Dioscin attenuates lupus nephritis in NZB/W F1 mice by decreasing NF-κB activation and NLRP3 inflammasome.

INTRODUCTION: . Dioscin, a natural steroid saponin, has anticancer, anti-inflammatory, anti-hyperlipidemic, and glycemic capabilities. This study focused on dioscin roles and its related mechanisms in experimental lupus nephritis. MATERIALS AND METHODS: . Lupus-prone NZB/W F1 mice were intragastrically administered with dioscin, prednisone or vehicle, and kidney, urine and blood samples were harvested after the mice were sacrificed. Proteinuria, blood urea nitrogen (BUN), creatinine, anti-dsDNA, IL-1ß, and IL-18 levels in serum as well as IFN-γ, IL-6, IL-17 and TNF-α levels in kidney tissues were assessed. Renal histopathology was examined through hematoxylin-eosin staining. IgG and C3 expression in kidney was evaluated using immunofluorescence staining. The number of glomerular F4/80-positive cells and NLRP3-positive cells was determined by immunohistochemical staining. The protein expression was examined by western blotting. RESULTS: . Dioscin alleviated lupus nephritis in NZB/W F1 mice. Dioscin declined serum anti-dsDNA level, prevented deposition of immune complexes in renal glomeruli, and inhibited the inflammatory response and infiltration of macrophages into mouse kidneys. Dioscin inhibited NF-κB and NLRP3 inflammasome in NZB/W F1 mice. CONCLUSIONS: . Dioscin ameliorates lupus nephritis through inhibition of NLRP3 inflammasome and NF-κB signaling.

Exogenous Serpin B1 restricts Immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis.

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation however, it's mechanism(s) of action are currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA and A2138 mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysomes.

Recombinant immune complexes as vaccines against infectious diseases.

New vaccine technologies are needed to combat many existing infections and prepare better for those that may emerge in the future. The conventional technologies that rely on protein-based vaccines are still severely restricted by the sparsity and poor accessibility of available adjuvants. One possible solution to this problem is to enhance antigen immunogenicity by a more natural means by complexing it with antibodies in the form of immune complexes (ICs). However, natural ICs are impractical as vaccines, and significant research efforts have been made to generate them in recombinant form, with plant bioengineering being at the forefront of these efforts. Here, we describe the challenges and progress made to date to make recombinant IC vaccines applicable to humans.

Corrigendum: Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

[This corrects the article DOI: 10.3389/fvets.2024.1368929.].

Effect of Telitacicept on Circulating Gd-IgA1 and IgA-Containing Immune Complexes in IgA Nephropathy.

Introduction: Telitacicept, a transmembrane activator and cyclophilin ligand interactor (TACI) fusion protein targeting B cell activating factor and a proliferation-inducing ligand (APRIL), has proven efficacy in treating Immunoglobulin A (IgA) nephropathy (IgAN). However, serum biomarkers that could predict the clinical response during the treatment remain unclear. Methods: Plasma samples from 24 participants in the phase 2 clinical trial were collected at baseline and after 4, 12, and 24 weeks; with 8 participants in the placebo group, 9 in the 160 mg group, and 7 in the 240 mg group. We measured the levels of galactose-deficient-IgA1 (Gd-IgA1), IgA-containing immune complexes, C3a, C5a, and sC5b-9. The association between the changes in these markers and proteinuria reduction was analyzed. Results: After 24 weeks of treatment, Gd-IgA1 decreased by 43.9% (95% confidence interval: 29.8%, 55.1%), IgG-IgA immune complex by 31.7% (14.4%, 45.5%), and poly-IgA immune complex by 41.3% (6.5%, 63.1%) in the 160 mg group; Gd-IgA1 decreased by 50.4% (38.6%, 59.9%), IgG-IgA immune complex decreased by 42.7% (29.5%, 53.4%), and poly-IgA immune complex decreased by 67.2% (48.5%,79.1%) in the 240 mg group. There were no significant changes in the circulatory C3a, C5a, or sC5b-9 levels during telitacicept treatment. Decreases in both plasma Gd-IgA1 and IgG-IgA or poly-IgA immune complexes were associated with proteinuria reduction. In turn, IgG-IgA or poly-IgA immune complexes showed a dose-dependent effect, consistent with proteinuria reduction during telitacicept treatment. Conclusion: Telitacicept lowered both circulating Gd-IgA1 and IgA-containing immune complexes, whereas IgA immune complex levels were more consistent with decreased proteinuria.

Clinical validation of circulating immune complexes for use as a diagnostic marker of canine leishmaniosis.

Introduction: Canine leishmaniosis (CanL) is a systemic disease that affects dogs. When multiplication of the parasite cannot be controlled, dogs consistently show high levels of antigen and IgG antibodies, which lead to the formation of circulating immune complexes (CIC). Timely intervention to reduce the parasite load and CIC levels is crucial for preventing irreversible organ damage. However, a diagnostic test to quantify CIC levels is currently lacking. Methods: In this real-world study, we aimed to examine the performance of a new ELISA to measure CIC levels in dogs naturally infected with Leishmania infantum. Thirty-four dogs were treated according to their clinical condition and followed for 360 days. Before (day 0) and after treatment (days 30, 90, 180, 270, and 360), all dogs underwent a physical examination, and blood samples were obtained for CBC, biochemical profile, serum protein electrophoresis and IFAT. Serum PEG-precipitated CIC were determined by ELISA. Results: Our results indicate higher CIC levels in dogs in advanced disease stages showing higher antibody titres (p < 0.0001, r = 0.735), anemia (p < 0.0001), dysproteinemia (p < 0.0001), and proteinuria (p = 0.004). Importantly, dogs responding well to treatment exhibited declining CIC levels (p < 0.0001), while in poor responders and those experiencing relapses, CIC were consistently elevated. CIC emerged as a robust discriminator of relapse, with an area under the curve (AUC) of 0.808. The optimal cut-off to accurately identify relapse was an optical density of 1.539. Discussion: Our findings suggest that declining CIC levels should be expected in dogs showing a favorable treatment response. Conversely, in dogs displaying a poor response and recurrent clinical relapses, CIC levels will be high, emphasizing the need for vigilant monitoring. These findings suggest that CIC could serve as a valuable biomarker for disease progression, treatment efficacy, and relapse detection in CanL. Our study contributes to enhancing diagnostic approaches for CanL and underscores the potential of CIC as a complementary tool in veterinary practice. As we move forward, larger studies will be essential to confirm these findings and establish definitive cut-offs for clinical application.

Identification of Four Mouse FcRn Splice Variants and FcRn-Specific Vesicles.

Research into the neonatal Fc receptor (FcRn) has increased dramatically ever since Simister and Mostov first purified a rat version of the receptor. Over the years, FcRn has been shown to function not only as a receptor that transfers immunity from mother to fetus but also performs an array of different functions that include transport and recycling of immunoglobulins and albumin in the adult. Due to its important cellular roles, several clinical trials have been designed to either inhibit/enhance FcRn function or develop of non-invasive therapeutic delivery system such as fusion of drugs to IgG Fc or albumin to enhance delivery inside the cells. Here, we report the accidental identification of several FcRn alternatively spliced variants in both mouse and human cells. The four new mouse splice variants are capable of binding immunoglobulins' Fc and Fab portions. In addition, we have identified FcRn-specific vesicles in which immunoglobulins and albumin can be stored and that are involved in the endosomal-lysosomal system. The complexity of FcRn functions offers significant potential to design and develop novel and targeted therapeutics.

Biodistribution of Drug/ADA Complexes: The Impact of Immune Complex Formation on Antibody Distribution.

The clinical use of therapeutic monoclonal antibodies (mAbs) for the treatment of cancer, inflammation, and other indications has been successfully established. A critical aspect of drug-antibody pharmacokinetics is immunogenicity, which triggers an immune response via an anti-drug antibody (ADA) and forms drug/ADA immune complexes (ICs). As a consequence, there may be a reduced efficacy upon neutralization by ADA or an accelerated drug clearance. It is therefore important to understand immunogenicity in biological therapies. A drug-like immunoglobulin G (IgG) was radiolabeled with tritium, and ICs were formed using polyclonal ADA, directed against the complementary-determining region of the drug-IgG, to investigate in vivo biodistribution in rodents. It was demonstrated that 65% of the radioactive IC dose was excreted within the first 24 h, compared with only 6% in the control group who received non-complexed 3H-drug. Autoradiographic imaging at the early time point indicated a deposition of immune complexes in the liver, lung, and spleen indicated by an increased radioactivity signal. A biodistribution study confirmed the results and revealed further insights regarding excretion and plasma profiles. It is assumed that the immune complexes are readily taken up by the reticuloendothelial system. The ICs are degraded proteolytically, and the released radioactively labeled amino acids are redistributed throughout the body. These are mainly renally excreted as indicated by urine measurements or incorporated into protein synthesis. These biodistribution studies using tritium-labeled immune complexes described in this article underline the importance of understanding the immunogenicity induced by therapeutic proteins and the resulting influence on biological behavior.

Exploring the Link between Hydrodynamic Size and Immunoglobulins of Circulating Immune Complexes in Rheumatoid Arthritis Patients.

The function of immune complexes in rheumatoid arthritis (RA) is related to their composition and size. Using dynamic light scattering (DLS), we investigated the link between the RA circulating immune complex (CIC) particles' size and the CIC immunoglobulin level. In this study, 30 RA patients and 30 healthy individuals were included. IgA, IgG, and IgM were found in all analyzed CICs, but more IgA and IgG were found in RA than in control CICs. In both control and RA CICs, DLS detected 50 particles that differed in size and clustered around two size groups: with a 7.5-164 nm radius and with a 342-1718 nm radius. An increased level of IgA in RA CICs, compared to control ones, was associated with more than 50% of CIC particles. In RA, compared to the control, a higher number of CICs with 28.2 nm, 531 nm, 712 nm, and 1718 nm particles and a lower number of CICs with 78.8 nm particles were detected. This particle distribution pattern did not reflect the changes in the CIC immunoglobulin level. Thus, RA elevated CIC IgA was linked with all these particles (except the 1718 nm particle), the IgM increase was linked with 43.8 nm and 712 nm particles, and the IgG increase was linked with the 712 nm particle only. This study provides the very first data on the association between CIC particles' size, CIC immunoglobulin level, and RA. It opens the possibility that the size of CICs determined by DLS can be used as a criterion in RA diagnosis or monitoring after a large-scale study confirmation.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Angiotensin II and post-streptococcal glomerulonephritis.

BACKGROUND: Post-streptococcal glomerulonephritis (PSGN) is a consequence of the infection by group A beta-hemolytic streptococcus. During this infection, various immunological processes generated by streptococcal antigens are triggered, such as the induction of antibodies and immune complexes. This activation of the immune system involves both innate and acquired immunity. The immunological events that occur at the renal level lead to kidney damage with chronic renal failure as well as resolution of the pathological process (in most cases). Angiotensin II (Ang II) is a molecule with vasopressor and pro-inflammatory capacities, being an important factor in various inflammatory processes. During PSGN some events are defined that make Ang II conceivable as a molecule involved in the inflammatory processes during the disease. CONCLUSION: This review is focused on defining which reported events would be related to the presence of this hormone in PSGN.

Vitamin D3 mitigates autoimmune inflammation caused by activation of myeloid dendritic cells in SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which defective T cells, immune complex deposition and other immune system alterations contribute to pathological changes of multiple organ systems. The vitamin D metabolite c is a critical immunomodulator playing pivotal roles in the immune system. Epidemiological evidence indicates that vitamin D deficiency is correlated with the severity of SLE. Our aim is to investigate the effects of 1,25(OH)2D3 (VitD3) on the activation of myeloid dendritic cells (mDCs) by autologous DNA-containing immune complex (DNA-ICs), and the effects of VitD3 on immune system balance during SLE. We purified DNA-ICs from the serum of SLE patients and isolated mDCs from normal subjects. In vitro studies showed that DNA-ICs were internalized and consumed by mDCs. VitD3 blocked the effects of DNA-ICs on RelB, IL-10 and TNF-α in mDCs. Further analysis indicated that DNA-ICs stimulated histone acetylation in the RelB promoter region, which was inhibited by VitD3. Knockdown of the histone deacetylase 3 gene (HDAC3) blocked these VitD3-mediated effects. Co-culture of mDCs and CD4+ T cells showed that VitD3 inhibited multiple processes mediated by DNA-ICs, including proliferation, downregulation of IL-10, TGF-ß and upregulation of TNF-α. Moreover, VitD3 could also reverse the effects of DNA-IC-induced imbalance of CD4+ CD127- Foxp3+ T cells and CD4+ IL17+ T cells. Taken together, our results indicated that autologous DNA-ICs stimulate the activation of mDCs in the pathogenesis of SLE, and VitD3 inhibits this stimulatory effects of DNA-ICs by negative transcriptional regulation of RelB gene and maintaining the Treg/Th17 immune cell balance. These results suggest that vitamin D may have therapeutic value for the treatment of SLE.

Formation and clearance of TNF-TNF inhibitor complexes during TNF inhibitor treatment.

BACKGROUND AND PURPOSE: Millions of patients with inflammatory diseases are treated with tumour necrosis factor (TNF) inhibitors (TNFi). Individual treatment response varies, in part related to variable drug clearance. The role of TNF-TNFi complexes in clearance of the different TNFi is controversial. Moreover, mechanistic insight into the structural aspects and biological significance of TNF-TNFi complexes is lacking. We hypothesized a role for Fc-mediated clearance of TNF-TNFi immune complexes. Therefore, we investigated circulating TNF-TNFi complexes upon treatment with certolizumab-lacking Fc tails-in comparison with adalimumab, golimumab, infliximab and etanercept. EXPERIMENTAL APPROACH: Drug-tolerant ELISAs were developed and used to quantify TNF during adalimumab, golimumab, etanercept, certolizumab and infliximab treatment in patients with inflammatory arthritis or ulcerative colitis for a maximum follow-up of 1 year. Effects on in vitro TNF production and Fc-mediated uptake of TNF-TNFi complexes were investigated for all five TNFi. KEY RESULTS: Circulating TNF concentrations were >20-fold higher during certolizumab treatment compared with adalimumab, reaching up to 23.1 ng·ml-1 . Internalization of TNF-TNFi complexes by macrophages depended on Fc valency, with efficient uptake for the full antibody TNFi (three Fc tails), but little or no uptake for etanercept and certolizumab (one and zero Fc tail, respectively). TNF production was not affected by TNFi. Total TNF load did not affect clearance rate of total TNFi. CONCLUSIONS AND IMPLICATIONS: Differences in TNFi structure profoundly affect clearance of TNF, while it is unlikely that TNF itself significantly contributes to target-mediated drug disposition of TNFi.

The elusive nature of the oligoclonal bands in multiple sclerosis.

Intrathecal immunoglobulin G (IgG) and oligoclonal bands (OCBs) detected in both the brain and cerebrospinal fluid (CSF) are seminal features of multiple sclerosis (MS). The presence of OCBs correlates with elevated disease burden and severity and supports the diagnosis of MS. Despite numerous investigations into the potential viral and autoantigen targets, the precise antigenic specificity of OCBs has remained elusive. We have little knowledge of the nature regarding these oligoclonal IgG bands. Here, we present compelling evidence highlighting the key findings that both OCBs and intrathecal IgG antibodies are under genetic control and that OCBs originate from clonal B-cells in both the periphery and CNS. We propose that MS OCBs are IgG immune complexes composed of IgG1 and IgG3 antibodies and that the pathological role of OCB stems from the IgG effector functions of these complexes, leading to demyelination and axonal injuries. We present additional evidence regarding the nature of MS OCBs: (1) disease-modifying therapies have been shown to affect CSF OCB; (2) OCBs have also been detected in several neuroinfectious diseases; (3) Epstein-Barr virus (EBV) has been particularly linked with MS pathogenesis, and its association with OCB is an important area of study. Although OCBs are closely associated with MS, more meticulously planned research is necessary to clarify the precise role of OCB in MS, both in terms of disease pathogenesis and diagnosis.

Colocalization of IgG and IgA Heavy Chains with Kappa and Lambda Light Chains in Glomerular Deposits of IgA Nephropathy Patients Using High-Resolution Confocal Microscopy and Correlation with Oxford MEST-C Scores.

Routine immunofluorescence microscopy of glomerular immunodeposits in IgA nephropathy shows IgA, C3, and lambda light chains, and sometimes IgG, IgM, and kappa light chains. However, a previous study using high-resolution confocal microscopy showed IgG in all IgA nephropathy cases, likely representing autoantibodies specific for galactose-deficient IgA1. Here, we used high-resolution confocal microscopy to examine the composition of glomerular immunodeposits and colocalization of kappa and lambda light chains with IgA or IgG heavy chains in kidney-biopsy samples from twenty patients with IgA nephropathy, seventeen without IgG, and nine with no or trace kappa light chains by routine immunofluorescence microscopy. IgG was detected in all biopsies by high-resolution confocal microscopy. Single-optical-plane images showed similar colocalization of IgG heavy chains with kappa and lambda light chains. Colocalization of IgA heavy chains was greater with lambda light chains than with kappa light chains. Colocalization of IgG heavy chain with kappa light chains was higher than with lambda light chains in biopsies with endocapillary hypercellularity and crescents, i.e., biopsies with active lesions. We confirmed the utility of high-resolution confocal microscopy to detect components of glomerular immunodeposits not apparent on routine immunofluorescence microscopy and for colocalization of different components, potentially clarifying the pathogenesis of IgA nephropathy.

Mechanisms of Long-Term Persistence of Mycoplasmas in Children with Asthma.

Improving the management of children with asthma associated with mycoplasma infection is important. Aim: To study the duration of the persistence of antigens, and DNA in a free state, in the structures of circulating immune complexes (CICs) and living cells of Mycoplasma pneumoniae (Mpn) and Mycoplasma hominis (Mh) in children with asthma. In total, 205 children with asthma from 1 to 14 years were observed. The reaction of aggregate-hemagglutination (AHAA), the direct immunofluorescence reaction (DIF), the reaction of the polymerase chain reaction (PCR), and the culture method were used. In addition, 47 children were re-examined 1.5 months after the treatment of mycoplasma infection with azithromycin. The number of samples positive for antigens and DNA in the free state and in the structures of CICs significantly decreased. Then, 50 blood serum samples containing Mh antigens, and 50 samples containing Mpn antigens were analyzed by culture method. Mh was isolated in 21 (65.5%) of 32 samples containing DNA. Mpn was isolated from antigen-positive samples in nine cases. The presented data indicate the long-term persistence of antigens, and DNA of mycoplasma cells in the free state, in the structure of CICs, as well as in the form of "microcolonies". A high level of CICs can be used to predict the course of the disease and the response to therapy.

Comparison of Three Lateral Flow Immunoassay Formats for the Detection of Antibodies against the SARS-CoV-2 Antigen.

Reliable detection of specific antibodies against pathogens by lateral flow immunoassay (LFIA) greatly depends on the composition of the detectable complex and the order of its assembly. We compared three LFIA formats for revealing anti-SARS-CoV-2 antibodies in sera with the following detected complexes in the analytical zone of the strip: antigen-antibodies-labeled immunoglobulin-binding protein (Scheme A); antigen-antibodies-labeled antigen (Scheme B); and immunoglobulin-binding protein-antibodies-labeled antigen (Scheme C). The lowest detection limit was observed for Scheme C, and was equal to 10 ng/mL of specific humanized monoclonal antibodies. When working with pooled positive sera, Scheme C had a detection limit 15 times lower than Scheme B and 255 times lower than Scheme A. Due to the high sensitivity of Scheme C, its application for the panel of human sera (n = 22) demonstrated 100% diagnostic specificity and sensitivity. These consistent results be useful for designing the format of LFIA serodiagnosis for other diseases.

INDICATORS OF DENTAL HEALTH AND LOCAL IMMUNITY IN YOUNG ADULTS WHO HAVE SUFFERED FROM CORONAVIRUS INFECTION.

OBJECTIVE: The aim: To determine the dental status and state of local immunity in young adults who have suffered from the coronavirus disease. PATIENTS AND METHODS: Materials and methods: The main group consisted of 30 people aged 20-22 years, who suffered from the coronavirus infection Covid19 6.1±1.2 months ago. The comparison group included 20 people who did not have a coronavirus infection. The control group consisted of 35 people, randomized by age and sex, who did not have signs of caries and periodontal tissue disease and did not have coronavirus disease. All patients were examined for dental status and local immunity. RESULTS: Results: The analysis of indicators of dental status revealed the possibility of the existence of a relationship between the signs of acute SARS-Cov2 viral infection and the development of caries and periodontal tissue diseases. Significant changes in the local immunity of the oral cavity were found in the examined patients, which had a pathogenetic influence on the development and progression of caries and periodontal tissue diseases: a significant increase in the level of Ig G, as well as a probable decrease in the concentration of SIg A relative to the comparison group, a probably higher normative value of pathogenic small- and medium-molecular CICs with a significant decrease in the level of physiological large-sized CICs relative to the comparison group, a decrease in the content of anti-inflammatory IL-4, as well as increased concentration of pro-inflammatory cytokines. CONCLUSION: Conclusions: Young adults who have suffered a coronavirus infection during the last 6 months have significantly higher caries prevalence, bleeding index, PMA index and hygiene index, halitosis, which indicates deeper tissue damage and tooth pathology with the formation of dentition defects than in the comparison group. Indicators of local immunity of the oral fluid have a deep and specific character.

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.

Characterization of anti-SARS-CoV-2 monoclonal antibodies focusing on antigen binding, neutralization, and FcγR activation via formation of immune complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Antibodies induced by SARS-CoV-2 infection or vaccination play pivotal roles in the body's defense against the virus; many monoclonal antibodies (mAbs) against SARS-CoV-2 have been cloned, and some neutralizing mAbs have been used as therapeutic drugs. In this study, we prepared an antibody panel consisting of 31 clones of anti-SARS-CoV-2 mAbs and analyzed and compared their biological activities. The mAbs used in this study were classified into different binding classes based on their binding epitopes and showed binding to the SARS-CoV-2 spike protein in different binding kinetics. A multiplex assay using the spike proteins of Alpha, Beta, Gamma, Delta, and Omicron variants clearly showed the different effects of variant mutations on the binding and neutralization activities of different binding classes of mAbs. In addition, we evaluated Fcγ receptor (FcγR) activation by immune complexes consisting of anti-SARS-CoV-2 mAb and SARS-CoV-2 pseudo-typed virus, and revealed differences in the FcγR activation properties among the binding classes of anti-SARS-CoV-2 mAbs. It has been reported that FcγR-mediated immune-cell activation by immune complexes is involved in the promotion of immunopathology of COVID-19; therefore, differences in the FcγR-activation properties of anti-SARS-CoV-2 mAbs are among the most important characteristics when considering the clinical impacts of anti-SARS-CoV-2 mAbs.