Search

Deoxy-hexose sugars, such as rhamnose and quinovose, and the dideoxy-hexoses colitose, abequose, and tyvelose are highly antigenic given that they are absent from animal glycoconjugates. To investigate the specificity of antibodies towards structurally similar carbohydrate epitopes found in bacteria, we synthesized trisaccharides containing colitose, abequose, and fucose motifs. Each trisaccharide was designed with a spacer ending with a primary amino group. These trisaccharide constructs were immobilized on O-succinimide coated glass slides alongside bacterial lipopolysaccharides (LPS) containing colitose, abequose, and fucose residues. We compared the recognition of the synthetic trisaccharides and natural LPS including structurally related epitopes by monoclonal and polyclonal antibodies targeting bacterial LPS. Additionally, we used arrays displaying the synthetic trisaccharides and natural LPS to assess the variability of IgA reactivity from breast milk samples towards the carbohydrate antigens. The results obtained underlined the cross-reactivity of polyclonal antibodies towards structurally related carbohydrate antigens and revealed a broad reactivity of breast milk-derived IgA towards the carbohydrate antigens tested. The significant cross-reactivity of antibodies towards structurally related LPS antigens may lead to false-positive detection of bacterial serotypes when used for diagnostic purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Immunoglobulin A (IgA) plays a crucial role in the human immune system, particularly in mucosal immunity. IgA antibodies that target the mucosal surface are made up of two to five IgA monomers linked together by the joining chain, forming polymeric molecules. These IgA polymers are transported across mucosal epithelial cells by the polymeric immunoglobulin receptor pIgR, resulting in the formation of secretory IgA (SIgA). This review aims to explore recent advancements in our molecular understanding of IgA, with a specific focus on SIgA, and the interaction between IgA and pathogen molecules., (© 2024 Federation of European Biochemical Societies.)

Background: Immunoglobulin A (IgA) plays a pivotal role in various immune responses, especially that of mucosal immunity. IgA is usually assembled into dimers with the contribution of J-chains. There are two N-glycosylation sites in human IgA1-Fc and one in the J-chain. There is no consensus as yet on the functional role of the N-glycosylation., Methods: To gain a better understanding of their role, we designed a series of IgA1-Fc mutants, which were expressed in the absence or presence of the J-chain., Results: IgA1-Fc without the J-chain, was predominantly expressed as a monomer, and in its presence dimers and some polymers appeared. N263 (Fc Cα2), N459 (Fc tailpiece) and N49 (J-chain) were shown to be site-specifically modified with N-glycans by mass spectrometry analysis. Mutant IgA1-Fc N459Q failed to form a proper dimer in the presence of the J-chain, instead higher-order aggregates appeared. Fluorescence experiments suggest that the N459-glycans cover a hydrophobic surface at the Fc tailpiece that prevents other Fc molecules from approaching the dimeric IgA. A thermofluor assay revealed that the N-glycans at N263 (Fc) and N49 (J-chain) both contribute in different ways to the thermal stability of the Fc-J-chain complex. NMR analysis of 13 C-labeled Fc suggests that the N459-glycan is relatively flexible while the N263-glycan is more rigid., Conclusions: We conclude that the N459-glycan of IgA1-Fc is essential for dimer formation and prevention of higher-order aggregates while those at N263 (Fc) and N49 (J-chain) stabilize the Fc-J-chain complex., General Significance: Site-specific role for N-glycan in molecular assembly is addressed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vaccination against the SARS-Cov-2 virus is an effective way to protect against the disease and the severe course of COVID-19. Forty-nine fully vaccinated with mRNA vaccines (BNT162b2 or mRNA-1273) SARS-CoV-2 infection-naïve volunteers aged 33-89 were enrolled in the study. Evaluation of the cellular and humoral immune response was performed within 1 to 3 months (T1) and 6-9 months (T2) after the second injection, and within 2-3 months (T3) after a booster dose. Additionally, a comparative analysis of the specific immune status was made between two age groups-below 60 (n = 22) and over 60 (n = 27) years. SARS-CoV-2-specific T-cell response was evaluated by IFN-γ-producing spot forming cells (SFCs) using a standardized ELISPOT assay. Virus neutralizing antibodies (VNA) against SARS-CoV-2 were measured by a blocking ELISA test and spike protein specific IgG (S-IgG) and IgA (S-IgA) antibodies-by semiquantitative ELISA. IFN-γ-producing SFCs, S-IgG, S-IgA and VNA significantly decreased 6-9 months after the second dose. After the third injection S-IgG and S-IgA markedly increased compared to T2 and reached the levels at T1. Of note, the highest values of VNA were observed at T3. No differences in the tested immune parameters were found between the two age groups. Data obtained showed that for a long period-6-9 months after a full course of immunization with mRNA vaccine, immune reactivity is present, but both cellular and humoral immune responses gradually decrease. The administration of a third dose mainly restores the specific humoral immune response against the SARS-CoV-2 virus., (© 2022 Wiley Periodicals LLC.)

We examined how summated training and match load measures relate to salivary immunological and hormonal profile changes in professional football players. Data were collected from 18 elite-level professional male football players from one English Championship team across a complete 40 wk competitive season. Daily training (micro-technology) and match (computerised tracking) measures of total, high-speed and high-metabolic load running distance and sprint, acceleration, deceleration and sRPE load were converted into exponentially weighted moving average "acute" (7d), "chronic" (28d) and acute:chronic composite load measures. Bi-weekly morning saliva samples were analysed for immunoglobulin-A, alpha-amylase, testosterone, cortisol and testosterone:cortisol. A two-stage data reduction technique using partial least squares modelling and a backward stepwise selection procedure determined the most parsimonious model for each salivary variable. Testosterone had non-linear relationships with chronic total ( P  = 0.015; Cohen's D: large), high-metabolic load ( P  = 0.001;small) and high-speed ( P  = 0.001;trivial) running distance and linear relationships with chronic sRPE ( P  = 0.002;moderate ↓) and acute:chronic high-speed running distance ( P  = 0.001; trivial ↑). Cortisol had a non-linear relationship with chronic high-speed running distance ( P  = 0.001;trivial). Testosterone:cortisol had non-linear relationships with chronic decelerations ( P  = 0.039;small) and chronic summated acceleration and deceleration load ( P  = 0.039;small). Non-linear relationships typically indicated optimal hormonal responses at squad mean loads. No load variables clearly related to salivary immunoglobulin-A or alpha-amylase changes. We conclude that chronic total and high-intensity load measures relate to hormonal changes and might be useful indicators of player readiness. Acute load variables were not related to immunological or hormonal changes and consequently, should not be used as surrogate measures of player readiness in isolation.

Objectives: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context., Methods: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available., Results: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies., Conclusions: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3-97.2%) compared to 91.7% (95% CI 77.5-98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3-100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AI post/pre ). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgG post/pre ). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay., (© 2022. The Author(s).)

A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination., Competing Interests: I have read the journal’s plicy and the authors of this manuscript have the following competing interests: BW holds a patent entitled ‘Enhancing serological assays via fusion proteins’ US10101327B2 describing the fusion protein based technology described in the COVID-19 Multiplex Assay. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

IgA Nephropathy (IgAN) is the main cause of primary glomerulonephritis, globally. This disease is associated with a wide range of clinical presentations, variable prognosis and a spectrum of histological findings. More than fifty years after its first description, this heterogeneity continues to complicate efforts to understand the pathogenesis. Nevertheless, involvement of the complement system in IgAN was identified early on. Dysfunction of the immunoglobulin A (IgA) system, the principal offender in this disease, including modification of isoforms and glycoforms of IgA1, the nature of immune complexes and autoantibodies to galactose deficient IgA1 might all be responsible for complement activation in IgAN. However, the specific mechanisms engaging complement are still under examination. Research in this domain should allow for identification of patients that may benefit from complement-targeted therapy, in the foreseeable future., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC) of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play a pivotal role in various immune responses, especially in mucosal immunity. Due to their structural complexity, 3D structural study of these molecules at atomic scale has been slow. With the emergence of cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC. Disulfide bridging and N -glycosylation on these molecules are also summarized. With the increasing information of structure-function relationships, IgA- and IgM-based monoclonal antibodies will be an effective option in the therapeutic field.

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA's structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Objectives: Serum autoantibody measurement aids in diagnosing and monitoring various autoimmune conditions. Defining autoantibody stability limits can improve laboratory process quality. Here, we define short-term stability in a refrigerator, long-term stability in a freezer, and the effect of freeze-thaw cycles to improve autoantibody testing procedures., Design and Methods: Seventy-nine residual serum samples were used to assess the stability of 11 autoantibodies (anti-dsDNA, anti-Ro52, anti-Ro60, anti-SSB, anti-RNP, anti-Sm, anti-aCL-IgG, anti-tTG-IgA, anti-tTG-IgG, anti-DGP-IgA, anti-DGP-IgG) and two screening assays (CTD screen, ENA7 screen) on the BIO-FLASH (Inova Diagnostics). Three storage conditions were assessed: 8 weeks at 2-8 °C, 12 months at -30 °C, and 6 freeze (-30 °C)-thaw cycles. The maximum permissible instability (MPI) for each autoantibody was set as 2x %CV, calculated as the weighted average CV from cumulative QC data over the study period., Results: By considering both mean percent difference (MPD) and mean absolute relative difference (MARD), all autoantibodies were stable for up to 8 weeks stored at 2-8 °C, except for CTD screen and anti-dsDNA. All autoantibodies were stable for up to 12 months stored at -30 °C, except ENA screen, anti-dsDNA, anti-DGP-IgA, anti-cardiolipin, and CTD screen. Lastly, all autoantibodies were stable for up to 6 freeze(-30 °C)-thaw cycles, except anti-RNP, anti-Ro60, anti-cardiolipin and anti-dsDNA., Conclusions: It is important to develop laboratory procedures derived from evidence-based stability limits. This study will aid laboratories in undertaking quality assurance and improvement initiatives to enhance autoantibody testing by ensuring appropriate storage conditions that consider defined sample stability limits., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and is a leading cause of renal failure. The disease mechanisms are not completely understood, but a higher abundance of galactose-deficient IgA is recognized to play a crucial role in IgAN pathogenesis. Although both types of human IgA (IgA1 and IgA2) have several N -glycans as post-translational modification, only IgA1 features extensive hinge-region O -glycosylation. IgA1 galactose deficiency on the O -glycans is commonly detected by a lectin-based method. To date, limited detail is known about IgA O- and N- glycosylation in IgAN., Methods: To gain insights into the complex O- and N -glycosylation of serum IgA1 and IgA2 in IgAN, we used liquid chromatography-mass spectrometry (LC-MS) for the analysis of tryptic glycopeptides of serum IgA from 83 patients with IgAN and 244 age- and sex-matched healthy controls., Results: Multiple structural features of N- glycosylation of IgA1 and IgA2 were associated with IgAN and glomerular function in our cross-sectional study. These features included differences in galactosylation, sialylation, bisection, fucosylation, and N- glycan complexity. Moreover, IgA1 O- glycan sialylation was associated with both the disease and glomerular function. Finally, glycopeptides were a better predictor of IgAN and glomerular function than galactose-deficient IgA1 levels measured by lectin-based ELISA., Conclusions: Our high-resolution data suggest that IgA O - and N- glycopeptides are promising targets for future investigations on the pathophysiology of IgAN and as potential noninvasive biomarkers for disease prediction and deteriorating kidney function., (Copyright © 2021 by the American Society of Nephrology.)

Salivary glycoproteins are known as an important barrier to inhibit influenza infection by presenting sialic acid (Sia) ligands that can bind with viral hemagglutination. Here, to further understand why pregnant women are more vulnerable to avian influenza virus (AIV), we investigated the alteration of protein sialylation in the saliva of women during pregnancy and postpartum, and its impact on the saliva binding affinity to AIV. Totally 1200 saliva samples were collected, the expression levels of terminal α2-3/6-linked Sia on salivary proteins were tested and validated, and the binding activities of salivary proteins were assessed against 3 strains of AIV and the H1N1 vaccine. Result showed that the expression of terminal α2-3-linked Sia in the saliva of women decreased dramatically during pregnancy compared to that of non-pregnancy control, especially for women in the second or third trimester (fold change = 0.53 and 0.37, p < 0.001). And their salivary protein binding ability to AIV declined accordingly. The variation of terminal α2-3-linked Sia on salivary MUC5B and IgA was consistent with the above results. This study indicates that the decrease of terminal α2-3-linked Sia on salivary glycoproteins of pregnant women affects their binding ability to AIV, which may provide new insights into AIV prevention and control., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Immunoglobulins A (IgA) include some of the most abundant human antibodies and play an important role in defending mucosal surfaces against pathogens. The unique structural features of the heavy chain of IgA subclasses (called IgA1 and IgA2) enable them to polymerize via the joining J-chain, resulting in IgA dimers but also higher oligomers. While secretory sIgA oligomers are dominant in milk and saliva, IgAs exist primarily as monomers in serum. No method currently allows disentangling the millions of unique IgAs potentially present in the human antibody repertoire. Obtaining unambiguous sequence reads of their hypervariable antigen-binding regions is a prerequisite for IgA identification. We here report a mass spectrometric method that uses electron capture dissociation (ECD) to produce straightforward-to-read sequence ladders of the variable parts of both the light and heavy chains of IgA1s, in particular, of the functionally critical CDR3 regions. We directly compare the native top-down ECD spectra of a heavily and heterogeneously N - and O -glycosylated anti-CD20 IgA1, the corresponding N -glycosylated anti-CD20 IgG1, and their Fab parts. We show that while featuring very different MS1 spectra, the native top-down ECD MS2 spectra of all four species are nearly identical, with cleavages occurring specifically within the CDR3 and FR4 regions of both the heavy and light chain. From the sequence-informative ECD data of an intact glycosylated IgA1, we foresee that native top-down ECD will become a valuable complementary tool for the de novo sequencing of IgA1s from milk, saliva, or serum.

Mental stress is closely connected with our physical and mental wellness. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. In this paper, we detect the secretory immunoglobulin A (sIgA), which is the candidate of salivary stress markers, with original electrochemical immunoassay: gold-linked electrochemical immunoassay (GLEIA). This biosensor is based on a sandwich-type immunosensor and adopts the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires a low sample volume (10 μL). In addition, the GLEIA show high sensitivity and selectivity. We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Furthermore, we obtained acceptable selectivity in the various solution with salivary proteins such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme, and mucin. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress markers in saliva.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent for the production system used to make the protein for this study in the cytoplasm of E. coli using a sulfhydryl oxidase and an isomerase is held by the University of Oulu: Method for producing natively folded proteins in a prokaryotic host (patent no. 9238817; date of patent January 19, 2016; inventor: L.W.R.)., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protective effect of Tagetes erecta flowers essential oils was investigated on oxidative stress, immune response, inflammation, and apoptosis against N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats. Essential oil were extracted from Tagetes erecta flowers and analyzed using gas chromatography-mass spectrometry (GC-MS). For observing a protective effect against MNNG induced gastric cancer, we divided rats into 4 groups (group A to D) having 10 rats in each group. Performed various experiments and measured a different parameters to investigate antioxidant activity, immune response, anti-inflammatory and anti-apoptotic activity. The levels of malondialdehyde were markedly increased in the presence of N-methyl-N'nitro-N-nitroguanidine, whereas, the antioxidant activities of superoxide dismutase, and catalase were lowered in the treated rats in contrast with the control. Intervention with TEEO to gastric cancer-induced rats upregulated the redox status and the activity of the immune system to decrease cancer risk. The proinflammatory cytokines (IL-6 and TNF-α) secretions that were induced by MNNG were markedly inhibited by TEEO. Administration of TEEO also significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling positive gastric cancer cells, expression of mRNA of caspase-3, and Bax. Whereas, the expression of Bcl-2 was increased. Additionally, downregulation of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and IκBα degradation and the nuclear factor-κB (NF-κB) p65 expression in tissues of the stomach of MNNG-induced-rats were markedly elevated due to TEEO. This suggested possession of TEEO with a protective shield against MNNG induced gastric cancer by the exertion of antioxidative stress, anti-apoptotic response, the anti-inflammatory response through Nrf2/HO-1, and NF-κB signaling pathways.

Background: Galactose-deficient IgA1 plays a key role in the pathogenesis of IgA nephropathy, the most common primary GN worldwide. Although serum levels of galactose-deficient IgA1 have a strong genetic component, the genetic link between this molecule and IgA nephropathy has not yet been clearly established., Methods: To identify novel loci associated with galactose-deficient IgA1, we performed a quantitative genome-wide association study for serum galactose-deficient IgA1 levels, on the basis of two different genome-wide association study panels conducted in 1127 patients with IgA nephropathy. To test genetic associations with susceptibility to IgA nephropathy, we also enrolled 2352 patients with biopsy-diagnosed IgA nephropathy and 2632 healthy controls. Peripheral blood samples from 59 patients and 27 healthy controls were also collected for gene expression analysis., Results: We discovered two loci, in C1GALT1 and GALNT12, that achieved genome-wide significance, explaining about 3.7% and 3.4% of variance in serum galactose-deficient IgA1 levels, respectively. We confirmed the previously reported association of C1GALT1 with serum galactose-deficient IgA1 levels, but with a different lead single-nucleotide polymorphism (rs10238682; β=0.26, P =1.20×10 -9 ); the locus we identified at GALNT12 (rs7856182; β=0.73, P =2.38×10 -9 ) was novel. Of more interest, we found that GALNT12 exhibits genetic interactions with C1GALT1 in both galactose-deficient IgA1 levels ( P =1.40×10 -2 ) and disease risk ( P =6.55×10 -3 ). GALNT12 mRNA expression in patients with IgA nephropathy was significantly lower compared with healthy controls., Conclusions: Our data identify GALNT12 as a novel gene associated with galactose-deficient IgA1 and suggest novel genetic interactions. These findings support a key role of genetically conferred dysregulation of galactose-deficient IgA1 in the development of IgA nephropathy., (Copyright © 2021 by the American Society of Nephrology.)

Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein-protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrometry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera. Molecular interaction analyses in silico implied that dimeric IgA and albumin interacted not only via disulfide bond formation, but also via noncovalent bonds. Disulfide bonds were predicted between Cys34 of albumin and Cys311 of IgA, resulting in an oxidized form of albumin. Furthermore, complex formation prolongs the half-life of IgA molecules in the IgA-albumin complex, leading to excessive glycation of IgA molecules and affects the accumulation of IgA in serum. These findings may demonstrate why complications such as hyperviscosity syndrome occur more often in patients with IgA dimer producing multiple myeloma.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies., Competing Interests: The authors have declared that no competing interests exist.

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.

Summary: Introduction and objective. Gastrointestinal complications are considered as one of the most common manifestations in patients with Common Variable Immunodeficiency (CVID). These complications can result from Small Intestinal Bacterial Overgrowth (SIBO). Hydrogen breath test is extensively used to diagnose SIBO. The objective of this study was to evaluate the prevalence of SIBO using the Hydrogen Breath Test (HBT) in patients with CVID. Materials and methods. Twenty-seven patients with CVID entered this cross-sectional study. Demographic and lower gastrointestinal symptoms were recorded in a check list. Hemoglobin level was measured in all patients. The concentration of IgA and IgG was assessed using nephelometry. Moreover, SIBO was detected by means of Glucose hydrogen breath test. Results. The mean (plus-minus SD) age of the patients was 35.25 (plus-minus 11.69) years. Twenty patients (74.1%) manifested at least one lower gastrointestinal symptom. The most frequent lower gastrointestinal manifestations were bloating (66.7%) and chronic diarrhea (40.7%), respectively. IgA level less than 10 mg/dl and IgG level less than 600 mg/dl were determined in 77.8% and 25.9% of patients, respectively. Positive HBT was detected in 40.7% (n = 11) of the patients. In the positive HBT group, bloating, chronic diarrhea and abdominal pain were the most common lower GI manifestations. There was no significant difference in terms of age, BMI, IgA level, and duration of CVID between the positive and negative HBT groups. The significant association of co-occurrence of anemia and abdominal pain with positive HBT (positive predictive value: 100%) might be considered as a clue to SIBO diagnosis. Conclusions. Regarding the high prevalence and non-specific manifestation of SIBO, it is suggested to consider concurrent symptoms in patients with CVID to manage the timely and precise diagnosis of SIBO.

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine., Competing Interests: The authors have declared that no competing interests exist.

The specific value of IgA Anti-β2glycoprotein I antibodies (aB2GP1) in the diagnosis and management of antiphospholipid syndrome (APS) is still controversial and a matter of active debate. The relevance of the IgA aB2GP1 isotype in the pathophysiology of APS has been increasingly studied in the last years. There is well know that subjects with multiple positive APS tests are at increased risk of thrombosis and/or miscarriage. However, these antibodies are not included in the 2006 APS classification criteria. Since 2010 the task force of the Galveston International Congress on APS recommends testing IgA aB2GP1 isotype in patients with APS clinical criteria in the absence of criteria antibodies. In this review, we summarize the molecular and clinical "state of the art" of the IgA aB2GP in the context of APS. We also discuss some of the characteristics that may help to evaluate the real value of the IgA aB2GP1 determination in basic research and clinical practice. The scientific community should be aware of the importance of clarifying the role of IgA aB2GP1 in the APS diagnosis.

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions., Competing Interests: SK, BP, AM, NH, KH, ET, BS No competing interests declared, (© 2020, Kumar Bharathkar et al.)

Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.

Human IgA could be from different isotypes (IgA1/IgA2) and/or isoforms (monomeric, dimeric, or secretory). Monomeric IgA mainly IgA1 are considered as an anti-inflammatory isotype whereas dimeric/secretory IgA have clearly dual pro- and anti-inflammatory effects. Here, we show that IgA isotypes and isoforms display different binding abilities to FcαRI, Dectin-1, DC-SIGN, and CD71 on monocyte-derived dendritic cells (moDC). We describe that IgA regulate the expression of their own receptors and trigger modulation of moDC maturation. We also demonstrate that dimeric IgA2 and IgA1 induce different inflammatory responses leading to cytotoxic CD8 + T cells activation. moDC stimulation by dimeric IgA2 was followed by a strong pro-inflammatory effect. Our study highlights differences regarding IgA isotypes and isoforms in the context of DC conditioning. Further investigations are needed on the activation of adaptive immunity by IgA in the context of microbiota/IgA complexes during antibody-mediated immune selection., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires 1,2 . Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice 3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.

Aims: To determine if an ELISA for measurement of IgA in equine serum could be used to measure concentrations of IgA in foal faeces and to determine correlations with concentrations in the milk of the dam. Methods: Faeces from 20 Welsh Cob and Welsh Pony foals and milk from their dams were collected within 12 hours (Day 0) and at 6 days after parturition (Day 6). On Day 6, faeces could not be collected from 2/20 foals, and milk samples could not be collected from 3/20 mares. An equine IgA ELISA validated for serum and plasma was used to measure concentrations of IgA in all samples in triplicate. The precision of the assay for each sample type was determined using modified CV. Results: IgA was not detectable in 7/20 Day 0 faecal samples and in 2/18 Day 6 faecal samples. For samples with detectable IgA, the mean modified CV was 10.5 (95% CI = 6.0-15.0)% for Day 0 faecal samples, and was 6.8 (95% CI = 4.3-9.4)% for Day 6 faecal samples. Median concentrations of IgA in faeces on Day 0 were lower than concentrations on Day 6 (0.7 mg/g vs . 37 mg/g dry matter; p = 0.003). Concentrations of IgA in milk and faeces on Day 6 were statistically correlated (r = 0.59; p = 0.006). Conclusions and clinical relevance: The IgA ELISA showed acceptable precision when used to estimate concentrations of IgA in foal faeces during the first week of life, but IgA could not be detected in 37% of meconium samples collected on Day 0. This assay may be useful for investigation of the role of maternal milk IgA in the gastrointestinal tract of neonatal foals, but further assessment of both accuracy and precision of the ELISA is required.

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Immunoglobulin (Ig) A is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralization) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated; by contrast, serum IgA is comparatively understudied. IgA binding to fragment crystallizable alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests that serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy., (© 2019 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

A common renal disease, immunoglobulin A (IgA) nephropathy (IgAN), is associated with glomerular deposition of IgA1-containing immune complexes. IgA1 hinge region (HR) has up to six clustered O-glycans consisting of Ser/Thr-linked N-acetylgalactosamine with β1,3-linked galactose and variable sialylation. IgA1 glycoforms with some galactose-deficient (Gd) HR O-glycans play a key role in IgAN pathogenesis. The clustered and variable O-glycans make the IgA1 glycomic analysis challenging and better approaches are needed. Here, we report a comprehensive analytical workflow for IgA1 HR O-glycoform analysis. We combined an automated quantitative analysis of the HR O-glycopeptide profiles with sequential deglycosylation to remove all but Gd O-glycans from the HR. The workflow was tested using serum IgA1 from healthy subjects. Twelve variants of glycopeptides corresponding to the HR with three to six O-glycans were detected; nine glycopeptides carried up to three Gd O-glycans. Sites with Gd O-glycans were unambiguously identified by electron-transfer/higher-energy collision dissociation tandem mass spectrometry. Extracted ion chromatograms of isomeric glycoforms enabled quantitative assignment of Gd sites. The most frequent Gd site was T 236 , followed by S 230 , T 233 , T 228 , and S 232 . The new workflow for quantitative profiling of IgA1 HR O-glycoforms with site-specific resolution will enable identification of pathogenic IgA1 HR O-glycoforms in IgAN.

Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.

Aims: Application of capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Background: Altered N-glycosylation of body fluid glycoproteins can be an effective indicator of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant immunoglobulin and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces, such as in saliva., Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders., Methods: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand, six structures were found in the serum samples not present in the saliva., Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The aim of this study was to estimate changes in selected physicochemical properties of saliva collected from users of electronic cigarettes. Methods: The study population consisted of 120 patients (40 users of electronic cigarettes, 40 smokers of traditional cigarettes and 40 non-smokers). Laboratory tests included verification of saliva amount of lysozyme, lactoferrin and IgA. Results: Among e-cigarette users, statistically significant differences were observed in values of lysozyme and lactoferrin; however, no statistically significant differences for the IgA value were found. In the group of traditional cigarette smokers, statistically significant differences were observed among all tested parameters in relation to the control group. In relation to IgA, statistically significant differences were found between e-cigarette users and traditional cigarette smokers, to the disadvantage of the latter. Conclusion: Saliva of e-cigarette users showed changes of antibacterial properties in comparison to the control group and traditional cigarette smokers. Further longitudinal studies on larger study groups should be conducted in order to assess the effect of observed changes in the antibacterial properties of saliva on oral health.

Immunoassays are widely used for detection and quantification of analytes in biological samples, but are vulnerable to analytical errors caused by interfering sample substances. Of particular interest are endogenous anti-animal antibodies that may bind to the immunoassay antibodies and cause erroneous test results. This phenomenon is a hazard to patient safety in both human and veterinary medicine. Here, we demonstrate that anti-mouse antibodies in dogs bind selectively to different regions of the murine IgG molecule, cross-react with IgG from different species, and consist of all major antibody classes present in canine serum (IgA, IgG and IgM). The antibody characteristics varied among individuals and their prevalence differed between two dog breeds. The selective binding to different IgG regions suggests that the antibodies might not originate from immunization through exposure to mice or other species. These findings show that canine anti-mouse antibodies are highly heterogeneous in nature and therefore require a combination of strategies to be counteracted.

Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N -glycan modifications of IgA1 and IgA2 on binding to the Fcα receptor (FcαRI), which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1), and IgA2m(2) variants in Nicotiana benthamiana ΔXT/FT plants lacking the enzymes responsible for generating nonhuman N -glycan structures. By coinfiltrating IgA with the respective glycan-modifying enzymes, we generated IgA carrying distinct homogenous N -glycans. We demonstrate that distinctly different N -glycan profiles did not influence antigen binding or the overall structure and integrity of the IgA antibodies but did affect their thermal stability. Using size-exclusion chromatography, differential scanning and isothermal titration calorimetry, surface plasmon resonance spectroscopy, and molecular modeling, we probed distinct IgA1 and IgA2 glycoforms for binding to four different FcαRI glycoforms and investigated the thermodynamics and kinetics of complex formation. Our results suggest that different N -glycans on the receptor significantly contribute to binding affinities for its cognate ligand. We also noted that full-length IgA and FcαRI form a mixture of 1:1 and 1:2 complexes tending toward a 1:1 stoichiometry due to different IgA tailpiece conformations that make it less likely that both binding sites are simultaneously occupied. In conclusion, N -glycans of human IgA do not affect its structure and integrity but its thermal stability, and FcαRI N -glycans significantly modulate binding affinity to IgA., (© 2019 Göritzer et al.)

Background: Although Lactobacillus casei Shirota (LcS) can benefit the immune status, the effects of LcS in the immune/inflammatory responses of marathon runners has never been evaluated. Therefore, here we evaluated the effect of daily ingestion of fermented milk containing or not LcS in the systemic and upper airway immune/inflammatory responses before and after a marathon., Methods: Forty-two male marathon runners ingested a fermented milk containing 40 billion of LcS/day (LcS group, n = 20) or placebo (unfermented milk, n = 22) during 30 days pre-marathon. Immune/inflammatory parameters in nasal mucosa and serum, as well as concentrations of secretory IgA (SIgA) and antimicrobial peptides in saliva, were evaluated before and after fermented milk ingestion, immediately, 72 h, and 14 d post-marathon., Results: Higher proinflammatory cytokine levels in serum and nasal mucosa, and also lower salivary levels of SIgA and antimicrobial peptides, were found immediately post-marathon in the placebo group compared to other time points and to LcS group. In opposite, higher anti-inflammatory levels and reduced neutrophil infiltration on nasal mucosa were found in the LcS group compared to other time points and to the placebo group., Conclusion: For the first time, it is shown that LcS is able to modulate the systemic and airways immune responses post-marathon.

Antenatal milk anti-influenza antibodies may provide additional protection to newborns until they are able to produce their own antibodies. To evaluate the relative abundance of milk, we studied the antibodies specific to influenza A in feeds and gastric contents and stools from preterm infants fed mother's own breast milk (MBM) and donor breast milk (DBM). Feed (MBM or DBM) and gastric contents (MBM or DBM at 1 h post-ingestion) and stool samples (MBM/DBM at 24 h post-ingestion) were collected, respectively, from 20 preterm (26-36 weeks gestational age) mother-infant pairs at 8-9 days and 21-22 days of postnatal age. Samples were analyzed via ELISA for anti-H1N1 hemagglutinin (anti-H1N1 HA) and anti-H3N2 neuraminidase (anti-H3N2 NA) specificity across immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) isotypes. The relative abundance of influenza A-specific IgA in feeds and gastric contents were higher in MBM than DBM at 8-9 days of postnatal age but did not differ at 21-22 days. Anti-influenza A-specific IgM was higher in MBM than in DBM at both postnatal times in feed and gastric samples. At both postnatal times, anti-influenza A-specific IgG was higher in MBM than DBM but did not differ in gastric contents. Gastric digestion reduced anti-H3N2 NA IgG from MBM at 21-22 days and from DBM at 8-9 days of lactation, whereas other anti-influenza A antibodies were not digested at either postnatal times. Supplementation of anti-influenza A-specific antibodies in DBM may help reduce the risk of influenza virus infection. However, the effective antibody dose required to induce a significant protective effect remains unknown.

Background: Glomerular deposition of IgA1 is a common feature of Henoch-Schönlein purpura nephritis (HSPN) and is indistinguishable from that seen in IgA nephropathy (IgAN). Serum IgA1 is abnormally O-glycosylated in IgA nephropathy, which may contribute to the development of glomerular injury. Abnormal O-glycosylated IgA1 was also detected in HSPN using lectin enzyme-linked immunosorbent assay; however, this method cannot provide the exact structural information of O-glycans. Mass spectrometry is an effective means of quantification of O-glycans, and there is no report to evaluate IgA1 O-glycans in HSPN using mass spectrometry., Materials and Methods: We investigated O-glycosylation profile in serum IgA1 from 7 HSPN recipients, 26 IgAN recipients, 25 recipients with other kidney diseases (OKDs), and 26 normal healthy donors using mass spectrometry., Results: Of the 14 GalNac-Gal combinations detected using mass spectrometry, the percentage of the only 6GalNAc-2Gal combination was significantly different between HSPN and IgAN. The percentage of GalNAc 3 in HSPN recipients was significantly higher than that in OKDs recipients and healthy donors (P = .0027 and P < .0001, respectively). Inversely, the percentage of GalNAc 5 in HSPN recipients was significantly lower than that in OKDs recipients and healthy donors (P = .0008, P < .0001, respectively). Moreover, the Gal content and the Gal/GalNAc ratio of HSPN recipients were significantly lower than OKDs recipients and healthy donors., Conclusions: Examination of Henoch-Schönlein purpura recipients revealed that the number of GalNAc fell and the Gal attachment to GalNAc was reduced compared to other kidney diseases and healthy donors. The IgA1 O-glycosylation profile of HSPN was very similar to that of IgAN., (Copyright © 2019 Elsevier Inc. All rights reserved.)

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.

Immunoglobulin A (IgA) is the most abundant antibody class present at mucosal surfaces. The production of IgA exceeds the production of all other antibodies combined, supporting its prominent role in host-pathogen defense. IgA closely interacts with the intestinal microbiota to enhance its diversity, and IgA has a passive protective role via immune exclusion. Additionally, inhibitory ITAMi signaling via the IgA Fc receptor (FcαRI; CD89) by monomeric IgA may play a role in maintaining homeostatic conditions. By contrast, IgA immune complexes (e.g., opsonized pathogens) potently activate immune cells via cross-linking FcαRI, thereby inducing pro-inflammatory responses resulting in elimination of pathogens. The importance of IgA in removal of pathogens is emphasized by the fact that several pathogens developed mechanisms to break down IgA or evade FcαRI-mediated activation of immune cells. Augmented or aberrant presence of IgA immune complexes can result in excessive neutrophil activation, potentially leading to severe tissue damage in multiple inflammatory, or autoimmune diseases. Influencing IgA or FcαRI-mediated functions therefore provides several therapeutic possibilities. On the one hand (passive) IgA vaccination strategies can be developed for protection against infections. Furthermore, IgA monoclonal antibodies that are directed against tumor antigens may be effective as cancer treatment. On the other hand, induction of ITAMi signaling via FcαRI may reduce allergy or inflammation, whereas blocking FcαRI with monoclonal antibodies, or peptides may resolve IgA-induced tissue damage. In this review both (patho)physiological roles as well as therapeutic possibilities of the IgA-FcαRI axis are addressed.

Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.

IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis, and disease recurrence often occurs after transplantation. On the other hands, Asymptomatic IgA deposition (IgAD) is occasionally observed in donated kidney. It is recognized that IgAD does not progress to IgAN, but the mechanism has not demonstrated yet. In IgAN, aberrant IgA1 O-glycan structure in the hinge region (HR) of serum IgA is suggested as one of the most convincing key mediators. However, little is known about IgA1 O-glycan structure in IgAD patients. Herein, we investigated the prevalence of IgAD in living renal transplant donors in our cohort. IgAD was observed in 21(13.0%) among 161 renal transplant donors and have statistically significant blood relationship with IgAN recipients (28.6% in relatives vs. 9.8% in non-relatives, respectively; p = 0.0073). Next, we evaluated the IgA1 O-glycan structure of serum IgA from IgAN recipients (n = 26), IgAD donors (n = 17), and non-IgAD helthy donors (n = 27) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The numbers of GalNAc and Gal and the Gal/GalNAc ratio in the HR of the IgAN recipients had significantly lower comparing to the IgAD and non-IgAD healthy donors. The decreased Gal/GalNAc ratio in IgAN recipients means the increased ratio of galactose-deficient IgA1. To the best of our knowledge, this is the first report to compare the O-glycan structures in IgAN recipients and IgAD donors using MALDI-TOF MS. We concluded that IgAD was more common in IgAN related donors. Overall, decreased GalNAc and Gal contents in HR could play a material pathogenic role in IgAN., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Galactose-deficient IgA1 (Gd-IgA1) that exposes GalNAc or sialylated GalNAc has been shown to be associated with disease activity of IgA nephropathy (IgAN). In a previous report, we established an enzyme-linked immunosorbent assay that measures human Gd-IgA1 using a specific monoclonal antibody KM55 (KM55 mAb), and showed that patients with IgAN contain a higher level of serum Gd-IgA1 than other types of renal diseases. Recently, we also found that the KM55 mAb specifically recognized the glomerular-deposited Gd-IgA1 in renal biopsy. In this study, we aimed to analyze the epitope of KM55 mAb using synthesized peptides corresponding to the hinge region of IgA1 with GalNAc moiety on putative glycosylated Ser/Thr residues, which are Thr225, Thr228, Ser230, Ser232, and Thr236. Binding analysis to single GalNAc-modified hinge region peptide of IgA1 showed that Thr225 with GalNAc is required for recognition of KM55. PST(GalNAC)PP motif was required for KM55 mAb to recognize hinge region peptide of IgA1 which is shown by binding assay with deletion peptide. This result was confirmed by binding of KM55 mAb against peptide with GalNAc at Thr233, which resulted in containing another PST(GalNAC)PP motif. Taken together, we concluded that the epitope of Gd-IgA1-specific KM55 mAb is PST(GalNAc)PP motif.

The N -glycosylation of immunoglobulin (Ig) G, the major antibody in the circulation of human adults, is well known for its influence on antibody effector functions and its alterations with various diseases. In contrast, knowledge on the role of glycans attached to IgA, which is a key immune defense agent in secretions, is very scarce. In this study we aimed to characterize the glycosylation of salivary (secretory) IgA, including the IgA joining chain (JC), and secretory component (SC) and to compare IgA and IgG glycosylation between human plasma and saliva samples to gain a first insight into oral cavity-specific antibody glycosylation. Plasma and whole saliva were collected from 19 healthy volunteers within a 2-h time window. IgG and IgA were affinity-purified from the two biofluids, followed by tryptic digestion and nanoLC-ESI-QTOF-MS(/MS) analysis. Saliva-derived IgG exhibited a slightly lower galactosylation and sialylation as compared to plasma-derived IgG. Glycosylation of IgA1, IgA2, and the JC showed substantial differences between the biofluids, with salivary proteins exhibiting a higher bisection, and lower galactosylation and sialylation as compared to plasma-derived IgA and JC. Additionally, all seven N -glycosylation sites, characterized on the SC of secretory IgA in saliva, carried highly fucosylated and fully galactosylated diantennary N -glycans. This study lays the basis for future research into the functional role of salivary Ig glycosylation as well as its biomarker potential.

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1-Fc domain (Fcα) at the C H 2-C H 3 junction and, except for C H 2 L257 and L258, all side-chain contacts are contributed by the C H 3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the FcαRI:Fcα interaction. Surprisingly, individual mutation of C H 2 residues L257 and L258, found at the periphery of the FcαRI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the C H 2-C H 3 junction (or its equivalent). Given the importance of residues near the C H 2-C H 3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fcα. Our simulations indicate that FcαRI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fcα, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that FcαRI binding to the Fcα C H 2-C H 3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy., Competing Interests: The authors declare no conflict of interest.

Aims: IgA nephropathy, the most common primary glomerulonephritis worldwide, is characterized by glomerular deposition of galactose-deficient IgA1 and elevated serum levels of this IgA1 glycoform. Current ELISA methods lack sensitivity to assess galactose deficiency using small amounts of IgA1, which limits studies in primary cells due to modest IgA1 production in isolated peripheral-blood lymphocytes., Methods: Lectin from Helix pomatia was conjugated to biotin or acridinium ester and used in ELISA to detect galactose deficiency of IgA1 using small amounts of IgA1., Results: Lectin conjugated to acridinium had an approximately a log-fold increased sensitivity compared with biotin-labeled lectin., Conclusions: The new method of using lectin from Helix pomatia conjugated to acridinium increased assay sensitivity, allowing future mechanistic studies with cultured primary cells.

The objective of this study was to determine the effects of dietary supplementation of Nigella sativa L. seeds, Rosmarinus officinalis L. leaves and their combination on rumen metabolism, nutrient intake and digestibility, growth performance, immune response and blood metabolites in Dorper lambs. Twenty-four entire male Dorper lambs (18.68 ± 0.6 kg, 4-5 months old) were randomly assigned to a concentrate mixture containing on a dry matter basis either, no supplement (control, T1), 1% R. officinalis leaves (T2), 1% N. sativa seeds (T3) or 1% R. officinalis leaves +1% N. sativa seeds (T4). The lambs had ad libitum access to urea-treated rice straw (UTRS) and were raised for 90 days. Supplemented lambs had greater (P < 0.05) intake of DM and UTRS than the control lambs. The T4 lambs had lower (P < 0.05) nutrient digestibility than those fed other treatments. Total and daily weight gain was greater (P < 0.05) in T2 lambs than those fed other diets. The T3 and T4 lambs had greater (P < 0.05) ruminal pH than the T1 and T2 lambs. Supplemented lambs had lower (P < 0.05) ruminal total volatile fatty acids, acetate, propionate, NH 3 -N and C18:0 than the control lambs. The T4 lambs had lower (P < 0.05) population of Fibrobacter succinogenes, Ruminococcus albus, methanogens and total protozoa compared with those fed other diets. Supplemented lambs had lower (P < 0.05) neutrophils, basophils and serum urea and greater (P < 0.05) serum IgA and IgG compared with the control lambs. The current results emphasised the variation in the efficacy of medicinal plants in ruminant nutrition.