Natural Immunoglobulin Research Articles

Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.

Malondialdehyde-specific natural IgM inhibit NETosis triggered by culprit site-derived extracellular vesicles from myocardial infarction patients.

Abstract Background and Aims Neutrophil extracellular traps (NETs) trigger atherothrombosis during acute myocardial infarction (AMI), but mechanisms of induction remain unclear. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE), which are targeted by specific natural immunoglobulin M (IgM), are increased at the culprit site in AMI. This study investigated EV as inducers of NETosis and assessed the inhibitory effect of natural anti-OSE–IgM in this process. Methods Blood from the culprit and peripheral site of ST-segment elevation myocardial infarction (STEMI) patients (n = 28) was collected, and myocardial function assessed by cardiac magnetic resonance imaging (cMRI) 4 ± 2 days and 195 ± 15 days post-AMI. Extracellular vesicles were isolated from patient plasma and cell culture supernatants for neutrophil stimulation in vitro and in vivo, in the presence of a malondialdehyde (MDA)-specific IgM or an isotype control. NETosis and neutrophil functions were assessed via enzyme-linked immunosorbent assay and fluorescence microscopy. Pharmacological inhibitors were used to map signalling pathways. Neutrophil extracellular trap markers and anti-OSE–IgM were measured by ELISA. Results CD45+ MDA+ EV and NET markers were elevated at the culprit site. Extracellular vesicles induced neutrophil activation and NET formation via TLR4 and PAD4, and mice injected with EV showed increased NETosis. Malondialdehyde-specific IgM levels were inversely associated with citH3 in STEMI patient blood. An MDA-specific IgM inhibited EV-induced NET release in vitro and in vivo. CD45+ MDA+ EV concentrations inversely correlated with left ventricular ejection fraction post-AMI. Conclusions Culprit site–derived EV induce NETosis, while MDA-specific natural IgM inhibit this effect, potentially impacting outcome after AMI.

Theories of immune recognition: Is anybody right?

The clonal selection theory (CST) is the centrepiece of the current paradigm used to explain immune recognition and memory. Throughout the past decades, the original CST had been expanded and modified to explain new experimental evidences since its original publication by Burnet. This gave origin to new paradigms that govern experimental immunology nowadays, such as the associative recognition of antigen model and the stranger/danger signal model. However, these new theories also do not fully explain experimental findings such as natural autoimmune immunoglobulins, idiotypic networks, low and high dose tolerance, and dual-receptor T and B cells. To make sense of these empirical data, some authors have been trying to change the paradigm of immune cognition using a systemic approach, analogies with brain processing and concepts from second-order cybernetics. In the present paper, we review the CST and some of the theories/hypotheses derived from it, focusing on immune recognition. We point out their main weaknesses and highlight arguments made by their opponents and believers. We conclude that, until now, none of the proposed theories can fully explain the totality of immune phenomena and that a theory of everything is needed in immunology.

A single-chain antibody construct with specificity of a natural IgM antibody reduces hepatic ischemia reperfusion injury in mice.

Natural immunoglobulin M (IgM) antibodies have been shown to recognize post-ischemic neoepitopes following reperfusion of tissues and to activate complement. Specifically, IgM antibodies and complement have been shown to drive hepatic ischemia reperfusion injury (IRI). Herein, we investigate the therapeutic effect of C2 scFv (single-chain antibody construct with specificity of a natural IgM antibody) on hepatic IRI in C57BL/6 mice. Compared with PBS-treated mice, C2 scFv-treated mice displayed almost no necrotic areas, significant reduction in serum ALT, AST and LDH levels, and significantly reduced in the number of TUNEL positive cells. Moreover, C2 scFv-treated mice exhibited a notable reduction in inflammatory cells after hepatic IRI than PBS-treated mice. The serum IL-6, IL-1β, TNF-α and MPC-1 levels were also severely suppressed by C2 scFv. Interestingly, C2 scFv reconstituted hepatic inflammation and IRI in Rag1-/- mice. We found that C2 scFv promoted hepatic cell death and increased inflammatory cytokines and infiltration of inflammatory cells after hepatic IRI in Rag1-/- mice. In addition, IgM and complement 3d (C3d) were deposited in WT mice and in Rag1-/- mice reconstituted with C2 scFv, indicating that C2 scFv can affect IgM binding and complement activation and reconstitute hepatic IRI. C3d expression was significantly lower in C57BL/6 mice treated with C2 scFv compared to PBS, indicating that excessive exogenous C2 scFv inhibited complement activation. These data suggest that C2 scFv alleviates hepatic IRI by blocking complement activation, and treatment with C2 scFv may be a promising therapy for hepatic IRI.

Dexamethasone Pretreatment Potentiates a Folic Acid-Functionalized Delivery System for Enhanced Lung Cancer Therapy.

Folic acid (FA) has been widely engineered to promote the targeted delivery of FA-modified nanoparticles (NPs) by recognizing the folate receptor α (FRα). However, the efficacy of FA-targeted therapy significantly varied with the abundance of FRα and natural immunoglobulin levels in different tumors. Therefore, a sequential therapy of dexamethasone (Dex)-induced FRα amplification and immunosuppression combined with FA-functionalized doxorubicin (DOX) micelles to synergistically suppress tumor proliferation was proposed in this study. In brief, a pH/reduction-responsive FA-functionalized micelle (FCSD) was obtained by grafting FA, derivatization-modified cholesterol, and 2,3-dimethylmaleic anhydride onto a chitosan oligosaccharide. The obtained FCSD/DOX NPs can effectively deliver DOX in tumors, and their targeting efficiency can be further improved with Dex pretreatment to decrease the immunoglobulin M (IgM) content in serum and amplify FRα levels on the surface of M109 cells. After internalization, charge reversal and disulfide bond breakage of FCSD vectors under the stimulation of tumor extracellular pH (pHe) and intracellular glutathione (GSH) would contribute to the disintegration of vectors and the rapid release of DOX. The sequential therapy that combined Dex pretreatment and targeted chemotherapy by FCSD/DOX NPs demonstrated superior tumor suppression compared with monotherapy, which is expected to provide a potential strategy for FRα-positive lung cancer patients.

Systematic evaluation of donor-KIR/recipient-HLA interactions in HLA-matched hematopoietic cell transplantation for AML

AbstractIn acute myeloid leukemia (AML), donor natural killer cell killer immunoglobulin–like receptors (KIR) and recipient HLA interactions may contribute to the graft-versus-leukemia effect of allogeneic hematopoietic cell transplantation (HCT). Analyses of individual KIR/HLA interactions, however, have yielded conflicting findings, and their importance in the HLA-matched unrelated donor (MUD) setting remains controversial. We systematically studied outcomes of individual donor-KIR/recipient-HLA interactions for HCT outcomes and empirically evaluated prevalent KIR genotypes for clinical benefit. Adult patients with AML (n = 2025) who received HCT with MUD grafts in complete remission reported to the Center for International Blood and Marrow Transplantation were evaluated. Only the donor-2DL2+/recipient-HLA-C1+ pair was associated with reduced relapse (hazard ratio [HR], 0.79; 95% confidence interval [CI], 0.67-0.93; P = .006) compared with donor-2DL2–/recipient-HLA-C1+ pair. However, no association was found when comparing HLA-C groups among KIR-2DL2+–graft recipients. We identified 9 prevalent donor KIR genotypes in our cohort and screened them for association with relapse risk. Genotype 5 (G5) in all recipients and G3 in Bw4+ recipients were associated with decreased relapse risk (HR, 0.52; 95% CI, 0.35-0.78; P = .002; and HR, 0.32; 95% CI, 0.14-0.72; P = .006; respectively) and G2 (HR 1.63, 95% CI, 1.15-2.29; P = .005) with increased relapse risk in C1-homozygous recipients, compared with other patients with the same ligand. However, we could not validate these findings in an external data set of 796 AML transplants from the German transplantation registry. Neither a systematic evaluation of known HLA-KIR interactions nor an empiric assessment of prevalent KIR genotypes demonstrated clinically actionable associations; therefore, these data do not support these KIR-driven strategies for MUD selection in AML.

Relationships between Parameters for Natural Antibodies, General Immunoglobulins, and Immunocompetent Blood Cells in Sheep in Postvaccination Immune Response

Relationships between Parameters for Natural Antibodies, General Immunoglobulins, and Immunocompetent Blood Cells in Sheep in Postvaccination Immune Response

Effective clearance of rituximab-resistant tumor cells by breaking the mirror-symmetry of immunoglobulin G and simultaneous binding to CD55 and CD20

Complement-dependent cytotoxicity (CDC), which eliminates aberrant target cells through the assembly and complex formation of serum complement molecules, is one of the major effector functions of anticancer therapeutic antibodies. In this study, we discovered that breaking the symmetry of natural immunoglobulin G (IgG) antibodies significantly increased the CDC activity of anti-CD20 antibodies. In addition, the expression of CD55 (a checkpoint inhibitor in the CDC cascade) was significantly increased in a rituximab-resistant cell line generated in-house, suggesting that CD55 overexpression might be a mechanism by which cancer cells acquire rituximab resistance. Based on these findings, we developed an asymmetric bispecific antibody (SBU-CD55 × CD20) that simultaneously targets both CD55 and CD20 to effectively eliminate rituximab-resistant cancer cells. In various cancer cell lines, including rituximab-resistant lymphoma cells, the SBU-CD55 × CD20 antibody showed significantly higher CDC activity than either anti-CD20 IgG antibody alone or a combination of anti-CD20 IgG antibody and anti-CD55 IgG antibody. Furthermore, the asymmetric bispecific antibody (SBU-CD55 × CD20) exhibited significantly higher CDC activity against rituximab-resistant cancer cells compared to other bispecific antibodies with symmetric features. These results demonstrate that enhancing CDC with an asymmetric CD55-binding bispecific antibody could be a new strategy for developing therapeutics to treat patients with relapsed or refractory cancers.

Antibody-Dependent Enhancement with a Focus on SARS-CoV-2 and Anti-Glycan Antibodies.

Antibody-dependent enhancement (ADE) is a phenomenon where virus-specific antibodies paradoxically cause enhanced viral replication and/or excessive immune responses, leading to infection exacerbation, tissue damage, and multiple organ failure. ADE has been observed in many viral infections and is supposed to complicate the course of COVID-19. However, the evidence is insufficient. Since no specific laboratory markers have been described, the prediction and confirmation of ADE are very challenging. The only possible predictor is the presence of already existing (after previous infection) antibodies that can bind to viral epitopes and promote the disease enhancement. At the same time, the virus-specific antibodies are also a part of immune response against a pathogen. These opposite effects of antibodies make ADE research controversial. The assignment of immunoglobulins to ADE-associated or virus neutralizing is based on their affinity, avidity, and content in blood. However, these criteria are not clearly defined. Another debatable issue (rather terminological, but no less important) is that in most publications about ADE, all immunoglobulins produced by the immune system against pathogens are qualified as pre-existing antibodies, thus ignoring the conventional use of this term for natural antibodies produced without any stimulation by pathogens. Anti-glycan antibodies (AGA) make up a significant part of the natural immunoglobulins pool, and there is some evidence of their antiviral effect, particularly in COVID-19. AGA have been shown to be involved in ADE in bacterial infections, but their role in the development of ADE in viral infections has not been studied. This review focuses on pros and cons for AGA as an ADE trigger. We also present the results of our pilot studies, suggesting that AGAs, which bind to complex epitopes (glycan plus something else in tight proximity), may be involved in the development of the ADE phenomenon.

Triazine herbicides exposure, natural immunoglobulin M antibodies, and fasting plasma glucose changes: Association and mediation analyses in general Chinese urban adults

The effects of triazine herbicides on glucose metabolism remain unclear. In this study, we aimed to assess the associations between serum triazine herbicides and glycemia-related risk indicators in general adults, and to evaluate the mediating role of natural immunoglobulin M antibodies (IgM) in the above associations among uninfected participants. We measured the concentrations of atrazine, cyanazine, and IgM in serum, as well as fasting plasma glucose (FPG), and fasting plasma insulin in 4423 adult participants from the Wuhan-Zhuhai cohort baseline population, enrolled in 2011–2012. Generalized linear models were used to evaluate the associations of serum triazine herbicides with glycemia-related risk indicators, and mediation analyses were performed to evaluate the mediating role of serum IgM in the above associations. The median levels of serum atrazine and cyanazine were 0.0237 μg/L and 0.0786 μg/L, respectively. Our study found significant positive associations of serum atrazine, cyanazine, and Σtriazine with FPG levels, risk of impaired fasting glucose (IFG), abnormal glucose regulation (AGR), and type 2 diabetes (T2D). Additionally, serum cyanazine and Σtriazine were found to be significant positive associated with the homeostatic model assessment of insulin resistance (HOMA-IR) levels. Significant negative linear relationships were observed in associations of serum IgM with serum triazine herbicides, FPG, HOMA-IR levels, the prevalence of T2D, and AGR (P < 0.05). Furthermore, we observed a significant mediating role by IgM in the associations of serum triazine herbicides with FPG, HOMA-IR, and AGR, with the proportions ranging from 2.96% to 7.71%. To ensure the stability of our findings, we conducted sensitivity analyses in normoglycemic participants and found that the association of serum IgM with FPG and the mediating role by IgM remained stable. Our results suggest that triazine herbicides exposure is positively associated with abnormal glucose metabolism, and decreasing serum IgM may partly mediate these associations.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

A B1a-natural IgG-neutrophil axis is impaired in viral- and steroid-associated aspergillosis.

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.

Molecular Mechanisms of Network Pharmacology-Based Immunomodulation of Huangqi (Astragali Radix)

Astragali Radix (Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, also known as Huangqi) has been used to improve stamina and immunity for thousands of years. However, the bioactive components and their mechanisms are still unclear, limiting its clinical application. In this research, for the first time, network pharmacology was used to investigate the molecular mechanisms of Huangqi and its bioactive components that play an important role in improving the immune system. An amount of 1,430 immunomodulation-related target proteins was collected from the human genome database, with 171 target proteins from 20 bioactive components, and resulted in 60 intersection target proteins. Subsequently, the networks of common target and protein-protein interaction (PPI) were analyzed. The results showed that Huangqi influenced the modulations of lymphocytes (B cells and T cells), natural killer cells, macrophages, cytokines, immunoglobulins, and immune signal transduction. To a certain limit, this study revealed the potential bioactive components and pharmacological mechanisms of Huangqi in immunomodulation. It was able to direct novel drug development for treating immune system deficiency.

Do departure and flight route decisions correlate with immune parameters in migratory songbirds?

Abstract Many songbirds migrate twice a year to exploit seasonally available resources. These journeys are energetically demanding and energy reserves of these small birds are limited. Accordingly, most of the time migrating is spent during stopovers that serve to rest, replenish resources and recover. While external influences, like prevailing weather conditions and resource availability, are well studied with regard to stopover behaviour and departure decisions, studies on how birds' individual physiological condition and stopover decisions may be linked are scarce. We used a large‐scale radio‐telemetry network covering the German Bight (SE North Sea) to study how birds' immunological constitution at stopover may correlate with departure and flight behaviour in five species of short‐ to medium‐distance migratory songbirds. We measured markers of the innate (bacterial killing activity, lysozyme concentration, natural antibodies and complement titres) and acquired immune function (immunoglobulin Y) in the birds' plasma. After sampling, we tracked the birds' behaviour in terms of stopover duration as well as flight routes and flight distances within the telemetry network after departure. We found that stopover durations were positively related to natural antibodies and immunoglobulin Y across species and to the bacterial killing activity solely in song thrushes in spring, while no relations became apparent in fall. Flight distances were linked positively to immunoglobulin Y concentrations in both spring and fall, though not in all of the investigated species. Coastal and offshore‐oriented routes were taken independent of the birds' immune status. Our study for the first time shows that migratory behaviour of songbirds in the wild is correlated with their immune status, not only during but also beyond stopover. Furthermore, birds with increased complement titres and Immunoglobulin Y concentrations, either due to recent infection or greater investment in their immune function, took additional time at their stopover sites, probably to reach their breeding grounds in good condition. During the less time‐constrained fall season, stopovers were generally prolonged, independent from the birds' immune status and any delays taken to improve immune status are most likely not detrimental in terms of fitness. Read the free Plain Language Summary for this article on the Journal blog.

Dairy-Based Probiotic-Fermented Functional Foods: An Update on Their Health-Promoting Properties

Numerous studies have shown a link between the consumption of fermented dairy foods and improved health outcomes. Since the early 2000s, especially probiotic-based fermented functional foods, have had a revival in popularity, mostly as a consequence of claims made about their health benefits. Among them, fermented dairy foods have been associated with obesity prevention and in other conditions such as chronic diarrhea, hypersensitivity, irritable bowel syndrome, Helicobacter pylori infection, lactose intolerance, and gastroenteritis which all are intimately linked with an unhealthy way of life. A malfunctioning inflammatory response may affect the intestinal epithelial barrier’s ability to function by interfering with the normal metabolic processes. In this regard, several studies have shown that fermented dairy probiotics products improve human health by stimulating the growth of good bacteria in the gut at the same time increasing the production of metabolic byproducts. The fermented functional food matrix around probiotic bacteria plays an important role in the survival of these strains by buffering and protecting them from intestinal conditions such as low pH, bile acids, and other harsh conditions. On average, cultured dairy products included higher concentrations of lactic acid bacteria, with some products having as much as 109/mL or g. The focus of this review is on fermented dairy foods and associated probiotic products and their mechanisms of action, including their impact on microbiota and regulation of the immune system. First, we discussed whey and whey-based fermented products, as well as the organisms associated with them. Followed by the role of probiotics, fermented-product-mediated modulation of dendritic cells, natural killer cells, neutrophils, cytokines, immunoglobulins, and reinforcement of gut barrier functions through tight junction. In turn, providing the ample evidence that supports their benefits for gastrointestinal health and related disorders.

Concentration and heritability of immunoglobulin G and natural antibody immunoglobulin M in dairy and beef colostrum along with serum total protein in their calves.

Immunoglobulin (Ig) G and natural antibody (NAb) IgM are passively transferred to the neonatal calf through bovine colostrum. Maternal IgG provides pathogen- or vaccine-specific protection and comprises about 85% of colostral Ig. NAb-IgM is less abundant but provides broad and nonspecific reactivity, potentially contributing to protection against the dissemination of pathogens in the blood (septicemia) in a calf’s first days of life. In the dairy and beef industries, failure of passive transfer (FPT) of colostral Ig (serum total protein [STP] <5.2 g/dL) is still a common concern. The objectives of this study were to: (1) compare colostral IgG concentrations and NAb-IgM titers between dairy and beef cows; (2) assess the effect of beef breed on colostral IgG; (3) compare passive transfer of colostral Ig in dairy and beef calves; and (4) estimate the heritability of colostral IgG and NAb-IgM. Colostrum was collected from Holstein dairy (n = 282) and crossbred beef (n = 168) cows at the University of Guelph dairy and beef research centers. Colostral IgG was quantified by radial immunodiffusion and NAb-IgM was quantified by an enzyme-linked immunosorbent assay. In dairy (n = 308) and beef (n = 169) calves, STP was estimated by digital refractometry. Beef cows had significantly greater colostral IgG (146.5 ± 9.5 standard error of the mean [SEM] g/L) than dairy cows (92.4 ± 5.2 g/L, P <0.01). Beef cows with a higher proportion of Angus ancestry had significantly lower colostral IgG (125.5 ± 5.8 g/L) than cows grouped as “Other” (142.5 ± 4.9 g/L, P = 0.02). Using the FPT cutoff, 13% of dairy and 16% of beef calves had FPT; still, beef calves had a significantly larger proportion with excellent passive transfer (STP ≥6.2 g/dL, P <0.01). The heritability of colostral IgG was 0.04 (±0.14) in dairy and 0.14 (±0.32) in beef. Colostral NAb-IgM titers in dairy (12.12 ± 0.22, log2 [reciprocal of titer]) and beef cows (12.03 ± 0.19) did not differ significantly (P = 0.71). The range of NAb-IgM titers was 9.18–14.60, equivalent to a 42-fold range in antibody concentration. The heritability of colostral NAb was 0.24 (±0.16) in dairy and 0.11 (±0.19) in beef cows. This study is the first to compare colostral NAb-IgM between dairy and beef cows. Based on the range in NAb-IgM titers and the heritability, selective breeding may improve colostrum quality and protection for neonatal calves in the early days of life.

B cells promote CD8 Tcell primary and memory responses to subunit vaccines.

SUMMARYThe relationship between B cells and CD4 T cells has been carefully studied, revealing a collaborative effort in which B cells promote the activation, differentiation, and expansion of CD4 T cells while the so-called “helper” cells provide signals to B cells, influencing their class switching and fate. Interactions between B cells and CD8 T cells are not as well studied, although CD8 T cells exhibit an accelerated contraction after certain infections in B-cell-deficient mice. Here, we find that B cells significantly enhance primary CD8 T cell responses after vaccination. Moreover, memory CD8 numbers and function are impaired in B-cell-deficient animals, leading to increased susceptibility to bacterial challenge. We also show that interleukin-27 production by B cells contributes to their impact on primary, but not memory, CD8 responses. Better understanding of the interactions between CD8 T cells and B cells may aid in the design of more effective future vaccine strategies.

Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase–like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde–2-piperidinol products, we evaluated the lysyl oxidase–like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde–2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.

Comprehensive comparison between 222 CTLA-4 haploinsufficiency and 212 LRBA deficiency patients: a systematic review

Cytotoxic T lymphocyte antigen 4 (CTLA-4) haploinsufficiency (CHAI) and lipopolysaccharide-responsivebeige-like anchor (LRBA) deficiency (LATAIE) are newly identified inborn errors of immunity with shared molecular pathomechanisms and clinical manifestations. In this review, we aimed to provide differential comparisons regarding demographic, clinical, immunological and molecular characteristics between these two similar conditions. A literature search was conducted in PubMed, Web of Science and Scopus databases and included studies were systematically evaluated. Overall, 434 (222 CHAI and 212 LATAIE) patients were found in 101 eligible studies. The CHAI patients were mainly reported from North America and western Europe, while LATAIE patients were predominantly from Asian countries. In CHAI, positive familial history (P<0·001) and in LATAIE, consanguineous parents (P<0·001) were more common. In CHAI patients the rates of granulomas (P<0·001), malignancies (P=0·001), atopy (P=0·001), cutaneous disorders (P<0·001) and neurological (P=0·002) disorders were higher, while LATAIE patients were more commonly complicated with life-threatening infections (P=0·002), pneumonia (P=0·006), ear, nose and throat disorders (P<0·001), organomegaly (P=0·023), autoimmune enteropathy (P=0·038) and growth failure (P<0·001). Normal lymphocyte subsets and immunoglobulins except low serum levels of CD9+ B cells (14·0 versus 38·4%, P<0·001), natural killer (NK) cells (21 versus 41·1%, P<0·001), immunoglobulin (Ig)G (46·9 versus 41·1%, P=0·291) and IgA (54·5 versus 44·7%, P=0·076) were found in the majority of CHAI and LATAIE patients, respectively. The most frequent biological immunosuppressive agents prescribed for CHAI and LATAIE patients were rituximab and abatacept, respectively. Further investigations into the best conditioning and treatment regimens pre- and post-transplantation are required to improve the survival rate of transplanted CHAI and LATAIE patients.

Natural immunoglobulin M-based delivery of a complement alternative pathway inhibitor in mouse models of retinal degeneration

PurposeAge-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. MethodsExpression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. ResultsB4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. ConclusionsData supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PrecisAMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.