Glomerular Immune Deposition in MPO-ANCA Associated Glomerulonephritis Is Associated With Poor Renal Survival.

Background: Rapidly progressive glomerulonephritis caused by antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is typically characterized as pauci-immune glomerulonephritis. However, immune complex (IC) deposition in the glomerulus has been reported in a growing number of studies. Here, we assess the presence of glomerular immune deposits alongside renal outcome in myeloperoxidase (MPO)-ANCA associated glomerulonephritis (MPO-ANCA GN). Methods: Clinical and histopathologic characteristics of 97 patients with MPO-ANCA GN classified by renal biopsy from January 2008 to December 2019 were extracted retrospectively from electronic medical records. The extent of immune deposits in the kidney (C3, C4, C1q, IgA, IgG, IgM) at diagnosis were analyzed by immunofluorescence (IF). Patients were followed up for a median period of 15 months. The response to treatment and outcomes of renal and histological lesion changes were also assessed. Results: In our study, 41% (40/97) of patients showed positive IF (≥2+) for at least one of the six immunoglobulin or complement components tested. Patients with IC deposits showed higher levels of serum creatinine (p=0.025), lower platelet counts (p=0.009), lower serum complement C3 (sC3) (≤790 ml/L) (p=0.013) and serum IgG (p=0.018) than patients with pauci-immune (PI) deposition at diagnosis. End-stage renal disease was negatively associated with eGFR (HR 0.885, 95% CI 0.837 to 0.935, p<0.0001), platelet count (HR 0.996, 95% CI 0.992 to 1.000, p=0.046) and serum globulin (HR 0.905, 95% CI 0.854 to 0.959, p=0.001). Patients with lower sC3 levels showed a worse renal outcome than the patients with normal sC3 at diagnosis (p=0.003). Analysis of the components of the renal deposits found that patients with IgG deposits exhibited a poorer renal outcome compared to patients that were IgG negative (p=0.028). Moreover, Bowman's capsule rupture occurred less frequently in patients with IgM deposition compared with IgM negative counterparts (p=0.028). Vascular lesions and granuloma-like lesions had been seen more frequently in cases with IgA deposition than those without IgA deposition (p=0.03 and 0.015, respectively). Conclusion: In conclusion, patients with immune complex deposits in the kidney showed less platelet count, lower sC3 and sIgG levels, and higher serum creatinine levels. Patients with low sC3 at initial and with continued low sC3 during the treatment displayed a trend toward poorer kidney survival. Moreover, the IC group showed a worse renal outcome than the PI group, further enforcing the present strategy of introducing complement targeted therapies in AAV.

Analysis of the B cell receptor repertoire in six immune-mediated diseases.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review.

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.

Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology.

BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.

Chain-specific antibodies for laminin-511.

Mouse monoclonal antibodies (mAbs) that bind to specific chains of laminin-511 (LM-511) have been developed. Antibody 2F12 binds to the LMα5 chain, 3G10 binds to the LMß1 chain and 3C12 binds to the LMγ1 chain. These antibodies can be used to purify LM-511, to detect LM-511 in cell extracts or to detect the location of LM-511 in tissue by immunohistochemistry. In combination, the antibodies recognize all three chains of LM-511 and combinations of the antibodies can be used to quantitate levels of LM-511 in physiological fluids. One of the antibodies (3G10) is a potent inhibitor of the activity of LM-511 in cell adhesion, spreading and proliferation assays.

Microcalorimetry of blood serum proteome: a modified interaction network in the multiple myeloma case.

Hereby we report on a novel approach in the study of multiple myeloma (MM), namely, differential scanning calorimetry (DSC) combined with serum protein electrophoresis. Distinct thermodynamic signatures describe the DSC thermograms of MM blood sera, in contrast to the unique profile found for healthy individuals. The thermal behavior of MM sera reflects a complex interplay between the serum concentration and isotype of the M protein and of albumin, and modified ligand- and/or protein-protein interactions, resulting in stabilization of globulins and at least a fraction of albumin. In all MM cases the 85 °C, transferrin-assigned transition is missing. A distinct feature of IgG isotype (κ and λ) DSC profiles only is the presence of a transition at 82 °C. A DSC-based classification of MM depicts two sets of melting patterns (MMt2 and MMt3 with two or three successive thermal transitions), and subsets within each set (MMt2(i) or MMt3(i), the subscript i = 1, 2 or 3 denotes the main transition being one of the three transitions). The results demonstrate the potential of DSC to monitor MM-related modifications of the serum proteome, even at low M protein concentrations, Bence Jones and importantly nonsecretory multiple myeloma cases, and prove DSC as a versatile tool for oncohematology.

Quantification of immune deposits in renal diseases.

In this series of renal diseases, in addition to semiquantitative scoring of direct immunofluorescein images, the immune deposits were quantified by image analysis. The aim of this study was to evaluate quantitative measurements for diagnosis and prognosis of renal immune complex diseases. Immunoglobulin A (IgA) nephropathy (n=27, 54%), membranous nephropathy (n=8, 16%), membranoproliferative glomerulonephritis (n=8, 16%), and systemic lupus erithematosus nephritis cases (n=7, 14%) were evaluated by semiquantitative scores (SS) for IgG, IgA, IgM, C3, C1q, λ, and κ. The quantitative measures, intensity, mean and total optical density (MOD and TOD) were determined by image analysis software. There was positive correlation between SS; and intensity as well as TOD for 199 positive stained images, but not between SS and MOD. TOD was important for determining SS by linear regression. When all of the cases were considered, creatinin at the time of biopsy was only slightly correlated with intensity and TOD of IgM. Intensity and TOD, but not SS of IgA was significantly increased in IgA nephropathy cases with adverse histopathologic prognostic features. In 4 cases (8%) only TOD allowed identification of the predominantly deposited antibody. TOD and intensity seems to have better correlation with prognostic histopathologic features than SS. TOD may be useful for determining predominant immune deposit, a feature important for diagnosis.

Generation and characterization of a high-affinity monoclonal antibody for MUC1 measurement in breast cancer.

Breast mucin is secreted by breast tumor cells and serves as a marker for breast cancer. Thus, antibodies against breast mucin will be valuable in the development of immunotherapy and laboratory diagnostic tests. Monoclonal antibodies (MAbs) against breast cancer-associated antigen were generated and characterized. Balb/c mice were immunized with breast cancer-associated antigen CA15-3, and subsequently splenocytes from immunized mice were fused with myeloma cells. After fusion, culture supernatants from hybridomas surviving HAT medium were screened by enzyme-linked immunosorbent assay (ELISA). A total of eight hybridomas producing MAbs against breast cancer showed significant levels of antibody activity against CA15-3. Two selected stable hybridomas were adapted into CELLine CL 350 bioreactors, and the MAbs produced were characterized for their subclass, specificity, and affinity. The MAbs were of high specificity and affinity as shown by ELISA. The MAbs produced may represent a powerful tool and are considered promising reagents for use in diagnosis and detection of early stage of the disease.

Generation and characterization of anti-chitinase monoclonal antibodies.

Class IV chitinase, an allergenic protein of Vitis vinifera (grape), was purified by anion exchange chromatography and used for immunization of Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells. Finally after three limiting dilutions, six stable clones were generated. Antibody isotyping showed that IgG(2a), IgG(2b), and IgM were produced by one, two, and three of the clones, respectively. All of the MAbs had kappa light chain. The affinities were in the range of 3 × 10(8) to 1.2 × 10(9) M(-1). The MAbs were specific for grape chitinase as confirmed by Western blotting. In conclusion, we successfully produced several MAbs against grape class IV chitinase, which could be used for assessment of this allergen in different grape cultivars.

Characterization of a monoclonal antibody to erythroid related factor.

We describe here a novel IgG monoclonal antibody to erythroid-related factor (ERAF), also known as alpha hemoglobin stabilizing protein (AHSP) and eryththroid differentiation related factor (EDRF). Our antibody named PCE 5 is an IgG(1) kappa chain and is to the peptide sequence MVTVVE ranked highly in our active site analysis and binds with high affinity to ERAF.

Patterns of monoclonal immunoglobulins and serum free light chains are significantly different in black compared to white monoclonal gammopathy of undetermined significance (MGUS) patients.

Monoclonal gammopathy of undetermined significance (MGUS), the precursor to multiple myeloma, is more common in blacks than whites. The serum free light chain (sFLC) assay is an important prognostic test in MGUS, but no study has evaluated sFLC levels and ratios in black MGUS patients. One-hundred and twenty-five black MGUS patients at two urban centers were compared to the white population of the Mayo Clinic. The median age for blacks was 73 years [41-94] and 75% were male. The M-protein isotype in blacks was 81% IgG, 13% IgA, 2% IgM, and 4% biclonal compared to 70%, 12%, 16%, and 2%, respectively, in whites, (P < 0.0005). The median M-protein concentration for blacks was 0.44 gm/dL (trace-2.33) compared to 1.2 gm/dl in whites. An abnormal sFLC ratio was present in 45% of black compared to 33% of white (P = 0.01) patients. Using the Mayo Clinic risk model, black patients had a significantly lower proportion of higher risk MGUS compared to whites: low 43%, low-intermediate 45%, high-intermediate 10%, and high 2% (P = 0.014). Black patients with MGUS have significantly different laboratory findings compared to whites. The biologic basis for these disparities and their effect on prognostic assessment is unknown. Prognostic models based on these biomarkers should be used cautiously in nonwhite populations.

Distribution and clinical features of primary immunodeficiency diseases in Chinese children (2004-2009).

Two hundred and one patients have been diagnosed with primary immunodeficiency diseases (PIDs) in our center from January 2004 to December 2009. The male-to-female ratio was 5.29:1. Spectrums of PIDs were as follows: predominantly antibody deficiency disease was the most common category (94 patients, 48.2%), followed by other well-defined immunodeficiency syndromes (40 patients, 20.5%), combined T and B cell immunodeficiencies (33 patients, 16.9%), congenital defects of phagocyte number and/or function (21 patients, 10.8%), and diseases of immune dysregulation (six patients, 3.1%). Agammaglobulinemia was the most frequent disease type. The median of diagnosis lag was 18.0 months. Pneumonia was the most common manifestation of PID patients. Some manifestations were prone to concentrate in certain diseases. As for therapy, 99 patients (50.8%) received intravenous immunoglobulin replacement therapy; 13 patients received hematopoietic stem cell transplantation and nine of them were still alive. In this study, we sought to describe and analyze the distribution, clinical features, and therapy methods of PIDs among children diagnosed in our country and to compare with reports from other countries and regions.

Antigen aggregation decides the fate of the allergic immune response.

Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis.

The reptiles are the last major group of jawed vertebrates in which the organization of the IGH locus and its encoded Ig H chain isotypes have not been well characterized. In this study, we show that the green anole lizard (Anolis carolinensis) expresses three Ig H chain isotypes (IgM, IgD, and IgY) but no IgA. The presence of the delta gene in the lizard demonstrates an evolutionary continuity of IgD from fishes to mammals. Although the germline delta gene contains 11 C(H) exons, only the first 4 are used in the expressed IgD membrane-bound form. The mu chain lacks the cysteine in C(H)1 that forms a disulfide bond between H and L chains, suggesting that (as in IgM of some amphibians) the H and L polypeptide chains are not covalently associated. Although conventional IgM transcripts (four C(H) domains) encoding both secreted and membrane-bound forms were detected, alternatively spliced transcripts encoding a short membrane-bound form were also observed and shown to lack the first two C(H) domains (VDJ-C(H)3-C(H)4-transmembrane region). Similar to duck IgY, lizard IgY H chain (upsilon) transcripts encoding both full-length and truncated (IgYDeltaFc) forms (with two C(H) domains) were observed. The absence of an IgA-encoding gene in the lizard IGH locus suggests a complex evolutionary history for IgA in the saurian lineage leading to modern birds, lizards, and their relatives.

Salivary anti-PGL IgM and IgA titers and serum antibody IgG titers and avidities in leprosy patients and their correlation with time of infection and antigen exposure.

The present work proposed to correlate serum antibody avidity and salivary antibody titers as parameters for time of infection and antigen exposure in a cohort study evaluating leprosy patients in different periods of treatment. Colorimetric enzyme-immunoassays for salivary antibodies, serum antibody IgG titers and avidities were performed in the samples. Anti-PGL-1 IgA and IgM salivary antibodies were significantly higher in multibacillar (MB-L) patients compared to normal controls (p<0.05), but not when compared to borderline tuberculoid (BT) or to paucibacillar (PB-L) patients (p>0.05). A good correlation was found between salivary anti-PGL-1 IgA and IgM levels in MB-L patients (r=0.41, p<0.01). Two out of 33 tested saliva samples from patients who had completed the drug regimen treatment presented positive salivary antibodies. Among non-treated patients, samples with low, medium or high serum IgG antibody avidity were found in similar frequencies. In patients under treatment, most of the serum samples showed low or medium IgG antibody avidity. The treated MB-L patients showed medium or high antibody avidity, except for two, who showed very low antibody avidity results. We suggest that salivary anti-PGL antibodies and serum IgG avidity could be useful for the indication of recent exposure or re-exposure to bacteria after chemotherapy.

Salivary anti-PGL IgM and IgA titers and serum antibody IgG titers and avidities in leprosy patients and their correlation with time of infection and antigen exposure

The present work proposed to correlate serum antibody avidity and salivary antibody titers as parameters for time of infection and antigen exposure in a co-hort study evaluating leprosy patients in different periods of treatment. Colorimetric enzyme-immunoassays for salivary antibodies, serum antibody IgG titers and avidities were performed in the samples. Anti-PGL-1 IgA and IgM salivary antibodies were significantly higher in multibacillar (MB-L) patients compared to normal controls (p<0.05), but not when compared to borderline tuberculoid (BT) or to paucibacillar (PB-L) patients (p>0.05). A good correlation was found between salivary anti-PGL-1 IgA and IgM levels in MB-L patients (r=0.41, p<0.01). Two out of 33 tested saliva samples from patients who had completed the drug regimen treatment presented positive salivary antibodies. Among non-treated patients, samples with low, medium or high serum IgG antibody avidity were found in similar frequencies. In patients under treatment, most of the serum samples showed low or medium IgG antibody avidity. The treated MB-L patients showed medium or high antibody avidity, except for two, who showed very low antibody avidity results. We suggest that salivary anti-PGL antibodies and serum IgG avidity could be useful for the indication of recent exposure or re-exposure to bacteria after chemotherapy.

Retrospective study of monoclonal gammopathies detected in the clinical laboratory of a Spanish healthcare district: 14-year series.

BACKGROUND: We studied the incidence, classification and isotype distribution of monoclonal gammopathies and M-protein detected between 1992 and 2005 inclusive in the clinical laboratory of a healthcare district in Madrid (Spain) with an average population of 280,574 inhabitants. METHODS: Serum electrophoresis was carried out on a cellulose acetate support up until 1997, and then using capillary zone electrophoresis systems, with M-protein identification carried out by agarose gel immunofixation. The age-adjusted incidences were standardized with respect to the WHO World Standard Population Distribution, based on the world average population between 2000 and 2025. The clinical diagnosis was recorded from the patient case history. RESULTS: M-protein was detected in a total of 537 patients; of these, 42 had been diagnosed before 1992, representing a 0.19% prevalence in our population. The mean age-adjusted incidence of monoclonal gammopathy was 10.72 per 100,000 inhabitants/year (SE 1.31), ranging from 4.85/100,000 in 1992 to 14.28/100,000 in 2003 and 2004. The median patient age at diagnosis was 73 years (range 25-96 years), with males accounting for 46.8% of all cases of monoclonal gammopathy, and 57.8% of all malignant monoclonal gammopathies. A total of 54.1% of the patients were clinically defined as presenting monoclonal gammopathy of undetermined significance, 31.3% presented multiple myeloma, and the remaining 14.6% presented malignant gammopathies. The most frequent M-protein isotype was IgG (55.8%), followed by IgA (20.8%) and IgM (13.6%). A total of 88% of the light chain M-proteins, 54% of isotype IgM, 51% of isotype IgA and 36% of isotype IgG were associated with B lymphoproliferative diseases. CONCLUSIONS: We conclude that the clinical laboratory should play an important role in the study of monoclonal gammopathies, since it is the only location where all M-protein patients are observed. On the other hand, studies of this type should be carried out over long-term periods, owing to the variations we have noted in the detection of M-proteins.

Sera from mice immunized with DNA encoding Porphyromonas gingivalis catalytic or adhesin part of HRgpA inhibit degradation of human fibronectin by HRgpA.

Gingipains are potent virulence factors of Porphyromonas gingivalis and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination could be a useful therapy for adult periodontitis. This study investigated the ability of antibodies raised against the catalytic part and the adhesin/haemagglutinin part of HRgpA to prevent haemagglutination and fibronectin degradation caused by P. gingivalis. We constructed two DNA vaccines, one containing the adhesin part of HRgpA and one with the catalytic part of HRgpA. BALB/c mice were immunized intramuscularly with either catalytic-part-encoding plasmids, adhesin-part-encoding plasmids or empty control plasmids. Sera from mice immunized with the catalytic vaccine or the adhesin vaccine each showed inhibition of human fibronectin degradation. A DNA vaccine encoding the adhesin or catalytic part of HRgpA induces responses that inhibit the degradation of molecules important for the structure and function of gingival and bone tissues.