Total IgA Research Articles

Evaluation of Celiac Disease Prevalence in Patients with ADHD (Attention Deficite/ Hyperactivity Disorder)

Background: Attention Deficit Hyperactivity Disorder (ADHD) is one of the most common behavioral disorders among children that has a significant impact on family and society. Recently since numerous cases of ADHD alongside celiac disease and improvement of behavioral symptoms of children with ADHD with gluten-free diet have been proposed, this study was conducted to determine the prevalence of celiac disease in children with ADHD based on serological findings in Sistan.
 Methods: The present study was performed on 76 children with a diagnosis of ADHD referred to the pediatric clinic of Amir Al-Momenin Hospital in Zabol. Demographic, clinical and paraclinical information such as age, sex and celiac laboratory diagnostic tests (anti-TTG and total IgA) were collected and the results were entered in the information form of each child and analyzed.
 Results: Among the 76 children participating in this study, 58 were male (76.3%) and the age range of patients was 12-2 years (6.9 ± 2.4). The results of the study showed that based on the results of celiac serology tests performed among children with ADHD, no cases of celiac disease were found. In this study, 26 children (34.21%) have anemia. However, the mean of anti-TTG and total IgA in children with anemia and children without anemia were not statistically significant (p value > 0.05).
 Conclusion: The results of the present study showed that based on serological findings, no cases of celiac disease were observed among children with ADHD.

Tobacco smokers as target group for complicated coronavirus infection

The aim of current study was to determine, retrospectively, possible correlations between smoking and the incidence, course severity, intubation rate, and mortality (by gender and age) in patients treated for complicated coronavirus infection in the internal medicine clinic at UMHATEM ”N. I. Pirogov” Sofia for the period 01.03.2020–31.12.2020. In a prospective study, the recovery period and immunogenesis in smokers and non-smokers within a one-year period after hospital discharge was investigated. The applied methods were: 1) computed tomography and blood gas analysis 2) chemiluminescent immunoassay for the qualitative determination of total IgM, IgA and IgG anti-SARS-CoV2 AB. Results showed that the part of non-smokers with a positive PCR test is significantly higher compared to the group of former and current smokers. The data obtained from the study confirmed that Covid infection is much more severe among smokers and former smokers with a higher levels of inflammatory markers noticed among the smoking group.

Peanut-Specific IgG4 and IgA in Saliva Are Modulated by Peanut Oral Immunotherapy

Antigen-specific immunoglobulin responses have yet to be studied at the oral mucosal surface during peanut oral immunotherapy (PnOIT). We aimed to quantify salivary peanut-specific IgG4 (PNsIgG4) and IgA (PNsIgA) and total IgG4 and IgA in participants from the Immune Tolerance Network's IMPACT study, a phase 2 PnOIT trial. Peanut-allergic children, aged 12 months to younger than 48 months at screening, were enrolled and randomized to PnOIT or placebo oral immunotherapy (OIT) for 134 weeks. Per-protocol analysis included 69 PnOIT and 23 placebo participants. Double-blind, placebo-controlled food challenges were conducted at weeks 134 and 160 to assess desensitization and remission, respectively. Saliva samples were collected at baseline and 30, 82, 134, and 160 weeks to quantify PNsIgG4, PNsIgA, and total IgG4 and IgA. Participants who received PnOIT experienced significant increases in PNsIgG4 in saliva, whereas participants on placebo did not (P < .01 at all time points). The PNsIgA/total IgA ratio was also significantly increased in participants treated with PnOIT when compared with those receiving placebo at 30 and 82 weeks (P < .05). During PnOIT, desensitized participants had increased PNsIgA that plateaued, whereas the not desensitized/no remission group did not change over time. Interestingly, when the PnOIT group was evaluated by clinical outcome, PNsIgA was higher at baseline in the not desensitized/no remission group than in the desensitized/remission group (P < .05). PnOIT induces substantial increases in allergen-specific IgG4 and IgA in saliva. These data provide insight into OIT-induced mucosal responses and suggest the utility of these easily obtained samples for biomarker development.

Decreased humoral immune response in the bronchi of rapid decliners with chronic obstructive pulmonary disease

BackgroundIdentification of COPD patients with a rapid decline in FEV1 is of particular interest for prognostic and therapeutic reasons.ObjectiveTo determine the expression of markers of inflammation in COPD patients with rapid functional decline in comparison to slow or no decliners.MethodsIn COPD patients monitored for at least 3 years (mean ± SD: 5.8 ± 3 years) for lung functional decline, the expression and localization of inflammatory markers was measured in bronchial biopsies of patients with no lung functional decline (FEV1% + 30 ± 43 ml/year, n = 21), slow (FEV1% ml/year, − 40 ± 19, n = 14) and rapid decline (FEV1% ml/year, − 112 ± 53, n = 15) using immunohistochemistry. ELISA test was used for polymeric immunoglobulin receptor (pIgR) quantitation “in vitro”.ResultsThe expression of secretory IgA was significantly reduced in bronchial epithelium (p = 0.011) and plasma cell numbers was significantly reduced in the bronchial lamina propria (p = 0.017) of rapid decliners compared to no decliners. Bronchial inflammatory cell infiltration, CD4, CD8, CD68, CD20, NK, neutrophils, eosinophils, mast cells, pIgR, was not changed in epithelium and lamina propria of rapid decliners compared to other groups. Plasma cells/mm2 correlated positively with scored total IgA in lamina propria of all patients. “In vitro” stimulation of 16HBE cells with LPS (10 μg/ml) and IL-8 (10 ng/ml) induced a significant increase while H2O2 (100 μM) significantly decreased pIgR epithelial expression.ConclusionThese data show an impaired humoral immune response in rapid decliners with COPD, marked by reduced epithelial secretory IgA and plasma cell numbers in the bronchial lamina propria. These findings may help in the prognostic stratification and treatment of COPD.

Lower Levels of ABO Anti-A and Anti-B of IgM, IgG and IgA Isotypes in the Serum but Not the Saliva of COVID-19 Convalescents.

Individuals with ABO type O, naturally possessing anti-A and anti-B antibodies in their serum, are underrepresented among patients infected with SARS-CoV-2 compared with healthy controls. The ABO antibodies might play a role in the viral transmission. Therefore, we aimed to quantify anti-A/anti-B, including their subclasses IgM, IgG and IgA, in the serum and saliva of Caucasians (n = 187) after mild COVID-19 to compare them with individuals who had never been infected with SARS-CoV-2. Two samples were collected within two months after the diagnosis (median days: 44) and two months later. ABO antibodies were determined by flow cytometry. Additionally, total IgA in saliva and antibodies specific to SARS-CoV-2 were tested by ELISA. COVID-19 convalescents had significantly lower levels of anti-A/anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). Interestingly, no significant differences were observed in saliva. ABO antibody levels remained stable over the period considered. No relation of ABO to the level of SARS-CoV-2-specific antibodies was observed. Total IgA was lower in convalescents than in controls (p = 0.038). Whereas ABO antibodies in the saliva may not contribute to the pathogenesis of COVID-19, individual pre-existing high serum concentrations of anti-A/anti-B may have a protective effect against SARS-CoV-2 infection.

Hydrolyzed chicken liver used as single source of animal protein in diet and its effect on cytokines, immunoglobulins, and fecal microbiota profile of adult dogs.

Dogs with food allergies and enteropathies may require hydrolyzed diets to prevent or reduce clinical signs, therefore the protein sources used in these diets must be previously characterized and evaluated in healthy dogs. This study aimed to investigate the effects of a hydrolyzed chicken liver powder-based diet (HCLP) versus a poultry by-product meal and bovine meat and bone meal-based diet (Control), on complete blood count (CBC), cytokine, immunoglobulins responses (assessed on days 0, 15, 30 and 45), and fecal microbiota (assessed on day 45) in healthy adult dogs. The CBC did not differ between diets (P>0.05), remaining within reference range. Total plasma IL-4 concentrations were decreased over time independent of the dietary treatment (P<0.001). Total plasma IgA decreased on day 30 compared to days 0 and 45 in dogs fed the control diet (P<0.001). Total plasma IgE concentrations were reduced on days 30 and 45 in dogs fed the control diet, and on days 15 vs 30 and 15 vs 45 in dogs fed HCLP diet (P = 0.001). The 16S rRNA gene sequencing showed similar species richness and abundances of phyla and genera between diets (P>0.05). β-diversity principal coordinate analysis plots demonstrated that HCLP group had a higher similarity than control. Based on our results, healthy adult dogs fed a HCLP based diet maintained normal values for hematological and immunological characteristics, and fecal microbiota after 45 days of feeding.

Description of a clinical case of seronegative celiac disease in a 5-year-old girl

Introduction. Celiac disease is an immuno-mediated systemic disease, which is defined as intolerance to gluten and the corresponding prolamins in the mucous membrane of the small intestine in genetically predisposed children, and is characterized by the development of atrophic enteropathy. The seronegative form of celiac disease, in which serological markers are not determined, but the development of appropriate symptoms and the effect of gluten-free diet (GFD) is observed, still remains a diagnostic and therapeutic dilemma.Objectives. Analysis of a clinical case of a child with seronegative celiac disease (SNCD).Results. The disease in a young child before the final diagnosis was severe due to chronic diarrhea and severe flatulence. The girl received outpatient treatment at the place of residence several times with a short-term positive effect, the condition worsened in dynamics: the child lost weight, stopped walking. At the age of 5, she was hospitalized in the gastroenterology department of the Republican Specialized Scientific and Practical Medical Center of Pediatrics, where, on the bases of normal values of antibodies to tissue transglutaminase and total IgA and IgG (анти-tTG IgA and G), changes in biopsies of the mucosa of the postbulary department of the duodenal bulb, characteristic of Marsh 3, and the presence of HLA DQ2, seronegative celiac disease was supposed to diagnose. The girl was prescribed GFD, symptomatic drug therapy was performed. The dynamics of the condition has improved. After 6 months, at the second visit, there were no gastrointestinal symptoms, physical development corresponded to age, what could help to confirm diagnosis of seronegative celiac disease.Conclusions. This clinical case in a girl with a classic form of celiac disease and severe atrophy of the mucosa of the bulbous small intestine, HLA DQ2, was accompanied by normal values of анти-tTG and total IgA. Treatment of a gluten-free diet was very effective: the girl fully compensated for weight deficiency and laboratory tests.

Frequency of celiac disease and distribution of HLA-DQ2/DQ8 haplotypes among siblings of children with celiac disease.

BACKGROUNDCeliac disease (CD) is a multifactorial disease, but genetic factors play a major role in its etiology. It has been known that human leucocyte antigen (HLA)-DQ2/DQ8 haplotypes are one of the most important predisposing genetic factors. The risk of developing CD in first-degree relatives and especially siblings of celiac patients is quite high because of having the same HLA haplotypes.AIMTo evaluate the frequency of CD and the distribution of the HLA-DQ2/DQ8 haplotypes in siblings of celiac patients.METHODSPatients with biopsy-proven CD and their siblings were included in the study; those who did not have HLA genotyping were excluded from the study. All siblings were on a gluten-containing diet. The HLA genotyping, tissue transglutaminase antibody IgA antibody test, and total IgA test were performed in all participants.RESULTSA total of 57 celiac patients and their 112 siblings were included in the study. The mean age of celiac patients and siblings were 10.30 ± 3.87 years and 9.90 ± 6.11 years, respectively. HLA-DQ2/DQ8 alleles were detected in 98.2% of patients with CD and 90.2% of siblings of celiac patients. HLA-DQ genotypes were present in all siblings diagnosed with CD. Tissue transglutaminase antibody IgA test was found to be positive in 16 siblings. CD was diagnosed in 12 siblings (10.7%) by intestinal biopsy.CONCLUSIONThe prevalence of CD was found to be 10.7% in siblings of celiac patients in our study. One-third of the siblings diagnosed with CD were asymptomatic. We detected HLA-DQ alleles in 98.2% of celiac patients and 100% in siblings diagnosed with CD. In addition, 1 of the 2 siblings was diagnosed with CD 1 year later and the other 4 years later. Therefore, we suggest that siblings of celiac patients should be followed up with clinical findings as well as HLA analysis and serological examination. Since the risk of developing CD is much higher in asymptomatic siblings, we recommend that siblings should be screened for CD even if they are asymptomatic.

B cell memory responses induced by foot-and-mouth disease virus-like particles in BALB/c mice

A challenging but critical question is that new foot-and-mouth disease (FMD) vaccines should be to induce B cell memory to provide antibodies for long-term protection. The maintenance of B cell memory is dependent on long-lived plasma cells (LLPCs) and memory B cells. We developed a chimeric FMDV virus-like particles (FMDV-VLPs), fusing VP1-VP4 into HBcAg. In our study, we investigated if or how long B cell memory was induced by FMDV-VLPs in mice. The data showed that FMDV-VLPs can induce memory humoral responses with a high level of total IgG1, IgG2a, IgA, and FMDV-specific IgG antibodies in serum. The persistence of antibody levels in serum could depend on LLPCs. The proportion of LLPCs in CD19+ cells in bone marrow exhibited a dynamic trend with two peaks at 28 days post-immunization (dpi) and 72 dpi, respectively. Additionally, the proportion of memory B cells in CD19+ cells in the spleen increased significantly both at 7 days post primary immunization and at 7 days post -boost immunization. Of note, LLPCs together with memory B cells contribute to the production of FMDV-specific IgG and IgG1. The changes of LLPCs and memory B cells may be related to TNF-α, IL-6 and, CXCL12. Taken together, FMDV-VLPs could induce B cells memory responses. A further understanding of the mechanisms that FMDV-VLPs how we can manipulate the induction and maintenance of memory B cells and LLPCs will promote vaccine design and likely address several challenges to develop FMDV new vaccines in the future.

NK cell spatial dynamics and IgA responses in gut-associated lymphoid tissues during SIV infections

HIV infection induces tissue damage including lymph node (LN) fibrosis and intestinal epithelial barrier disruption leading to bacterial translocation and systemic inflammation. Natural hosts of SIV, such as African Green Monkeys (AGM), do not display tissue damage despite high viral load in blood and intestinal mucosa. AGM mount a NK cell-mediated control of SIVagm replication in peripheral LN. We analyzed if NK cells also control SIVagm in mesenteric (mes) LN and if this has an impact on gut humoral responses and the production of IgA known for their anti-inflammatory role in the gut. We show that CXCR5 + NK cell frequencies increase in mesLN upon SIVagm infection and that NK cells migrate into and control viral replication in B cell follicles (BCF) of mesLN. The proportion of IgA+ memory B cells were increased in mesLN during SIVagm infection in contrast to SIVmac infection. Total IgA levels in gut remained normal during SIVagm infection, while strongly decreased in intestine of chronically SIVmac-infected macaques. Our data suggest an indirect impact of NK cell-mediated viral control in mesLN during SIVagm infection on preserved BCF function and IgA production in intestinal tissues.

A Randomized Controlled Trial of Dietary Rice Bran Intake on Microbiota Diversity, Enteric Dysfunction, and Fecal Secretory IgA in Malian and Nicaraguan Infants

ABSTRACTBackgroundMalnutrition and diarrhea are leading causes of death in children aged <5 y. Rice bran is a nutrient-dense prebiotic available globally.ObjectivesThe objective of this secondary analysis was to evaluate the effects of daily rice bran supplementation on environmental enteric dysfunction (EED) markers, total fecal secretory IgA (sIgA), and microbiota in infants at high risk of malnutrition.MethodsSix-month-old Malian and Nicaraguan infants were randomly assigned to control or daily rice bran supplementation cohorts (1 to 5 g/d). Feces were collected monthly for 6 mo to evaluate fecal sIgA, markers of EED, and microbiota diversity. Statistical methods included linear mixed models, generalized mixed models, Spearman correlation, and Wilcoxon rank-sum tests.ResultsSix-month-old Malian infants had significantly elevated sIgA (4.0× higher, P < 0.001), fecal myeloperoxidase (31.6× higher, P < 0.001), fecal α1-antitrypsin (1.8× higher, P = 0.006), and lower fecal neopterin (0.13× higher, P < 0.001) than the age-matched Nicaraguan infants. In the Nicaraguan rice bran cohort from 6 to 12 mo of age, there was a significant decrease in sIgA concentrations (0.4×, P < 0.05) and a correlation between sIgA and the EED marker α1-antitrypsin (0.523, P < 0.0001) at 12 mo of age. In Malian infants, daily rice bran ingestion resulted in decreased EED scores (0.71×, P = 0.02) and a stable sIgA concentration over time. The rice bran group of Malian infants also had correlation between sIgA and the EED marker neopterin (0.544, P < 0.001) at 12 mo of age and a significant (P < 0.05) increase in microbiota α-diversity at a younger age (9 mo with rice bran compared with 10 mo in control group), which supports earlier microbiota maturation.ConclusionsThese results support rice bran as a functional food ingredient targeting gut mucosa in children at high-risk of malnutrition.

Dietary supplementation of sulfur amino acids improves intestinal immunity to Eimeria in broilers treated with anti-interleukin-10 antibody.

Oral antibody to interleukin-10 (anti-IL-10) enhances the intestinal immune defense against Eimeria. The sulfur amino acids methionine and cysteine (M+C) play essential roles in inducing and maintaining protective immune responses during intestinal infections. Hence, increased dietary M+C may support the anti-IL-10-induced intestinal immunity to Eimeria. Broilers (n = 640) were arranged in a 2 × 2 × 2 factorial design with 2 levels of each of the 3 main factors: dietary standardized ileal digestible (SID) M+C levels (0.6% or 0.8%), dietary anti-IL-10 supplementation (with or without), and coccidiosis challenge (control or challenge). Briefly, the broilers were supplied with either 0.6% or 0.8% SID M+C, each with or without anti-IL-10 (300 μg/kg), from d 10 to 21. On d 14, broilers from each diet were gavaged with either PBS or Eimeria. The resulting Eimeria infection induced fecal oocyst shedding and intestinal lesions. Broilers fed 0.8% SID M+C (main effects, P ≤ 0.05) had decreased feed-to-gain ratio, increased duodenum and cecum luminal anti-Eimeria IgA titers, and decreased fecal oocyst counts, when compared to 0.6% SID M+C. The supplementation of anti-IL-10 (main effects, P ≤ 0.05) increased cecum luminal total IgA concentration and decreased cecum lesions. Interactions (P ≤ 0.05) were detected for growth performance and cecum luminal IFN-γ. Briefly, the highest body weight gain and feed intake were reached in PBS-gavaged broilers fed 0.8% SID M+C with no anti-IL-10 and in Eimeria-challenged broilers fed 0.8% SID M+C with anti-IL-10. In Eimeria-infected broilers, anti-IL-10 increased intestinal luminal IFN-γ and body weight gain only at 0.8% SID M+C. Collectively, anti-IL-10 increased intestinal luminal IFN-γ levels, decreased cecum lesions and restored growth only when fed with adequate amounts of sulfur amino acids. Our findings underscore the importance of providing sufficient essential nutrients to support the anti-IL-10 induced immunity against coccidiosis.

Antibodies against SARS-CoV-2 after natural infection in healthcare workers and clinical characteristics as putative antibody production prediction

IntroductionThere is a need for detailed data on early antibody responses against SARS-CoV-2 as this may contribute to the prediction of the clinical course of COVID-19 and the optimization of convalescent plasma treatment. This study aims to gain insight into developing antibodies to SARS-CoV-2 in health care workers (HCWs) infected in the first wave of the SARS-CoV-2 pandemic in the Netherlands. Materials and methodsIn this retrospective analysis, sera from PCR-confirmed COVID-19 positive HCWs are included at the time of the initial PCR (T = 0, n = 95) and at least 21 days after the initial serum (T ≥ 21, n = 133). This study assesses correlations between qualitative total Ig, IgM, IgA, IgG, and quantitative anti-S-RBD antibody responses and participant characteristics. ResultsHigher Ct values were associated with higher antibody positivity rates for total Ig (OR 1.261 (95% CI 1.095–1.452)), IgM (OR 1.373 (95% CI 1.125–1.675)), and IgA (OR 1.222 (95% CI 1.013–1.475)). Gender was predictive of IgM and IgA antibody positivity rates at T = 0 (OR 0.018 (95% CI 0.001–0.268)) and (OR 0.070 (95% CI 0.008–0.646)). At T ≥ 21, a substantial proportion of HCWs developed IgM (103/133; 77.4%) and total Ig (128/133; 96.2%) antibodies. IgA and IgG seroconversions were observed in only 51.1% (67/131) and 55.7% (73/131) of HCWs. Anti-S-RBD responses were higher when the interval between onset of symptoms and sampling was longer. ConclusionThe findings of this study give insight into early antibody responses and may have implications for the selection of convalescent plasma donors and the further development of monoclonal antibody treatment.

A Randomized, Triple-Blind, Placebo-Controlled Clinical Trial Investigating Two Dietary Supplements on Supporting Immune Function in Healthy Adults in a Flu Vaccine Model

ObjectivesTo examine the role of two dietary supplements, UP360 and UP446, on supporting immune function in response to influenza vaccination. The ingredients in UP360 and UP446 have been shown to activate the immune system, reduce inflammation and oxidative stress and protect against lung damage. The primary outcome assessed changes in blood lymphocytes (CD3+, CD4+, CD8+, CD45+, TCRγδ+, CD3-CD16 + 56+) and immunoglobulins (IgG, IgM, IgA) between UP360 or UP446 vs. placebo.MethodsThis randomized, triple-blind, placebo-controlled, parallel study consisted of a 56-day intervention period with a 28-day pre-vaccination period, influenza vaccination on Day 28 and 28-day post-vaccination period. At baseline, Day 28 and 56, lymphocyte populations, Igs, cytokines, oxidative stress markers and influenza-specific Igs were measured in blood, and a quality of life (QoL) questionnaire was completed. The WURSS-24 was completed daily to assess upper respiratory tract infections (URTIs). Participants were healthy men and women ages 40–80 who had not received their flu vaccine and were willing to maintain their lifestyle habits during the study.ResultsIn the post-vaccination period, supplementation with UP360 increased the percentage of TCRγδ+ cells compared to placebo (p < 0.001). Total IgA and IgG levels were increased with UP446 supplementation vs. placebo (p ≤ 0.023). UP360 and UP446 groups had increases of 15.6% and 19.4%, respectively, in influenza B-specific IgG in the post-vaccination period (p ≤ 0.0006). Serum glutathione peroxidase (GSH-Px) was increased in the pre-vaccination period in both UP360 and UP446 groups (p ≤ 0.0270). Despite these changes in immune and antioxidant markers, there were no between-group differences in severity, frequency, or duration of URTI symptoms or QoL.ConclusionsThese results suggest supplementation with UP360 or UP446 was beneficial in mounting a robust humoral response following vaccination. Increasing GSH-Px in the pre-vaccination period may be beneficial in mitigating the oxidative stress over and above that induced following influenza vaccination and/or viral infection. There were no changes in subjective measures of URTI illness or QoL, demonstrating QoL of these participants was not negatively impacted by supplementation.Funding SourcesUnigen Inc.

Cholecystokinin Outcome on Markers of Intestinal IgA Antibody Response.

Cholecystokinin 8 (CCK8) is an entero-octapeptide that participates in crosstalk with components of intestinal immunity via the CCK receptor (CCKR), but its role in modulation of the IgA response is not fully known under physiological conditions. Male eight-week-old BALB/c mice each were intraperitoneally injected once during 7 days with CCK8, devazapide (CCKR1 antagonist), L365,260 (CCKR2 antagonist) or vehicle (sham group). In intestinal lavages, total and secretory IgA (SIgA) were determined by ELISA; in lamina propria, IgA+ B lymphocytes and IgA+ plasma cells were analyzed by flow cytometry; mRNA levels of polymeric immunoglobulin receptor (pIgR) in epithelial cells and α chain, interleukins (ILs) in lamina propria cells were assessed by qRTPCR. Regarding the sham conditions, IgA+ plasma-cell percentage and IL-2, IL-5, IL-10 and transforming growth factor-β (TGF-β) mRNA levels were either increased by CCK8 or decreased by both CCKR antagonists. For IgA/SIgA responses, IL-4/IL-6 mRNA levels were decreased by all drugs and pIgR mRNA was increased by CCK8 and reduced by L365,260. IgA+ B cell percentage and α chain mRNA levels were elicited by CCK8 and L365,260. Data suggested a presumable differential role of CCK/CCKR on the IgA-response; outcome of L365,260 on the elicitation of IgA+ B cells and α chain mRNA needs further examination.

Assessment of M-protein quantification using capillary electrophoresis and immunosubtraction-based integration in clinical samples with low M-protein concentrations

ObjectivesQuantification of monoclonal protein (M-protein) by serum protein electrophoresis (SPE) is indispensable for diagnosing and monitoring monoclonal gammopathies. However, quantification of small and beta migrating M-proteins is challenging because of overlapping non-immunoglobulin and/or polyclonal immunoglobulin protein fractions. We compared a new integration method based on immunosubtraction (IS-CE) using capillary zone electrophoresis (CZE) against the routine method, which includes a combination of perpendicular drop (0.4%), corrected perpendicular drop (1%) and tangent skimming (98.5%). Design & methodsThe proposed method of M-protein quantification involves calculating the difference in area under the curve between the SPE and a class-specific IS-CE trace. We analyzed the difference in estimated M-protein concentrations obtained with the new method and routine integration methods using 913 samples. For IgA M-proteins at < 10 g/L, the estimated M-protein concentrations were compared with the total IgA concentration. ResultsThe median M-protein concentration of 913 consecutive samples was 6.2 g/L (IQR 2.1–14.3 g/L). The median and median % difference between the two integration methods was 0.68 g/L (IQR 0.01–1.55 g/L) and 10.9% (IQR 0.18–38.7%), showing a larger estimated M-protein concentration with the new method. More than 25% difference was observed in 38% of the samples and was associated with lower total protein concentration, lower M-protein concentration, IgA and IgM heavy chain isotypes, and beta- or beta-gamma migration. When 161 samples with IgA M-protein < 10 g/L were compared against total IgA concentration, the median bias of the new method was smaller compared to that of the existing method (-0.95 g/L vs. −1.3 g/L, P < 0.0001). ConclusionsThe use of IS based integration using CZE and IS-CE is promising especially for small and beta migrating M-proteins.

Recurrence of IgA nephropathy after kidney transplantation: experience from the Swiss transplant cohort study

BackgroundRecurrence of IgA nephropathy (IgAN) after kidney transplantation occurs in about 30% of patients. The relevance of recurrence for the long-term graft survival is expected to increase, since graft survival continues to improve.MethodsIn a nested study within the Swiss Transplant Cohort Study the incidence of IgAN recurrence, predictive factors, graft function and graft and patient survival were evaluated. Serum concentration of total IgA, total IgG, Gd-IgA1 and IgA-IgG immune complex were measured using ELISA-based immunologic assays.ResultsBetween May 2008 and December 2016, 28 women and 133 men received their kidney allograft for end-stage kidney disease due to IgAN in Switzerland. Over a median follow-up time of 7 years after transplantation, 43 out of 161 patients (26.7%) developed an IgAN recurrence, of which six (13.9%) had an allograft failure afterwards and further four patients (9.3%) died. During the same follow-up period, 6 out of 118 patients (5%) each experienced allograft failure or died without prior IgAN recurrence. After 11 years the risk for IgAN recurrence was 27.7% (95%-CI: 20.6–35.3%). Renal function was similar in patients with and without recurrence up to 7 years after transplantation, but worsened thereafter in patients with recurrence (eGFR median (interquartile range) at 8 years: 49 ml/min/1.73m2 (29–68) vs. 60 ml/min/1.73m2 (38–78)). Serum concentration of total IgA, total IgG, Gd-IgA1 and IgA-IgG immune complex within the first year posttransplant showed no significant effect on the recurrence of IgAN. Younger recipients and women had a higher risk of recurrence, but the latter only in the short term.ConclusionsOur study showed a recurrence risk of 28% at 11 years after transplantation, which is consistent with previous literature. However, the predictive value of known biomarkers, such as serum Gd-IgA1 and IgA-IgG IC, for IgAN recurrence could not be confirmed.

FC049: Nefecon® (Budesonide) is an Epigenetic Modifier in IGA Nephropathy

Abstract BACKGROUND AND AIMS In recent years, the dysregulated expression of microRNAs (miRs)—small molecules of noncoding RNA that epigenetically fine-tune post-transcriptional gene expression—has been increasingly implicated in the pathogenesis of IgA nephropathy (IgAN). Extracellular miRs, contained within extracellular vesicles such as exosomes and microvesicles, are thought to function as important intercellular and interorgan modes of communication in health and disease. In the current study, next-generation sequencing (NGS) and subsequent real-time quantitative polymerase chain reaction (RT-qPCR) validation were performed to identify serum miRs associated with a high risk of future kidney function decline in IgAN. We then went on to determine whether treatment with Nefecon® 16 mg/day for 9 months was capable of modifying the serum levels of miRs associated with high-risk IgAN. This study was supported by a grant from Calliditas Therapeutics AB. METHOD NGS was performed by Qiagen (Germany) on serum from patients with IgAN (10 at high risk and 10 at low risk of progression) or membranous nephropathy (MN, n=10), as well as healthy individuals (HS, n=10). Sequencing data were validated by RT-qPCR using TaqMan miR PCR assays in independent sets of serum samples (20 per cohort). Using the Total Exosome Isolation (from serum) reagent (Thermo Fisher Scientific, UK), extracellular exosomes were isolated from the serum of participants of the NEFIGAN trial from the placebo group (n=40) and Nefecon® 16 mg/day group (n=40) at the start of treatment (SOT) and end of treatment (EOT). RNA was isolated and levels of miRs-483-5p and -122-5p were measured by RT-qPCR. The NEFIGAN trial (NCT01738035) was a randomized, double-blind, placebo-controlled Phase 2b trial designed to assess the safety and efficacy of Nefecon® a novel targeted-release formulation of budesonide, designed to deliver the drug to the distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. RESULTS miRs-483-5p and -122-5p were significantly overexpressed in the serum of patients with IgAN at high risk of progression compared with those who had stable IgAN or HS, but not compared with patients with MN. Both miRs were also found to be significantly increased in extracellular exosomes in high-risk IgAN compared with low-risk IgAN, HS and MN. Interestingly, levels of exosomal miRs-483-5p and -122-5p did not correlate with total serum IgA, IgA1, galactose-deficient IgA1 or IgA–IgG immune complex levels, suggesting a novel mechanism of action. Treatment with 16 mg/day of Nefecon® led to a significant reduction in the exosomal levels of miR-122-5p, while no change in miR-483-5p levels was observed in the treated group. CONCLUSION These data reveal a dysregulation of systemic exosomal miR expression in IgAN, and demonstrate for the first time that it is possible to pharmacologically modify the miR content of circulating exosomes in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively downregulated exosomal miR-122-5p, while having no effect on exosomal miR-483-5p levels, arguing against a generalized ‘anti-miR’ effect of Nefecon®. Studies are ongoing to both precisely delineate the cellular source(s) of these exosomes and determine the downstream impact of these changes on glomerular and tubulointerstitial biology.

Reduced humoral response 3 months following BNT162b2 vaccination in SARS-CoV-2 uninfected residents of long-term care facilities.

BackgroundSARS-CoV-2 vaccination is the most effective strategy to protect older residents of long-term care facilities (LTCF) against severe COVID-19, but primary vaccine responses are less effective in older adults. Here, we characterised the humoral responses of institutionalised seniors 3 months after they had received the mRNA/BNT162b2 vaccine.Methodsplasma levels of SARS-CoV-2-specific total IgG, IgM and IgA antibodies were measured before and 3 months after vaccination in older residents of LTCF. Neutralisation capacity was assessed in a pseudovirus neutralisation assay against the original WH1 and later B.1.617.2/Delta variants. A group of younger adults was used as a reference group.Resultsthree months after vaccination, uninfected older adults presented reduced SARS-CoV-2-specific IgG levels and a significantly lower neutralisation capacity against the WH1 and Delta variants compared with vaccinated uninfected younger individuals. In contrast, COVID-19-recovered older adults showed significantly higher SARS-CoV-2-specific IgG levels after vaccination than their younger counterparts, whereas showing similar neutralisation activity against the WH1 virus and an increased neutralisation capacity against the Delta variant. Although, similarly to younger individuals, previously infected older adults elicit potent cross-reactive immune responses, higher quantities of SARS-CoV-2-specific IgG antibodies are required to reach the same neutralisation levels.Conclusionsalthough hybrid immunity seems to be active in previously infected older adults 3 months after mRNA/BNT162b2 vaccination, humoral immune responses are diminished in COVID-19 uninfected but vaccinated older residents of LTCF. These results suggest that a vaccine booster dose should be prioritised for this particularly vulnerable population.

The development of a gut-specific immune response to an oral antigen is dependent on α4β7 mediated lymphocyte trafficking

Abstract Objective Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab. Methods An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry. Results MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group. Conclusion α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.