IgM Complexes Research Articles

Immunoglobulin M-based local production in skin-associated lymphoid tissue of flounder (Paralichthys olivaceus) initiated by immersion with inactivated Edwardsiella tarda

Fish skin, the mucosal site most exposed to external antigens, requires protection by an efficient local mucosal immune system. The mucosal reserve of IgM is recognized as an immune strategy that blocks pathogen invasion to maintain homeostasis, whereas the mechanism of skin-associated local IgM production induced by mucosal antigens is not well know. In this study, we found that the skin of flounder (Paralichthys olivaceus) was equipped with the immune cellular and molecular basis for processing mucosal antigens and triggering local specific responses, i.e., CD4+ Zap-70+ T cells, CD4− Zap-70+ T/NK cells, IgM+ MHCII+ B cells, PNA+ MHCII+ antigen-presenting cells, UEA-1+ WGA+ and UEA-1+ WGA− antigen-sampling cells, as well as secreted IgM and pIgR, as demonstrated by indirect immunofluorescence assay using different antibodies and lectins. After immersion immunization with inactivated Edwardsiella tarda, qPCR assay displayed up-regulation of immune-related genes in flounder skin. Flow cytometry analysis and EdU labeling demonstrated that the mucosal inactivated vaccine induced local proliferation and increased amounts of cutaneous IgM+ B cells. Skin explant culture proved the local production of specific IgM in the skin, which could bind to the surface of E. tarda. ELISA, laser scanning confocal microscopy, and Western blot revealed that, in addition to the elevated IgM levels, pIgR protein level was significantly up-regulated in skin tissue and surface mucus containing the pIgR (secretory component, SC)-tetrameric IgM complex, indicating that mucosal vaccine stimulated up-regulation of IgM and pIgR, which were secreted as a complex into skin mucus to exert the protective effects as secretory IgM. These findings deepen the understanding of IgM-based local responses in the mucosal immunity of teleosts, which will be critical for subsequent investigation into the protective mechanism of mucosal vaccines for fish health.

A-138 Implications of native protein conformation in the IgM quantification: total Ig vs HLC methods comparison

Abstract Background For IgM monoclonal gammopathies such as Waldenström Macroglobulinemia, the diagnosis, risk stratification and monitoring response to therapy is based on the detection and quantification of the secreted IgM monoclonal protein. Accurate IgM measurement is challenging due to its complex nature and the occurrence in higher polymeric structures (i.e., pentamers or hexamers), as well as heterogeneity and inherent subjectivity of the selected common methodologies. The recently developed Heavy/Light chain assay (Binding Site, part of Thermofisher Scientific) allows the separate determination of the IgM-Kappa and IgM-Lambda molecules, offering an alternative method to the serum electrophoresis (SPE) with reported higher sensitivity (Ref. Boyle E. et al, Clin Cancer Research 2016). In this study we explore the effect of the molecular structure on 3 different analytical techniques for accurate IgM measurements Methods 214 serum samples from patients (age median 63yo) under follow-up at our hospital were tested for: IgG, IgA, and IgM levels (immunoturbidimetry, either on a Beckman Coulter AU5800 analyser; Nyon, Switzerland; or a Cobas c 702 system, Roche Diagnostics); Hevylite assay (on an Optilite, Binding Site); and SPE and Immunofixation (Capillarys 3 Octa and Hydrasys 2, Sebia Inc.). The effect of the molecular conformation was evaluated through sample depolymerization with N-Acetylcysteine (NAC) according to the manufacturer’s instruction (Sebia Inc.) and IgM quantification by: tIgM (immunoturbidimetric total Ig); red-Ig (idem after NAC reduction); and, HLC-Ig (i. e. sum of the IgM-K+IgM-L heavy/light chain pair measured by Hevylite (Binding Site)). Results Total-Ig, red-Ig, and HLC-Ig returned similar results for IgG and IgA immunoglobulins (Kruskal-Wallis test, p-value > 0.05), and Passing Bablok regression analysis showed good agreement between the three methods, except for higher IgA levels. In the later, both HLC-IgA levels red-IgA values were higher than total-IgA. Regarding the IgM, PB regression revealed a significant proportional bias, with obtained values approx. 2,5x higher by red-IgM and approx. 1,4x by HLC-IgM methods than tIgM (red-IgM=-5.5+2.42*tIgM, Spearman’s r=0.995; HLC-IgM=-0,77+1.34*tIgM, Spearman’s r=0.978; red-IgM=-6.29+1.82*HLC-IgM, Spearman’s r=0.98). So, both red-IgM and HLC-IgM results are higher than total IgM. Of note, 1 sample presented antigen excess, resulting in unusually low IgM levels by tIgM but high red-IgM and high HLC-IgM levels coherent with the patient’s clinical condition. 75 samples had monoclonal protein measurable by SPE, showing good correlation with the three methods, however with greater proportional errors between measurements with other techniques Conclusions IgM molecular conformational status may affect the epitopes exposed and, therefore, available for the different assays’ recognition. Higher IgM complexes depolymerization into monomeric conformation results in higher IgM levels, which agree better with the Hevylite than with the total IgM measured previous NAC reduction, reflecting the impact of the native protein conformation in the IgM precise detection. Although also affected by the conformational status, Hevylite IgM results were closer to the reduced IgM levels than the obtained by the total IgM assay, therefore offering a sensitive assay that does not need a reduction treatment for a more accurate IgM quantification, optimizing the laboratory workflow

Antibody feedback regulation.

Antibodies are able to up- or downregulate antibody responses to the antigen they bind. Two major mechanisms can be distinguished. Suppression is most likely caused by epitope masking and can be induced by all isotypes tested (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgE). Enhancement is often caused by the redistribution of antigen in a favorable way, either for presentation to B cells via follicular dendritic cells (IgM and IgG3) or to CD4+ T cells via dendritic cells (IgE, IgG1, IgG2a, and IgG2b). IgM and IgG3 complexes activate complement and are transported from the marginal zone to follicles by marginal zone Bcells expressing complement receptors. IgE-antigen complexes are captured by CD23+ B cells in the blood and transported to follicles, delivered to CD8α+ conventional dendritic cells, and presented to CD4+ T cells. Enhancement of antibody responses by IgG1, IgG2a, and IgG2b in complex with proteins requires activating FcγRs. These immune complexes are captured by dendritic cells and presented to CD4+ T cells, subsequently helping cognate B cells. Endogenous feedback regulation influences the response to booster doses of vaccines and passive administration of anti-RhD antibodies is used to prevent alloimmunization of RhD-negative women carrying RhD-positive fetuses.

Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b

Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.

Low levels of squamous cell carcinoma antigen–IgM complexes in serum are predictors of better survival in patients with liver cirrhosis

Low levels of squamous cell carcinoma antigen–IgM complexes in serum are predictors of better survival in patients with liver cirrhosis

Biomarkers of IgA vasculitis nephritis in children.

Henoch–Schönlein purpura is a systemic vasculitis characterized by IgA deposits, which target the skin, joints, and kidneys, among other organs. In children, prognosis is often good but little is known about biomarkers of pediatric nephritis. We hypothesized that biological markers, including cytokines, immunoglobulins, IgA-immune complexes, IgA glycosylation and neutrophil gelatinase-associated lipocalin (NGAL), may discriminate IgA vasculitis (IgAV) pediatric patients with renal involvement from those without renal involvement. Fifty children at the time of IgAV rash between 2010 and 2015 were prospectively enrolled and compared to 21 controls. All patients were assessed for clinical and biological parameters at the time of diagnosis, including the levels of cytokines, immunoglobulins, immune complexes, IgA glycosylation and NGAL in serum and urine. Among IgAV patients, 33 patients exhibited nephritis (IgAV-N) and 17 children were without nephritis (IgAV-woN). The serum level of galactose-deficient (Gd)-IgA1 (p<0.01) and the urinary concentrations of IgA, IgG, IgM, IL-6, IL-8, IL-10, IgA-IgG complexes and IgA-sCD89 complexes (p<0.001 for all) were higher in the IgAV-N patients than in the IgAV-woN patients. Among those markers, urinary IgA and IgM had the highest AUC (0.86 and 0.87 respectively, p<0.0001). This prospective cohort study furthers our understanding of the pathophysiology of IgAV. We identified biomarkers that are able to distinguish patients initially with or without nephritis. To conclude, serum Gd-IgA1 and urinary IgA, IgG, IgM, IL-6, IL-8, IL-10, and IgA-IgG and IgA-sCD89 complexes could identify IgAV pediatric patients with renal involvement at the time of diagnosis.

Value of biomarkers for predicting immunoglobulin A vasculitis nephritis outcome in an adult prospective cohort.

Henoch-Schönlein purpura, more recently renamed immunoglobulin A vasculitis (IgAV), is a systemic vasculitis characterized by IgA deposits. The current markers used to assess IgAV inaccurately evaluate the risk of nephritis occurrence and its long-term outcomes. The current study assessed biomarkers of nephritis outcomes. This French multicentre prospective study enrolled 85 adult patients at the time of disease onset. Patients were assessed for clinical and biological parameters and re-examined after 1 year. Immunoglobulins, cytokines, IgA glycosylation, IgA complexes and neutrophil gelatinase-associated lipocalin (NGAL) concentrations were assessed in blood and urine. We identified 60 patients with IgAV-related nephritis (IgAV-N) and 25 patients without nephritis (IgAV-woN). At the time of inclusion (Day 1), the serum levels of galactose-deficient IgA1 (Gd-IgA1) and urinary concentrations of IgA, IgG, IgM, NGAL, interleukin (IL)-1β, IL-6, IL-8, IL-10, IgA-IgG and IgA-sCD89 complexes were higher in the IgAV-N patients than in the IgAV-woN patients (P < 0.005 for all comparisons). After follow-up (1 year), 22 patients showed a poor outcome. Among the tested markers, urine IgA at disease onset adequately reclassified the risk of poor outcome over conventional clinical factors, including estimated glomerular filtration rate, proteinuria and age (continuous net reclassification improvement = 0.72, P = 0.001; integrated discrimination improvement = 0.13, P = 0.009) in IgAV patients. Taken together, these results showed that serum Gd-IgA1 and urinary IgA, IgG, IgM, NGAL, IL-1β, IL-6, IL-8, IL-10, IgA-IgG and IgA-sCD89 complexes were associated with nephritis in IgAV patients. Urinary IgA level may improve patient risk stratification for poor outcome.

A peptide extension dictates IgM assembly

Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-μ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension.

Vascularite cutanée induite par l’amiodarone

A wide variety of drugs can cause cutaneous vasculitis. Herein we report a case of immune complex vasculitis induced by amiodarone. A 57-year-old patient reported a recent history of pruritus associated with large erythematous, inflammatory, necrotic plaques localized on the lower limbs and back. These cutaneous lesions had appeared less than 2months after initiation of amiodarone for supra-ventricular arrhythmia. Histological and direct immunofluorescence examinations of a skin biopsy sample revealed vasculitis with the presence of IgM and C3immune complexes in vessels. The remaining laboratory tests were unremarkable (in particular, cryoglobulin and autoantibody tests were negative). The patient himself attributed his symptoms to the recent administration of amiodarone and spontaneously stopped the drug without medical advice. No other therapy was prescribed. Following drug withdrawal, the lesions that had been present for more than 4months completely disappeared. No recurrence occurred after follow-up of over 6months. The diagnosis of amiodarone-induced vasculitis was retained. Fewer than 10cases of amiodarone-induced vasculitis have been reported in the medical literature. It is not known whether this entity is rare, under-diagnosed or under-reported.

Circulating SCCA–IgM complex is a useful biomarker to predict the outcome of therapy in hepatocellular carcinoma patients

Introduction: Hepatocellular carcinoma (HCC) develops in about 3–4% of cirrhotic patients every year. The squamous cell carcinoma antigen (SCCA) has been found elevated in liver cancer specimens by immunohistochemistry, and detected in complex with IgM (SCCA-IgM) in the serum of patients with HCC. The aim of this study was to evaluate the ability of serological SCCA-IgM levels to predict the efficacy of HCC therapy.Materials and methods: From April 2012 to April 2014, 131 patients with a new diagnosis of HCC were enrolled. The HCC diagnosis was made according to the EASL guidelines. The patients were staged and treated according to the BCLC Staging System: BCLC stages A and B were treated with locoregional therapy, and BCLC stage C was treated with Sorafenib. Response to therapy was evaluated according to the mRECIST criteria. Serum SCCA-IgM levels were determined by a commercially available ELISA kit at basal time (T0) and after one month of treatment (T1).Results: At baseline and one month into therapy, SCCA-IgM levels were significantly lower (p value <.05) in patients who responded to therapy compared to those who did not respond (median SCCA-IgM level [25th + 75th percentile] at T0:115.1 AU/mL [50.0 + 174.4] vs. 149.1 AU/mL [111.3 + 198.8]; median SCCA-IgM level [25th + 75th percentile] at T1: 113.4 AU/mL [50.0 + 194.2] vs. 170.6 AU/mL [111.7 + 344.2]).Conclusion: Our study suggests that the SCCA-IgM determination could be helpful in predicting the response to therapy in patients with HCC.

Delivery route determines the presence of immune complexes on umbilical cord erythrocytes

Objective: Umbilical cord blood offers a unique opportunity to study the basal level of immunoglobulin complexes. This study aims to determine the presence of immune complexes and complement deposition on erythrocytes from umbilical cord blood from normal, full-term pregnancies. Methods: In vitro pre-formed IgA, IgG, and IgM complexes were used as positive control for flow cytometry detection, and for C3d deposition. Blood samples (34) of umbilical cord blood taken from vaginal and cesarean deliveries were tested for the presence of immunoglobulin complexes. Results: Fourteen samples from vaginal deliveries and 20 samples from cesarean deliveries were assessed. IgG and IgM complexes were detected on erythrocytes, whereas no IgA complexes or complement deposition was observed. Interestingly, the percentage of IgG complexes was higher on erythrocytes from vaginal delivery samples compared to those from cesarean deliveries. No other associations between immune complexes and other maternal or newborn variables were found. Conclusions: IgG and IgM complexes seem to be normally present on umbilical cord erythrocytes. Erythrocytes from vaginal deliveries have a higher percentage of IgG complexes present compared to that from cesarean deliveries. Since no C3d activity was detected, these complexes are non-pathological and should be part of the newborn’s initial innate immune response.

Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals.

Cryoglobulins (circulating immune complexes of polyclonal IgG, monoclonal IgM, and rheumatoid factor) are detected in the circulation of 40% to 60% of patients with chronic hepatitis C virus infection, and cryoglobulinemic vasculitis (CV) is observed in approximately 10% of patients. We aimed to assess the clinical and immune effects of direct-acting antiviral treatment. We performed a prospective study of 64 patients with HCV infection with circulating cryoglobulins receiving direct-acting antiviral therapy at a single center in Barcelona, Spain, from January 2014 through April 2016. Patients were classified as having CV (n= 35) or asymptomatic circulating cryoglobulins (ACC, n= 29). Clinical response was considered complete if a patient's Birmingham Vasculitis Activity Score (version 3) was 0, or if all affected organs improved 12 weeks after the end of therapy. A complete immunologic response (CIR) was defined as no detection of circulating cryoglobulins and normalized levels of complement and/or rheumatoid factor. Clinical manifestations of CV included purpura (65%), weakness (70%), arthralgia (31%), myalgia (20%), peripheral neuropathy (50%), and renal involvement (20%). At baseline, patients with CV had significantly higher levels of rheumatoid factor and lower levels of C4 complement than patients with ACC, whereas cryocrits were similar between groups (3.2% vs 2.6%). Overall, 60 patients (94%) had a sustained viral response 12 weeks after therapy. Among patients with CV, the median Birmingham Vasculitis Activity Score (version 3) decreased from 9 (range, 2-31) to 3 (range, 0-12) (P < .001). Twenty-five patients with CV (71%) achieved a complete clinical response. Immune-suppressive therapy was reduced for 4of 13 patients and withdrawn for 6 of 13. Overall, 48% of patients achieved a CIR. A low baseline cryocrit level (<2.7%) was the only factor associated with CIR (odds ratio, 9.8; 95% confidence interval, 2.2-44; P= .03). Viral eradication was associated with clinical improvement in most patients with CV. Markers of immune activation, including circulating cryoglobulins, persisted in 52% of patients with CV or ACC, despite a sustained viral response 12 weeks after therapy. A longer follow-up period after viral eradication might be necessary to ensure a normal immune response.

Role of IgM and angiotensin II Type I receptor autoantibodies in local complement activation in placental ischemia-induced hypertension in the rat.

Preeclampsia is characterized by development of hypertension during pregnancy and reduced placental perfusion. Previous studies in a rat model of placental ischemia-induced hypertension demonstrated that inhibiting complement activation attenuated increased maternal blood pressure with C3a and C5a identified as the important products of complement activation. Given that in other forms of ischemia both natural IgM and antigen antibody complexes initiate complement activation, we hypothesized that placental ischemia exposes neoepitopes recognized by IgM to cause local complement activation and hypertension. Alternatively, we postulated that autoantibody to angiotensin II Type 1 receptor (AT1-AA) interacts with AT1 receptors to cause complement activation. Since complement activation occurs in kidney and placenta in preeclampsia, we used immunohistochemistry to determine IgM deposition and local complement activation in each organ (C3 deposition), and quantitative real-time polymerase chain reaction (qRT-PCR) to quantitate mRNA for endogenous regulators of complement activation CD55, CD59 and Complement receptor 1-related gene/protein y (Crry). On gestation day (GD)14.5, timed pregnant Sprague Dawley rats underwent Sham surgery or placement of clips on inferior abdominal aorta and ovarian arteries to create placental ischemia using the reduced utero-placental perfusion pressure (RUPP) model. As previously reported, RUPP surgery increased mean arterial pressure and circulating C3a on GD19.5. In placenta, IgM and C3 deposition increased, whereas mRNA for complement regulators Crry and CD59 decreased along with Crry protein in RUPP compared to Sham treated animals. In kidney, IgM deposition increased in animals subjected to RUPP vs Sham surgery without a significant change in C3 deposition and coincident with an increase in mRNA for CD55 and CD59. The AT1 receptor antagonist losartan prevents placental ischemia-induced hypertension as well as AT1-AA interaction with AT1 receptors. However, losartan did not attenuate complement activation as measured by circulating C3a or placental C3 deposition. Importantly, our studies indicate that following placental ischemia, complement activation is not due to AT1-AA but is associated with IgM deposition. These studies suggest a role for natural antibodies interacting with placental ischemia-induced neoepitopes to activate complement and contribute to hypertension.

Squamous cell carcinoma antigen-IgM is associated with hepatocellular carcinoma in patients with cirrhosis: A prospective study

BackgroundSquamous cell carcinoma antigen (SCCA)-IgM complex has been described as a promising tool to identify patients with progressive liver disease at higher risk of hepatocellular carcinoma (HCC) development in retrospective studies. AimTo assess the clinical value of this biomarker in patients with cirrhosis in a prospective study. MethodsPatients with overt cirrhosis were prospectively evaluated at 6-month intervals for HCC development and decompensation with clinical examination, liver ultrasound, α-fetoprotein measurement. SCCA-IgM was measured in serum by immunoenzymatic assay. Median follow-up duration was 52 months (range 12–68 months). Results70 patients (26% male; mean age 56±10 years) were enrolled. The main aetiological factors were alcohol (44%) and hepatitis C (34%). Baseline values of SCCA-IgM were significantly higher in patients who developed HCC. Positivity of the biomarker at baseline was associated with a significantly shorter HCC-free survival, while α-fetoprotein (cut off >20ng/ml) was not significant. SCCA-IgM positivity and hepatitis C were significant prognostic factors for HCC development. The biomarker was not associated with the development of clinical complications of cirrhosis. ConclusionThis prospective study demonstrates that in patients with cirrhosis SCCA-IgM is associated with HCC development and may be useful for clinical management of cirrhotic patients at higher risk of HCC development.

P214: Study of sympathetic skin response in diabetic patients with soft tissue rheumatism of the hand

P214: Study of sympathetic skin response in diabetic patients with soft tissue rheumatism of the hand

Antibodies that bind complex glycosaminoglycans accumulate in the Golgi

Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) gene junction of the variable (Vλx) editor L chain. When paired with the anti-DNA heavy chain, VH56R, the Vλx variants yield antibodies with differing specificities, including glycosaminoglycan reactivity. Our results implicate these specificities in the evasion of receptor editing through intracellular sequestration of IgM and the release of insoluble IgM complexes. Our findings can be extrapolated to human L chains and have implications for understanding a latent component of the Ig repertoire that could exert pathogenic and protective functions.

AB0695 Anti-prothrombin/phosphatidyl-serine complex autoantibodies in antiphospholipid syndrome: Preliminary data using a new ELISA method

BackgroundNew emerging anti-phospholipid (aPL) assays could improve our ability in diagnosing anti-phospholipid syndrome (APS). However, the availability of aPL assays in routine laboratory practice is limited and present available assays...

Identification of CMTM7 as a Transmembrane Linker of BLNK and the B-Cell Receptor

BLNK is a pivotal adaptor protein in the signal transduction pathway from the IgM class B-cell receptor. BLNK is phosphorylated by Syk and binds various signaling intermediates, leading to cellular events including MAP-kinase activation, culminating in cellular activation. It remains unclear how BLNK is initially recruited to the surface IgM (sIgM) complex to which Syk is also recruited. Here we show that CMTM7, a tetra-spanning membrane protein of unknown function, co-localized with clathrin and sIgM at the plasma membrane. RNA-interference-mediated knockdown of CMTM7 expression in B cells resulted in an impairment of sIgM-ligation-induced tyrosine phosphorylation of BLNK, which was due to an impaired interaction of BLNK and Syk, and in a failure to activate JNK and ERK, but not upstream kinases such as Src-family kinases and Syk. CMTM7 was bound to BLNK in a membrane fraction, and their association was augmented after sIgM ligation. Exogenous CMTM7 or a mutant with an N-terminal deletion (ΔN), but not one with a C-terminal deletion (ΔC) that is defective in membrane localization, were able to restore BLNK-Syk binding, BLNK phosphorylation and ERK activation in the CMTM7-knockdown B cells. In addition, CMTM7 and the ΔN, but not the ΔC, were constitutively associated with sIgM, and this binding was required for BLNK recruitment to sIgM. From these data, we conclude that CMTM7 functions to link sIgM and BLNK in the plasma membrane, to recruit BLNK to the vicinity of Syk, and to initiate the BLNK-mediated signal transduction.

Alleviation of IgM Monoclonal Protein Interference in Nephelometric Assays by Sample Treatment With Reducing Agent in a Chaotropic Salt Solution: Table 1

In some patients, the presence of monoclonal IgM proteins can cause substantial false elevation in nephelometric assays. A 59-year-old woman with previously unrecognized Waldenström macroglobulinemia had high serum viscosity. Nephelometric analysis revealed elevated measured concentrations of IgG, IgA, and IgM. Pretreatment with a reducing salt solution yielded resolution of cross-linked immunoglobulins into a distinct IgM κ monoclonal protein band by immunofixation electrophoresis. Measured quantitative immunoglobulin measurements in treated and untreated specimens were compared. An approximately 50% drop in IgG and IgA concentrations was observed in the presence of reducing agent, while IgM concentrations were unchanged. We report that the addition of reducing agent solution can alleviate artifactual elevation of nephelometric determination of IgG and IgA concentrations. These results are consistent with dissociation of higher order IgM complexes, leading to reduction of nonspecific light scattering by IgM complexes in IgA and IgG nephelometric reactions.

CD22 serves as a receptor for soluble IgM

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FcγRIIB).