Normal Human Serum Research Articles

Simultaneous electrochemical sensing of dopamine and uric acid with the aids of chemometric methods

It is important to simultaneously detect dopamine (DA) and uric acid (UA) for health monitoring and disease diagnostics. Studies shows that bimetallic catalysts with synergistic effect have a better catalytic performance in the simultaneous determination. Herein, a composite of N-doped CoFe on carbon matrix doped with ▪ (CoFe/▪/NC) was prepared by pyrolyzing a metal-organic framework. Then the composite was modified on glassy carbon electrodes to simultaneously detect DA and UA. The study of double potential step chronoamperometry showed that the diffusion coefficients and the catalytic rate constants for UA were less than those of DA. Since the electrochemical signals related to DA and UA interfere with each other, the concentration calibration curve of one component would distort if the concentration of coexisting component varied. The simultaneous detection was executed via differential pulse voltammetry with the aid of chemometric methods. The models established by partial least squares regression analysis have little help for the simultaneous detection, especially for the concentration of UA in the samples. The models build by back propagation artificial neural network can realize the simultaneous determination with only 4 neurons in three-layer network. DA and UA in normal human serum were detected by the CoFe/▪/NC electrode with the aids of chemometrics.

Cael-101 Enhances the Clearance of Light Chain Fibrils and Intermediate Aggregates By Phagocytosis

Background Light chain amyloidosis (AL) is a rare, systemic disease characterized by amyloid fibril deposition in various organs due to overproduction of light chains from plasma cell dyscrasia (PCD). Current treatment strategies target PCD rather than clearing amyloid deposits directly from organs. CAEL-101 is a chimeric monoclonal IgG1 antibody that targets these amyloid fibrils. It binds to a cryptic epitope at the N-terminus of both kappa (κ) and lambda (λ) light chain misfolded proteins. In this study we tested the role of CAEL-101 in mediating the phagocytosis of synthetic fibrils and their intermediates by macrophages and neutrophils, as a way of elucidating its mechanism of action. Methods Two human VL proteins, named LEN and AL09, were recombinantly expressed in mammalian cell systems. Synthetic LEN and AL09 fibrils were generated using purified recombinant VL protein. Fibril formation was assessed by Thioflavin T (ThT) fluorescence emission fold change over time, change in radius of protein sample via Dynamic Light Scattering (DLS), and Transmission Electron Microscopy (TEM). The binding of CAEL-101 to fibrils was determined using an immunoassay performed on an MSD® platform and SPR (Biacore™ T200). To generate fibril intermediates (light chain aggregates), recombinant AL09 VL protein was processed per a fibril formation protocol. Samples were taken at 48- and 72-hour timepoints wherein the ThT signal had not yet achieved saturation. DLS readings showed these samples to be smaller in size than samples acquired with fully formed synthetic fibrils. TEM revealed samples to be aggregated and lacking a beta-pleated fibrillar structure. Phagocytosis of synthetic fibrils and intermediates were demonstrated using PMA differentiated THP1 macrophages and neutrophils isolated from healthy donors. Synthetic fibrils and intermediates were labeled with pHrodo™ iFL Red STP Ester dye and incubated with various dilutions of CAEL-101, IgGk1 isotype control, and 10% normal human serum (NHS). Fucoidan, a macrophage scavenger receptor A inhibitor, was included in assays using THP-1 cells. An Fc blocking reagent was included to evaluate whether the observed phagocytosis is FcγR dependent. Phagocytosis assays were also done with complement depleted human serum to evaluate the impact of complement activation on CAEL-101 mediated amyloid clearance. Phagocytosis at 3hr timepoint was assessed using an Incucyte® S3 system, as indicated by presence of pHrodo signal emitted from cells. Results CAEL-101 bound to synthetic fibrils dehydrated onto an MSD plate with an EC 50 of 6.346E-010 M for LEN fibrils and 9.21E-07 M for AL09 fibrils. CAEL-101 bound to LEN fibrils via SPR with an affinity of 2.66E-11 KD (M) and to AL09 fibrils with 3.62E-07 KD (M) at pH 7.4. Phagocytosis assays demonstrated that incubation of synthetic fibrils with CAEL-101 led to statistically significant enhancement of phagocytosis in both the differentiated THP-1 cells and neutrophils, as compared to phagocytosis observed with IgGk1 isotype control antibody or fibril alone. Phagocytosis was enhanced in the presence of 10% normal human serum compared to complement depleted serum. At 3hr timepoint, CAEL-101 significantly enhanced LEN fibril phagocytosis (p<0.05, two-way ANOVA) at antibody concentrations of 0.41 nM and above, with no phagocytosis observed above background in the presence of Fc block. CAEL-101 significantly enhanced the phagocytosis of intermediate AL09 light chain aggregates (48- and 72- hour timepoint collections), as compared with IgGk1 isotype control antibody or fibril alone. Conclusions The current in-vitro study demonstrated that CAEL-101 binds to light chain amyloid fibrils and upon binding causes the phagocytosis of fibrils via macrophages and neutrophils in a FcγR dependent manner. The phagocytotic activity is further enhanced in the presence of complement. In addition, for the first time we demonstrated that CAEL-101 was able to phagocytose intermediate soluble light chain aggregates, known precursors of amyloid fibril formation.

Mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) and its application in RNA detection

Some non-coding RNAs are abnormally expressed during the occurrence and development of diseases, so it is necessary to develop analytical methods that can specifically and sensitively detect them. In typical CRISPR/Cas12a system, a complete crRNA that can recognize single-stranded or double-stranded DNA is necessary to activate its trans-cleavage activity, which limits its direct application in RNA detection. Here, we prospectively find that slicing the facilitated crRNA in the typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, and a mini crRNA-mediated CRISPR/Cas12a system (MCM-CRISPR/Cas12a) was proposed based on this. This system can detect non-coding RNA to pM-level (10 pM for miRNA-21). To expand the application of this system, we combined it with HCR and CHA to establish a detection platform for non-coding RNA. The results show that the proposed method can specifically detect RNA to fM-level (2.5 fM for miRNA-21, 8.98 fM for miR-128–3p, and 81.6 fM for lncRNA PACER). The spiked recovery rates of miRNA-21, miR-128–3p, and lncRNA PACER in normal human serum were in range from 104.7 to 109.4 %, indicating the proposed method owns good applicability. In general, this MCM-CRISPR/Cas12a system further breaks the limitations of the typical CRISPR/Cas12a system that cannot be directly used for non-coding RNA detection. Besides, its combination with HCR and CHA achieves highly sensitive detection of non-coding RNA.

Downregulation of TRPC6 regulates ERK1/2 to prevent sublytic C5b‑9 complement complex‑induced podocyte injury through activating autophagy.

Idiopathic membranous nephropathy (IMN) is a common glomerular disease, in which 50-60% of patients can progress to end-stage renal disease within 10-20 years, seriously endangering human health. Podocyte injury is the direct cause of IMN. Sublytic C5b-9 complement complex induces damage in podocytes' structure and function. In sublytic C5b-9 treated podocytes, the expression of canonical transient receptor potential 6 (TRPC6) is increased. However, the specific mechanism of TRPC6 in sublytic C5b-9 treated podocytes is unclear. The present study aimed to reveal the effect and mechanism of TRPC6 on sublytic C5b-9-induced podocytes. Normal human serum was stimulated using zymosan to form C5b-9. A lactate dehydrogenase release assay was used to examine C5b-9 cytotoxicity in podocytes. The RNA and protein expression levels were analyzed using reverse transcription-quantitative PCR, western blotting and immunofluorescent assay, respectively. Cell Counting Kit-8 assay and flow cytometry were carried out to test the viability and apoptosis of podocytes, respectively. Transmission electron microscopy was used to observe autophagic vacuole. F-actin was tested through phalloidin staining. Sublytic C5b-9 was deposited and TRPC6 expression was boosted in podocytes stimulated through zymosan activation serum. Knockdown of TRPC6 raised the viability and reduced the apoptosis rate of sublytic C5b-9-induced podocytes. Meanwhile, transfection of small-interfering (si)TRPC6 facilitated autophagy progression and enhanced the activation of cathepsin B/L in sublytic C5b-9-induced podocytes. The phosphorylation level of ERK1/2 was receded in siTRPC6 and sublytic C5b-9 co-treated podocytes. Moreover, the addition of the ERK1/2 activator partially reversed the effect of TRPC6 inhibition on sublytic C5b-9-induced podocytes. TRPC6 knockdown reduced the damage of sublytic C5b-9 to podocytes by weakening the ERK1/2 phosphorylation level to activate autophagy. These results indicated that targeting TRPC6 reduced the injury of sublytic C5b-9 on podocytes.

Modeling complement activation on human glomerular microvascular endothelial cells.

Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Nodal-paranodal antibodies in HIV-immune mediated radiculo-neuropathies: Clinical phenotypes and relevance.

The frequency of nodal-paranodal antibodies in HIV-infected patients with chronic immune-mediated radiculo-neuropathies (IMRN) has not been previously described. HIV-infected patients who met the inclusion criteria for chronic IMRN were screened for immunoglobulin G (IgG) antibodies directed against nodal (neurofascin (NF)186) and paranodal (NF155, contactin-1 (CNTN1) and contactin-associated protein(Caspr1)) cell adhesion molecules, using a live, cell-based assay. To explore potential pathogenicity, binding of human IgG to myelinated co-cultures was assessed by incubation with patients' sera positive for nodal or paranodal antibodies. Normal human serum was added as a source of complement to assess for complement activation as a mechanism for myelin injury. Twenty-four HIV-infected patients with IMRN were included in the study, 15 with chronic inflammatory demyelinating polyneuropathy (CIDP), 4 with ventral root radiculopathies (VRR), and 5 with dorsal root ganglionopathies (DRG). Five patients with CIDP had combined central and peripheral demyelination (CCPD). Three patients (12.7%) tested positive for neurofascin IgG1 antibodies in the following categories: 1 patient with VRR was NF186 positive, and 2 patients were NF155 positive with DRG and mixed sensory-motor demyelinating neuropathy with optic neuritis, respectively. The frequency of nodal-paranodal antibodies is similar among IMRN regardless of HIV status. Interpretation of the results in the context of HIV is challenging as there is uncertainty regarding pathogenicity of the antibodies, especially at low titres. Larger prospective immune studies are required to delineate pathogenicity in the context of HIV, and to establish a panel of antibodies to predict for a particular clinical phenotype.

Detection of CYFRA 21-1 in human serum by an electrochemical immunosensor based on UiO-66-NH2@CMWCNTs and CS@AuNPs.

In this study, an electrochemical immunosensor was constructed to detect the cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) in human serum. CYFRA 21-1 is the most sensitive tumor marker of non-small cell lung cancer (NSCLC), its content in normal human serum should be less than 3.3ng/mL. When lung cancer cells dissolve or die, a myriad of CYFRA 21-1 is released into a tumor patient's blood circulation, and its serum content elevates strikingly. Consequently, detecting CYFRA 21-1 by an electrochemical biosensor is expected to provide a new method for the early detection and prevention of lung cancer. In this study, a composite of UiO-66-NH2 and carboxylated multi-walled carbon nanotubes (CMWCNTs) was used as the substrate material of a sensor; the resulting sensor had a large specific surface area and strong electrical conductivity. Moreover, gold nanoparticles (AuNPs) were used to bind to antibodies through an Au-S bonds. Also, a supersensitive detection of CYFRA 21-1 was achieved through the specific bindings of antigens and antibodies. Under optimal detection conditions, the change of current signal intensity of the immunosensor was proportional to the logarithm of CYFRA 21-1 concentration and had a linear relation in the range of 0.005-400ng/mL, while the detection limit was 1.15pg/mL (S/N=3). The proposed immunosensor had high precision, stability, and selectivity. More importantly, the sensor was been successfully applied to detect CYFRA 21-1 in human serum with high recovery, providing a new method for early screening and dynamic monitoring of lung cancer.

Porous carbon nanosheets derived from two-dimensional Fe-MOF for simultaneous voltammetric sensing of dopamine and uric acid

It is of great significance for electrochemical sensors to simultaneously detect dopamine (DA) and uric acid (UA) related to biological metabolism. In this work, two-dimensional (2D) porous carbon nanosheets (CNS) was prepared as electrocatalysts to improve the sensitivity, the selectivity, and the detection limit of the simultaneous detection. First, 2D amorphous iron-metal organic frameworks (Fe-MOF) was synthesized with Fe3+ and terephthalic acid via a facile wet chemistry method at room temperature. And then, CNS was prepared by pyrolysis and pickling of Fe-MOF. CNS had large specific surface area, good electrical conductivity and lots of carbon defects. The response currents of the CNS modified electrode was larger than those of the control electrodes in the simultaneous determination. The simultaneous determination was measured via differential pulse voltammetry to reduce the effect of capacitive currents on quantitative analysis. The CNS modified electrodes showed high sensitivity and low detection limit for the simultaneous detection of DA and UA. The modified electrodes have been successfully used to detect DA and UA in normal human serum.

Metabolomics analyses of serum metabolites perturbations associated with Naja atra bite.

Naja atra bite is one of the most common severe snakebites in emergency departments. Unfortunately, the pathophysiological changes caused by Naja atra bite are unclear due to the lack of good animal models. In this study, an animal model of Naja atra bite in Guangxi Bama miniature pigs was established by intramuscular injection at 2 mg/kg of Naja atra venom, and serum metabolites were systematically analyzed using untargeted metabolomic and targeted metabolomic approaches. Untargeted metabolomic analysis revealed that 5045 chromatographic peaks were obtained in ESI+ and 3871 chromatographic peaks were obtained in ESI-. Screening in ESI+ modes and ESI- modes identified 22 and 36 differential metabolites compared to controls. The presence of 8 core metabolites of glutamine, arginine, proline, leucine, phenylalanine, inosine, thymidine and hippuric acid in the process of Naja atra bite was verified by targeted metabolomics significant difference (P<0.05). At the same time, during the verification process of the serum clinical samples with Naja atra bite, we found that the contents of three metabolites of proline, phenylalanine and inosine in the serum of the patients were significantly different from those of the normal human serum (P<0.05). By conducting functional analysis of core and metabolic pathway analysis, we revealed a potential correlation between changes in key metabolites after the Naja atra bite and the resulting pathophysiological alterations, and our research aims to establish a theoretical foundation for the prompt diagnosis and treatment of Naja atra bite.

Human embryonic stem cells secrete macrophage migration inhibitory factor: A novel finding.

Macrophage migration inhibitory factor (MIF) is expressed in a variety of cells and participates in important biological mechanisms. However, few studies have reported whether MIF is expressed in human Embryonic stem cells (ESCs) and its effect on human ESCs. Two human ESCs cell lines, H1 and H9 were used. The expression of MIF and its receptors CD74, CD44, CXCR2, CXCR4 and CXCR7 were detected by an immunofluorescence assay, RT-qPCR and western blotting, respectively. The autocrine level of MIF was measured via enzyme-linked immunosorbent assay. The interaction between MIF and its main receptor was investigated by co-immunoprecipitation and confocal immunofluorescence microscopy. Finally, the effect of MIF on the proliferation and survival of human ESCs was preliminarily explored by incubating cells with exogenous MIF, MIF competitive ligand CXCL12 and MIF classic inhibitor ISO-1. We reported that MIF was highly expressed in H1 and H9 human ESCs. MIF was positively expressed in the cytoplasm, cell membrane and culture medium. Several surprising results emerge. The autosecreted concentration of MIF was 22 ng/mL, which was significantly higher than 2 ng/mL-6 ng/mL in normal human serum, and this was independent of cell culture time and cell number. Human ESCs mainly expressed the MIF receptors CXCR2 and CXCR7 rather than the classical receptor CD74. The protein receptor that interacts with MIF on human embryonic stem cells is CXCR7, and no evidence of interaction with CXCR2 was found. We found no evidence that MIF supports the proliferation and survival of human embryonic stem cells. In conclusion, we first found that MIF was highly expressed in human ESCs and at the same time highly expressed in associated receptors, suggesting that MIF mainly acts in an autocrine form in human ESCs.

Serum components influence antibody reactivity to glycan and DNA antigens

We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.

CD4 T lymphocyte subsets display heterogeneous susceptibility to apoptosis induced by serum from patients with systemic lupus erythematosus

BackgroundSerum from systemic lupus erythematosus (SLE) patients has been shown to induce T-lymphocyte (TL) apoptosis. Given that different cells of the immune system display different sensitivity to apoptosis, we set to evaluate the in vitro effect of SLE serum on regulatory T-cells (Treg), Th17, Th1 and Th2 from SLE patients and healthy controls.MethodsPeripheral blood mononuclear cells from SLE patients or normal controls were exposed to a pool of sera from SLE patients or normal controls. Annexin V was used to label cells in apoptosis or necrosis. Annexin V-labeled Treg, Th17, Th1 and Th2 cells were determined using flow cytometry.ResultsTotal CD3 + and CD4 + cells from SLE patients showed higher frequency of spontaneous apoptosis/necrosis, whereas Th1 cells from SLE patients presented reduced spontaneous apoptosis/necrosis rate as compared with cells from controls. Incubation with SLE serum induced increased frequency of apoptotic/necrotic CD3 + , CD4 + and Th2 cells from normal controls or from SLE patients as compared with cultures incubated with normal human serum (NHS) or without human serum at all. Incubation with SLE serum did not increase the apoptosis/necrosis rate in Th1 or Th17 cells. Treg cells from SLE patients were more prone to apoptosis/necrosis induced by SLE serum than Treg cells from normal individuals. Th1, Th2, and Th17 cells presented increased apoptosis rates in cultures without human serum.ConclusionOur findings indicate that the serum of patients with active SLE stimulates apoptosis of CD4 + T cells in general and exhibit differentiated effects on CD4 + T-cell subsets.

Triple-recognition strategy for one-pot detection of single nucleotide variants by aligner-mediated cleavage-triggered exponential amplification

The detection of single nucleotide variants (SNVs) is important for the diagnosis and treatment of cancer. To date, researchers have devised several methods to detect SNVs, but most of them are complex and time-consuming. To improve SNVs detection specificity and sensitivity, we developed a triple-recognition strategy, which facilitates aligner-mediated cleavage-triggered exponential amplification (Trec-AMC-EXPAR) for the rapid, specific, and one-pot detection of SNV. Under optimized conditions, Trec-AMC-EXPAR detected two clinically significant SNVs, PIK3CAH1047R and EGFR L858R within 80 min, with a reliable detection of 0.1% SNV in the wide type, which is lower than that of allele-specific PCR (AS-PCR) for detecting SNV. Finally, by spiking into normal human serum samples, mutants mixed with the wild-type targets in different ratios were analyzed, resulting in the relative standard deviation (RSD) of recovery ratios <3%. The findings suggested the potential application of Trec-AMC-EXPAR in clinical disease diagnosis. In summary, the proposed Trec-AMC-EXPAR technique provides a novel fast and convenient method for one-pot detection of SNV with high sensitivity and specificity.

Regulatable Complement Inhibition of the Alternative Pathway Mitigates Wet Age-Related Macular Degeneration Pathology in a Mouse Model.

Risk for developing age-related macular degeneration (AMD) is linked to an overactive complement system. In the mouse model of laser-induced choroidal neovascularization (CNV), elevated levels of complement effector molecules, including complement C3, have been identified, and the alternative pathway (AP) is required for pathology. The main soluble AP regular is complement factor H (fH). We have previously shown that AP inhibition via subretinal AAV-mediated delivery of CR2-fH using a constitutive promoter is efficacious in reducing CNV. Here we ask whether the C3 promoter (pC3) effectively drives CR2-fH bioavailability for gene therapy. Truncated pC3 was used to generate plasmids pC3-mCherry/CR2-fH followed by production of corresponding AAV5 vectors. pC3 activation was determined in transiently transfected ARPE-19 cells stimulated with H2O2 or normal human serum (+/- antioxidant or humanized CR2-fH, respectively). CNV was analyzed in C57BL/6J mice treated subretinally with AAV5-pC3-mCherry/CR2-fH using imaging (optical coherence tomography [OCT] and fundus imaging), functional (electroretinography [ERG]), and molecular (protein expression) readouts. Modulation of pC3 in vitro is complement and oxidative stress dependent, as shown by mCherry fluorescence. AAV5-pC3-CR2-fH were identified as safe and effective using OCT and ERG. CR2-fH expression significantly reduced CNV compared to mCherry and was correlated with reduced levels of C3dg/C3d in the retinal pigment epithelium/choroid fraction. We conclude that complement-dependent regulation of AP inhibition ameliorates AMD pathology as effectively as using a constitutive promoter. The goal of anticomplement therapy is to restore homeostatic levels of complement activation, which might be more easily achievable using a self-regulating system.

Novel recombinant proteins and peptides from Clonorchis sinensis and Opisthorchis viverrini for liver fluke exposure ELISA

Human serum samples from individuals living in Vietnam and Taiwan suspected of past Clonorchis sinensis or Opisthorchis viverrini infection were screened using several novel peptides and recombinant liver fluke proteins to determine if any consistent patterns could be discerned and used as the basis for future liver fluke ELISA development. Absorbance values at 405 nm were compared to those of pooled unexposed normal human serum and analyzed for statistical significance. The data exhibited some interesting patterns consistent with egg antigen sequestration in the gut possibly leading to lower serum antibody levels and potential regional exposure differences between Vietnamese and Taiwanese subjects. In particular, antibodies against Cathepsin B and B2 peptides, as well as a partial Cahedrin Domain peptide may be elevated in some Taiwanese serum samples while antibodies against recombinant Clonorchis egg protein and Hepatocellular Carcinoma Peptide Antigen 59 may be elevated in some samples from both Taiwan and Vietnam. The data appear to suggest that some of the novel recombinant protein and peptide antigens selected and tested herein warrant further study with larger sample sizes as possible targets for detecting anti-liver fluke antibodies by ELISA from humans suspected of liver fluke infections.

Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean+ 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

#5885 HYPOXIA INDUCES COMPLEMENT ACTIVATION ON HUMAN KIDNEY EPITHELIAL CELLS

Abstract Background and Aims Ischemia-reperfusion injury is an inevitable event associated with kidney transplantation (Tx) and has a major impact on short- and long-term graft survival. Proximal tubular epithelial cells are both a source of complement during Tx and a target of complement activation. The normal kidney has a capacity to protect itself from complement activation through cellular expression of complement regulatory proteins. In this study we investigated whether hypoxia and reoxygenation increase C3 deposition and alter complement regulatory protein expression (CD46, CD59 and CD55). Method In vitro, HKC-8 renal proximal tubular epithelial cells were subjected to 24h of hypoxia (1% O2) and then reoxygenated for 4h (O2 = 21%) in the presence of 40% normal human serum. qRT-PCR was used to estimate the level of VEGF-A (as a positive control), C3, CD46, CD59 and CD55 expression. Similar analysis was performed in untreated, normoxic cells. Immunofluorescence staining was used to determine C3 deposition on the cell surface and intensity quantified by ImageJ. Experiments were repeated three times. Results qRT-PCR analysis of HKC-8 cells in hypoxia revealed significant increases in the expression of VEGF-A after 24h (p = 0.0113). C3 expression following hypoxia and reoxygenation increased insignificantly. Increased expression of CD59 with the presence of serum was observed in epithelial cells (p = 0.026). However, no significant change was seen in the expression of CD55 and CD46. Reoxygenation in human serum led to significantly greater deposition of C3 on hypoxic cells compared with normoxic HKC-8 cells (Figure 1, 2). Conclusion Our preliminary data suggests that hypoxia and reoxygenation significantly increases C3 deposition on the proximal tubular cell surface. CD59, as one of the regulators of complement activation is upregulated at mRNA level after hypoxia followed by 4h reperfusion in the presence of serum. These data demonstrate that hypoxia and reoxygenation activate complement system with upregulation of CD59 as a cell defence. However, expression of CD46 and CD55 do not change. Further study is needed to identify the impact of hypoxia on complement regulation in primary epithelial cells and kidney tissues.

Silver chalcogenide loaded V2CTx MXene-molecularly imprinted polymer-based novel ratiometric sensor for the early predictive cancer marker: L-Fucose

Fucosylated-haptoglobin (Hpt) and urinary free L-fucose (FU) are primary markers in the progression of tumor and metastasis-related to cancer of the liver (Hepatocellular carcinoma), pancreatic, prostate, colon and oral cancer. The p-aminobenzoic acid (PABA)-FU containing molecularly imprinted polymer (MIP) cavities were accessed to detect FU molecules with signal amplifying layer of novel silver selenide (Ag2Se) doped vanadium carbide (V2CTx) MXene. The selection of suitable monomers through theoretical calculations and optimization of factors involved in the fabrication of a MIP-based sensor via Taguchi orthogonal array (5 × 5) was abundant in improvising the sensor performance. Ratiometry of the oxidation response of FU (IFU) and Ag2Se@V2CTx (IM) played a vital role in eliminating the interference of serum proteins which includes Hpt (undigested), thus sample pretreatment is not required. The extraordinary selectivity with remarkable LOD (1.85 μM) was achieved for FU detection in 100 mM PBS (pH = 6.0) and structurally mimicking interferents failed to show an impactful response against the sensor. Urinary-free FU (spiked) and serum FU (unspiked and spiked) were analyzed in which the LOD of urinary-free FU was as low as 2.22 μM. In addition, normal human serum showed 440.2 μM of FU and the %recovery of FU-spiked samples was between 98.0 and 101.5%.