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• Transfer of RA antibody (B09) to mice induces pain without joint inflammation. • B09 is retained in the dorsal root ganglia where it binds satellite glia cells. • Fcγ receptors are critical for B09 antibody-induced pain-like behavior development. • B09 alters the transcriptional profile of satellite glia and neurons in the DRG. Joint pain is one of the most debilitating symptoms of rheumatoid arthritis (RA) and patients frequently rate improvements in pain management as their priority. RA is hallmarked by the presence of anti-modified protein autoantibodies (AMPA) against post-translationally modified citrullinated, carbamylated and acetylated proteins. It has been suggested that autoantibody-mediated processes represent distinct mechanisms contributing to pain in RA. In this study, we investigated the pronociceptive properties of monoclonal AMPA 1325:01B09 (B09 mAb) derived from the plasma cell of an RA patient. We found that B09 mAb induces pain-like behavior in mice that is not associated with any visual, histological or transcriptional signs of inflammation in the joints, and not alleviated by non-steroidal anti-inflammatory drugs (NSAIDs). Instead, we found that B09 mAb is retained in dorsal root ganglia (DRG) and alters the expression of several satellite glia cell (SGC), neuron and macrophage-related factors in DRGs. Using mice that lack activating FcγRs, we uncovered that FcγRs are critical for the development of B09-induced pain-like behavior, and partially drive the transcriptional changes in the DRGs. Finally, we observed that B09 mAb binds SGC in vitro and in combination with external stimuli like ATP enhances transcriptional changes and protein release of pronociceptive factors from SGCs. We propose that certain RA antibodies bind epitopes in the DRG, here on SGCs, form immune complexes and activate resident macrophages via FcγR cross-linking. Our work supports the growing notion that autoantibodies can alter nociceptor signaling via mechanisms that are at large independent of local inflammatory processes in the joint. [ABSTRACT FROM AUTHOR]

In this review we discuss an underexposed mechanism in the adaptive immune system where B cell and T cell immunity collaborate. The main function of B cell immunity is the generation of antibodies which are well known for their high affinity and antigen-specificity. Antibodies can bind antigens in soluble form making so-called immune complexes (ICs) or can opsonize antigen-exposing cells or particles for degradation. This leads to well-known effector mechanisms complement activation, antibody-dependent cytotoxicity and phagocytosis. What is less realized is that antibodies can play an important role in the targeting of antigen to dendritic cells (DCs) and thereby can drive T cell immunity. Here we summarize the studies that described this highly efficient process of antibody-mediated antigen uptake in DCs in vitro and in vivo. Only very low doses of antigen can be captured by circulating antibodies and subsequently trapped by DCs in vivo. We studied the handling of these ICs by DCs in subcellular detail. Upon immune complex engulfment DCs can sustain MHC class I and II antigen presentation for many days. Cell biological analysis showed that this function is causally related to intracellular antigen-storage compartments which are functional endolysosomal organelles present in DCs. We speculate that this function is immunologically very important as DCs require time to migrate from the site of infection to the draining lymph nodes to activate T cells. The implications of The implications of these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed. these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed. [ABSTRACT FROM AUTHOR]

Cardiovascular disease remains the leading cause of mortality worldwide. Cardiomyocytes are irreversibly lost due to cardiac ischemia secondary to disease. This leads to increased cardiac fibrosis, poor contractility, cardiac hypertrophy, and subsequent life-threatening heart failure. Adult mammalian hearts exhibit notoriously low regenerative potential, further compounding the calamities described above. Neonatal mammalian hearts, on the other hand, display robust regenerative capacities. Lower vertebrates such as zebrafish and salamanders retain the ability to replenish lost cardiomyocytes throughout life. It is critical to understand the varying mechanisms that are responsible for these differences in cardiac regeneration across phylogeny and ontogeny. Adult mammalian cardiomyocyte cell cycle arrest and polyploidization have been proposed as major barriers to heart regeneration. Here we review current models about why adult mammalian cardiac regenerative potential is lost including changes in environmental oxygen levels, acquisition of endothermy, complex immune system development, and possible cancer risk tradeoffs. We also discuss recent progress and highlight conflicting reports pertaining to extrinsic and intrinsic signaling pathways that control cardiomyocyte proliferation and polyploidization in growth and regeneration. Uncovering the physiological brakes of cardiac regeneration could illuminate novel molecular targets and offer promising therapeutic strategies to treat heart failure. • Heart Regenerative potential varies across phylogeny and ontogeny. • Blood oxygen level, endothermy acquisition, complex immune systems, and cancer risk may influence regenerative capacity. • Extrinsic and intrinsic pathways and factors regulate cardiomyocyte renewal and cardiac regeneration. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) is a chronic complication of systemic lupus erythematosus (SLE). At present, no drugs are capable of delaying the progression of LN without a risk of serious side effects. There is thus a pressing need for further studies of LN pathogenesis to identify novel therapeutic targets and aid in the development of new approaches to treating this debilitating disease. In this study, a multi-omics approach was used to characterize the pathogenesis of LN and to identify disease-related targets, ultimately leading to the identification and validation of Yin Yang 1 (YY1) as a promising therapeutic target in LN. A rapid approach to efficiently screening for candidate YY1 ligands was implemented using drug databases that established rebamipide as a YY1 antagonist suitable for use in the management of LN. Specifically, the YY1 antagonist activity of rebamipide was found to regulate lymphocyte activity, reduce autoantibody production, limit immune complex deposition, and suppress macrophage activation while improving symptoms in a murine model of LN. Results supportive of a similar pathologic mechanism of action were also obtained when analyzing renal tissue sections from LN patients, underscoring the potential clinical significance of YY1 and its antagonist rebamipide, suggesting that rebamipide may have positive effects on lymphocytes and may improve symptoms in treated patients. This study provides a robust foundation for further research focused on the pathogenesis and treatment of LN. [Display omitted] [ABSTRACT FROM AUTHOR]

Autoantibodies are immunoglobulin proteins produced by autoreactive B cells responding to self-antigens. Extracellular vesicles (EVs) are membranous structures released by almost all types of cells and extensively distributed in various biological fluids. Studies have indicated that EVs loaded with self-antigens not only play important roles in antigen presentation and autoantibody production but can also form functional immune complexes with autoantibodies (termed EV autoantibodies). While numerous papers have summarized the production and function of pathogenic autoantibodies in diseases, especially autoimmune diseases, reviews on EV autoantibodies are rare. In this review, we outline the existing knowledge about EVs, autoantibodies, and EV antigens, highlighting the formation of EV autoantibodies and their functions in autoimmune diseases and cancers. In conclusion, EV autoantibodies may be involved in the occurrence of disease(s) and also serve as potential non-invasive markers that could help in the diagnosis and/or prognosis of disease. Additional studies designed to define in more detail the molecular characteristics of EV autoantibodies and their contribution to disease are recommended. • The formation and existence of EV autoantibodies in different diseases. • EV autoantibodies as biomarkers for diseases. • The functions of EV autoantibodies in different diseases. [ABSTRACT FROM AUTHOR]

Tubulointerstitial lesions could also be prominent in lupus nephritis, and the pathogenesis of tubulointerstitial lesions may be different from glomerular lesions. Previous studies have showed that plasma antibodies against modified /monomeric C-reactive protein (mCRP) are associated with renal tubulointerstitial lesions in patients with lupus nephritis, and amino acid (aa) 199–206 was one of the major epitopes of mCRP. However, the role of anti-mCRP 199–206 antibodies in the pathogenesis of tubulointerstitial lesions in lupus nephritis is unknown. A total of 95 patients with renal biopsy-proven lupus nephritis were enrolled in this study. Plasma levels of anti-mCRP 199–206 antibodies were screened by enzyme-linked immunosorbent assay (ELISA). A lupus prone mouse model was immunized using peptides mCRP 199–206 to explore the potential role of anti-mCRP 199–206 antibodies in tubulointerstitial lesions. The mechanism of anti-mCRP 199–206 antibodies damage to renal tubular epithelial cells was investigated in vitro. Plasma antibodies against mCRP 199–206 were associated with renal tubulointerstitial lesions and prognosis in patients with lupus nephritis. Immunization with peptides mCRP 199–206 in lupus prone mice could aggravate tubulointerstitial lesions and drive tubulointerstitial inflammation and fibrosis. Anti-mCRP 199–206 antibodies could activate the TGF-β1/Smad3 signal pathway and induce tubular damage by binding with CRP. Circulating antibodies against mCRP 199–206 could be a biomarker to reveal tubulointerstitial lesion, and participate in the pathogenesis of tubulointerstitial lesions, which might provide a potential therapeutic target for lupus nephritis. • The antibodies against mCRP 199–206 could be abiomarker to reveal tubulointerstitial lesion and predict prognosis in LN patients. • Immunization with mCRP 199–206 peptides drive tubulointerstitial inflammation and fibrosis in lupus prone mice. • The anti-mCRP 199–206 antibodies were able to recognize mCRP and form immune complexes, which might provide a potential therapeutic target for LN. [ABSTRACT FROM AUTHOR]

[Display omitted] Detailed evaluations of the antigen and antibody interaction rate and strength of the immune complex formed are very important for medical and bioanalytical applications. These data are crucial for the development of sensitive and fast immunosensors suitable for continuous measurements. Therefore, combined spectroscopic ellipsometry (SE) and quartz crystal microbalance with dissipation (QCM-D) technique (SE/QCM-D) was used for the evaluation: (i) of covalent immobilization of SARS-CoV-2 nucleocapsid protein (SCoV2-N) on QCM-D sensor disc modified by self-assembled monolayer based on 11-mercaptoundecanoic acid and (ii) interaction of immobilized SCoV2-N with specific polyclonal anti-SCoV2-N antibodies followed by immune complex formation process. The results show that the SCoV2-N monolayer is rigid due to the low energy dissipation registered during the QCM-D measurement. In contrast, the anti-SCoV2-N layer produced after interaction with the immobilized SCoV2-N formed a soft and viscous layer. It was determined, that the sparse distribution of SCoV2-N on the surface affected the spatial arrangement of the antibody during the formation of immune complexes. The hinge-mediated flexibility of the antibody Fab fragments allows them to reach the more distantly located SCoV2-N and establish a bivalent binding between proteins in the formed SCoV2-N/anti-SCoV2-N complex. It was noted that the SE/QCM-D method can provide more precise quantitative information about the flexibility and conformational changes of antibody during the formation of the immune complex on the surface over time. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) are web-like structures composed of cytoplasmic contents, DNA chromatin and various granular proteins released by neutrophils in response to viruses, bacteria, immune complexes and cytokines. Studies have shown that NETs can promote the occurrence, development and metastasis of tumors. In this paper, the mechanism underlying the formation and degradation of NETs and the malignant biological behaviors of NETs, such as the promotion of tumor cell proliferation, epithelial mesenchymal transition, extracellular matrix remodeling, angiogenesis, immune evasion and tumor-related thrombosis, are described in detail. NETs are being increasingly studied as therapeutic targets for tumors. We have summarized strategies for targeting NETs or interfering with NET-cancer cell interactions and explored the potential application value of NETs as biomarkers in cancer diagnosis and treatment, as well as the relationship between NETs and therapeutic resistance. [ABSTRACT FROM AUTHOR]

Antibodies mediate the majority of their effects in the extracellular domain, or in intracellular compartments isolated from the cytosol. Under a growing list of circumstances, however, antibodies are found to gain access to the cytoplasm. Cytosolic immune complexes are bound by the atypical antibody receptor TRIM21, which mediates the rapid degradation of the immune complexes at the proteasome. These discoveries have informed the development of TRIM-Away, a technique to selectively deplete proteins using delivery of antibodies into cells. A range of related approaches that elicit selective protein degradation using intracellular constructs linking antibody fragments to degradative effector functions have also been developed. These methods hold promise for inducing the degradation of proteins as both research tools and as a novel therapeutic approach. Protein aggregates are a pathophysiological feature of neurodegenerative diseases and are considered to have a causal role in pathology. Immunotherapy is emerging as a promising route towards their selective targeting, and a role of antibodies in the cytosol has been demonstrated in cell-based assays. This review will explore the mechanisms by which therapeutic antibodies engage and eliminate intracellularly aggregated proteins. We will discuss how future developments in intracellular antibody technology may enhance the therapeutic potential of such antibody-derived therapies. [ABSTRACT FROM AUTHOR]

The cytosolic antibody receptor and RING E3 ligase TRIM21 targets intracellular, antibody-coated immune complexes for degradation and activates the immune system. Here we review how TRIM21 degrades diverse targets and how this activity can be exploited in molecular biology and for the development of new therapeutics. In addition, we compare what is known about TRIM21′s mechanism to other TRIM proteins and RING E3 ligases. [ABSTRACT FROM AUTHOR]

The complement system consists of three pathways (alternative, classical, and lectin) that play a fundamental role in immunity and homeostasis. The multifunctional role of the complement system includes direct lysis of pathogens, tagging pathogens for phagocytosis, promotion of inflammatory responses to control infection, regulation of adaptive cellular immune responses, and removal of apoptotic/dead cells and immune complexes from circulation. A tight regulation of the complement system is essential to avoid unwanted complement-mediated damage to the host. This regulation is ensured by a set of proteins called complement regulatory proteins. Deficiencies or malfunction of these regulatory proteins may lead to pro-thrombotic hematological diseases, renal and ocular diseases, and autoimmune diseases, among others. This review focuses on the importance of two complement regulatory proteins of the alternative pathway, Factor H and properdin, and their role in human diseases with an emphasis on: (a) characterizing the main mechanism of action of Factor H and properdin in regulating the complement system and protecting the host from complement-mediated attack, (b) describing the dysregulation of the alternative pathway as a result of deficiencies, or mutations, in Factor H and properdin, (c) outlining the clinical findings, management and treatment of diseases associated with mutations and deficiencies in Factor H, and (d) defining the unwanted and inadequate functioning of properdin in disease, through a discussion of various experimental research findings utilizing in vitro, mouse and human models. [ABSTRACT FROM AUTHOR]

[Display omitted] • Antibodies Trastuzumab and Pertuzumab are effective in treating HER2-positive breast cancer. • A cryoEM structure of the HTP ternary complex allows detailed epitope-paratope views. • The structure suggests a further basis for the synergistic effect of trastuzumab and pertuzumab. • The data are available for further improvement of cryoEM software for flexible complexes. Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images. [ABSTRACT FROM AUTHOR]

Recent research has demonstrated the immunomodulatory potential of Panax notoginseng in the treatment of chronic inflammatory diseases and cerebral hemorrhage, suggesting its significance in clinical practice. Nevertheless, the complex immune activity of various components has hindered a comprehensive understanding of the immune-regulating properties of Panax notoginseng , impeding its broader utilization. This review evaluates the effect of Panax notoginseng to various types of white blood cells, elucidates the underlying mechanisms, and compares the immunomodulatory effects of different Panax notoginseng active fractions, aiming to provide the theory basis for future immunomodulatory investigation. [Display omitted] [ABSTRACT FROM AUTHOR]

Proteins from the C1q domain-containing (C1qDC) family recognize self-, non-self-, and altered-self ligands and serves as an initiator molecule for the classical complement pathway as well as recognizing immune complexes. In this study, C1qDC gene family members were identified and analyzed in grass carp (Ctenopharyngodon idellus). Members of the C1q subfamily were cloned, and their response to infection with the grass carp virus was investigated. In the grass carp genome, 54 C1qDC genes and 67 isoforms have been identified. Most were located on chromosome 3, with 52 shared zebrafish homologies. Seven substantially differentially expressed C1qDC family genes were identified in the transcriptomes of cytokine-induced killer (CIK) cells infected with grass carp reovirus (GCRV), all of which exhibited sustained upregulation. The opening reading frames of grass carp C1qA, C1qB , and C1qC , belonging to the C1q subfamily, were determined to be 738, 732, and 735 base pairs, encoding 245, 243, and 244 amino acids with molecular weights of 25.81 kDa, 25.63 kDa and 26.16 kDa, respectively. Three genes were detected in the nine collected tissues, and their expression patterns were similar, with the highest expression levels observed in the spleen. In vivo after GCRV infection showed expression trends of C1qA , C1qB , and C1qC in the liver, spleen, and kidney. An N-type pattern in the liver and kidney was characterized by an initial increase followed by a decrease, with the highest expression occurring during the recovering period, and a V-type pattern in the spleen with the lowest expression levels during the death period. In vitro, after GCRV infection showed expression trends of C1qA, C1qB , and C1qC, and this gradually increased within the first 24 h, with a notable increase observed at the 24 h time point. After CIK cells incubation with purified recombinant proteins, rC1qA, rC1qB, and rC1qC for 3 h, followed by GCRV inoculation, the GCRV replication indicated that rC1qC exerted a substantial inhibitory effect on viral replication in CIK cells after 24 h of GCRV inoculation. These findings offer valuable insights into the structure, evolution, and function of the C1qDC family genes and provide a foundational understanding of the immune function of C1q in grass carp. • 54C1qDC genes with 67 isoforms were identified from the genome of grass carp. • 7 upregulated C1qDC family genes were identified in the transcriptome of CIK infected with GCRV. • Genes from the C1q subfamily, namely C1qA, C1qB, and C1qC, were cloned. • The mRNA expression patterns of C1qA, C1qB, and C1qC were elucidated both in vivo and in vitro. • C1qC may play a crucial role of inhibiting the replication of GCRV. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) is a severe complication of systemic lupus erythematosus, with high mortality rates despite medical advancements. The complexity of its pathogenesis, including the pivotal role of podocytes — kidney-localized cells — remains a challenge, lacking effective treatments and biomarkers. Recent studies highlight the significant contribution of these cells to LN's development, particularly through their immune-related functions and interaction with other kidney cells. This new understanding opens possibilities for targeted therapies aimed at these cellular mechanisms. This review aims to summarize these recent developments, shedding light on the intricate involvement of podocytes in LN and potential avenues for innovative treatments. • LN affects 40% of SLE patients, targeting podocytes. • Podocytes form a barrier; their injury leads to proteinuria. • Innate and adaptive immunity roles in LN. • LN involves immune complex responses, damaging podocytes. • Future LN treatments focus on immune podocytes. [ABSTRACT FROM AUTHOR]

Cancer and autoimmune diseases are the two devastating conditions that together constitute a leading health problem worldwide. The rising burden of these disorders in the developing world demands a multifaceted approach to address the challenges it poses. Understanding the root causes and specific molecular mechanisms by which the progression of the diseases takes place is need of the hour. A strong inflammatory background and common developmental pathways, such as activation of immune cells, proliferation, increased cell survival and migration which are controlled by growth factors and inflammatory cytokines have been considered as the critical culprits in the progression and complications of these disorders. Enzymes are the potential immune modulators which regulate various inflammatory events and can break the circulating immune complexes via macrophages production. In the current manuscript, we have uncovered the possible role of proteolytic enzymes in the pathogenesis and progression of cancer and autoimmune diseases. In the light of the available scientific literature, we advocate in-depth comprehensive studies which will shed light towards the role of proteolytic enzymes in the modulation of inflammatory responses in cancer and autoimmune diseases together. [ABSTRACT FROM AUTHOR]

• Systemic lupus erythematosus (SLE) is an auto-inflammatory disease that is characterized by chronic and widespread inflammation. • IL-35, the newest member of the IL-12 family, is an immunosuppressive and anti-inflammatory cytokine secreted mainly by regulatory cells. • IL-35 appears to hold therapeutic and diagnostic potential in cancer and autoimmune diseases. • IL-35 can consider as a preferential therapeutic or diagnostic candidate in SLE management. It is well known that systemic lupus erythematosus (SLE) is an auto-inflammatory disease that is characterized by chronic and widespread inflammation. The exact pathogenesis of SLE is still a matter of debate. However, it has been suggested that the binding of autoantibodies to autoantigens forms immune complexes (ICs), activators of the immune response, in SLE patients. Ultimately, all of these responses lead to an imbalance between anti-inflammatory and pro-inflammatory cytokines, resulting in cumulative inflammation. IL-35, the newest member of the IL-12 family, is an immunosuppressive and anti-inflammatory cytokine secreted mainly by regulatory cells. Structurally, IL-35 is a heterodimeric cytokine, composed of Epstein-Barr virus-induced gene 3 (EBI3) and p35. IL-35 appears to hold therapeutic and diagnostic potential in cancer and autoimmune diseases. In this review, we summarized the most recent associations between IL and 35 and SLE. Unfortunately, the comparative review of IL-35 in SLE indicates many differences and contradictions, which make it difficult to generalize the use of IL-35 in the treatment of SLE. With the available information, it is not possible to talk about targeting this cytokine for the lupus treatment. So, further studies would be needed to establish the clear and exact levels of this cytokine and its related receptors in people with lupus to provide IL-35 as a preferential therapeutic or diagnostic candidate in SLE management. [ABSTRACT FROM AUTHOR]

To provide recommendations for managing hypersensitivity adverse events (HAEs) to an injectable enzyme substitution therapy (pegvaliase, a PEGylated phenylalanine ammonia lyase enzyme) in adult patients with phenylketonuria (PKU). Eight European academic immunology experts with a broad range of experience in hypersensitivity, anaphylaxis, and/or drug reactions, and two geneticists from the USA with pegvaliase experience convened for two advisory board meetings. Efficacy, safety, and immunological profile of pegvaliase were discussed with the objective of developing recommendations for the clinical management of HAEs associated with pegvaliase treatment. Based on available immunogenicity data, it was concluded that pegvaliase induces a Type III hypersensitivity reaction, causing HAEs with peak event rates during induction/titration and a decline over time during maintenance therapy. The decline in HAEs with longer duration of therapy was considered to likely be driven by anti-drug antibody affinity maturation, reduced immune complex formation, and decreased complement activation over time. Immunology and PKU experts unanimously supported that the use of an induction, titration, and maintenance dosing regimen and implementation of several risk mitigation strategies contributed to the improvement of tolerability over time. Key risk mitigation strategies utilized in the Phase 3 clinical trials such as premedication with H1-receptor antagonists, allowance for a longer titration period after an HAE, patient education, and requirement to carry auto-injectable adrenaline (epinephrine) should be continued in clinical practice. A tool for administration of auto-injectable adrenaline in patients using pegvaliase was suggested. It was added that after the occurrence of a severe HAE a temporary dose reduction is more likely to improve tolerability than treatment interruption. Overall, it was agreed that pegvaliase has a generally tolerable safety profile in adults with PKU. Importantly, the risk mitigation strategies utilized in the clinical trials were considered to support the continued use of key strategies for management in the commercial setting, such as a slow induction/titration dosing paradigm and premedication with H1-receptor antagonists. However, physicians and patients need to be aware of the risk of HAEs associated with pegvaliase; presence of a trained observer during early treatment may be beneficial in certain circumstances, and a requirement to carry auto-injectable adrenaline is recommended. Because pegvaliase offers the possibility to normalize diet, while maintaining blood phenylalanine within the recommended therapeutic range, safe use of this medication in the clinical setting is important. Ongoing monitoring of long-term clinical safety of patients on pegvaliase treatment in the commercial setting was recommended. [ABSTRACT FROM AUTHOR]

Microparticles (MPs) are small extracellular vesicles released from apoptotic or activated cells through a blebbing process. MPs express surface molecules from their parental cells and they bind IgG to form circulating immune complexes (MP-ICs) in patients with systemic lupus erythematosus (SLE). Through investigation of MP size, IgG expression, content of nucleic acids and mitochondrial molecules, we hypothesized that unrecognized particle populations can be identified in SLE. We investigated 327 well-characterized SLE patients and 304 controls divided into two sets (280/280 and 47/24). We measured MPs by flow cytometry using a gating strategy to encompass small (0.2–0.7 μm) and large (0.7–3.0 μm) MPs. Nucleic acids were labeled with SYTO 13 and mitochondria with MitoTracker. Expression of mitochondria markers TOM-20 and Hexokinase 1 and the presence of IgG was investigated. MPs staining with SYTO 13 were more frequent in 280 SLE patients compared to 280 controls. In 47 SLE patients, levels of large MPs were elevated compared to 24 controls. The majority of large MPs contained mitochondria (mitoMPs). The number of mitoMPs associated positively with high disease activity, anti-dsDNA antibodies and pro-inflammatory cytokines. Patients with active lupus nephritis had higher levels of mitoMPs and IgG-positive mitoMPs. Blood of patients with SLE contain a previously unrecognized population of circulating large MPs with bound IgG and mitochondrial proteins. Levels of these particles are related to several measures of active SLE, suggesting that these structures may have a role in disease pathogenesis. • Blood in SLE shows increased levels of microparticles containing nucleic acids. • Microparticles in SLE display IgG as well as nucleic acids. • A sub-population of microparticles in SLE is larger in size than 0.7 μm. • The majority of the large microparticles contain mitochondrial molecules. • The level of microparticles with mitochondria is associated with features of SLE. [ABSTRACT FROM AUTHOR]

Here, we explore the serum levels of anti-oxidized lipid autoantibodies as well as immune complexes in patients with SLE and determine their correlation with disease. Serum levels of oxidized-LDL immune complexes, autoantibodies to dsDNA, ox-LDL, MDA-LDL, 9-HODE, 13-HODE and POVPC were detected by ELISA in 64 SLE patients and 9 healthy controls. Active SLE patients exhibited increased serum levels of autoantibodies compared to healthy controls, including anti-MDA-LDL-IgG (p =.003), anti-ox-LDL-IgG (p =.004), anti-9-HODE-IgG (p =.001), anti-13-HODE-IgG (p =.0003), anti-POVPC-IgG (p =.001) and ox-LDL-IC (p =.003). Serum anti-ox-LDL-IgG was positively correlated with SLEDAI (r = 0.34; p =.01), and negatively with C3 (r = −0.40; p =.01). Anti-9-HODE-IgG and anti-POVPC-IgG were positively correlated with SLEDAI and negatively with C4. Active SLE patients exhibit significantly increased serum levels of IgG anti-oxidized-lipid autoantibodies. Coordinated elevation of oxidized lipids, autoantibodies to these lipids, and immune complexes of these lipid-antibody components could potentially serve as pathogenic drivers and serum markers of SLE disease activity. [ABSTRACT FROM AUTHOR]

Acquired von Willebrand syndrome (AVWS) is a rare, potentially fatal bleeding disorder caused by low activity of von Willebrand factor (VWF) in patients without congenital deficiency. The majority of adult cases are associated with hematological malignancy, including lymphoproliferative (48%) or myeloproliferative (15%) disorders (Federici et al., 2000). Both qualitative and quantitative defects occur, due to antibody-mediated clearance or functional interference, increased proteolysis, absorption to malignant cells or platelets, or increased shear stress due to valvular defects or mechanical vascular devices (Tiede et al., 2011). The predominant mechanism for decreased or absent VWF in malignancy is autoantibodies that are inhibitory to VWF function or shorten VWF survival (Kumar et al., 2002 [ 3 ]). Antibody-mediated clearance occurs through inactivating antibody directed towards VWF, antibody binding the non-active sites of VWF, and nonspecific antibodies that form circulating immune complexes with VWF, enhancing clearance by the reticuloendothelial system (Mannucci et al., 1984). Bleeding may be very difficult to treat due to reduced half-life of VWF-concentrates. [ABSTRACT FROM AUTHOR]

Rapid and versatile methods are needed for evaluation of immunogenicity in early safety studies. The present work presents a generic, simple and easy to use sandwich enzyme-linked immunosorbent assay for quasi-quantitative measurement of circulating immune complexes (CICs) formed by anti-drug antibodies (ADAs) in complex with human IgG in mouse plasma. The assay is suitable for evaluating the presence of in vivo formed CICs in mice exposed to human IgG antibodies independent of target and IgG subtype. The assay is established using commercially available antibodies, and calibrated using CIC mimics based on bis(sulfosuccinimidyl)suberate conjugated human and mouse IgG. The development and qualification process of the generic methodology is described and include acceptance criteria, stability, sensitivity, drug tolerance, spike recovery, precision and cut point determination. In order to demonstrate assay performance, its use is exemplified by quantifying CICs in mice administered with a fully human anti -TNF-α IgG 1 antibody (adalimumab) or a humanized anti -trinitrophenol (TNP) IgG 4 antibody. Results show a well-qualified reproducible assay set-up with adequate sensitivity, easy discrimination between positive and negatives and quasi-quantitative measurement of ADA-human IgG CICs in mice administered with each of two different human/humanized IgG antibodies. [ABSTRACT FROM AUTHOR]

Adult immunoglobulin A vasculitis (IgAV) is an immune complex small vessel vasculitis. So far, the involvement of T cells in this pathology has been poorly studied. The aim of this study was to analyze T-cell homeostasis as well as cytokine and chemokine concentrations in the blood and tissues of IgAV patients. T cells, cytokine and chemokine concentrations were analyzed in peripheral blood using flow cytometry and multiplex assays. T-cell infiltrates in the kidney and the skin were characterized by immunohistochemistry. This study prospectively included 44 adult patients with biopsy-proven IgAV and 24 age- and sex-matched healthy controls. We observed reduced proportions of circulating CXCR5-and CXCR3-expressing memory CD4 T cells at diagnosis but normal values at remission. The plasma levels of Th1-related cytokines (IL-12, IL-27 and IFNγ) and of the TFH-related cytokine, IL-21, were paradoxically not reduced in patients. We observed increased plasma concentrations of the CXCR5 ligand, CXCL13, and of the CXCR3 ligands, CXCL10/11, suggesting a potential relocation of the corresponding T cells into inflamed tissues. We then confirmed the recruitment of CXCR3-expressing T cells into the skin and kidneys. In the skin, T-cell infiltrates mainly co-localized with damaged dermal small vessels. Finally, patients with the largest kidney T-cell infiltrates were also those with the highest proteinuria. Altogether, our results strongly suggest that, in IgAV patients, CXCL10/11 orchestrate the recruitment of CXCR3-expressing T cells in injured tissues, contributing to tissue damage and disease activity. • Proportions of circulating CXCR5-and CXCR3-expressing memory T cells are decreased in adult patients with IgA vasculitis at diagnosis. • Plasma concentrations of CXCL10, CXCL11 and CXCL13 are increased in adult patients with IgA vasculitis. • CXCL10/11 may orchestrate the recruitment of CXCR3-expressing T cells into injured tissues. • In the kidneys, the extent of T-cell infiltrates correlates with the activity of the disease (proteinuria). [ABSTRACT FROM AUTHOR]

Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents. Image 1 • B. malayi proteins with potential as epidemiological markers and diagnostic/therapeutic/vaccine candidates were revealed. • B. malayi microfilaria-specific antigens in the circulating ICs were investigated for the first time. • Detection of the ICs can be used for assessing efficacy of anti-microfilaremic drugs. • The IC-forming proteins might play role in the host immune evasion. [ABSTRACT FROM AUTHOR]

Abstract Viperin is recognized as an antiviral protein that is stimulated by interferon, viral exposures, and other pathogenic molecules in vertebrate. In this study, a viperin homolog in the Big-belly seahorse (Hippocampus abdominalis ; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity in vitro. The HaVip coding sequence encodes a 348 amino acid polypeptide with predicted molecular weight of 38.48 kDa. Sequence analysis revealed that HaVip comprises three main domains: the N-terminal amphipathic α-helix, a radical S-adenosyl- l -methionine (SAM) domain, and a conserved C-terminal domain. Transfected GFP-tagged HaVip protein was found to localize to the endoplasmic reticulum (ER). Overexpressed-HaVip in FHM cells was found to significantly reduce viral capsid gene expression in VHSV infection in vitro. Under normal physiological conditions, HaVip expression was ubiquitously detected in all 14 examined tissues of the seahorse, with the highest expression observed in the heart, followed by skin and blood. In vivo studies showed that HaV ip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and Streptococus iniae challenges caused a significant increase in expression of HaVip in all the analyzed tissues. The obtained results suggest that HaVip is involved in the immune system of the seahorse, triggering antiviral and antibacterial responses, upon viral and bacterial pathogenic infections. Highlights • Identified and characterized viperin from big-belly seahorse. • The HaVip gene was ubiquitously expressed in unchallenged-fish tissues. • Modulated HaVip transcription pattern revealed its contribution in the immune response. • Overexpression of HaVip in FHM cells showed the antiviral activity. [ABSTRACT FROM AUTHOR]

Abstract Objective Neutrophil extracellular traps (NET), produced by activated polymorphonuclear neutrophils (PMN), are supposed to play a role in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease characterized by anti-citrullinated protein antibodies (ACPA). Indeed, NET contain citrullinated autoantigens and some RA autoantibodies recognize NET. However, the mechanisms by which NET trigger or perpetuate the inflammatory process in RA are hitherto not elucidated. We hypothesized that, in addition to citrullination, NET might also contain stimulatory proteins and directly activate inflammatory target cells, as PMN and macrophages. Methods NET antigenic and inflammatory properties were analyzed in 157 healthy donors (HD) and RA patients, the largest analysis reported so far. Primary PMN and monocyte-derived macrophages were isolated and immunoglobulin G (IgG) purified. NET were induced (NETosis), isolated and quantified. NET antigenicity was analyzed by fluorescence microscopy. PMN and macrophages were stimulated with NET with/without ACPA, C1q, LL-37 or lipopolysaccharide (LPS) and cell activation was estimated by flow cytometry and ELISA. Results PMN from RA patients produced more NET than HD PMN. We next dissected how NET mechanistically affect inflammatory cells. Particularly, we show for the first time that RA and HD NET activated both resting macrophages and PMN, but importantly RA NET were more stimulatory, leading to secretion of inflammatory cytokines and up-regulation of HLA/CD86/CD11b. IgG from ACPA-positive RA patients specifically recognized RA and even HD NET. Nevertheless, NET-induced cell activation occurs independently of immune complex formation with ACPA. Likewise, endosomal acidification was not required. Notably, we also report that complement C1q increased the NET stimulatory activity on macrophages only, due to higher expression of C1q receptors, which was further supported by the LL-37 antimicrobial peptide. In contrast, NET specifically inhibited interleukin (IL)-6 secretion by LPS-activated macrophages and not PMN, especially with C1q/LL-37. This inhibition was not mediated by NET-derived proteases or LPS neutralization and was associated with the simultaneous induction of IL-10 secretion. Conclusion We show that NET possess both pro- and anti-inflammatory properties depending on target cells, their activation levels and C1q/LL-37. Thus, independently of ACPA, NET modulate RA chronic inflammation via this new dual activity we identified. In addition, NET may trigger autoimmunity in RA as ACPA recognize NET antigens but not non-activated PMN. Therefore, we conclude that excess of NETosis together with enhanced NET activity participate to RA pathogenesis at different levels. Graphical abstract Dual activity of NET. Activated PMN enter NETosis in response to some stimuli. In RA, PMN are significantly more sensitive to NETosis triggering. These NET directly activate (pink arrows) steady state PMN (left) and macrophages (MΦ, right), leading to strong IL-8, TNF and IL-6 secretion (three important cytokines in RA) but low IL-10 induction, i.e. a pro-inflammatory profile. In addition, RA NET are significantly more stimulatory than normal NET. Although NET are recognized by ACPA, the resulting immune complexes do not clearly enhance PMN and MΦ response to NET. Likewise, endosomal TLR are probably not involved in NET sensing. Regarding MΦ, NET-induced activation is specifically increased in the presence of DNA-binding molecules like C1q and LL-37 (blue arrows). C1q in combination with LL-37 carry NET to PMN and MΦ which express surface C1q and LL-37 receptors (C1qR, LL-37-R). Because MΦ express much more C1qR than PMN, MΦ (and not PMN) activation by NET is enhanced by C1q or C1q/LL-37. On the contrary, NET induce an anti-inflammatory profile in strongly-activated MΦ but not PMN (green arrows). Indeed, NET specifically (partly) inhibit secretion of the pro-inflammatory cytokine IL-6 by MΦ in response to LPS, especially in the presence of both C1q and LL-37, and inversely enhance IL-10 secretion by those MΦ. Image 1 Highlights • NET are pro-inflammatory and directly activate RESTING neutrophils and macrophages. • NET from RA patients are more stimulatory than NET from healthy donors. • On the contrary, NET are anti-inflammatory on strongly ACTIVATED macrophages only. • C1q enhances NET regulatory activities on macrophages, due to high C1q receptors expression, especially with LL-37. • NET have a dual immunoregulatory activity depending on target cells, without requirement of ACPA. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • PMPs generated in vitro resemble circulating vesicles of patients with RA. • PMPs contain citrullinated peptides and form immune complexes (PMPs-ICs). • PMPs-ICs induce proinflammatory cytokines and migration receptors in monocytes. • PMPs-ICs induce a tolerogenic response in monocytes from RA patients. Abstract Patients with rheumatoid arthritis (RA) have increased amount of platelet-derived microparticles (PMPs) positive for citrullinated peptides (CPs) that form immune complexes (PMPs-ICs). Monocytes are important inflammatory mediators that play a role in the clearance of PMPs-ICs. We aimed to generate PMPs-ICs in vitro and determine its effect on monocytes from patients with RA and healthy individuals (HI). PMPs from patients showed platelet markers, mitochondria content, and phosphatidylserine exposure similar to PMPs from HI. However, patients had a higher frequency of IgG+ and CPs+ vesicles than HI. PMPs-ICs generated in vitro were similar to the circulating vesicles of patients with respect to IgG- and CPs-positivity. PMPs-ICs induced pro-inflammatory cytokines and CX3CR1 expression in monocytes from HI, and IL-10 and CD36 upregulation in monocytes from patients. These results suggest that PMPs-ICs induce activation of monocytes, with a pro-inflammatory response in HI and a more tolerant response in cells of patients with RA. [ABSTRACT FROM AUTHOR]

• Increase in D-dimer (DD) is associated with coagulopathy in severe COVID-19. • Immune Complexes (IC) of SARS CoV-2 with human plasma anti-spike IgG were formed. • Monocyte-derived M1 and M2 macrophages activated with DD + IC produce PGE2 and IL-6. • DD + IC activated M2 but not M1 macrophages produce TNFα, IL-1β, and IL-10 cytokines. • DD + IC contribute to inflammatory response in lung macrophages in severe COVID-19. Lung macrophages are the first line of defense against invading respiratory pathogens including SARS-CoV-2, yet activation of macrophage in the lungs can lead to hyperinflammatory immune response seen in severe COVID-19. Here we used human M1 and M2 polarized macrophages as a surrogate model of inflammatory and regulatory macrophages and explored whether immune complexes (IC) containing spike-specific IgG can trigger aberrant cytokine responses in macrophages in the lungs and associated lymph nodes. We show that IC of SARS-CoV-2 recombinant S protein coated with spike-specific monoclonal antibody induced production of Prostaglandin E2 (PGE2) in non-polarized (M0) and in M1 and M2-type polarized human macrophages only in the presence of D-dimer (DD), a fibrinogen degradation product, associated with coagulopathy in COVID-19. Importantly, an increase in PGE2 was also observed in macrophages activated with DD and IC of SARS-CoV-2 pseudovirions coated with plasma from hospitalized COVID-19 patients but not from healthy subjects. Overall, the levels of PGE2 in macrophages activated with DD and IC were as follows: M1≫M2>M0 and correlated with the levels of spike binding antibodies and not with neutralizing antibody titers. All three macrophage subsets produced similar levels of IL-6 following activation with DD+IC, however TNFα, IL-1β, and IL-10 cytokines were produced by M2 macrophages only. Our study suggests that high titers of spike or virion containing IC in the presence of coagulation byproducts (DD) can promote inflammatory response in macrophages in the lungs and associated lymph nodes and contribute to severe COVID-19. [ABSTRACT FROM AUTHOR]

In this work, a dual-mode immunoassay to detect Ochratoxin A (OTA) was developed relied on p -nitrophenyl phosphate (PNPP) and 2-aminoterephthalic acid (PTA-NH 2). This proposed immunoassay based on microplate immunoenzyme technique used alkaline phosphatase (ALP) as a label for forming immune complexes. The ALP was used to catalyze the dephosphorization of PNPP to generate yellow p -nitrophenol (PNP). Since the absorption wavelength of PNP overlapped with the fluorescence emission of PTA-NH 2 , the fluorescence of PTA-NH 2 can be quenched by PNP. The color of PNP and the fluorescence of PTA-NH 2 were used as double signals to detect OTA. In this dual-mode immunoassay, the limit of detection (LOD) and the linear detection range of fluorescence mode were 0.57 ng/mL (about 0.57 μg/kg) and 1.56–50.00 ng/mL (about 1.56–50.00 μg/kg), respectively. For absorbance mode, the LOD and linear range were 0.07 ng/mL (about 0.07 μg/kg) and 0.39–25.00 ng/mL (about 0.39–25.00 μg/kg), respectively. Both detection modes showed good selectivity for OTA. The recoveries rates of fluorescence mode and absorption mode in maize flours were 91.80–128.85% and 100.10–114.00%, respectively. • The dual-mode immunoassay was developed based on PNPP and PTA-NH 2. • This assay based on microplate immunoenzyme technique used ALP as a label for forming immune complexes. • The mutual verification of the two modes improved the accuracy of analysis method. • The dual-mode immunoassay had a wide linear range and low LOD. [ABSTRACT FROM AUTHOR]

Abstract The complement system is a complex and autoregulated multistep cascade at the interface of innate and adaptive immunity. It is activated by immune complexes or apoptotic cells (classical pathway), pathogen-associated glycoproteins (lectin pathway) or a variety of molecular and cellular surfaces (alternative pathway). Upon activation, complement triggers the generation of proteolytic fragments that allow the elimination of the activating surface by enhancing inflammation, opsonization, phagocytosis, and cellular lysis. Moreover, complement efficiently discriminates self from non-self surfaces by means of soluble and membrane-bound complement regulators which are critical for innate self-tolerance. Complement deficiency or dysfunction disturb complement homeostasis and give rise to diseases as diverse as bacterial infections, autoimmunity, or renal and neurological disorders. Research on complement-targeted therapies is an expanding field that has already improved the prognosis of severe diseases such as atypical Haemolytic Uremic syndrome or Paroxysmal Nocturnal Haemoglobinuria. Therefore, complement analysis and monitoring provides valuable information with deep implications for diagnosis and therapy. In addition to its important role as an extracellular defense system, it has now become evident that complement is also present intracellularly, and its activation has profound implications for leukocyte survival and function. In this review, we summarize the essential, up-to-date information on the use of complement as a diagnostic and therapeutic tool in the clinics. [ABSTRACT FROM AUTHOR]

The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response. [ABSTRACT FROM AUTHOR]

Neuropsychiatric systemic lupus erythematosus (NPSLE) is often difficult to diagnose and distinguish from other diseases, because no NPSLE-specific antibodies have been identified. We developed a novel proteomic strategy for identifying and profiling antigens in immune complexes in the cerebrospinal fluid (CSF), and applied this strategy to 26 NPSLE patients. As controls, we also included 25 SLE patients without neuropsychiatric manifestations (SLE), 15 with relapsing remitting multiple sclerosis (MS) and 10 with normal pressure hydrocephalus (NPH). We identified immune complexes of suprabasin (SBSN) in the CSF of the NPSLE group. The titer of anti-SBSN antibodies was significantly higher in the CSF of the NPSLE group compared to those of the SLE, MS and NPH groups. Microarray data showed that the senescence and autophagy pathways were significantly changed in astrocytes exposed to anti-SBSN antibodies. Our findings indicate that SBSN could be a novel autoantibody for the evaluation of suspected NPSLE. [ABSTRACT FROM AUTHOR]

Objective In systemic lupus erythematosus (SLE) patients, excessive formation of neutrophil extracellular traps (NETs) is observed and their degradation is impaired. In vitro, immune complexes (ICx) trigger NET formation while NET-derived DNA is a postulated autoantigen for anti-nuclear autoantibodies (ANAs), found in SLE. Based on these self-perpetuating mechanisms in SLE, this study investigates whether interfering with ICx formation using a combination of rituximab (RTX) and belimumab (BLM) could decrease NET formation and ameliorate disease. Methods A phase 2A, open-label, single arm proof-of-concept study was performed wherein 16 SLE patients with severe, refractory disease were treated with a combination of CD20-mediated B-cell depletion with rituximab and sustained inhibition of B-cell activating factor BlyS with belimumab. Besides safety, the study's endpoints were chosen to address the concept of autoantibodies in relation to excessive NET formation. Results We demonstrated a surge of BlyS levels upon RTX-mediated B-cell depletion which was abrogated by subsequent BLM treatment. As such, therapeutic intervention with RTX + BLM led to specific reductions in ANAs and regression of excessive NET formation. RTX + BLM appeared to be safe and achieved clinically significant responses: low lupus disease activity state was achieved in 10 patients, renal responses in 11 patients and concomitant immunosuppressive medication was tapered in 14 out of the 16 patients. Conclusions This study provides novel insights into clinical beneficence of reducing excessive NET formation in SLE by therapeutic targeting ANA production with RTX + BLM. Altogether putting forward a new treatment concept that specifically ameliorates underlying SLE pathophysiology. Trial registration ClinicalTrials.gov NCT02284984 . [ABSTRACT FROM AUTHOR]

Lupus nephritis is an immune-complexes mediated glomerulonephritis. Vascular lesions and endothelial cell injuries are common in lupus nephritis and important for renal damage. However, the precise mechanisms by which immune complexes lead to endothelial cell injuries are still unclear. Autophagy is a conserved metabolic process and shows protective roles in many cell types and diseases. In present study, we investigated whether immune complexes could affect autophagy and participate in endothelial dysfunctions. Heat-aggregated gamma globulin (HAGG) was used to substitute immune complexes. Glomerular endothelial cells (GECs) were incubated with HAGG and autophagy-related markers were evaluated. Results showed that HAGG suppressed autophagy in GECs, through Akt/mTOR-dependent pathway. The combination of HAGG and tumor necrosis factor-alpha suppressed autophagy in GECs and further decreased cell viabilities. The suppressed effects of HAGG on GECs autophagy and viability, especially under inflammatory microenvironment, may provide new views for explaining the mechanisms of renal impairments in lupus nephritis. [ABSTRACT FROM AUTHOR]

Immune complexes (IC) containing predominantly malondialdehyde-LDL and the corresponding autoantibodies (MDA-LDL IC) predict acute cardiovascular events, while IC rich in oxidized LDL (oxLDL IC) predict cardiovascular disease progression. Our objective was to determine mechanisms that could explain these prognostic differences. We compared the effects of the interaction of oxLDL, MDA-LDL and the corresponding IC with human macrophages focusing on apoptosis, metalloproteinases, and proinflammatory cytokines. MDA-LDL IC induced higher degrees of apoptosis, higher levels of caspase-3 expression, and increased expression and release of MMP-1 and TNF compared to MDA-LDL, oxLDL, and oxLDL IC. The pro-apoptotic effects of MDA-LDL IC were inhibited by blocking TNFR 1 or FcγRI. Blocking FcγRI abrogated the induction and expression of MMPs and proinflammatory cytokines by MDA-LDL IC. In conclusion, the interaction of MDA-LDL IC with FcγRI triggers macrophage apoptosis and increased expression and release of TNF and MMP-1, which can lead to the rupture of unstable plaques. [ABSTRACT FROM AUTHOR]

The pentraxin C-reactive protein (CRP) is a pentameric protein now known to be able to undergo dissociation into a monomeric, modified isoform, referred to as mCRP. In carefully assessing the bioactivities of each isoform, mCRP has strong pro-inflammatory activities while pCRP has mild anti-inflammatory activities. Systemic lupus erythematosus (SLE) is a disease characterized by a vast number of autoantibodies, including anti -CRP autoantibodies which have been associated with SLE disease activity and lupus nephritis. The origin of these autoantibodies is currently unknown. Extracellular vesicles (EVs) have been implicated in SLE pathogenesis as they can expose nuclear antigens on their outside surface, thereby being a potential adjuvant for the generation of autoantibodies. Herein, we studied exposure of both pCRP and mCRP on EVs in SLE plasma and the implications of each in disease activity, organ damage and clinical manifestations. We used flow cytometry to detect CRP isoforms on EV surfaces in 67 well-characterized SLE patients and 60 sex- and age-matched healthy controls. Autoantibodies against mCRP were measured using ELISA. We found an abundance of both pCRP and mCRP on SLE EVs compared to controls. Furthermore, mCRP+ but not pCRP+ EVs were elevated in patients with active disease and in anti -CRP positive patients. The proportions of mCRP+ EVs were lower in patients with acquired organ damage, especially in patients with lupus nephritis (LN), and displayed an inverse relationship with disease duration in LN and patients with active disease. Speculatively, these data suggest EV-bound mCRP as a relevant factor in SLE pathogenesis, which could contribute to development of anti -CRP autoantibodies by stimulating an immune response. [Display omitted] • Monomeric CRP (mCRP) is present on extracellular vesicles (EVs) in active SLE. • Presence of anti -mCRP autoantibodies coincide with mCRP on EVs in SLE. • mCRP-opsonized EVs may co-stimulate autoantibody generation against mCRP. [ABSTRACT FROM AUTHOR]

Monogenic lupus, a distinctive variant of systemic lupus erythematosus (SLE), is characterized by early onset, family-centric clustering, and heightened disease severity. So far, over thirty genetic variations have been identified as single-gene etiology of SLE and lupus-like phenotypes. The critical role of these gene mutations in disrupting various immune pathways is increasingly recognized. In particular, single gene mutation-driven dysfunction within the innate immunity, notably deficiencies in the complement system, impedes the degradation of free nucleic acid and immune complexes, thereby promoting activation of innate immune cells. The accumulation of these components in various tissues and organs creates a pro-inflammatory microenvironment, characterized by a surge in pro-inflammatory cytokines, chemokines, reactive oxygen species, and type I interferons. Concurrently, single gene mutation-associated defects in the adaptive immune system give rise to the emergence of autoreactive T cells, hyperactivated B cells and plasma cells. The ensuing spectrum of cytokines and autoimmune antibodies drives systemic disease manifestations, primarily including kidney, skin and central nervous system-related phenotypes. This review provides a thorough overview of the single gene mutations and potential consequent immune dysregulations in monogenic lupus, elucidating the pathogenic mechanisms of monogenic lupus. Furthermore, it discusses the recent advances made in the therapeutic interventions for monogenic lupus. • Monogenic mutations directly trigger SLE onset. • Dysregulated immune responses in monogenic lupus. • Unraveling insights into pathogenesis and treatment strategies. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Circulating immune complexes (CICs) size in rheumatoid arthritis (RA) could be an emerging criterion in disease diagnosis. • The CICs size of RA patients' sera can be determined by dynamic light scattering (DLS). • DLS identified RA-specific, but also age-specific distribution of CICs size. • Zeta potential revealed an RA-related decrease in the colloidal stability of CICs. • DLS could be a method for CIC size analysis in IC-mediated diseases. [ABSTRACT FROM AUTHOR]

Lupus nephritis affects up to 70% of patients with systemic lupus erythematosus and is an important treatable cause of kidney failure. Cardinal features of lupus nephritis include loss of self-tolerance, production of autoantibodies, immune complex deposition and immune-mediated injury to the kidney, resulting in increased cell proliferation, apoptosis, and induction of inflammatory and fibrotic processes that destroy normal nephrons. The production anti-dsDNA antibodies is a cardinal feature in lupus and their level correlates with disease activity. In addition to the formation of immune complexes thereby triggering complement activation, how anti-dsDNA antibodies home to the kidney and induce pathological processes in the renal parenchyma remain to be fully elucidated. Data from our laboratory and other investigators show that the properties of anti-dsDNA antibodies vary between patients and change over time, and that anti-dsDNA antibodies could bind directly to integral cell surface molecules such as annexin II or α-actinin, or indirectly through chromatin material deposited on the cell surface. The binding of anti-dsDNA antibodies to mesangial cells and proximal renal tubular epithelial cells triggers downstream inflammatory and fibrotic pathways, which include the activation of the PKC and MAPK signaling pathways, increased secretion of pro-inflammatory cytokines and matrix protein deposition that contribute to pathological processes in the renal parenchyma. [ABSTRACT FROM AUTHOR]

In addition to contributing to immune exclusion at mucosal surfaces, secretory IgA (SIgA) made of polymeric IgA and secretory component is able to selectively reenter via microfold cells into Peyer's patches (PPs) present along the intestine and to associate with dendritic cells (DCs) of the CD11c + CD11b + MHCII + F4/80 − CD8 − phenotype in the subepithelial dome region and the draining mesenteric lymph nodes (MLNs). However, the nature of the receptor(s) for SIgA on murine PP and MLN DCs is unknown. We find that glycosylated secretory component moiety and polymeric IgA are both involved in the specific interaction with these cells. Using blocking antibodies and competition experiments, we identify Dectin-1 and specific intercellular adhesion molecule-3 grabbing non-integrin receptor 3 (SIGNR3) as receptors for SIgA. While SIgA-commensal immune complexes (ICs) contribute to local homeostasis upon interaction with mucosal DCs, the picture is less clear for pathogenic agents. We find that in comparison with incubation of Shigella flexneri alone, association of the enteropathogen with SIgA prompts freshly isolated DCs from PPs and MLNs to invert the production of pro- versus non-inflammatory cytokines/chemokines. The sum of the data suggests that in contrast to IgG-based ICs boosting immune reactivity of antigen-presenting cells, SIgA produced during an ongoing immune response can, in addition to its known function of immune exclusion, modulate mucosal DC conditioning via specific interaction with Dectin-1 and SIGNR3. [ABSTRACT FROM AUTHOR]

Structure-based vaccine design depends on extensive structural analyses of antigen–antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5 Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants. [ABSTRACT FROM AUTHOR]

Phase plates in cryo-electron tomography (cryoET) improve contrast, increasing the ability to discern separate molecules and molecular complexes in dense biomolecular environments. Here, we applied this new technology to the activation of the human complement system. Binding of C1 to antigen-antibody complexes initiates a cascade of proteolytic events that deposits molecules onto adjacent surfaces and terminates with the formation of membrane-attack-complex (MAC) pores in the targeted membranes. We imaged steps in this process using a Volta phase plate mounted on a Titan Krios equipped with a Falcon-II direct electron detector. The data show patches of single-layer antibodies on the surface and C1 bound to antibody platforms, with ca. ∼4% of instances where C1r and C1s proteases have dissociated from C1, and potentially instances of C1 transiently interacting with its substrate C4 or product C4b. Next, extensive deposition of C4b and C3b molecules is apparent, although individual molecules cannot always be properly distinguished with the current methods. Observations of MAC pores include formation of both single and composite pores, and instances of potential soluble-MAC dissociation upon failure of membrane insertion. Overall, application of the Volta phase plate cryoET markedly improved the contrast in the tomograms, which allowed for individual components to be more readily interpreted. However, variability in the phase shift induced by the phase-plate during the course of an experiment, together with incomplete sampling during tomogram acquisition, limited the interpretability of the resulting tomograms. Our studies exemplify the potential in studying molecular processes with complex spatial topologies by phase-plate cryoET. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cortisol level can be used as a biomarker for stress and other disorders. • Anti-cortisol (17) Fab fragment with a high affinity for cortisol was discovered. • The first crystal structure of anti-cortisol Fab fragment was determined. • X-ray structures with glucocorticoids were solved to understand the binding specificity. • The structural basis for the cross-reactivity assists the immunoassay development. Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders. Therefore, the accurate quantification of cortisol levels in body fluids is essential for clinical diagnosis. In this article, we describe the isolation of recombinant anti-cortisol antibodies with high affinity for cortisol and discover their cross-reactivity with other glucocorticoids. To describe the cortisol binding site and elucidate the structural basis for the binding specificity, the high-resolution crystal structures of the anti-cortisol (17) Fab fragment in the absence of glucocorticoid (2.00 Å) and the presence of cortisol (2.26 Å), corticosterone (1.86 Å), cortisone (1.85 Å) and prednisolone (2.00 Å) were determined. To our knowledge, this is the first determined crystal structure of a cortisol-specific antibody. The recognition of cortisol is driven by hydrophobic interactions and hydrogen bonding at the protein–ligand interface coupled with a conformational transition. Comparison of ligand-free and ligand-bound structures showed that the side chains of residues Tyr58-H and Arg56-H can undergo local conformational changes at the binding site, most likely prior to the binding event via a conformational selection mechanism. Compared to other anti-steroid antibody–antigen complexes, (17) Fab possesses a structurally unique steroid binding site, as the H3 loop from the CDR area has only a minor contribution, but framework residues have a prominent contribution to hapten binding. [ABSTRACT FROM AUTHOR]

White shrimp Litopenaeus vannamei receiving fucoidan at 2, 6, and 10 μg g −1 after 0–144 h or 0–120 h were examined for immune parameters (haemograms, phenoloxidase activity, respiratory burst, and superoxide dismutase activity), proliferation of haemocyte in the haematopoietic tissue (HPT), gene expression, and phagocytic activity and clearance efficiency to Vibrio alginolyticus . Immune parameters and mitotic index of HPT increased after 3–24 h, reached their maxima after 48–72 h, and returned to background values after 144 h. Transcripts of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), prophenoloxidase (proPO) I, proPO II, astakine, and haemocyte homeostasis-associated protein (HHAP) were up-regulated to a maximum after 48–72 h and returned to background values after 144 h. Phagocytic activity and clearance efficiency to V. alginolyticus increased after 12 h, reached its maximum after 48 h, and continued to remain higher after 120 h. In another experiment, shrimp receiving fucoidan after 48 h and 144 h were respectively challenged with V. alinolyticus at 6 × 10 6 colony-forming units (cfu) shrimp −1 or challenged with WSSV at 1.2 × 10 5 copies shrimp −1 and then placed in seawater. The survival rate of shrimp receiving fucoidan was significantly higher than in controls. In conclusion, shrimp receiving fucoidan showed a proliferation of HPT, increased immune parameters, and up-regulated transcripts of LGBP, PX, proPO I, proPO II, astakine, and HHAP after 48 h. Shrimp receiving fucoidan exhibited a defense against V. alginolyticus and WSSV, even after immune parameters recovered to background levels. [ABSTRACT FROM AUTHOR]

Considerable evidence is available supporting the atherogenic role of immune complexes (IC) formed by modified forms of LDL and their corresponding antibodies in humans and other species. In this study, we assessed the effect of IgG F(ab′) 2 fragments of murine anti-mouse oxLDL, which binds oxLDL forming IC that cannot interact with Fcγ receptors, on the development of atherosclerosis in diabetic LDL receptor-deficient (LDLR −/−) mice. Immunohistochemical study showed that treatment with the F(ab′) 2 fragments for 8 weeks significantly reduced the content of macrophages and interleukin 6 expression in atherosclerotic lesions. Furthermore, histological study showed that treatment with the same F(ab′) 2 fragments significantly reduced atherosclerotic lesions in diabetic LDLR −/− mice. Taken together, this study demonstrated for the first time that F(ab′) 2 fragments of anti-oxLDL IgG inhibited vascular inflammation and atherogenesis in diabetic LDLR −/− mice and uncovered a possible new avenue for therapy in patients at high risk to progress to cardiovascular complications. [ABSTRACT FROM AUTHOR]

IgA nephropathy (IgAN) is the leading cause of primary glomerulonephritis in the world. The disease is characterized by the presence of IgA-containing immune complexes in the circulation and in mesangial deposits with ensuing glomerular injury. Although in humans there are two IgA subclasses, only IgA1 molecules are involved. The exclusivity of participation of IgA1 in IgAN prompted extensive structural and immunological studies of the unique hinge region (HR) of IgA1, which is absent in otherwise highly homologous IgA2. HR of IgA1 with altered O -glycans serves as an antigen recognized by autoantibodies specific for aberrant HR glycans leading to the generation of nephritogenic immune complexes. However, there are several unresolved questions concerning the phylogenetic origin of human IgA1 HR, the structural basis of its antigenicity, the origin of antibodies specific for HR with altered glycan moieties, the regulatory defects in IgA1 glycosylation pathways, and the potential approaches applicable to the disease-specific interventions in the formation of nephritogenic immune complexes. This review focuses on the gaps in our knowledge of molecular and cellular events that are involved in the immunopathogenesis of IgAN. [ABSTRACT FROM AUTHOR]

The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies. [ABSTRACT FROM AUTHOR]

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3 + T cells and CD14 + macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types ( V b ) were determined relative to the initial cell volume ( V 0 ) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water ( L p ) and to four different cryoprotective agent (CPA) solutions ( P s ) at room temperature were also measured. Results indicated V b values of 0.516 V 0 and 0.457 V 0 for mucosal T cells and macrophages, respectively. L p values at room temperature were 0.196 and 0.295 μm/min/atm for T cells and macrophages, respectively. Both cell types had high P s values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10 −3 cm/min), but transport of the fourth CPA, glycerol, occurred 50–150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells. [ABSTRACT FROM AUTHOR]