Serum Immune Complexes Research Articles

Association Between Cytokeratin 19-Specific IgG and Neutrophil Activation in Asthma.

Patients with non-eosinophilic asthma (NEA) are less responsive to anti-inflammatory drugs and suffer from frequent asthma exacerbations. The pathogenic mechanism of NEA is not fully understood; however, the roles of monocytes and autoimmune mechanisms targeting airway epithelial cell (AEC) antigens have been proposed. The effects of monocyte extracellular traps (MoETs) on cytokeratin 19 (CK19) production in AECs, as well as the impact of CK19-specific immunoglobulin (Ig) G on neutrophil and monocyte activation, were investigated both in vivo and in vitro. Sixty asthmatic patients and 15 healthy controls (HCs) were enrolled, and the levels of serum immune complexes containing CK19-specific IgG and neutrophil extracellular trap (NET)-specific IgG were measured using enzyme-linked immunoassay. MoETs induced CK19 and CK19-specific IgG production. Furthermore, the levels of serum CK19-specific IgG were significantly higher in the NEA group than in the eosinophilic asthma group. Among patients with NEA, asthmatics with high levels of CK19-specific IgG had higher levels of myeloperoxidase and NET-specific IgG than those with low levels of CK19-specific IgG (P = 0.020 and P = 0.017; respectively). Moreover, the immune complexes from asthmatics with high CK19-specific IgG enhanced NET formation and reactive oxygen species production (neutrophil activation), which were suppressed by N-acetylcysteine and anti-CD16 antibody treatment. These findings suggest that circulating CK19 and CK19-specific IgG may contribute to NET formation, leading to airway inflammation and steroid resistance in NEA.

Dynamic light scattering analysis of immune complexes in sera of rheumatoid arthritis patients

The size of circulating immune complexes (CICs) in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. This study analyzed size and electrokinetic potential of CICs from RA patients, healthy young adults, and RA patients age-matched controls aiming to establish their unique CIC features. Pooled CIC of 30 RA patients, 30 young adults, and 30 RA group's age-matched controls (middle-aged and oldеr healthy adults), and in vitro IgG aggregates from pooled sera of 300 healthy volunteers were tested using dynamic light scattering (DLS). Size distribution of CIC in healthy young adults exhibited high polydispersity. RA CIC patients and their age-matched control showed distinctly narrower size distributions compared with young adults. In these groups, particles clustered around two well-defined peaks. Particles of peak 1 were 36.1 ± 6.8 nm in RA age-matched control, and 30.8 ± 4.2 nm in RA patients. Particles of peak 2 of the RA age-matched control's CIC was 251.7 ± 41.2 nm, while RA CIC contained larger particles (359.9 ± 50.5 nm). The lower zeta potential of RA CIC, compared to control, indicated a disease-related decrease in colloidal stability. DLS identified RA-specific, but also age-specific distribution of CIC size and opened possibility of becoming a method for CIC size analysis in IC-mediated diseases.

Iron Status Alters Murine Systemic Lupus Erythematosus

The effect of Fe status on murine systemic lupus erythematosus was investigated. Weanling female MRL/MPJ-lpr/lpr mice (systemic lupus erythematosus strain) were fed diets with the following levels (mg Fe/kg diet): 3 (severely deficient), 10 (moderately deficient), 35 (control) and 250 (supplemented). A fifth group was pair fed the control diet in the amounts consumed by the severely deficient group. C3H/Hej mice fed the same diets were used as nonlupus controls. Anemia was more severe in severely deficient mice than in all other MRL groups and C3H severely deficient mice. Incidence of skin lesions was highest in MRL severely and moderately deficient mice compared with pair-fed, control and supplemented mice. By 22 wk of age, mortality was higher in supplemented and severely deficient mice than in moderately deficient, pair-fed and control MRL mice. Anti-dsDNA activity in serum was not altered by Fe. In a second experiment, kidney function was examined in mice fed severely deficient, control, supplemented and pair-fed diets. Urine protein concentration was highest in supplemented mice at 14 wk of age. Serum urea nitrogen was significantly higher in MRL severely deficient mice than in pair-fed and control mice at 18 wk of age. Glomerular filtration rate, measured by creatinine clearance, was significantly lower in MRL severely deficient mice than in pair-fed and Fe supplemented mice at 16 wk of age and pair-fed and control mice at 18 wk of age. Renal histopathology was more severe in Fe supplemented mice than in pair-fed and control mice, and more severe in severely deficient and pair-fed mice than in control mice. Fluorescent staining of kidneys with anti-Ig G and anti-C3 fluorescein-conjugated antibodies was most intense in severely deficient mice, and the concentration of circulating immune complexes in serum was significantly higher in severely deficient mice than in all other groups. These data demonstrate that systemic lupus erythematosus in MRL/MPJ-lpr/lpr mice is altered by dietary iron.

Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity.

B cells drive the production of autoreactive antibody-secreting cells (ASCs) in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Sjögren's syndrome, causing long-term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibodies. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-1 could deplete ASCs in autoimmune conditions in vivo and in vitro. Use of a BMI-1 inhibitor in both mouse and human autoimmune settings was investigated. Lyn -/- mice, a model of SLE, were treated with the BMI-1 small molecule inhibitor PTC-028, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-028 on ASCs derived from Sjögren's syndrome patients was evaluated. BMI-1 inhibition significantly decreased splenic and bone marrow ASCs in Lyn -/- mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG3, immune complexes and anti-DNA IgG. PTC-028 was also efficacious in reducing ex vivo plasma cell survival from both Sjögren's syndrome patients and age-matched healthy donors. These data provide evidence that inhibiting BMI-1 can deplete ASC in a variety of contexts and thus BMI-1 is a viable therapeutic target for antibody-mediated autoimmune diseases.

Immunological Indicators of Pink Salmon Oncorhynchus during the Marine Phase of their Life Cycles

A comparative study of some parameters of nonspecific immunity in sexually mature individuals of pink salmon and chum salmon caught on June 6–July 1, 2018 by an epipelagic trawl in the open waters of the northwestern part of the Pacific Ocean was carried out. The level of antimicrobial properties of blood serum and nonspecific immune complexes in the tissues of the kidney, spleen and liver and the proportion of immunodeficient individuals were studied. Significant differences in the antimicrobial properties of blood serum and immunodeficient individuals were found. These differences are probably due to species characteristics, as well as the pre-spawning period of the annual cycle in pink salmon.

Immune complexome analysis of a rich variety of serum immune complexes identifies disease-characteristic immune complex antigens in systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular endothelial dysfunction and skin fibrosis. Recently, the presence and pathogenic role of immune complexes (ICs) of SSc patients were reported. However, the identities of antigens in these ICs are unknown. Therefore, we examined ICs in the serum of SSc patients to elucidate SSc pathogenesis. In this study, IC concentrations in serum samples from SSc and systemic lupus erythematosus (SLE) patients were measured by C1q enzyme-linked immunosorbent assays; immune complex analysis was used for comprehensive identification and comparison of antigens incorporated into ICs (IC-antigens). The expression patterns of SSc-specific IC-antigens in skin sections were investigated by immunohistochemistry. Compared with SLE patients who developed disease because of IC deposition, SSc patients had a greater number of IC-antigens and a smaller difference in IC concentrations, suggesting that SSc pathogenesis is affected by the proteins present in ICs. In contrast, the IC concentration and number of IC-antigens did not significantly differ according to the clinical phenotype of SSc. We identified 478 IC-antigens in SSc patients, including multiple RNAP II-associated proteins that were targeted by antibodies previously associated with SSc pathogenesis. The most frequently detected RNAP II-associated protein, RNA polymerase II transcription subunit 30 (MED30), was strongly expressed at lesion sites and reportedly regulates endothelial differentiation. Therefore, increased expression of MED30 in lesions may have an antigenic effect, and MED30 function may be impaired or inhibited by IC formation. RNAP II-associated proteins may SSc pathogenesis through mechanisms such as the MED30 pathway.

Atacicept Reduces Serum Immune Complex Levels in Patients With IgA Nephropathy (IgAN)

Atacicept Reduces Serum Immune Complex Levels in Patients With IgA Nephropathy (IgAN)

Sensitive, Color-Indicating and Labeling-Free Multi-Detection Cholesteric Liquid Crystal Biosensing Chips for Detecting Albumin.

A novel device for cholesteric liquid crystal (CLC)-based microfluidic chips, accommodated in a polydimethylsiloxane material, was invented. In this device, the reorientation of the CLCs was consistently influenced by the surface of the four channel walls and adjacent CLCs. When the inside of the microchannel was coated with the alignment layer, the CLCs oriented homeotropically in a focal conic state under cross-polarizers. Once antigens had bound onto antibodies immobilized onto the orientation sheet-coated channel walls, the light intensity of the CLC molecules converted from a focal conic state to a bright planar state caused by disrupting the CLCs. By means of utilizing pressure-propelling flow, the attachment of antigen/antibody to the CLCs should be detectable within consecutive sequences. The multi-microfluidic CLC-based chips were verified by measuring bovine serum albumin (BSA) and immune complexes of pairs of BSA antigen/antibody. We showed that the multiple microfluidic immunoassaying can be used for measuring BSA and pairs of antigen/antibody BSA with a detection limit of about 1 ng/mL. The linear range is 0.1 μg/mL–1 mg/mL. A limit of immune detection of pairs of BSA antigens/antibodies was 10 ng/mL of BSA plus 1000 ng/mL of the anti-BSA antibodies was observed. According to this innovative creation of immunoassaying, an unsophisticated multi-detection device with CLC-based labeling-free microfluidic chips is presented.

Innovative approaches to improve serodiagnosis of Strongyloides stercoralis infection

Strongyloidiasis is a helminthiasis of neglected condition that has no gold standard parasitological diagnosis due to the intermittent release of larvae in feces. This study aimed to use an scFv (single chain variable fragment) obtained by Phage Display, previously validated to detect immune complexes in serum samples from individuals infected with Strongyloides stercoralis by enzyme-linked immunosorbent assay (ELISA). Now the ability of scFv to detect the immune complexes was verified by immunofluorescence, flow cytometry using magnetic beads and surface plasmon resonance (SPR). As ELISA, the SPR, immunofluorescence and flow cytometry demonstrated the ability of scFv to detect immune complexes in sera from individuals with strongyloidiasis and discriminate them from sera of individuals with other parasitic diseases and healthy individuals. Besides de conventional ELISA, the novel approaches can also be promptly applied as auxiliary diagnostic tools to the existing parasitological method for accurate diagnosis of human strongyloidiasis.

Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

COVID-19: Therapeutics and Their Toxicities.

SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is causing the COVID-19 pandemic. There is no current standard of care. Clinicians need to be mindful of the toxicity of a wide variety of possibly unfamiliar substances being tested or repurposed to treat COVID-19. The United States Food and Drug Administration (FDA) has provided emergency authorization for the use of chloroquine and hydroxychloroquine. These two medications may precipitate ventricular dysrhythmias, necessitating cardiac and electrolyte monitoring, and in severe cases, treatment with epinephrine and high-doses of diazepam. Recombinant protein therapeutics may cause serum sickness or immune complex deposition. Nucleic acid vaccines may introduce mutations into the human genome. ACE inhibitors and ibuprofen have been suggested to exacerbate the pathogenesis of COVID-19. Here, we review the use, mechanism of action, and toxicity of proposed COVID-19 therapeutics.

A method combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem-mass spectrometry to detect circulating immune complexes between therapeutic monoclonal antibodies and anti-drug antibodies in animals

Therapeutic monoclonal antibodies can potentially induce unwanted immune responses, resulting in the production of anti-drug antibodies (ADAs). The binding of ADAs to drugs and subsequent formation of immune complexes (ICs) can trigger various responses, dependent on the size, concentration, and subclass of ADAs. To better understand the impact of ADAs on pharmacokinetics, pharmacodynamics, and toxicological profiles, a bioanalytical method was developed for the detection of ICs between human monoclonal immunoglobulin G (IgG) and ADAs in biological samples. Regarding the experimental procedure, in brief, the human antibody-specific ICs and unbound human antibody in biological samples are separated through blue native polyacrylamide gel electrophoresis (BN-PAGE). The target fractions are then cut from the gel, followed by in-gel trypsin digestion and subsequent liquid chromatography tandem-mass spectrometry (LC-MS/MS) to monitor the human IgG-specific peptide. This method was able to detect various types of human antibodies with a lower limit of detection of 10 μg/mL in monkey serum. The assay performance for the detection of ICs was demonstrated using spiked samples, and pre-incubated ICs in monkey serum were clearly detected. Taken together, these findings indicate that our method enables a semi-quantitative analysis for estimating the ratio of human antibody included ICs in comparison to the total antibody. This method was successfully applied to an in vivo study using mice, and the data helped explain the unexpectedly rapid clearance of a humanized antibody due to the formation of large ICs. The combination of the separation of ICs by BN-PAGE and the detection of the human IgG-specific peptide by LC-MS/MS is a useful general bioanalytical approach for the detection of ICs in animals.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection.

Definitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

Immunomodulatory Effect of Eriocitrin in Experimental Animals with Benzo(a)Pyrene-induced Lung Carcinogenesis.

Lung carcinogenesis is one of the main sources of cancer-related mortality globally and it is estimated that nearly 1 million people die from it every year. The 5-year survival rate of lung carcinogenesis is reported at just 15%. The aim of the current research was to investigate the immunomodulatory effect of eriocitrin against benzo(a)pyrene [B(a) P]-induced lung tumorigenesis in Swiss albino mice. The lung sarcoma was provoked through oral gavage of B(a)P (50 mg/kg body weight) two times/week for four weeks. CEA, lung weight, lipid peroxidation (LPO), body weight, immuno-globulin (IgG, IgA, and IgM), tumor incidence, serum marker enzymes (LDH, AHH, λ-GT, and 5'-NTs), hematological counts (leucocytes, lymphocytes, neutrophils, absolute numbers of lymphocytes and neutrophils), antioxidants (SOD and CAT), inflammatory modulators (IL-1β, IL-6, and TNF-α), immune complexes (avidity index, phagocyte index, NBT reduction, and SIC) and histopathological changes were analyzed. Moreover, the status of apoptosis proteins (Bax, caspase-9, and caspase-3) and cell proliferative protein (cyclin D1 and cyclin A) expression was determined by Western blot and PCNA by immunohistochemical analysis. B(a)P-challenged cancer-bearing mice exhibited augmented levels of lipid peroxidation, tumor incidence, lung weight, CEA, serum marker enzymes, IgA, SIC, cell proliferative markers, and inflammatory cytokines with concurrent decrease in body weight, antioxidant levels, hematological counts, immunoglobulins, immune complexes, and apoptotic protein expression. The eriocitrin treatments caused significant reversion of all these marker to previous levels. Overall, the results propose the immunomodulatory prospective of eriocitrin against B(a) P-induced lung carcinogenesis on Swiss albino mice.

Conditional Upregulation of IFN-α Alone Is Sufficient to Induce Systemic Lupus Erythematosus.

The cause of systemic lupus erythematosus (SLE) is unknown. IFN-α has been suggested as a causative agent of SLE; however, it was not proven, and to what extent and how IFN-α contributes to the disease is unknown. We studied the contribution of IFN-α to SLE by generating inducible IFN-α transgenic mice and directly show that conditional upregulation of IFN-α alone induces a typical manifestation of SLE in the mice not prone to autoimmunity, such as serum immune complex, autoantibody against dsDNA (anti-dsDNA Ab), and the organ manifestations classical to SLE, such as immune complex-deposited glomerulonephritis, classical splenic onion-skin lesion, alopecia, epidermal liquefaction, and positive lupus band test of the skin. In the spleen of mice, activated effector CD4 T cells, IFN-γ-producing CD8 T cells, B220+CD86+ cells, and CD11c+CD86+ cells were increased, and the T cells produced increased amounts of IL-4, IL-6, IL-17, and IFN-γ and decreased IL-2. In particular, activated CD3+CD4-CD8- double-negative T cells positive for TCRαβ, B220, CD1d-teteramer, PD-1, and Helios (that produced increased amounts of IFN-γ, IL-4, IL-17, and TNF-α) were significantly expanded. They infiltrated into kidney and induced de novo glomerulonephritis and alopecia when transferred into naive recipients. Thus, sole upregulation of IFN-α is sufficient to induce SLE, and the double-negative T cells expanded by IFN-α are directly responsible for the organ manifestations, such as lupus skin disease or nephritis.

Influence of Marmix premix on the state of lipid peroxidation and indices of non-specific resistance of the organism of pregnant mares with microelementosis

We observed the processes of lipid peroxidation, characterized by increased content of intermediate and its end products (diene conjugates, lipid hydroperoxides, malonic dialdehyde), in the blood of the pregnant mares. The changes of the data of indicators in the organism of the mares at the 9–11 months of pregnancy were established. The level of diene conjugates in the blood of animals was 3.6 ± 0.12 μmol/l, 33.3 and 44.0%, which was higher compared to non-pregnant mares and mares at 4th month of pregnancy. The concentration of lipid hydroperoxides in the blood of mares increased by 100% and by 42.9% in the blood of non-pregnant mares and mares at 4th month of pregnancy. The concentration of malondialdehyde in the blood of mares increased by 75.0%, 51.2% and 25.0% compared to non-pregnant mares and mares at 4th and 7th month of pregnancy, respectively. The results of our research showed that the mineral-vitamin premix Marmix had a positive influence on the state of lipid peroxidation products, contributed to their lowering in the blood of pregnant mares, which demonstrated antioxidant action and stimulated the function of the immune system. It was established that the bactericidal and lysozyme activity of the blood serum and the concentration of circulating immune complexes increases in the mares with lack of trace elements. It was shown that feeding mares during the 9–11 months of pregnancy with the mineral-vitamin premix Marmix had a positive influence on the state of humoral factors of nonspecific resistance. In particular, the concentration of bactericidal activity of serum increased by 31.0%, lysozyme activity of blood serum – 45.4% and contributed to the decrease of the concentration of circulating immune complexes in serum by 3.8 times. The use of mineral-vitamin premix Marmix during 60 days in feeding pregnant mares caused a restoration of the clinical status, a decrease in the content of lipid peroxidation products.

Influence of cryptosporidiosis agent on biochemical and immunological parameters of blood of calves

Cows, young cattle and calves of black-and-bark breeds, spontaneously affected by the causative agent Cryptosporidium parvum, were selected for the research. Blood for research was taken from calves in the morning before feeding from the jugular vein. Biochemical parameters of blood serum were determined using a biochemical analyzer of the closed type. Changes in the activity of serum enzymes in the initial stages of the disease are not specific, they indicate a compensatory reaction of liver cells and biliary tract. Effect of Cryptosporidium in calves body accompanied by severe disorders that affect the metabolism of proteins, carbohydrates and enzymes. These changes lead to structural changes in the organs at the cellular level. Serum infested animals significantly reduced total protein content of 25.5% (P < 0.001), albumin – by 14.6% (P < 0.01), the concentration of glucose in – 21.2% (P < 0.01), carotene content – 12.6% (P < 0.05), the level of calcium and phosphorus – 17.5 and 18% (P < 0.05), respectively, and increased total bilirubin content by 23% (P < 0.001), indicating significant violations of the protein synthesis and detoxification processes in the liver and the tension in the metabolism of their body. Reduced glucose concentration in blood serum is due to the fact that in the body of sick animals there have been increased costs for maintaining the energy needs of its own organism. In conducting studies for 35 days in animals in the experimental group noted an increase in the concentration of circulating immune complexes by 10.5% relative to control. Increasing the concentration of circulating immune complexes in serum of calves, patients with cryptosporidiosis, indicates the presence of specific interaction antigen-antibody and decrease the activity of the humoral link of the immune system. In the determination of immunosuppressive proteins – serum cords in serum of calves, it was found that at 7, 14 days their concentration was within the physiological limits. Increasing the concentration of circulating immune complexes in serum of calves, patients with cryptosporidiosis, indicates the presence of specific interaction antigen-antibody and decrease the activity of the humoral link of the immune system.

Icariin alleviates murine lupus nephritis via inhibiting NF-κB activation pathway and NLRP3 inflammasome

AimsLupus nephritis (LN) is a kidney inflammatory disease caused by systemic lupus erythematosus (SLE). Both NF-κB activation and NLRP3 inflammasome activation are implicated in LN pathogenesis, suggesting they are potential targets for LN treatment. Icariin, which is isolated from Chinese medicine Horny Goat Weed (Ying Yang Huo), has been shown to have anti-inflammation activity, and inhibit activations of both NF-κB and NLRP3 inflammasome. In present study, the effects of icariin on LN were evaluated in MRL/lpr mice. Main methodsWe treated MRL/lpr mice with icariin for 8 weeks and then analyzed the renal function and kidney pathology. We monitored the levels of anti-dsDNA antibody and the deposition of immune complex after icariin treatment. We also detected the macrophage infiltration, NF-κB activation, NLRP3 inflammasome activation and inflammatory cytokine TNF-α production in MRL/lpr mice after icariin treatment. Key findingsWe found that MRL/lpr mice treated with icariin displayed significantly attenuated the renal disease. Icariin-treated mice showed significantly reduced serum anti-dsDNA antibody level and immune complex deposition. Icariin inhibited NF-κB activation and TNF-α production in MRL/lpr mice. Icariin inhibited CCL2 production and macrophage infiltration in MRL/lpr mice. Finally, icariin suppressed NLRP3 inflammasome activation and IL-1β production in MRL/lpr mice. SignificanceIcariin alleviated murine lupus nephritis via inhibiting NF-κB activation and NLRP3 inflammasome activation.

Selective, sensitive and comprehensive detection of immune complex antigens by immune complexome analysis with papain-digestion and elution

Comprehensive identification and profiling of antigens in immune complexes (ICs) in biological fluids, such as serum and cerebrospinal fluid, is useful for developing early diagnostic markers and specific treatments for many diseases. We have developed a method, designated “immune complexome analysis”, to comprehensively identify the antigens in ICs. In this method, we first purify ICs from biological fluid by using Protein G- or Protein A-coated beads, then these ICs are subjected to tryptic digestion on the beads and subsequent analysis using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We previously used this method to find specific antigens in circulating ICs (CIC-antigens) in serum for autoimmune diseases, infectious disease and cancers. However, this method detects not only CIC-antigens but also antibodies and proteins bound non-specifically to the beads, which restricts the detection of minor peptides released by the digestion of CIC-antigens whose amounts are generally much less than antibodies and the proteins. To selectively detect CIC-antigens with enhanced sensitivity, in this study we compared three methods (Method A, direct tryptic digestion on the beads; Method B, low-pH elution and tryptic digestion; Method C, papain-digestion, elution, and tryptic digestion) and examined which method selectively elutes CIC-antigens from CICs bound to the beads and selectively detects CIC-antigens using nano-LC-MS/MS. We also compared three types of CIC-capturing beads (Protein G-coated magnetic beads, Protein A-coated magnetic beads and Proceptor™-sepharose beads) to examine if parallel use of these beads aids the comprehensive detection of CIC-antigens in immune complexome analysis. Comparison showed that Method C provided the most selective and sensitive detection of CIC-antigens, without interference by antibodies and proteins non-specifically bound to the beads. In addition, using three types of beads allowed the examination of a wide range of CIC-antigens in immune complexome analysis. Therefore, combining Method C with three types of beads should allow the selective and sensitive identification of IC-antigens present in biological fluids from patients with a variety of diseases. The identification of IC-antigens may lead to the development of diagnostic methods and protocols for specific treatments for these diseases.

Number of individual ACPA reactivities in synovial fluid immune complexes, but not serum anti-CCP2 levels, associate with inflammation and joint destruction in rheumatoid arthritis

IntroductionIndividual patients with rheumatoid arthritis (RA) show divergent specific anti-citrullinated protein/peptide antibodies (ACPA) patterns, but hitherto no individual ACPA specificity has consistently been linked to RA pathogenesis. ACPA are also...