Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection.

Ebola virus (EBOV) remains a public health threat. We performed a longitudinal study of B cell responses to EBOV in four survivors of the 2014 West African outbreak. Infection induced lasting EBOV-specific immunoglobulin G (IgG) antibodies, but their subclass composition changed over time, with IgG1 persisting, IgG3 rapidly declining, and IgG4 appearing late. Striking changes occurred in the immunoglobulin repertoire, with massive recruitment of naive B cells that subsequently underwent hypermutation. We characterized a large panel of EBOV glycoprotein-specific monoclonal antibodies (mAbs). Only a small subset of mAbs that bound glycoprotein by ELISA recognized cell-surface glycoprotein. However, this subset contained all neutralizing mAbs. Several mAbs protected against EBOV disease in animals, including one mAb that targeted an epitope under evolutionary selection during the 2014 outbreak. Convergent antibody evolution was seen across multiple donors, particularly among VH3-13 neutralizing antibodies specific for the GP1 core. Our study provides a benchmark for assessing EBOV vaccine-induced immunity.

Subclass-switched anti-spike IgG3 oligoclonal cocktails strongly enhance Fc-mediated opsonization.

Antibodies play a central role in the immune defense against SARS-CoV-2. Emerging evidence has shown that nonneutralizing antibodies are important for immune defense through Fc-mediated effector functions. Antibody subclass is known to affect downstream Fc function. However, whether the antibody subclass plays a role in anti-SARS-CoV-2 immunity remains unclear. Here, we subclass-switched eight human IgG1 anti-spike monoclonal antibodies (mAbs) to the IgG3 subclass by exchanging their constant domains. The IgG3 mAbs exhibited altered avidities to the spike protein and more potent Fc-mediated phagocytosis and complement activation than their IgG1 counterparts. Moreover, combining mAbs into oligoclonal cocktails led to enhanced Fc- and complement receptor-mediated phagocytosis, superior to even the most potent single IgG3 mAb when compared at equivalent concentrations. Finally, in an in vivo model, we show that opsonic mAbs of both subclasses can be protective against a SARS-CoV-2 infection, despite the antibodies being nonneutralizing. Our results suggest that opsonic IgG3 oligoclonal cocktails are a promising idea to explore for therapy against SARS-CoV-2, its emerging variants, and potentially other viruses.

Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

High-Throughput Glycan Profiling of Human Serum IgG Subclasses Using Parallel Reaction Monitoring Peptide Bond Fragmentation of Glycopeptides and Microflow LC-MS.

LC-MS-based N-glycosylation profiling in four human serum IgG subclasses (IgG1, IgG2, IgG3, and IgG4) often requires additional affinity-based enrichment of specific IgG subclasses, owing to the high amino acid sequence similarity of Fc glycopeptides among subclasses. Notably, for IgG4 and the major allotype of IgG3, the glycopeptide precursors share identical retention time and mass and therefore cannot be distinguished based on precursor or glycan fragmentation. Here, we developed a parallel reaction monitoring (PRM)-based method for quantifying Fc glycopeptides through combined transitions generated from both glycosidic and peptide bond fragmentation. The latter enables the subpopulation of IgG3 and IgG4 to be directly distinguished according to mass differences without requiring further enrichment of specific IgG subclasses. In addition, a multinozzle electrospray emitter coupled to a capillary flow liquid chromatograph was used to increase the robustness and detection sensitivity of the method for low-yield peptide backbone fragment ions. The gradient was optimized to decrease the overall run time and make the method compatible with high-throughput analysis. We demonstrated that this method can be used to effectively monitor the relative levels of 13 representative glycoforms, with a good limit of detection for individual IgG subclasses.

Clinical, serological and genetic predictors of response to immunotherapy in anti-IgLON5 disease.

Anti-IgLON5 disease is a newly defined clinical entity characterized by a progressive course with high disability and mortality rate. While precise pathogenetic mechanisms remain unclear, features characteristic of both autoimmune and neurodegenerative diseases were reported. Data on immunotherapy are limited, and its efficacy remains controversial. In this study, we retrospectively investigated an anti-IgLON5 disease cohort with special focus on clinical, serological and genetic predictors of the immunotherapy response and long-term outcome. Patients were recruited from the GENERATE (German Network for Research on Autoimmune Encephalitis) registry. Along with clinical parameters, anti-IgLON5 immunoglobulin (Ig)G in serum and CSF, anti-IgLON5 IgG1-4, IgA and IgM in serum, neurofilament light chain and glial fibrillary acidic protein in serum as well as human leukocyte antigen-genotypes were determined. We identified 53 patients (symptom onset 63.8 ± 10.3 years, female:male 1:1.5). The most frequent initial clinical presentations were bulbar syndrome, hyperkinetic syndrome or isolated sleep disorder [at least one symptom present in 38% (20/53)]. At the time of diagnosis, the majority of patients had a generalized multi-systemic phenotype; nevertheless, 21% (11/53) still had an isolated brainstem syndrome and/or a characteristic sleep disorder only. About one third of patients [28% (15/53)] reported subacute disease onset and 51% (27/53) relapse-like exacerbations during the disease course. Inflammatory CSF changes were evident in 37% (19/51) and increased blood-CSF-barrier permeability in 46% (21/46). CSF cell count significantly decreased, while serum anti-IgLON5 IgG titre increased with disease duration. The presence of human leukocyte antigen-DRB1*10:01 [55% (24/44)] was associated with higher serum anti-IgLON5 IgG titres. Neurofilament light chain and glial fibrillary acidic protein in serum were substantially increased (71.1 ± 103.9 pg/ml and 126.7 ± 73.3 pg/ml, respectively). First-line immunotherapy of relapse-like acute-to-subacute exacerbation episodes resulted in improvement in 41% (11/27) of patients and early initiation within the first 6 weeks was a predictor for therapy response. Sixty-eight per cent (36/53) of patients were treated with long-term immunotherapy and 75% (27/36) of these experienced no further disease progression (observation period of 20.2 ± 15.4 months). Long-term immunotherapy initiation during the first year after onset and low pre-treatment neurofilament light chain were significant predictors for a better outcome. In conclusion, subacute disease onset and early inflammatory CSF changes support the primary role of autoimmune mechanisms at least at initial stages of anti-IgLON5 disease. Early immunotherapy, prior to advanced neurodegeneration, is associated with a better long-term clinical outcome. Low serum neurofilament light chain at treatment initiation may serve as a potential biomarker of the immunotherapy response.

Biological basis of critical illness subclasses: from the bedside to the bench and back again.

Critical illness syndromes including sepsis, acute respiratory distress syndrome, and acute kidney injury (AKI) are associated with high in-hospital mortality and long-term adverse health outcomes among survivors. Despite advancements in care, clinical and biological heterogeneity among patients continues to hamper identification of efficacious therapies. Precision medicine offers hope by identifying patient subclasses based on clinical, laboratory, biomarker and 'omic' data and potentially facilitating better alignment of interventions. Within the previous two decades, numerous studies have made strides in identifying gene-expression based endotypes and clinico-biomarker based phenotypes among critically ill patients associated with differential outcomes and responses to treatment. In this state-of-the-art review, we summarize the biological similarities and differences across the various subclassification schemes among critically ill patients. In addition, we highlight current translational gaps, the need for advanced scientific tools, human-relevant disease models, to gain a comprehensive understanding of the molecular mechanisms underlying critical illness subclasses.

The Systematics of the Trematoda.

The platyhelminth class Trematoda comprises two subclasses with largely disparate species diversity, with the small Aspidogastrea with c.80 species and the speciose Digenea with c.18,000 species, which has attracted much effort towards our understanding of evolutionary relationships among suprageneric taxa. This chapter focuses on insights into the classification of the Digenea, that have become apparent from our advanced understanding of both morphological and molecular data. The field of molecular systematics of the Digenea has experienced significant advances over the past 15 years. Phylogenetic analyses of sequence data predominantly from the 18S and 28S rRNA genes have incorporated a considerable diversity of taxa, thus increasing the accuracy of phylogenetic inferences at higher taxonomic levels. As a result, the status of long-standing supraspecific taxa has been revised, new higher-level taxa have been defined, and inferences made in association with morphological and life-cycle evidence. A substantial effort has been made towards a classification reflecting a natural system of the Digenea by considering morphological evidence in conjunction with phylogenies inferred from molecular data; this has resulted in considerable congruence. However, limited taxon sampling in the phylogeny of the Digenea still remains relevant, especially in relation to some higher-level taxa, and an outline of these omissions is presented. A framework that has led to robust estimates of phylogeny is outlined, and the application of advanced morphological and molecular approaches in digenean taxonomy and systematics is illustrated using the most comprehensively studied digenean superfamilies.

A Nationwide Study of the Delayed Diagnosis and the Clinical Manifestations of Predominantly Antibody Deficiencies and CTLA4-Mediated Immune Dysregulation Syndrome in Greece.

Background and Objectives: Predominantly antibody deficiencies (PAD) represent the most common type of primary immunodeficiencies in humans, characterized by a wide variation in disease onset, clinical manifestations, and outcome. Considering that the prevalence of PAD in Greece is unknown, and there is limited knowledge on the clinical and laboratory characteristics of affected patients, we conducted a nationwide study. Materials and Methods: 153 patients (male/female: 66/87; median age: 43.0 years; range: 7.0-77.0) diagnosed, and followed-up between August 1979 to September 2023. Furthermore, we classified our cohort into five groups according to their medical history, immunoglobulin levels, and CTLA4-mutational status: 123 had common variable immunodeficiency (CVID), 12 patients with "secondary" hypogammaglobulinemia due to a previous B-cell depletion immunotherapy for autoimmune or malignant disease several years ago (median: 9 years, range 6-14) displaying a typical CVID phenotype, 7 with combined IgA and IgG subclass deficiencies, 5 patients with CVID-like disease due to CTLA4-mediated immune dysregulation syndrome, and 6 patients with unclassified hypogammaglobulinemia. Results: We demonstrated a remarkable delay in PAD diagnosis, several years after the onset of related symptoms (median: 9.0 years, range: 0-43.0). A family history of PAD was only present in 11.8%, with the majority of patients considered sporadic cases. Most patients were diagnosed in the context of a diagnostic work-up for recurrent infections, or recurrent/resistant autoimmune cytopenias. Interestingly, 10 patients (5.6%) had no history of infection, diagnosed due to either recurrent/resistant autoimmunity, or during a work-up of their medical/family history. Remarkable findings included an increased prevalence of lymphoproliferation (60.1%), while 39 patients (25.5%) developed bronchiectasis, and 16 (10.5%) granulomatous disease. Cancer was a common complication in our cohort (25 patients, 16.3%), with B-cell malignancies representing the most common neoplasms (56.7%). Conclusion: Our findings indicate the necessity of awareness about PAD and their complications, aiming for early diagnosis and the appropriate management of affected patients.

Plasmablasts in previously immunologically naïve COVID-19 patients express markers indicating mucosal homing and secrete antibodies cross-reacting with SARS-CoV-2 variants and other beta-coronaviruses.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin ß1, only some integrin ß7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

Identification of metabolism-related subtypes and feature genes in Alzheimer's disease.

BACKGROUND: Owing to the heterogeneity of Alzheimer's disease (AD), its pathogenic mechanisms are yet to be fully elucidated. Evidence suggests an important role of metabolism in the pathophysiology of AD. Herein, we identified the metabolism-related AD subtypes and feature genes. METHODS: The AD datasets were obtained from the Gene Expression Omnibus database and the metabolism-relevant genes were downloaded from a previously published compilation. Consensus clustering was performed to identify the AD subclasses. The clinical characteristics, correlations with metabolic signatures, and immune infiltration of the AD subclasses were evaluated. Feature genes were screened using weighted correlation network analysis (WGCNA) and processed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Furthermore, three machine-learning algorithms were used to narrow down the selection of the feature genes. Finally, we identified the diagnostic value and expression of the feature genes using the AD dataset and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: Three AD subclasses were identified, namely Metabolism Correlated (MC) A (MCA), MCB, and MCC subclasses. MCA contained signatures associated with high AD progression and may represent a high-risk subclass compared with the other two subclasses. MCA exhibited a high expression of genes related to glycolysis, fructose, and galactose metabolism, whereas genes associated with the citrate cycle and pyruvate metabolism were downregulated and associated with high immune infiltration. Conversely, MCB was associated with citrate cycle genes and exhibited elevated expression of immune checkpoint genes. Using WGCNA, 101 metabolic genes were identified to exhibit the strongest association with poor AD progression. Finally, the application of machine-learning algorithms enabled us to successfully identify eight feature genes, which were employed to develop a nomogram model that could bring distinct clinical benefits for patients with AD. As indicated by the AD datasets and qRT-PCR analysis, these genes were intimately associated with AD progression. CONCLUSION: Metabolic dysfunction is associated with AD. Hypothetical molecular subclasses of AD based on metabolic genes may provide new insights for developing individualized therapy for AD. The feature genes highly correlated with AD progression included GFAP, CYB5R3, DARS, KIAA0513, EZR, KCNC1, COLEC12, and TST.

Proliferative Glomerulonephritis With Hidden Monotypic IgG3κ Deposits: A Case Report.

Rare cases of immunoglobulin G (IgG)-dominant immune complex-mediated glomerulonephritis demonstrate immunoglobulin subclass restriction without light chain restriction. Some of these cases may represent proliferative glomerulonephritis with monotypic immunoglobulin deposits (PGNMID) in which monotypic immunoglobulin is obscured by coexisting polytypic immunoglobulin. However, rigorous demonstration of this possibility is lacking to date. Here, we describe a case of IgG3-restricted immune complex-mediated glomerulonephritis without light chain restriction that apparently "transformed" into IgG3κ-PGNMID in a subsequent biopsy. We demonstrate, using several ancillary techniques, including use of the newly described antibodies directed against the conformational epitope at the junctions of heavy and light chains (HLC-IF), that the first biopsy likely represents IgG3κ-PGNMID in which monotypic IgG3κ was hidden by polytypic IgM. This case underscores the need to consider PGNMID in a differential diagnosis of IgG-dominant immune complex-mediated glomerulonephritis without light chain restriction and highlights the potential utility of IgG subclass staining and HLC-IF in such cases to detect monotypic immunoglobulin that may be obscured by coexisting IgM and/or IgA deposits.

Identification of non-neutralizing anti-factor X autoantibodies in three Japanese cases of autoimmune acquired factor X deficiency.

INTRODUCTION: Autoimmune factor X (FX or F10) deficiency (AiF10D) is an extremely rare acquired haemorrhagic disorder characterized by a severe reduction in FX activity due to autoantibodies against FX. AIM: Anti-FX autoantibodies were investigated in four patients with suspected AiF10D, and their properties were analysed. METHODS AND RESULTS: Anti-FX auto antibodies in plasma were detected by ELISA with three of four cases. One case of anti-FX autoantibody negativity was later diagnosed as AL-amyloidosis. IgG1 and IgG3 coexisted in all anti-FX autoantibodies of the three patients with AiF10D (cases X1, X2, and X3). Western blot analysis showed that the antibodies were bound to the FX light chain for cases X2 and X3, but the binding was weak for case X1. When the fusion proteins of a secretory luciferase with full-length FX or its γ-carboxylated glutamic acid (Gla) domain were added to the plasma of the three patients, both fusion proteins were immunoprecipitated as antigen-antibody complexes. Contrarily, the latter fusion protein produced in the presence of warfarin demonstrated a decrease in the collection rate, suggesting that their autoantibodies recognized the light chain and regions containing Gla residues. Since all three patients were essentially negative for FX inhibitors, it was concluded that the anti-FX autoantibodies for these cases were predominantly non-neutralizing. The concentration of the FX antigen also significantly reduced in these patients, suggesting that anti-FX autoantibodies promote the clearance of FX. CONCLUSION: Immunological anti-FX autoantibody detection is highly recommended to ensure that AiF10D cases are not overlooked, and to start necessary immunosuppressive therapies.

Using imlifidase to elucidate the characteristics and importance of anti-GBM antibodies produced after start of treatment.

BACKGROUND: Autoantibodies are common in glomerulonephritis, but the clinical benefit of rapid elimination has not been determined, even in anti-glomerular basement membrane (GBM) disease. Even less is known about the importance of autoantibody characteristics, including epitope specificity and immunoglobulin G (IgG) subclass distribution. We aimed to address this by characterizing the autoantibody profile in anti-GBM patients: we utilized samples from the GOOD-IDES-01 (treating GOODpasture's disease with Imunoglobulin G Degrading Enzyme of Streptococcus pyogenous) (ClinicalTrials.gov identifier: NCT03157037) trial , where imlifidase, which cleaves all IgG in vivo within hours, was given to 15 anti-GBM patients. METHODS: In the GOOD-IDES-01 trial, plasmapheresis was (re)started if anti-GBM antibodies rebounded. Serum samples were collected prospectively for 6 months and analyzed for anti-GBM epitope specificity using recombinant constructs of the EA and EB epitopes, IgG subclass using monoclonal antibodies, and anti-neutrophil cytoplasmic antibodies (ANCA). The results were correlated with clinical data. RESULTS: Patients with a rebound (n = 10) tended to have lower eGFR at 6 months (11 vs 34 mL/min/1.73 m2, P = .055), and patients with dialysis at 6 months had a higher EB/EA ratio at rebound (0.8 vs 0.5, P = .047). Moreover, two patients demonstrated increasing epitope restriction and several patients displayed a shift in subclass distribution at rebound. Six patients were double positive for ANCA. ANCA rebound was seen in 50% of patients; only one patient remained ANCA positive at 6 months. CONCLUSIONS: In this study, rebound of anti-GBM antibodies, especially if directed against the EB epitope, was associated with a worse outcome. This supports the notion that all means should be used to eliminate anti-GBM antibodies. In this study ANCA was removed early and long-term by imlifidase and cyclophosphamide.

Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study.

BACKGROUND: The previous study investigated whether the functions of small, medium, and large high density lipoprotein (S/M/L-HDL) are correlated with protein changes in mice. Herein, the proteomic and functional analyses of high density lipoprotein (HDL) subclasses were performed in humans and rats. METHODS: After purifying S/M/L-HDL subclasses from healthy humans (n = 6) and rats (n = 3) using fast protein liquid chromatography (FPLC) with calcium silica hydrate (CSH) resin, the proteomic analysis by mass spectrometry was conducted, as well as the capacities of cholesterol efflux and antioxidation was measured. RESULTS: Of the 120 and 106 HDL proteins identified, 85 and 68 proteins were significantly changed in concentration among the S/M/L-HDL subclasses in humans and rats, respectively. Interestingly, it was found that the relatively abundant proteins in the small HDL (S-HDL) and large HDL (L-HDL) subclasses did not overlap, both in humans and in rats. Next, by searching for the biological functions of the relatively abundant proteins in the HDL subclasses via Gene Ontology, it was displayed that the relatively abundant proteins involved in lipid metabolism and antioxidation were enriched more in the medium HDL (M-HDL) subclass than in the S/L-HDL subclasses in humans, whereas in rats, the relatively abundant proteins associated with lipid metabolism and anti-oxidation were enriched in M/L-HDL and S/M-HDL, respectively. Finally, it was confirmed that M-HDL and L-HDL had the highest cholesterol efflux capacity among the three HDL subclasses in humans and rats, respectively; moreover, M-HDL exhibited higher antioxidative capacity than S-HDL in both humans and rats. CONCLUSIONS: The S-HDL and L-HDL subclasses are likely to have different proteomic components during HDL maturation, and results from the proteomics-based comparison of the HDL subclasses may explain the associated differences in function.

The value of PLA2R antigen and IgG subclass staining relative to anti-PLA2R seropositivity in the differential diagnosis of membranous nephropathy.

BACKGROUND: The diagnostic performance of PLA2R and IgG subclass staining of kidney biopsies relative to anti-PLA2R seropositivity in the differentiation of primary and secondary membranous nephropathy (pMN, sMN) was examined. Besides PLA2R staining - which has a lower specificity than anti-PLA2R antibody serology - there is insufficient knowledge to decide which IgG1-4 subtype immunohistological patterns (IgG4-dominance, IgG4-dominance/IgG1-IgG4-codominance or IgG4-dominance/IgG4-codominance with any IgG subtype) could be used to distinguish between pMN and sMN. METHODS: 87 consecutive Hungarian patients (84 Caucasians, 3 Romas) with the biopsy diagnosis of MN were classified clinically as pMN (n = 63) or sMN (n = 24). The PLA2R and IgG subclass staining was part of the diagnostic protocol. Anti-PLA2R antibodies were determined by an indirect immunofluorescence test in 74 patients with disease activity. RESULTS: For pMN, the sensitivity of anti-PLA2R seropositivity was 61.1%, and the specificity was 90.0%; and similar values for PLA2R staining were 81.0%, and 66.7%, respectively. In all stages of pMN, IgG4-dominance was the dominant subclass pattern, while the second most frequent was IgG3/IgG4-codominance. The sensitivity and specificity scores were: IgG4-dominance 52.2% and 91.7%, IgG4-dominance/IgG3-IgG4-codominance 76.2% and 87.5%, IgG4-dominance/IgG1-IgG4-codominance 64.2% and 75%, and IgG4-dominance/codominance with any IgG subclass 92.1% and 70.8%, respectively. Anti-PLA2R seropositivity, glomerular PLA2R, and IgG4-dominance/codominance significantly correlated with each other. The IgG4 subclass was rarely encountered in sMN. CONCLUSION: In our series, IgG4-dominance had the highest specificity in the differentiation of MN, just as high as that for anti-PLA2R seropositivity. The specificity values of PLA2R staining and IgG4-dominance/codominance with any IgG subclass or IgG4-dominance/IgG1-IgG4 codominance were ≤ 75%. Apart from IgG4 dominance, IgG4-dominance/IgG3-IgG4-codominance also had good statistical value in differentiating pMN from sMN. As IgG subclass switching during the progression of pMN was not the feature of our cohort, pMN in Hungarian patients is presumed to be an IgG4-related disorder right from the start. Although anti-PLA2R seropositivity has become the cornerstone for diagnosing pMN, if a kidney biopsy evaluation is conducted, besides the staining of PLA2R antigen, the evaluation of IgG subclasses provides relevant information for a differential diagnosis. Even in cases with IgG4-dominance, however, malignancy should be thoroughly checked.

Immunoglobulin G deposition on proximal tubules and the tubular basement membrane in acute tubular injury complicated with focal segmental glomerulosclerosis (FSGS): A possible prediction tool for subclinical FSGS.

Immunofluorescent deposition of immunoglobulin G (IgG) in the tubular basement membrane (TBM) has been evaluated in the diagnosis of various diseases; however, few studies have investigated the immunofluorescence of acute tubular injury (ATI). Herein, we attempted to clarify IgG expression in the proximal tubular epithelium and TBM in ATI due to various causes. Patients with ATI with nephrotic-range proteinuria, including focal segmental glomerulosclerosis (FSGS, n = 18) and minimal change nephrotic syndrome (MCNS, n = 8), ATI with ischemia (n = 6), and drug-induced ATI (n = 7), were enrolled. ATI was evaluated by light microscopy. CD15 and IgG double staining and IgG subclass staining were performed to evaluate immunoglobulin deposition in the proximal tubular epithelium and TBM. IgG deposition was identified in the proximal tubules only in the FSGS group. Furthermore, IgG deposition in the TBM was observed in the FSGS group showing severe ATI. IgG3 was predominantly deposited by the IgG subclass study. Our results indicate that IgG deposition in the proximal tubular epithelium and TBM suggests the leaking of IgG from the glomerular filtration barrier and its reabsorption by proximal tubules, which may predict disruption of the glomerular size barrier, including subclinical FSGS. FSGS with ATI should be included as a differential diagnosis when IgG deposition in TBM is observed.

HDL Isolated by Immunoaffinity, Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct.

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.

Defining Clinical and Immunological Predictors of Poor Immune Responses to COVID-19 mRNA Vaccines in Patients with Primary Antibody Deficiency.

Immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines in primary antibody deficiencies (PADs) are largely unknown. We investigated antibody and CD4+ T-cell responses specific for SARS-CoV-2 spike protein (S) before and after vaccination and associations between vaccine response and patients' clinical and immunological characteristics in PADs. The PAD cohort consisted of common variable immune deficiency (CVID) and other PADs, not meeting the criteria for CVID diagnosis (oPADs). Anti-S IgG, IgA, and IgG subclasses 1 and 3 increased after vaccination and correlated with neutralization activity in HCs and patients with oPADs. However, 42% of CVID patients developed such responses after the 2nd dose. A similar pattern was also observed with S-specific CD4+ T-cells as determined by OX40 and 4-1BB expression. Patients with poor anti-S IgG response had significantly lower levels of baseline IgG, IgA, CD19+ B-cells, switched memory B-cells, naïve CD8+ T-cells, and a higher frequency of EM CD8+ T-cells and autoimmunity compared to patients with adequate anti-S IgG responses. Patients with oPADs can develop humoral and cellular immune responses to vaccines similar to HCs. However, a subset of CVID patients exhibit impairment in developing such responses, which can be predicted by the baseline immune profile and history of autoimmunity.

Anti-PD1/PDL1 IgG subclass distribution in ten cancer types and anti-PD1 IgG4 as biomarker for the long time survival in NSCLC with anti-PD1 therapy.

BACKGROUND: Antibodies targeting programmed cell death-1(PD1) and its ligand (PDL1) have revolutionized cancer therapy. However, little is known about the preexisted anti-PD1/PDL1 autoantibodies (AAbs) distribution in multiple cancer types, nor is their potential biomarker role for anti-PD1 therapy. METHOD: Plasma anti-PD1/PDL1 AAb IgG and subclasses (IgG1-4) were detected by enzyme-linked immune sorbent assay (ELISA) in 190 cancer patients, covering 10 cancer types (lung, breast, esophageal, colorectal, liver, prostatic, cervical, ovarian, gastric cancers and lymphoma), the comprehensive correlation of AAbs with multiple clinical parameters was analyzed. We further tested these AAbs in 76 non-small cell lung cancer (NSCLC) samples receiving anti-PD1 therapy, the association of AAbs level with survival was analyzed and validated in an independent cohort (n = 32). RESULTS: Anti-PD1/PDL1 AAb IgG were globally detected in 10 types of cancer patients. IgG1 and IgG2 were the major subtypes for anti-PD1/PDL1 AAbs. Correlation analysis revealed a distinct landscape between various cancer types. The random forest model indicated that IgG4 subtype was mostly associated with cancer. In discovery cohort of 76 NSCLC patients, high anti-PD1 IgG4 was associated with a reduced overall survival (OS, p = 0.019), not progression-free survival (PFS, p = 0.088). The negative association of anti-PD1 IgG4 with OS was validated in 32 NSCLC patients (p = 0.032). CONCLUSION: This study reports for the first time the distribution of preexisted anti-PD1/PDL1 AAb IgG and subclasses across 10 cancer types. Moreover, the anti-PD1 AAb IgG4 subclass was identified to associate with OS, which may serve as a potential biomarker for anti-PD1 therapeutic survival benefit in NSCLC patients.

Overexpression of IgG2 in patients resembling IgG4-related disease with normal IgG4.

IgG4-Related Disease (IgG4-RD) results from tissue infiltration by IgG4-expressing plasma cells and lymphocytes, leading to fibrosis and organomegaly. Clinical presentation is remarkably variable according to organ involvement, and high IgG4 serum concentration, initially considered a diagnostic hallmark of IgG4-RD, tends to be forgone as an indispensable criterion for its diagnosis; it can indeed be absent in some patients, highlighting the diversity of presentation of this dysimmune condition. Nevertheless, elevation of IgG4 serum concentration in suggestive settings remains an argument in favour of IgG4-RD, and while other IgG subclasses can be elevated, this biological feature lacks any diagnostic value. We retrospectively studied 9 patients (5 females, 4 males, 31-81 years old) for whom a diagnosis of IgG4-RD had been considered, based on clinical, imaging or histological criteria, but appeared to display abnormally high serum IgG2 while IgG4 levels were normal. Increased serum IgG1 in one case and increased IgG3 in another one were also noticed. Immunohistochemical analyses of intracellular immunoglobulins could be performed on tissue lymph node biopsies from 2 patients, which demonstrated strong infiltration with IgG2-expressing plasma cells. Thus, overexpression of IgG2 subclass may highlight cases of dysimmune disorders resembling IgG4-RD, although the disease trigger might be different, notably infectious. We suggest measuring all serum IgG subclass levels in patients with features consistent with IgG4-RD.