELISA. Peripheral blood mononuclear cells were isolated and tested by ELISpot for circulating IgM ANCA B-cells. To determine whether infection affected IgM ANCA levels, we studied non-AAV patients with bacterial endocarditis or Staphylococcus aureus bacteraemia and measured IgM ANCA levels over time. IgM ANCA is detectable in both healthy individuals and patients with AAV. Circulating IgM MPO-ANCA B cells were identified by ELISpot. In the presence of infection, we could not find a significant change in IgM ANCA levels. We report the presence of low-level specific IgM ANCA in the sera of healthy individuals and in patients with ANCA-associated vasculitis. Bacterial infection did not affect the level of IgM ANCA in this small study.

A novel chemiluminescent immunoassay based on original acridinium ester labels as better solution for diagnosis of human toxoplasmosis than conventional ELISA test

Toxoplasma gondii infection is one of the most common human zoonosis. Laboratory diagnosis of this disease is mainly based on the results of serological methods detecting specific antibodies in the patient’s sera. In this study we aimed to evaluate the performance of a chemiluminescence immunoassay (CLIA) based on the use of a novel immunochemical reagent in the form of the conjugate of original acridinium label (AL) attached to secondary antibody (IgG-AL) and SAG2-GRA1-ROP1L chimeric antigen for T. gondii specific antibodies detection. The CLIA test was compared with conventional ELISA, which was based on the same recombinant antigen and differed only in terms of the detection methodology of immune complexes. The new CLIA assay proved to be more sensitive and better differentiated sera of patients with T. gondii infection from sera of healthy individuals, being a promising alternative to more labor, cost-demanding and less versatile ELISA as screening test in toxoplasmosis diagnostics.

A serological study of antibodies to Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in the sera of healthy individuals collected two decades apart.

Sera collected from healthy individuals from the general population in the Czech Republic during repeated cross-sectional surveys were analyzed. Samples collected in the same six districts in two time periods, 1978-1989 and 2001, were compared. The study subjects were divided into six age categories between 10 and 59 years. Overall, 434 samples from 1978-1989 and 270 samples from 2001 were screened for Anaplasma phagocytophilum (AP) and Borrelia burgdorferi sensu lato (BB). The anti-AP positivity rates were 13.1% and 11.5% in the first and second period, respectively, and did not differ significantly between the periods (P = 0.559). The anti-BB antibodies were detected in 33.9% and 14.8% of study subjects, respectively. The positivity rates were significantly lower in the second period (P<0.001). No considerable changes were observed in the sex distribution of positive findings between the two periods. The highest positivity rates of anti-AP antibodies were found in the 10-14 year age group: 16.0% in 1978-1989 and 16.7% in 2001. The age distribution of the anti-AP antibody positivity rates did not change substantially (P = 0.872). In 1978-1989, the lowest anti-BB antibody positivity rate (26.7%) was found in the 10-14 year age group, with a gradual increase with age to 41.1% in 50-59 year-olds. In 2001, the positivity rate in the 10-14 year age group was 26.2% and was not significantly different from that in the first period (P = 0.955). However, the positivity rates in the older age groups 15-59 years decreased significantly (P<0.001) and varied between 8.3% and 15.1%.

Cytokine Profiling of Serum Allows Monitoring of Disease Progression in Inclusion Body Myositis.

Inclusion body myositis is a late onset inflammatory myopathy lacking reliable serum biomarkers for diagnosis and for disease progression. To identify diagnostic and predictive biomarkers, cytokine profiling is used to assess the potential of cytokines to discriminate between cases and controls and to assess whether treatment with methotrexate can influence biomarkers associated with disease progression. The diagnostic and follow-up potential of 48 cytokines was tested using Bioplex-assay and ELISA in sera of healthy individuals, IBM patients and patients with other neuromuscular disorders. Ten cytokines (TRAIL, IL-8, MIF, MCP-1, LIF, IP-10, IFN-α2, MIG, bNGF and IL-3) were identified to be good to excellent markers to discern IBM patients from healthy controls. Three cytokines (IP-10, Eotaxin and SDF1A) changed significantly upon methotrexate treatment as compared with the natural clinical course. Muscle strength loss was associated with changes in IL-8 and SDF1A levels. IFN-γ levels were only associated with survival of IBM patients before correction for multiple comparisons. Cytokine profiling can discriminate IBM patients from healthy controls and other neuromuscular disorders. Immunosuppression with methotrexate affects cytokine levels in IBM. IL-8 and SDF1A could serve as biomarkers for disease progression.

Sialic acid level is significantly elevated in IgM enriched protein fraction in sera of cancer patients

ABSTRACTSerum sialic acid (SA) levels are important for diagnosis, follow-up, and mechanistic analysis of malignant diseases. However, little is known about the levels of SA bound to serum IgM. Here, we isolated IgM from sera of healthy individuals and patients with cancer using DEAE chromatography and 8% polyethylene glycol precipitation. In this fraction, which contained partially purified IgM (recovery; 52%; purity: 25%), SA was quantified with fluorescence detection-HPLC (detection limit: 0.08 μM). SA levels in the IgM-enriched fraction was significantly higher in cancer patients (104 ± 27 µM) than in healthy individuals (81 ± 11 µM; P = 0.003).

Study of Antigenic properties of Human immunodeficiency Virus NEF protein Recombinant Analog

Protein p27/p25 is a product of HIV nef gene and is a multifunctional factor of HIV pathogenicity. Antigenic properties of recombinant NEF polypeptide which includes N-terminal fragment of HIV-1 p27 protein fused to E. coli ß-galactosidase were studied by serological and virological methods. The interaction was shown between recombinant NEF antigen and serums of HIV-positive individuals by ELISA, Western blot and line immunoassay. There was no interaction with sera of healthy individuals. The antigen specificity of recombinant antigen was shown in the reactions with commercial HIV-1 p27/p25 protein analog and with polyclonal antibodies to synthetic peptides corresponding to N-, C-terminal regions of HIV-1 p27/p25 protein. The interaction between IgG of rabbits immunized with recombinant NEF antigen and viral antigens was shown by indirect immunofluorescence and neutralizing assays. Thus it was proven the possibility of using recombinant NEF protein as an antigen for diagnostic and experimental purposes.

Transcriptome analysis of blood stasis syndrome in subjects with hypertension

ObjectiveTo screen for mRNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension. MethodsThis study involved groups of patients with hypertension and blood stasis, including those with Qi deficiency, Qi stagnation, cold retention and heat retention; as well as hypertensive patients without blood stasis and healthy individuals. Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome. Total RNA was extracted from these cells and assessed by a high-throughput sequencing method (Solexa) and digital gene expression. Differentially expressed genes among these six groups were compared using whole genome sequences, and mRNAs associated with blood stasis syndrome identified. Differences in gene use and gene ontology function were analyzed. Genes enriched significantly and their pathways were determined, as were network interactions, and encoded proteins. Gene identities were confirmed by real-time polymerase chain reactions. ResultsCompared with cells cultured in sera of the blood stasis groups, those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences, respectively in stasis-related genes. Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and Jun proto-oncogene (JUN). Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress. Cells cultured in sera of patients with blood stasis and Qi deficiency, Qi stagnation, heat retention, and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome. The comparison showed differences in expression of 28,28,34, and 32 specific genes, respectively. ConclusionThe pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and JUN genes.

Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans.

Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

Detection of SENV Virus in Healthy, Hepatitis B- and Hepatitis C-Infected Individuals in Yazd Province, Iran.

Background:SEN virus (SENV) is the latest virus proposed as a cause of unknown hepatitis cases. Among nine detected genotypes of the virus, genotypes D and H are more frequent in hepatitis cases of unknown origin. The aim of this study was to determine the frequency of SENV-D and SENV-H genotypes in the sera of healthy individuals and hepatitis B and C patients. Methods:Totally, 200 serum samples from healthy individuals as well as 50 hepatitis B and 50 hepatitis C patients were collected. Anti-HCV (hepatitis C virus), anti-human immunodeficiency virus, hepatitis B surface antigen and anti-HBV (hepatitis B virus) core antigen were detected, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Viral DNA was subjected to nested PCR. Fisher's exact and unpaired ANOVA tests were used for statistical analyses. Results:SENV was detected in 90%, 66%, and 46% of the healthy individuals HBV and HCV-positive individuals, respectively. The frequency of SENV and its two genotypes were significantly lower in hepatitis B and hepatitis C patients (P<0.01). Also, the frequency of SENV-H was higher than SENV-D in all studied groups. In SENV-positive HBV patients, the level of ALT and AST enzymes were significantly less than SENV-negative patients (P<0.05). It was the same for SENV-H-negative and -positive cases. Conclusions:The levels of liver enzymes were significantly lower in HBV patients co-infected with SENV compared to HBV patients (P<0.05), indicating a positive impact of the virus in liver pathology by decreasing liver damage and thus decreasing the liver enzymes.

Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease.

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galβ(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs from sera of healthy individuals (NHS anti-α-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Galα(1,3)Galβ(1,4)GlcNAcα ∼20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Galβ(1,4)GlcNAcα and the monosaccharide GlcNAcα or GlcNAcβ conjugated to BSA are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Galα moiety for recognition by Ch anti-α-Gal Abs and the lack of Abs against nonself Galβ(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galβ(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galβ(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galβ(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1β in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n=27) and GBM (n=28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p<0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-FcγRIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1β (IL1β) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1β neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies.Biological significanceA complex antibody microarray based serum marker profiling identified a three-marker panel — LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1β which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1β as a potential candidate for developing targeted therapy.

Blood circulating microparticle species in relapsing–remitting and secondary progressive multiple sclerosis. A case–control, cross sectional study with conventional MRI and advanced iron content imaging outcomes

BackgroundAlthough multiple sclerosis (MS) is thought to represent an excessive and inappropriate immune response to several central nervous system (CNS) autoantigens, increasing evidence also suggests that MS may also be a neurovascular inflammatory disease, characterized by endothelial activation and shedding of cell membrane microdomains known as ‘microparticles’ into the circulation. ObjectiveTo investigate the relationships between these endothelial biomarkers and MS. MethodsWe examined the relative abundance of CD31+/PECAM-1, CD51+CD61+ (αV–β3) and CD54+ (ICAM-1) bearing microparticles in sera of healthy individuals, patients with relapsing–remitting MS, and secondary-progressive MS. We also investigated the correlation among circulating levels of different microparticle species in MS with conventional MRI (T2- and T1-lesion volumes and brain atrophy), as well as novel MR modalities [assessment of iron content on susceptibility-weighted imaging (SWI)-filtered phase]. ResultsDifferences in circulating microparticle levels were found among MS groups, and several microparticle species (CD31+/CD51+/CD61+/CD54+) were found to correlate with conventional MRI and SWI features of MS. ConclusionThese results indicate that circulating microparticles' profiles in MS may support mechanistic roles for microvascular stress and injury which is an underlying contributor not only to MS initiation and progression, but also to pro-inflammatory responses.

Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS.

Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS.