Serum Of Healthy Volunteers Research Articles

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Sensitivity of Extracellular Vesicles from Human Blood Serum to Various Detergents

Blood exosomes and microvesicles, collectively known as small extracellular vesicles (sEV), are vesicles about 100-150 nm in size. Small EV are involved in various aspects of signaling in the body; in addition, they can serve as markers of various pathologies. For biochemical studies, vesicle solubilization is often required. We tested the ability of various detergents to dissolve membranes of the sEV. Small EV were isolated from the blood serum of healthy volunteers by gel filtration on Sepharose CL-2B and tried to solubilize them using the anionic detergent DOC (sodium deoxycholate), non-ionic detergent Brij 35 (polyoxyethyleneglycol dodecyl ether), zwitterionic detergent CHAPS (3 - [(3-chloramidopropyl) dimethylammonio] -1-propanesulfonate), and cationic detergent CTAB (cetyl trimethylammonium bromide). The concentration of sEV in the solution was determined by dynamic light scattering. We find DOC is the most effective for sEV solubilization.

Interplay between antioxidant activity, health and disease

The article discusses the relationship between oxidative stress (OS) and pathological conditions, the possibilities and benefits of estimating OS considering the integral antioxidant activity (AOA) as an OS criterion, and using a simple accessible hybrid potentiometric method (HPM) with a mediator system for AOA monitoring. The results of AOA of blood serum in healthy volunteers and patients with various diseases are presented. Preliminary reference values are found. The lower levels of AOA of blood serum in patients with different diseases in comparison with the control group are observed. The potential mechanisms of changes in the AOA levels and it’s clinical significance are discussed from the position of biointerfaces interplay. With AOA equal or greater than 1.40 mmol-eq l–1 the person is healthy, the range from 0.95 to 1.40 mmol-eq l–1 indicates that the patient is at risk and needs to undergo a further medical examination. When AOA blood serum is below 0.95 mmol-eq l–1, detailed diagnostics and relevant treatment are required. The findings allow suggesting that the approach determine antioxidant/oxidant activity of biological fluids holds considerable promise for monitoring OS; it opens up new opportunities in expanding the use of analytical chemistry in medicine.

Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection.

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis.

Single Triglyceride-Rich Meal Destabilizes Barrier Functions and Initiates Inflammatory Processes of Endothelial Cells.

Postprandial hypertriglyceridemia is an independent risk factor for cardiovascular disease. The aim of this study was to assess the effects of a single fat-rich meal on barrier functions and inflammatory status on human umbilical vascular endothelial cells (HUVECs), furthermore we assess the effects of mixture of palmitic acid and 25-hydroxycholesterol (PA +25OHCH) on integrity of endothelial cells and their inflammatory properties. HUVECs were induced with serum of healthy volunteers taken before, and 3 h after, the consumption of a meal with a standardized daily required dose of fats. In addition, endothelial cells were induced with PA +25OHCH (800 μM/L+10 μg/mL). Total cholesterol, triglycerides (TGs), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high sensitivity c-reactive protein, and glucose were measured at fasting and postprandially. HUVEC integrity was measured in the RTCA-DP xCELLigence system. mRNA expression of interleukin (IL)-33, IL-32, intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), CX3C-chemokine, vascular endothelial growth factor (VEGF) occludin, and VE-cadherin was analyzed by real-time polymerase chain reaction. Viability and apoptosis were assessed in flow cytometry. The level of VEGF and IL-33 in fasting and postprandial serum was assessed by enzyme-linked immunosorbent assay (ELISA). Three hours after consumption of a fatty meal, all patients displayed increased levels of TGs and Toll-like receptors (110 ± 37 mg/dL versus 182 ± 64 mg/dL P < 0.05) (24 ± 11 mg/dL versus 42 ± 14 mg/dL P < 0.05). Postprandial serum and PA +25OHCH caused >20% decrease of HUVEC integrity than fasting serum (P < 0.001). HUVEC disintegration was accompanied by a decrease of occludin mRNA expression as compared with fasting serum (P < 0.05). The fatty meal affected neither VE-cadherin mRNA expression nor its apoptosis (P > 0.05). Mixture of PA +25OHCH caused decrease of VE-cadherin mRNA expression as compared with fasting serum (P < 0.01). PA +25OHCH did not affect HUVEC apoptosis (P > 0.05). Postprandial serum and PA +25OHCH caused increase of IL-33, MCP-1, ICAM-1, IL-32, VEGF, and CX3C-chemokine mRNA expression as compared with fasting serum (P < 0.05). Moreover, level of VEGF in fatty serum was significantly higher (P < 0.001). Postprandial lipemia after a single fatty meal may destabilize the endothelial barrier and initiate inflammatory processes.

Abstract 1205: Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S., outranking breast and colorectal cancer by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the cancer genome atlas shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhances expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy. Methods: Level of secreted VIP in 1) serum of healthy volunteers, 2) untreated PDAC patients, 3) cell culture supernatant from murine melanoma, breast and PDAC cells and 4) serum from mice bearing these cell lines were tested via VIP specific enzyme immunoassay. Effect of inhibiting VIP signaling in-vivo was tested using VIPhyb, an antagonist for VIP receptor. Mice were subcutaneously inoculated with MT5 or luciferase transfected KPC (KPC luc), PDAC cell lines and treated with VIPhyb and/or anti-PD1 treatment after the tumors were palpable. The treatment regimen involved administering 10µg VIPhyb subcutaneously, every day and 200µg anti-PD1 or IgG2a, intraperitoneally, every three days, for 10 days. The tumors were measured using calipers or imaged via IVIS imaging system. The mice were sacrificed when tumors ulcerated or when they reached IACUC endpoint. Immunohistochemistry was performed on tumor tissue sections harvested at the time of sacrifice for expression of VIP, CD8 and DAPI. Results: Consistent with the human genome atlas, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from untreated human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing MT5 or KPC luc PDAC cells with the combination of VIPhyb and anti-PD1 produced complete and durable regression of tumors in 20% of the mice and delayed tumor progression in both tumor models. Further, upon analysis of the tumor tissues sections, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1. Conclusion: VIP is a novel immune checkpoint pathway in PDAC. Blocking VIP signaling during anti-PD1 therapy, shows improved survival and delayed tumor progression in mouse models of PDAC. Experiments are underway to understand the mechanism of action, as well as, improving the potency of VIPhyb for enhanced clinical outcome. Citation Format: Sruthi Ravindranathan, Yiwen Li, Shuhua Wang, Mohammad Y. Zaidi, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Targeting vasoactive intestinal peptide signaling to enhance pancreatic cancer responsiveness to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1205.

Serum nesfatin-1 as a marker of systemic inflammation in rheumatoid arthritis.

The purpose of this study was to determine the level of nesfatin-1 (NF-1) in the blood serum of healthy volunteers and patients with rheumatoid arthritis (RA) to establish the threshold for normal values of this parameter and to reveal the relationship between the level of NF-1 and clinical manifestations of RA. We examined 170 people, of which 110 patients with RA and 60 donors who made up the comparison group. The mean level of serum nesfatin-1 in healthy subjects was 31.61 ± 3.17 ng/ml (M ± σ). The level of normal values of nesfatin-1 in healthy individuals, defined as M ± 2σ, was from 25.27 to 37.95 ng/ml. These studies showed the relationship between the concentration of NF-1 and the severity of clinical manifestations of RA. We found that a higher serum level of NF-1 was characteristic of patients with a more severe clinical course of the disease. The data obtained indicate that high level of NF-1 positively correlates with higher concentrations of C-reactive protein and ESR. This data indirectly proves the proinflammatory effect of NF-1 and confirms the hypothesis about the primary role of systemic inflammation in the pathogenesis of RA.

Cyclophilin B promotes cell proliferation, migration, invasion and angiogenesis via regulating the STAT3 pathway in non-small cell lung cancer

Lung cancer is the most common type of cancer and has become the leading cause of cancer-associated mortality worldwide. It has been reported that expression of Cyclophilin B was greatly elevated in the pancreatic cancer patient sera as compared with the healthy volunteer sera. This study aimed to investigate the role and regulatory mechanism of CypB in NSCLC progression. The expression levels of CypB was detected in NSCLC samples and cell lines by ELISA, western blot and immunohistochemistry assay. In addition, CCK8, colony formation, scratch and transwell assays were used to evaluate the proliferation, migration and invasion of A549 cells with CypB silencing. The expression of angiogenesis related proteins and pathway-related factors were detected by western blot. In NSCLC samples, CypB expression was upregulated. The expression of CypB was significantly reduced in the siRNA-cyclophilin B group. In addition, CypB silencing inhibited cell proliferation, migration and invasion. The expression of angiogenesis related proteins and pathway-related factors have also changed significantly. These findings suggested that CypB silencing may suppress the proliferation, invasion, migration and angiogenesis of A549 cells via inhibiting STAT3 pathway.

Effects of modest iron loading on iron indices in healthy individuals.

Intravenous iron administration is typically indicated in individuals who have iron deficiency refractory to oral iron. However, in certain chronic disease states such as heart failure, it may be beneficial to administer intravenous iron to individuals who are not strictly iron deficient. The purpose of this study was to define a dose-response relationship between clinical indices of iron status and modest loading with intravenous iron in healthy, iron-replete participants. This was a double-blind, controlled study involving 18 male participants. Participants were block-randomized 2:1 to the iron and saline (control) groups. Participants in the iron group received 3.75 mg/kg body wt up to a maximum of 250 mg of intravenous iron, once a month for 6 mo, provided that their ferritin remained measured <300 µg/l within the week before a dose was due and their transferrin saturation remained <45%. Otherwise they received a saline infusion, as did the control participants. Iron indices were measured monthly during the study. The pulmonary vascular response to sustained hypoxia and total hemoglobin mass were measured before, at 3 mo (hemoglobin mass only), and at 6 mo as variables that may be affected by iron loading. Serum ferritin was robustly elevated by intravenous iron by 0.21 µg·l-1·mg-1 of iron delivered (95% confidence interval: 0.15-0.26 µg·l-1·mg-1), but the effects on all other iron indices did not reach statistical significance. The pulmonary vascular response to sustained hypoxia was significantly suppressed by iron loading at 6 mo, but the hemoglobin mass was unaffected. We conclude that the robust effect on ferritin provides a quantitative measure for the degree of iron loading in iron-replete individuals.NEW & NOTEWORTHY There has been an increasing interest in administering intravenous iron to patients to alter their iron status. Here, we explore various indices of iron loading and show that in healthy volunteers serum ferritin provides a robust indicator of the amount of iron loaded, with a value of 21 µg/l increase in ferritin per 100 mg of iron loaded.

A novel prognostic marker and immunogenic membrane antigen: prohibitin (PHB) in pancreatic cancer.

BackgroundPrevious study, using immunoblotting with IgG and membrane proteins, identified prohibitin (PHB) as a potential immunogenic membrane antigen. Now, investigate PHB expression and biological functions in pancreatic cancer.MethodsPHB expression was analysed in PDAC cell lines, normal pancreas tissues, cancer tissues, PDAC patient sera and healthy volunteer sera using QRT-PCR, Western blotting, IHC, and ELISA, and a survival analysis and a COX regression analysis were performed. Low and high PHB expression levels were accomplished using RNA interference technology and gene transfer techniques. Cell proliferation, migration, and invasion, apoptosis-related proteins were assessed 48 h after transfection.ResultsPHB was generally expressed in the 8 tested PDAC cell lines. PHB was significantly increased in PDAC tissues and negatively correlated with overall survival (p < 0.01). PHB was an independent prognostic factor in PDAC (p < 0.01). PHB was increased in PDAC patient sera (p < 0.01). siRNA-PHB decreased cell growth, migration and invasion. However, PHB overexpression resulted in the opposite effects. Western blotting and Flow cytometric analysis revealed apoptosis inhibition in siRNA-PHB PDAC cells.ConclusionsPHB plays a key role in modulating the malignant phenotype and apoptosis induction, which may be a novel prognostic predictor and a candidate for targeted therapy against PDAC.

Bioinformatics-based interaction analysis of miR-92a-3p and key genes in tamoxifen-resistant breast cancer cells

The abnormal expression of miR-92a-3p was detected in multiple cancers. However, the biological role and underlying mechanism of miR-92a-3p in tamoxifen-resistant cells are still unknown. The main objective of our study was to find potential miR-92a-3p regulating pathways involved in tamoxifen resistance and to construct their regulatory network using bioinformatics. Four gene expression profiles were retrieved from GEO database and the GEO2R tool was used for analysis. GSE41922 and GSE42072 were applied to investigate aberrant miR-92a-3p expression in breast cancer serum and tissue. We found that miR-92a-3p expression was higher in breast cancer serum or tissue than in healthy volunteer serum or adjacent normal tissue, and high expression of miR-92a-3p could predict poor prognosis of breast cancer patients. In our qRT-PCR validation, we found that miR-92a-3p was upregulated in tamoxifen-resistant cells. MiR-92a-3p might play a role in tamoxifen resistance. In order to find the relationship between miR-92a-3p and some key genes and their potential molecular mechanisms in tamoxifen-resistant cells. The microarray data GSE26459 and GSE28267 were analyzed to determine the differentially expressed genes (DEGs) or miRNAs (DEMs). Furthermore, the related long non-coding RNAs (lncRNAs) were screened with starBase v2.0. Finally,microRNA.org,miRDB, targetminer and targetscan were applied to predict the targets of miR-92a-3p. Through analysis, we find that miR-92a-3p may be used as a potential biomarker for early detection of cancer and monitoring the efficacy of endocrine therapy.

MicroRNA-411 promoted the osteosarcoma progression by suppressing MTSS1 expression.

MicroRNAs (miRNAs) play crucial roles in the progression of different tumors. In our study, we investigated the expression and roles of miR-411 in human osteosarcoma. In this study, we first confirmed that the miR-411 expression was higher in the serum of patients with osteosarcoma than in the serum of healthy volunteers. In addition, we found that the miR-411 expression was upregulated in the osteosarcoma tissues compared to that in the matched normal bone tissues. We also demonstrated that the miR-411 expression was upregulated in the four osteosarcoma cell lines. Elevated expression of miR-411 promoted osteosarcoma cell proliferation and migration. Moreover, we identified that metastasis suppressor protein 1 (MTSS1) was a direct target gene of miR-411 in the osteosarcoma cell. We also demonstrated that the MTSS1 expression was downregulated in the osteosarcoma tissues compared to that in the matched normal bone tissues. In addition, MTSS1 expression level was inversely correlated with miR-411 expression in the osteosarcoma tissues. Furthermore, elevated expression of miR-411 enhanced the osteosarcoma cell proliferation and migration through inhibiting the MTSS1 expression. These data suggested that miR-411 played as oncogene in the osteosarcoma partly by inhibiting the MTSS1 expression.

Design, expression and evaluation of novel chimeric protein constructed from colorectal tumor‐associated antigen

There were 134,000 new diagnosis and 49,000 deaths in 2016 due to colorectal cancer. Similar to most cancers, early diagnosis increases the chance of successful treatment. Detection of tumor-associated antigens or the immune response against such markers is one of the most common methods of diagnosis. In that regard, we aimed to design and express a chimeric protein from the most common tumor-associated antigens in colorectal cancer and assess its ability to detect the immune response in comparison with the parental tumor-associated antigens in patient's sera. Through bioinformatics approaches a chimeric protein from carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19-9) was designed and expressed in E. coli (BL21DE3). Proper folding, expression levels and immune reactivity were assessed by western blot, ELISA and immunohistochemistry. Recombinant proteins functionality and immune reactivity were confirmed by ELISA and Western blot. Results showed that recombinant CEA, recombinant CA19.9 and chimeric protein of CEA- CA19.9 have strong reactivity with antibodies in the sera of colorectal cancer patients, whereas no reactivity was seen with the sera of healthy volunteers. Significantly stronger immune reactivity was seen with the chimeric protein than each of the CEA or CA19.9 alone. Overall, it was concluded that the designed recombinant proteins in this study could be used to detect autoantibodies produced against the colorectal tumor-associated antigens. The chimeric CEA-CA19.9 protein shows a stronger reactivity with the sera antibodies of colorectal cancer patients that CEA or CA19.9 alone.

Evaluation of Serum Apolipoprotein E as a Potential Biomarker for Pharmacological Therapeutic Efficacy Monitoring in Dopamine Dictated Disease Spectrum of Schizophrenia and Parkinson's disease: A Preliminary Study.

Aim of the Study:Parkinson’s disease and schizophrenia are disease end points of dopaminergic deficit and hyperactivity, respectively, in the mid brain. Accordingly, current medications aim to restore normal dopamine levels, overshooting of which results in adverse effects of psychosis and extra-pyramidal symptoms, respectively. There are currently no available laboratory tests to guide treatment decisions or help predict adverse side effects of the drugs. The aim was to therefore explore the possibility of using apolipoprotein E as a biomarker to monitor pharmacological intervention in dopamine dictated states of Parkinson’s disease and schizophrenia for optimum therapy.Methods:Naïve and treated, Parkinson’s disease and schizophrenic patients were recruited from neurology and psychiatry clinics. Serum of healthy volunteers was collected as controls. Serum concentrations of apolipoprotein E was estimated by enzyme-linked immunosorbent assay (ELISA). Pathway analysis was carried out to delineate the interactions of apolipoprotein E in Parkinson’s disease and schizophrenia.Results:Apolipoprotein E levels are higher in Parkinson’s disease patients as compared with schizophrenic samples (P < .05). Also, post-treatment apolipoprotein E levels in both disease states were at par with levels seen in healthy controls. The interactions of apolipoprotein E validate the results and place the differential expression of the protein in Parkinson’s disease and schizophrenia in the right perspective.Conclusion:Apolipoprotein E concentration across the dopaminergic spectrum suggests that it can be pursued not only as a potential biomarker in schizophrenia and Parkinson’s disease, but can also be an effective tool for clinicians to determine efficacy of drug-based therapy.

Significance of Cu/Zn-Superoxide Dismutase Levels in Hemodialysis Patients: A Mini Review

Cu/Zn-superoxide dismutase (Cu/Zn-SOD) is an enzyme that is ubiquitously present in the cytoplasm and causes dismutation of superoxide radicals, therefore Cu/Zn-SOD is primarily used as an antioxidant marker. Levels of Cu/Zn-SOD are higher in the serum of hemodialysis patients than in serum of healthy volunteers. The increase of serum Cu/Zn-SOD levels is related to the decrease of kidney function with aging and arteriosclerosis in hemodialysis patients. Moreover, infection, vascular puncture, and hemostasis may be related to the increase in serum Cu/Zn-SOD levels. As it is associated with numerous factors in hemodialysis patients, Cu/Zn-SOD may serve as a complex marker for arteriosclerosis, vascular, and inflammatory conditions. It is important to investigate various agents that decrease serum Cu/Zn-SOD levels to improve the life-span of hemodialysis patients.

The nicotinamide-phosphoribosiltransferase as a marker of systemic inflammation under osteoarthrosis.

The purpose of study is to establish the level of visfatin (Nampt, nicotinamide-phosphoribosiltransferase) in blood serum of healthy volunteers for determination of the level of normal values of of the given parameter specific to healthy people. Besides, the given parameter also was analyzed in patients with osteoarthritis. The sampling consisted of 152 individuals. Out of them, 92 patients with primary osteoarthritis and 60 healthy individuals. The level of Nampt in blood serum of healthy people was detected by using commercial test-systems (RaiBiotech, cat. № EIA-VIS-1). The average level of Nampt in blood serum of healthy people made up to 2,43±0,17 ng/ml (M ± m). The level of normal values of Nampt in healthy people detected as М ± 2σ, made up to from 0 to 5.07 ng/ml. The study data proved the relationship between concentration of Nampt ans expression of clinical manifestations of osteoarthritis. It is demonstrated that higher level of Nampt in blood serum was typical for patients with more severe clinical course of disease. The obtained data testify that high level of Nampt correlates positively with higher concentrations of C-reactive protein and ESR that indirectly testifies in behalf of anti-inflammatory character of action of Nampt and substantiates hypothesis about primary role of systemic inflammation in pathogenesis of osteoarthritis.

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture.

BackgroundNonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing.MethodsThirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF–MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann–Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC–MS/MS, nephelometry, western blotting or ELISA assays.ResultsFrom the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF–MS using CM10 and IMAC-Cu2+ ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls.ConclusionTwo proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1019-1) contains supplementary material, which is available to authorized users.

9,10-Phenanthrenequinone as a mass-tagging reagent for ultra-sensitive liquid chromatography–tandem mass spectrometry assay of aliphatic aldehydes in human serum

9,10-Phenanthrenequinone (PQ) was successfully used as a new mass-tagging reagent for sensitive labeling of aliphatic aldehydes (C3-C10) prior liquid chromatography-electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS). This reagent could overcome the drawbacks of previous amine or hydrazine-based reagents, such as lower sensitivity, formation of two stereoisomeric reaction products for each single analyte, need for longer derivatization time, and poor reactivity with aliphatic aldehydes. The PQ-aldehyde derivatives exhibited intense [M+H]+ and a common product ion with ESI in the positive-ion mode. The derivatives were monitored at the transition of [M+H]+→m/z 231.9 with detection limits from 4.0 to 100 pM (signal to noise ratio=3). 3-Phenylpropanal was used as an internal standard (IS) and the separation of the eight aldehydes and IS was achieved in less than 10min employing gradient elution with methanol and ammonium formate buffer (20mM, pH 4.0). The method employed salting out liquid–liquid extraction for aliphatic aldehydes form serum for the first time with excellent recoveries (92.6–110.8%). The developed method was validated and applied for quantification of the target aldehydes in serum of healthy volunteers (n=14).

Improved Gas Chromatographic Determination of Guanidino Compounds Using Isovaleroylacetone and Ethyl Chloroformate as Derivatizing Reagents.

An improved GC method in terms of sensitivity and decrease in the analysis time has been developed for the analysis of eight guanidino compounds: guanidine (G), methylguanidine (MG), creatinine (CTN), guanidinoacetic acid (GAA), guanidinobutyric acid (GBA), guanidinopropionic acid (GPA), argenine (Arg), and guanidinosuccinic acid (GSA), using isovaleroylacetone (IVA) and ethyl chloroformate (ECF) as derivatizing reagents. The separation was obtained from column HP-5 (30 m × 0.32 mm i.d.) with film thickness of 0.25 μm within 11 min. The linear calibrations were obtained with 0.5 to 50 μg/mL with coefficient of determination (R(2)) within 0.9969 - 0.9998. Limits of detections (LODs) were within 5 - 140 ng/mL. The derivatization, separation and determination was repeatable (n = 6) with relative standard deviation (RSD) within 1.2 - 3.1%. The guanidino compounds were determined in deproteinized serum of healthy volunteers and uremic patients within below LOD to 8.8 μg/mL and below LOD to 43.99 μg/mL with RSD within 1.4 - 3.6%. The recovery of guanidino compounds calculated by standard addition from serum was within 96.1 - 98.9%, with RSD 1.4 - 3.6%.

Abstract A035: Pharmacokinetics and pharmacodynamics of the immunotherapeutic TLR-9 agonist MGN1703 – conclusions by comparison of data from clinical trials with healthy volunteers and cancer patients

Abstract Background: The synthetic DNA-based immunomodulator MGN1703 is a member of the new dSLIM® family of TLR-9 agonists, comprising covalently closed dumbbell-like DNA molecules which consist entirely of natural DNA with two single-stranded CG-containing loops separated by a double-stranded stem. MGN1703 exerts its function by activating the innate and both arms of the adaptive immune system. MGN1703 has already shown a good safety profile and benefit for patients compared to placebo in maintenance therapy of metastatic colorectal carcinoma (mCRC) after fist-line induction therapy in a phase 2 clinical trial (MGN1703-C02, IMPACT). Methods: Following preclinical studies in mice and monkeys, a human clinical program was performed, including a phase 1 trial (MGN1703-C01), on a variety of solid tumors, and the double-blinded, placebo-controlled phase 2 IMPACT trial with mCRC patients. Additionally, a single dose crossover, placebo-controlled phase 1 cardiac safety trial, MGN1703-C04, was conducted to assess cardiac and general safety, pharmacokinetics (PK) and pharmacodynamics (PD) in healthy volunteers (HV). Fourteen HV were randomized and 13 subjects completed the study receiving a subcutaneous dose of 60 mg MGN1703 during one treatment period and a single dose of placebo during the other. Results: After single dose administration of MGN1703 standard PK parameters were determined in the MGN1703-C04 trial: mean Tmax (14h), AUC0-t (5000 ng•hr/mL) and shape of the curve of MGN1703 concentration in serum. These were similar to single dose data obtained from cancer patients, where a mean Tmax of 10h and a mean AUC0-t of 5340 ng•hr/mL have been observed. Notably, multiple doses resulted in similar levels in serum of cancer patients, without accumulation of MGN1703. PD was mainly evaluated by analyzing levels of the chemokine IP-10 in serum of the HV as an equivalent of innate immune activation. Elevated IP-10 levels in the serum of HV were shown peaking at 24-48 hours after dosing with MGN1703. This is clearly in line with expected time frame of immune activation subsequent to MGN1703 appearance in the serum of the HV. Timing and levels of IP-10 elevation are in keeping with data from the peripheral blood of cancer patients, where in addition to IP-10 also the subsequent activation of innate immune cells – especially the up-regulation of CD169 on monocytes – as a consequence of MGN1703 administration was observed. Conclusions: The data show obvious similarities between healthy volunteers and cancer patients with respect to PK and PD and also confirm the appearance of the appropriate immunomodulatory signature downstream of MGN1703 appearance. This supports the twice weekly application scheme of current immunotherapy studies with MGN1703, e.g. the ongoing phase 3 IMPALA trial in mCRC patients. Further data will allow for evaluation of a possible association with clinical parameters in cancer patients. Citation Format: Manuel Schmidt, Kerstin Kapp, Detlef Oswald, Matthias Schroff, Burghardt Wittig, Alfredo Zurlo. Pharmacokinetics and pharmacodynamics of the immunotherapeutic TLR-9 agonist MGN1703 – conclusions by comparison of data from clinical trials with healthy volunteers and cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A035.