Each N-glycan on human IgA and J-chain uniquely affects oligomericity and stability.

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 13C-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.

Durability of humoral and cell-mediated immune response after SARS-CoV-2 mRNA vaccine administration.

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.

IgA rheumatoid factor in rheumatoid arthritis.

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.

Ultrasensitive detection of salivary SARS-CoV-2 IgG antibodies in individuals with natural and COVID-19 vaccine-induced immunity.

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 (AIpost/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 IgGpost/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.

Development of a quantitative COVID-19 multiplex assay and its use for serological surveillance in a low SARS-CoV-2 incidence community.

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.

Summated training and match load predictors of salivary immunoglobulin-A, alpha-amylase, testosterone, cortisol and T:C profile changes in elite-level professional football players: A longitudinal analysis.

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.

3D Structures of IgA, IgM, and Components.

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.

Increased in vitro neutralizing activity of SARS-CoV-2 IgA1 dimers compared to monomers and IgG.

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.

Is complement the main accomplice in IgA nephropathy? From initial observations to potential complement-targeted therapies.

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.

Pregnancy-associated decrease of Siaα2-3Gal-linked glycans on salivary glycoproteins affects their binding ability to avian influenza virus.

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.

Sample stability of autoantibodies: A tool for laboratory quality initiatives.

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.

O- and N-Glycosylation of Serum Immunoglobulin A is Associated with IgA Nephropathy and Glomerular Function.

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.

Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation.

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.

Interaction between GALNT12 and C1GALT1 Associates with Galactose-Deficient IgA1 and IgA Nephropathy.

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; ß=0.26, P=1.20×10-9); the locus we identified at GALNT12 (rs7856182; ß=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.

Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma.

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.

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

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.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

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.

Modulatory effect of Tagetes erecta flowers essential oils via Nrf2/HO-1/NF-κB/p65 axis mediated suppression of N-methyl-N'nitro-N-nitroguanidine (MNNG) induced gastric cancer in rats.

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.

Systemic and intestinal porcine epidemic diarrhea virus-specific antibody response and distribution of antibody-secreting cells in experimentally infected conventional pigs.

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.

Can concurrent lower gastrointestinal manifestations help the timely diagnosis of small intestinal bacterial overgrowth in CVID patients?.

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.