Plasma IgG Research Articles

Autoimmune hepatitis displays distinctively high multi-antennary sialylation on plasma N-glycans compared to other liver diseases

BackgroundChanges in plasma protein glycosylation are known to functionally affect proteins and to associate with liver diseases, including cirrhosis and hepatocellular carcinoma. Autoimmune hepatitis (AIH) is a liver disease characterized by liver inflammation and raised serum levels of IgG, and is difficult to distinguish from other liver diseases. The aim of this study was to examine plasma and IgG-specific N-glycosylation in AIH and compare it with healthy controls and other liver diseases.MethodsIn this cross-sectional cohort study, total plasma N-glycosylation and IgG Fc glycosylation analysis was performed by mass spectrometry for 66 AIH patients, 60 age- and sex-matched healthy controls, 31 primary biliary cholangitis patients, 10 primary sclerosing cholangitis patients, 30 non-alcoholic fatty liver disease patients and 74 patients with viral or alcoholic hepatitis. A total of 121 glycans were quantified per individual. Associations between glycosylation traits and AIH were investigated as compared to healthy controls and other liver diseases.ResultsGlycan traits bisection (OR: 3.78 [1.88–9.35], p-value: 5.88 × 10− 3), tetraantennary sialylation per galactose (A4GS) (OR: 2.88 [1.75–5.16], p-value: 1.63 × 10− 3), IgG1 galactosylation (OR: 0.35 [0.2–0.58], p-value: 3.47 × 10− 5) and hybrid type glycans (OR: 2.73 [1.67–4.89], p-value: 2.31 × 10− 3) were found as discriminators between AIH and healthy controls. High A4GS differentiated AIH from other liver diseases, while bisection associated with cirrhosis severity.ConclusionsCompared to other liver diseases, AIH shows distinctively high A4GS levels in plasma, with potential implications on glycoprotein function and clearance. Plasma-derived glycosylation has potential to be used as a diagnostic marker for AIH in the future. This may alleviate the need for a liver biopsy at diagnosis. Glycosidic changes should be investigated further in longitudinal studies and may be used for diagnostic and monitoring purposes in the future.

Non-cross-reactive epitopes dominate the humoral immune response to COVID-19 vaccination - kinetics of plasma antibodies, plasmablasts and memory B cells.

COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs). Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay. The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination. Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

Effect of in ovo feeding of xylobiose and xylotriose on plasma immunoglobulin, cecal metabolites production, microbial ecology, and metabolic pathways in broiler chickens

BackgroundDietary supplementation of xylooligosaccharides (XOS) has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites. But no study investigated and compared the effect of in ovo feeding of xylobiose (XOS2) and xylotriose (XOS3) in chickens. This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens. A total of 144 fertilized chicken eggs were divided into three groups: a) non-injected control (CON), b) XOS2, and c) XOS3. On the 17th embryonic day, the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85% normal saline through the amniotic sac. After hatching, the chicks were raised for 21 d. Blood was collected on d 14 to measure plasma immunoglobulin. Cecal digesta were collected for measuring short-chain fatty acids (SCFA) on d 14 and 21, and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21.ResultsThe results were considered significantly different at P < 0.05. ELISA quantified plasma IgA and IgG on d 14 chickens, revealing no differences among the treatments. Gas chromatography results showed no significant differences in the concentrations of cecal SCFAs on d 14 but significant differences on d 21. However, the SCFA concentrations were lower in the XOS3 than in the CON group on d 21. The cecal metagenomics data showed that the abundance of the family Clostridiaceae significantly decreased on d 7, and the abundance of the family Oscillospiraceae increased on d 21 in the XOS2 compared to the CON. There was a reduction in the relative abundance of genus Clostridium sensu stricto 1 in the XOS2 compared to the CON on d 7 and the genus Ruminococcus torques in both XOS2 and XOS3 groups compared to the CON on d 21. The XOS2 and XOS3 groups reduced the genes for chondroitin sulfate degradation I and L-histidine degradation I pathways, which contribute to improved gut health, respectively, in the microbiome on d 7. In contrast, on d 21, the XOS2 and XOS3 groups enriched the thiamin salvage II, L-isoleucine biosynthesis IV, and O-antigen building blocks biosynthesis (E. coli) pathways, which are indicative of improved gut health. Unlike the XOS3 and CON, the microbiome enriched the pathways associated with energy enhancement, including flavin biosynthesis I, sucrose degradation III, and Calvin-Benson-Bassham cycle pathways, in the XOS2 group on d 21.ConclusionIn ovo XOS2 and XOS3 feeding promoted beneficial bacterial growth and reduced harmful bacteria at the family and genus levels. The metagenomic-based microbial metabolic pathway profiling predicted a favorable change in the availability of cecal metabolites in the XOS2 and XOS3 groups. The modulation of microbiota and metabolic pathways suggests that in ovo XOS2 and XOS3 feeding improved gut health during the post-hatch period of broilers.

High levels of anti–factor VIII immunoglobulin G4 and immunoglobulin G total are associated with immune tolerance induction failure in people with congenital hemophilia A and high-responding inhibitors

BackgroundImmune tolerance induction (ITI) is the treatment of choice to eradicate neutralizing anti–factor (F)VIII alloantibodies (inhibitors) in people with inherited hemophilia A. However, it is not successful in 10% to 40% of the cases. The biological mechanisms and biomarkers associated with ITI outcome are largely unknown. ObjectivesThe aim of this study was to investigate the association of plasma cytokines (interferon-γ, tumor necrosis factor, interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, and IL-17A), chemokines (IL-8/CXCL8, RANTES/CCL5, MIG/CXCL9, MCP-1/CCL2, and IP-10/CXCL10), and anti-FVIII immunoglobulin (Ig) G total, IgG1, and IgG4 with ITI outcome. MethodsIn this cross-sectional analysis of the Brazilian Immune Tolerance Study, we assessed plasma levels of anti-FVIII IgGs using an enzyme-linked immunosorbent assay with plasma-derived FVIII and recombinant FVIII as target antigens, immobilized in microplates. ResultsWe assayed 98 plasma samples of moderately severe and severe (FVIII activity, <2%) people with hemophilia A after completion of a first ITI course. Levels of anti–recombinant FVIII IgG total and IgG4 were higher in people with hemophilia A who failed ITI (IgG total optical density [OD], 0.37; IQR, 0.15-0.73; IgG4 OD, 2.19; IQR, 0.80-2.52) than in those who had partial (IgG total OD, 0.03; IQR, 0.00-0.14; IgG4 OD, 0.39; IQR, 0.09-1.11; P < .0001 for both) or complete success (IgG total OD, 0.04; IQR, 0.00-0.07; IgG4 OD, 0.07; IQR, 0.06-0.40; P < .0001 for both). Plasma cytokines, chemokines, and anti-FVIII IgG1 were not associated with ITI outcome. ConclusionOur results show that high levels of plasma anti-FVIII IgG4 and IgG total are associated with ITI failure.

Next-generation B cell ImmunoSpot™ assays permit in-depth assessment of the B cell response elicited by SARS-CoV-2 infection and/or COVID-19 mRNA vaccination

Abstract The affinity distribution of antigen-specific Bmem in the body is a critical variable that defines an individual’s ability to rapidly generate protective antibody specificities. Moreover, defining the Ig class/subclass usage of such antigen-specific Bmem is also critical since it forecasts the anamnestic response to be engaged upon antigen re-encounter. Here, we leveraged multiplexed ImmunoSpot to comprehensively define the magnitude and Ig class/subclass usage of the SARS-CoV-2 S-specific Bmem following infection and/or multiple COVID-19 mRNA vaccinations. Beyond evidencing past SARS-CoV-2 infection more reliably than assessment of plasma IgG reactivity against the nucleocapsid protein, direct assessment of Bmem provided insights into their affinity distribution and cross-reactivity profiles against emerging variants such as Delta and Omicron. Following stimulation of PBMC to transition resting Bmem into antibody-secreting cells, our studies entailed tracking of the S-specific Bmem compartment in subjects as their immune histories became increasingly more complex. Of note, our next-generation ImmunoSpot assay approaches evidenced improved functional affinity at single-cell resolution. Collectively, such B cell ImmunoSpot assays offer tremendous value for future B cell immune monitoring efforts owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization and suitability for regulated testing.

Nasal vaccination of triple-RBD scaffold protein with flagellin elicits long-term protection against SARS-CoV-2 variants including JN.1

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic. Here, we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain (RBD) scaffold protein (3R-NC) adjuvanted with a flagellin protein (KFD) (3R-NC + KFDi.n). In mice, the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year. This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG- and IgA-producing plasma cells, alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant. Based upon these preclinical findings, an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2 (IAV) vaccine. With a favorable safety profile, the 3R-NC + KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions. To meet the challenge of frequently emerged variants, we further designed an updated triple-RBD scaffold protein with mutated RBD combinations, which can induce adaptable antibody responses to neutralize the newly emerging variants, including JN.1. Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection.

P140 IgG Autoantibodies with Increased Affinity for Epithelial Cells in Severe Systemic Sclerosis

Abstract Background/Aims In health IgG immunoglobulins form a network through interaction with cell surface proteins and controlled by regulatory cells, whereas in autoimmune diseases IgG responses become skewed towards higher affinity and potentially damaging responses against self-antigens. Moreover, recent studies have demonstrated activation of the epithelial layer of skin in systemic sclerosis (SSc), implicating keratinocytes as a target of the autoimmune process. We investigated the presence of plasma IgG autoantibodies directed against keratinocytes and sought correlation with clinical parameters in well-characterized patients. Methods Diffuse SSc (dcSSc), limited SSc (lcSSc) and healthy control (HC) plasma samples (each n = 30), were assayed in an anti-epithelial cell antibody assay (AEpCA), using fixed keratinocytes to evaluate binding of IgG . Clinical characteristics of patients with positive versus negative AEpCA were compared. The capacity for SSc IgG to activate epithelial cells was assessed using keratinocytes in vitro. Results Preliminary serial dilution assay of SSc and HC plasma (n = 10) showed enhanced affinity of SSc IgG for keratinocytes (ANOVA p &amp;lt; 0.001 for overall effect SSc vs HC IgG p &amp;lt; 0.0048 for dose effect)(Figure 1A). Dilution of 1/80 gave optimal differential binding in the AEpCA assay (corrected p &amp;lt; 0.027 for SSc vs HC at 1/80). Further HC, dcSSc, and lcSSc plasma samples (each n = 30) were screened, demonstrating increased AEpCA titre in SSc (Figure 1B) (Kruskall-Wallace test p &amp;lt; 0.014 for lcSSc vs HC, p &amp;lt; 0.041 for dcSSc vs HC). SSc patients with positive AEpCA had higher skin scores (mRSS) (AEPCA neg 5(0-24); AEpCA pos 9(0-40) p &amp;lt; 0.0032), higher frequency of ATA antibody (AEpCA neg ATA pos 6/30; AEpCA pos ATA pos 13/30, chi squared p = 0.052) more interstitial lung disease (AEPCA neg 7/30, AEpCA pos 13/30, p NS)(Figure 1C) and trend towards higher use of immunosuppression. SSc but not HC IgG activated keratinocytes in vitro, triggering IL-1α release. Conclusion In this study, we have shown evidence of IgG autoantibodies with increased affinity for keratinocytes in the plasma of SSc patients. Positive testing for the AEpCA was associated with more severe skin disease and lung involvement. The perturbed IgG network in such patients could be activating through common epithelial cells antigens in the skin and lung. Disclosure C. Moezinia: None. V. Wong: None. J. Watson: None. L. Nagib: None. S. Lopez Garces: None. H. Lopez: None. G. Martin: None. S. Zhang: None. B. Ahmed Abdi: None. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Stroke and myocardial infarction induce neutrophil extracellular trap release disrupting lymphoid organ structure and immunoglobulin secretion

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer’s patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3–10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA+ cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients.

Genetics of immune response to Epstein-Barr virus: prospects for multiple sclerosis pathogenesis.

Epstein-Barr virus (EBV) infection has been advocated as a prerequisite for developing multiple sclerosis (MS) and possibly the propagation of the disease. However, the precise mechanisms for such influences are still unclear. A large-scale study investigating the host genetics of EBV serology and related clinical manifestations, such as infectious mononucleosis (IM), may help us better understand the role of EBV in MS pathogenesis. This study evaluates the host genetic factors that influence serological response against EBV and history of IM and cross-evaluates them with MS risk and genetic susceptibility in the Swedish population. Plasma IgG antibody levels against EBV nuclear antigen-1 [EBNA-1, truncated = amino acids (aa) (325-641), peptide = aa(385-420)] and viral capsid antigen p18 (VCAp18) were measured using bead-based multiplex serology for 8744 MS cases and 7229 population-matched control subjects. The MS risk association for high/low EBV antibody levels and history of IM was compared to relevant clinical measures along with sex, age at sampling, and associated HLA allele variants. Genome-wide and HLA allele association analyses were also performed to identify genetic risk factors for EBV antibody response and IM history. Higher antibody levels against VCAp18 [odds ratio (OR) = 1.74, 95% confidence interval (CI) = 1.60-1.88] and EBNA-1, particularly the peptide (OR = 3.13, 95% CI = 2.93-3.35), were associated with an increased risk for MS. The risk increased with higher anti-EBNA-1 IgG levels up to 12× the reference risk. We also identified several independent HLA haplotypes associated with EBV serology overlapping with known MS risk alleles (e.g. DRB1*15:01). Although there were several candidates, no variants outside the HLA region reached genome-wide significance. Cumulative HLA risk for anti-EBNA-1 IgG levels, particularly the peptide fragment, was strongly associated with MS. In contrast, the genetic risk for high anti-VCAp18 IgG levels was not as strongly associated with MS risk. IM history was not associated with class II HLA genes but negatively associated with A*02:01, which is protective against MS. Our findings emphasize that the risk association between anti-EBNA-1 IgG levels and MS may be partly due to overlapping HLA associations. Additionally, the increasing MS risk with increasing anti-EBNA-1 levels would be consistent with a pathogenic role of the EBNA-1 immune response, perhaps through molecular mimicry. Given that high anti-EBNA-1 antibodies may reflect a poorly controlled T-cell defence against the virus, our findings would be consistent with DRB1*15:01 being a poor class II antigen in the immune defence against EBV. Last, the difference in genetic control of IM supports the independent roles of EBNA-1 and IM in MS susceptibility.

Proteomics validate circulating GDF-15 as an independent biomarker for COVID-19 severity.

Growth differentiation factor 15 (GDF-15) was originally described as a stress-induced cytokine, and a biomarker of aging and cardiovascular diseases. We hypothesized that circulating GDF-15 would be associated with COVID-19 disease severity. Herein, we explored this hypothesis in a large cohort of COVID-19 patients. Blood samples were collected from 926 COVID-19 adult patients and from 285 hospitalized controls from the Biobanque Québécoise de la COVID-19 (BQC19). COVID-19 severity was graded according to the WHO criteria. SOMAscan proteomics assay was performed on 50µL of plasma. ELISA were performed on 46 selected participants with left-over plasma to validate differences in plasma GDF-15 levels. Statistical analyses were conducted using GraphPad Prism 9.0 and SPSS. P values < 0.01 were considered significant. Proteomics showed that plasma GDF-15 levels were higher in COVID-19 patients compared to hospitalized controls. GDF-15 levels increased with COVID-19 severity. COVID-19 patients presenting with comorbidities including diabetes, cancer, chronic obstructive pulmonary disease (COPD) and cardiovascular disease had higher GDF-15 levels. ELISA revealed significant elevation of GDF-15 until 30 days after hospitalization. Plasma GDF-15 elevation was correlated with older age. Moreover, GDF-15 levels correlated with pro-inflammatory cytokine interleukin-6 (IL-6) and inflammation marker C-reactive protein (CRP) as well as soluble levels of its putative receptor CD48. No association was established between anti-SARS-CoV-2 IgG levels and plasma GDF-15 levels. This study confirms GDF-15 as a biomarker for COVID-19 severity. Clinical evaluation of GDF-15 levels could assist identification of persons at high-risk of progressing to severe disease, thus improving patient care.

Antigen-specific Fab profiling achieves molecular-resolution analysis of human autoantibody repertoires in rheumatoid arthritis

The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.

Chitosan-LeoA-DNA Nanoparticles Promoted the Efficacy of Novel LeoA-DNA Vaccination on Mice Against Helicobacter pylori.

More than half of the world's population is infected with Helicobacter pylori (H. pylori), which may lead to chronic gastritis, peptic ulcers, and stomach cancer. LeoA, a conserved antigen of H. pylori, aids in preventing this infection by triggering specific CD3+ T-cell responses. In this study, recombinant plasmids containing the LeoA gene of H. pylori are created and conjugated with chitosan nanoparticle (CSNP) to immunize BALB/c mice against the H. pylori infection. We used the online Vaxign tool to analyze the genomes of five distinct strains of H. pylori, and we chose the outer membrane as a prospective vaccine candidate. Afterward, the proteins' immunogenicity was evaluated. The DNA vaccine was constructed and then encapsulated in CSNPs. The effectiveness of the vaccine's immunoprotective effects was evaluated in BALB/c mice. Purified activated splenic CD3+ T cells are used to test the anticancer effects in vitro. Nanovaccines had apparent spherical forms, were small (mean size, 150-250 nm), and positively charged (41.3 ± 3.11mV). A consistently delayed release pattern and an entrapment efficiency (73.35 ± 3.48%) could be established. Compared to the non-encapsulated DNA vaccine, vaccinated BALB/c mice produced higher amounts of LeoA-specific IgG in plasma and TNF-α in splenocyte lysate. Moreover, BALB/c mice inoculated with nanovaccine demonstrated considerable immunity (87.5%) against the H. pylori challenge and reduced stomach injury and bacterial burdens in the stomach. The immunological state in individuals with GC with chronic infection with H. pylori is mimicked by the H. pylori DNA nanovaccines by inducing a shift from Th1 to Th2 in the response. In vitro human GC cell development is inhibited by activated CD3+ T lymphocytes. According to our findings, the H. pylori vaccine-activated CD3+ has potential immunotherapeutic benefits.

The association between plasma IgG N-glycosylation and neonatal hypoxic-ischemic encephalopathy: a case-control study.

Hypoxic-ischemic encephalopathy (HIE) is one of severe neonatal brain injuries, resulting from inflammation and the immune response after perinatal hypoxia and ischemia. IgG N-glycosylation plays a crucial role in various inflammatory diseases through mediating the balance between anti-inflammatory and pro-inflammatory responses. This study aimed to explore the effect of IgG N-glycosylation on the development of HIE. This case-control study included 53 HIE patients and 57 control neonates. An ultrahigh-performance liquid chromatography (UPLC) method was used to determine the features of the plasma IgG N-glycans, by which 24 initial glycan peaks (GPs) were quantified. Multivariate logistic regression was used to examine the association between initial glycans and HIE, by which the significant parameters were used to develop a diagnostic model. Though receiver operating characteristic (ROC) curves, area under the curve (AUC) and 95% confidence interval (CI) were calculated to assess the performance of the diagnostic model. There were significant differences in 11 initial glycans between the patient and control groups. The levels of fucosylated and galactosylated glycans were significantly lower in HIE patients than in control individuals, while sialylated glycans were higher in HIE patients (p < 0.05). A prediction model was developed using three initial IgG N-glycans and fetal distress, low birth weight, and globulin. The ROC analysis showed that this model was able to discriminate between HIE patients and healthy individuals [AUC = 0.798, 95% CI: (0.716-0.880)]. IgG N-glycosylation may play a role in the pathogenesis of HIE. Plasma IgG N-glycans are potential noninvasive biomarkers for screening individuals at high risk of HIE.

Autoantibodies against acetylcholine receptors are increased in archived serum samples from patients with schizophrenia

Previous studies have demonstrated that the levels of IgG against neurotransmitter receptors are increased in patients with schizophrenia. Genome-wide association (GWA) studies of schizophrenia confirmed that 108 loci harbouring over 300 genes were associated with schizophrenia. Although the functional implications of genetic variants are unclear, theoretical functional alterations of these genes could be replicated by the presence of autoantibodies. This study examined the levels of plasma IgG antibodies against four neurotransmitter receptors, CHRM4, GRM3, CHRNA4 and CHRNA5, using an in-house ELISA in 247 patients with schizophrenia and 344 non-psychiatric controls. Four peptides were designed based on in silico analysis with computational prediction of HLA-DRB1 restricted and B-cell epitopes. The relationship between plasma IgG levels and psychiatric symptoms, as defined by the Operational Criteria Checklist for Psychotic Illness and Affective Illness (OPCRIT), were examined. The results showed that the levels of plasma IgG against peptides derived from CHRM4 and CHRNA4 were significantly increased in patients with schizophrenia compared with control subjects, but there was no significant association of plasma IgG levels with any symptom domain or any specific symptoms. These preliminary results suggest that CHRM4 and CHRNA4 may be novel targets for autoantibody responses in schizophrenia, although the pathogenic relationship between increased serum autoantibody levels and schizophrenia symptoms remains unclear.

Machine learning framework to extract the biomarker potential of plasma IgG N-glycans towards disease risk stratification

Effective management of chronic diseases and cancer can greatly benefit from disease-specific biomarkers that enable informative screening and timely diagnosis. IgG N-glycans found in human plasma have the potential to be minimally invasive disease-specific biomarkers for all stages of disease development due to their plasticity in response to various genetic and environmental stimuli. Data analysis and machine learning (ML) approaches can assist in harnessing the potential of IgG glycomics towards biomarker discovery and the development of reliable predictive tools for disease screening. This study proposes an ML-based N-glycomic analysis framework that can be employed to build, optimise, and evaluate multiple ML pipelines to stratify patients based on disease risk in an interpretable manner. To design and test this framework, a published colorectal cancer (CRC) dataset from the Study of Colorectal Cancer in Scotland (SOCCS) cohort (1999–2006) was used. In particular, among the different pipelines tested, an XGBoost-based ML pipeline, which was tuned using multi-objective optimisation, calibrated using an inductive Venn-Abers predictor (IVAP), and evaluated via a nested cross-validation (NCV) scheme, achieved a mean area under the Receiver Operating Characteristic Curve (AUC-ROC) of 0.771 when classifying between age-, and sex-matched healthy controls and CRC patients. This performance suggests the potential of using the relative abundance of IgG N-glycans to define populations at elevated CRC risk who merit investigation or surveillance. Finally, the IgG N-glycans that highly impact CRC classification decisions were identified using a global model-agnostic interpretability technique, namely Accumulated Local Effects (ALE). We envision that open-source computational frameworks, such as the one presented herein, will be useful in supporting the translation of glycan-based biomarkers into clinical applications.

Viral DNAemia and DNA Virus Seropositivity and Mortality in Pediatric Sepsis

Sepsis is a leading cause of pediatric mortality. Little attention has been paid to the association between viral DNA and mortality in children and adolescents with sepsis. To assess the association of the presence of viral DNA with sepsis-related mortality in a large multicenter study. This cohort study compares pediatric patients with and without plasma cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), human herpesvirus 6 (HHV-6), parvovirus B19 (B19V), BK polyomavirus (BKPyV), human adenovirus (HAdV), and torque teno virus (TTV) DNAemia detected by quantitative real-time polymerase chain reaction or plasma IgG antibodies to CMV, EBV, HSV-1, or HHV-6. A total of 401 patients younger than 18 years with severe sepsis were enrolled from 9 pediatric intensive care units (PICUs) in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Collaborative Pediatric Critical Care Research Network. Data were collected from 2015 to 2018. Samples were assayed from 2019 to 2022. Data were analyzed from 2022 to 2023. Death while in the PICU. Among the 401 patients included in the analysis, the median age was 6 (IQR, 1-12) years, and 222 (55.4%) were male. One hundred fifty-four patients (38.4%) were previously healthy, 108 (26.9%) were immunocompromised, and 225 (56.1%) had documented infection(s) at enrollment. Forty-four patients (11.0%) died in the PICU. Viral DNAemia with at least 1 virus (excluding TTV) was detected in 191 patients (47.6%) overall, 63 of 108 patients (58.3%) who were immunocompromised, and 128 of 293 (43.7%) who were not immunocompromised at sepsis onset. After adjustment for age, Pediatric Risk of Mortality score, previously healthy status, and immunocompromised status at sepsis onset, CMV (adjusted odds ratio [AOR], 3.01 [95% CI, 1.36-6.45]; P = .007), HAdV (AOR, 3.50 [95% CI, 1.46-8.09]; P = .006), BKPyV (AOR. 3.02 [95% CI, 1.17-7.34]; P = .02), and HHV-6 (AOR, 2.62 [95% CI, 1.31-5.20]; P = .007) DNAemia were each associated with increased mortality. Two or more viruses were detected in 78 patients (19.5%), with mortality among 12 of 32 (37.5%) who were immunocompromised and 9 of 46 (19.6%) who were not immunocompromised at sepsis onset. Herpesvirus seropositivity was common (HSV-1, 82 of 246 [33.3%]; CMV, 107 of 254 [42.1%]; EBV, 152 of 251 [60.6%]; HHV-6, 253 if 257 [98.4%]). After additional adjustment for receipt of blood products in the PICU, EBV seropositivity was associated with increased mortality (AOR, 6.10 [95% CI, 1.00-118.61]; P = .049). The findings of this cohort study suggest that DNAemia for CMV, HAdV, BKPyV, and HHV-6 and EBV seropositivity were independently associated with increased sepsis mortality. Further investigation of the underlying biology of these viral DNA infections in children with sepsis is warranted to determine whether they only reflect mortality risk or contribute to mortality.

Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention

Objective Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. Methods Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. Results VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. Discussion The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20–30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. Conclusion These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids’ therapeutic potential against toxicant-triggered autoimmune responses.

P052 Mucosal IgG-like naive B-cell expansion and maturation contribute to ulcerative colitis pathogenesis

Abstract Background A higher abundance of colonic naive B-cells and IgG plasma cells is implicated in the pathogenesis of ulcerative colitis (UC) during active inflammatory episodes. However, B-cell inhibition by targeting CD20 was found to be ineffective in achieving remission in UC patients indicating that the causal role of the B-cell expansion in UC is not well understood. In this study, we performed single cell RNA-sequencing (scRNA-seq) on mucosal tissue where we interrogated the B-cells in inflamed and non-inflamed UC colon biopsies. We subsequently explored the pathogenic role of B-cells in colitis mouse models. Methods We collected biopsies from the colon of 5 UC patients with active inflammation and 5 UC patients showing no inflamed mucosa. Three mucosal biopsies were collected from the same colon location for each patient and processed for scRNA-seq. We acquired usable transcriptomes of 21.475 cells. In a T-cell transfer colitis mice model, we evaluated the effects of the co-transfer of naive B-cells. Both CD4+ T and naive B-cells were transferred into SCID mice through intraperitoneal injection in ratios of 2:1, 1:1 and 1:2 (T:B ratio). The colitis course was followed for 35 days. Results Differential abundance analysis relative to all immune cells confirmed a higher abundance of naive B-cells (p= 0.03) and IgG plasma B-cells (p= 0.002) in inflamed relative to non-inflamed UC patient tissue, whereas no differences in abundance of transitional B-cells and memory B-cells were found. Pseudo-time analysis indicated a higher rate of maturation in the naive B-cells in inflamed tissue. This was evidenced by higher expression of IGHG1 (p= 0.025) and IGHG3 (p&amp;lt; 0.001) in naive B-cells in inflamed tissue. In a T-cell transfer model, we co-transferred naive B-cells to the CD4+ T-cell pool into SCID mice in various ratios (2:1, 1:1 and 1:2 T to B-cells respectively) and T-cell only control. Significant increase in disease severity was observed in a 1:2 T:B ratio based on body weight loss, increase of colon density, mouse colitis histology index and histological changes by disrupted mucosa. This observation was associated to significant elevated IgG concentration in serum and colon and B-cell presence in colon through immunohistochemistry. Conclusion We observed significantly higher abundance of naive B-cells and IgG plasma cells in inflamed relative to non-inflamed UC colon tissue, where naive B-cells were shown to be more mature and IgG-like in inflamed condition. Notably, transferring increased ratio of naive B-cells resulted in elevated disease severity in a colitis model, demonstrating its pathogenic role. Our findings suggest that specific targeting of the IgG-like naive B-cell may be beneficial for UC patients.

Dose-response of inactivated yeast in diets of late gestating and lactating gilts on immunoglobulin transfer and offspring preweaning growth performance.

Fifty gilts (initial body weight [BW] 190.7 ± 4.2kg) were recruited on day 85 of gestation and were used until day 19 of lactation to assess the dose-response of inactivated yeast via hydrolyzation (HY) inclusion on offspring growth and immunoglobulin (Ig) transfer prior to weaning. Gilts were assigned to one of the 5 experimental diets: a control with no HY (HY0) or inclusion of 0.25% (HY0.25), 0.5% (HY0.5), 1.0% (HY1.0), or 1.2% (HY1.2) HY. Gilts were weighed on days 85 and 110 of gestation and days 1 and 19 (weaning) after farrowing. Offspring were weighed on days 1 and 19 of age. On lactation day 1 (approximately 24h after farrowing), colostrum, gilt plasma, and plasma from 2 median BW piglets were collected and on day 19, plasma from each gilt and 2 median BW piglets per litter were collected for determination of Ig concentrations. Contrast statements were used to assess the linear, quadratic, cubic, and quartic effects of HY inclusion. The inclusion of HY had minimal effects on gilt BW or litter characteristics at birth (total number born and born alive, piglet birth weight). Lactation average daily feed intake of the gilts tended to increase then decrease with increasing HY inclusion (quadratic; P = 0.085). Piglet preweaning average daily gain (linear, quadratic, and quartic; P < 0.05) and BW at weaning (quadratic and quartic; P < 0.05) increased then decreased with increasing HY inclusion. On lactation day 1, colostrum and gilt plasma Ig concentrations were not affected by dietary treatment (P > 0.10) but piglet IgA and IgM decreased then increased with HY inclusion level (cubic; P < 0.05). On lactation day 19, piglet plasma IgG tended to increase with HY inclusion (linear; P = 0.099). In summary, increasing HY inclusion in late gestating and lactating gilt diets improved immune transfer in the first 24h after birth and piglet preweaning growth rates and BW at weaning. Therefore, maternal feeding of HY could be used as a strategy to improve offspring immunocompetence and BW at weaning, with possible carryover benefits for the postweaning phase.