Regulation of the orientation of the IgG and IgM molecule at the interface

Pathological immunoglobulins (IgG from patients with multiple myeloma and IgM from patients with Waldenstr?m macroglobulinemia) have been shown to possess hydrophylic-lipophylic balance (HLB) which differed from normal Ig HLB. HLB deficiency in pathological proteins was due to the increase of hydrophobic area at the surface of protein globe, which was the reason for different normal and abnormal Ig orientations at the aqueous NaCl solution--air interface. The normal IgG and IgM had horizontal orientation while abnormal ones had vertical orientation. Both normal and abnormal Ig changed their orientation in monolayers as a result of sodium deoxycholate processing. The change in orientation depended on protein molecules interaction with single molecules or micelles of sodium deoxycholate.     

Changes in the orientation of mouse myeloma immunoglobulin G in monolayers after treatment with sodium deoxycholate

Molecules of normal mouse IgG are oriented horizontally in monolayers at air-water interface unlike the molecules of mouse IgG1 kappa secreted by MOPC-21 myeloma which have vertical orientation. Sodium desoxycholate processing of both preparations at concentrations below the critical micelle concentration resulted in abnormal IgG1 kappa preservation and normal IgG acquisition of vertical orientation in monolayers. When sodium desoxycholate was used for IgG modification at concentration higher than the critical micelle concentration both normal and abnormal IgG had horizontal orientation in monolayers.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

Identical nature of the differences between normal and myeloma IgG in the mouse and man determined by the monolayer method

The surface denaturation kinetics of mouse normal IgG and IgGl kappa secreted by myeloma MOPC-21 was studied in monomolecular layers at the air-water interface. Based on the denaturation kinetics data the orientation of the native IgG molecules was determined relative to the interface surface, which turned out to be horizontal for normal IgG and vertical for myelomic ones. As regards the orientation in the monolayers and the rate of surface denaturation, the mouse normal IgG were found to be similar to normal IgG from other species. Like human myelomic IgG, MOPC-21 IgGl kappa differed from normal IgG in both the orientation and lesser native structure stability.     

Normal cellular and humoral immunologic parameters in the baboon (Papio ursinus) compared to human standards

Normal adult Chacma baboons were investigated with reference to their basic immunological parameters, including serum immunoglobulin concentrations, proportions of T- and B-lymphocytes in the peripheral blood, and lymphocyte response to activators. As rabbit antihuman antisera to immunoglobulins were used for the serum immunoglobulin determinations, cross-reactivity between human and baboon immunoglobulins was evaluated, and it was found that human and baboon IgG and IgM were both completely cross-reactive, while IgA was partially cross-reactive. The serum immunoglobulin concentrations, proportions of T- and B-lymphocytes, and response to activators were found to be similar to those of man. These findings indicate that the Chacma baboon would be a useful and relevant model for the study of cellular and humoral immunology.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Immunofluorescence analysis of IgA binding by human mononuclear cells in blood and lymphoid tissue

The nature of IgA-binding cells and their tissue distribution was examined by an indirect immunofluorescence assay with the use of IgA1 and IgA2 paraproteins and fluorochrome- or biotin-labeled F(ab')2 fragments of idiotype-specific antibodies. The frequency of IgA-binding mononuclear cells was approximately 13% in blood and spleen samples but less than 1% in tonsil samples. IgA binding could be visualized by flow immunocytometry on monocyte/macrophages, but not on T and B cells. IgA polymers were bound better than IgA dimers and monomers. Nonhomologous IgA myelomas of both IgA1 and IgA2 subclasses inhibited the IgA-binding to monocytes, whereas aggregated normal serum IgG, IgM paraproteins, and an IgG myeloma did not. IgA binding was relatively insensitive to changes in temperature or cation concentration. IgA-binding monocytes were found in IgA-deficient patients at the same frequency as in normal individuals. The results indicate that monocytes constitutively express class-specific binding sites for both IgA1 and IgA2 molecules.     

Effect of the heterogeneity of persons vaccinated, with respect to the state of their immune system, on the intensity of the immune response

Prior to the immunization of children aged 3-7 years with parotitis vaccine the state of their immune system was evaluated by the determination of the concentration of IgM, IgG and IgA, the ratio and absolute numbers of T- and B-lymphocytes, the intensity of the blast transformation of lymphocytes. This study revealed that the group of immunized children was essentially heterogeneous with respect to the state of their immune system: in 79.9% of the children all immunological characteristics were normal, in 20.1% of the children some deviations from the characteristics considered to be normal for their age (gammopathy, eosinophilia, the deficiency of T- and B-lymphocytes, a decrease in the intensity of their blast transformation, the leftward shift of the leukocytic formula) were noted. The intensity of humoral immune response in children with deviations from normal characteristics peculiar to their age was significantly lower than in children with normal immunological characteristics. On the basis of these results, the immunological rehabilitation of the risk group among the children to be immunized is recommended.     

Serum N-Glycans: A New Diagnostic Biomarker for Light Chain Multiple Myeloma

The aim of this study was to evaluate the diagnostic and differential diagnostic power of serum N-glycans for light chain multiple myeloma (LCMM). A total of 167 cases of subjects, including 42 LCMM, 42 IgG myeloma, 41 IgA myeloma, and 42 healthy controls were recruited in this study. DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) was applied to determine the quantitive abundance of serum N-glycans. The core fucosylated, bisecting and sialylated modifications were analyzed in serum of LCMM patients (n=20) and healthy controls (n=20) randomly selected from the same cohort by lectin blot. Moreover, serum sialic acid (SA) level was measured by enzymatic method. We found two N-glycan structures (NG1A2F, Peak3; NA2FB, Peak7) showed the optimum diagnostic efficacy with area under the ROC curve (AUC) 0.939 and 0.940 between LCMM and healthy control. The sensitivity and specificity of Peak3 were 88.1% and 92.9%, while Peak7 were 92.9% and 97.6%, respectively. The abundance of Peak3 could differentiate LCMM from IgG myeloma with AUC 0.899, sensitivity 100% and specificity 76.2%, and Peak7 could be used to differentiate LCMM from IgA myeloma with AUC 0.922, sensitivity 92.9% and specificity 82.9%. Serum SA level was significantly higher in patients with LCMM than that in healthy controls. Moreover, the decreased core fucosylation, bisecting and increased sialylation characters of serum glycoproteins were observed in patients with LCMM. We concluded that serum N-glycan could provide a simple, reliable and non-invasive biomarker for LCMM diagnosis and abnormal glycosylation might imply a new potential therapeutic target in LCMM.     

Complement-binding activity of myeloma and normal immune complexes

The comparison of complement-fixing capacity of simulated immune complexes formed by normal IgG and IgG, isolated from serum of patients with multiple myeloma, has been performed. In both cases a non-linear dependence of complement-fixing capacity on the complex molecular mass was demonstrated, it being higher for myeloma proteins. Complement supplementation to high molecular complexes leads to their collapse, with normal immune complexes destroyed at lower molecular masses. Heat-aggregation of myeloma immunoglobulins leads to the formation of simulated immune complexes of lower molecular mass compared to normal proteins.     

Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4(+) T cells or CD8(+) T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens.     

Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzyme-neutralizing antibodies in both serum and secretory IgA, and also in serum IgG

Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces.     

68例多发性骨髓瘤的临床特点及实验室检查回顾性分析

目的：探讨68例多发性骨髓瘤MM患者的临床首发症状,实验室相关检查特点。方法：回顾分析我院2002年1月-2012年3月期间68例MM患者临床表现及实验室相关资料。结果：68例MM患者中临床表现具有多样性,细胞形态学检查中骨髓瘤细胞比例大于20％的占76％,骨髓瘤细胞比例小于20％的占24％,骨髓中成熟红细胞呈缗钱状排列者占78％,血清蛋白电泳中53例患者出现M蛋白,免疫球蛋白IgG增高15例,IgA增高11例,轻链型4例,X线检查结果48例提示骨质有异常改变。结论：临床表现和实验室检查对明确诊断MM及早期治疗都有很重要的意义。     

Cellular and humoral immunodeficiency in children with vaccine-associated paralytic poliomyelitis

Markers of humoral and cellular immunity in 16 patients with vaccine-associated paralytic poliomyelitis (VAPP) were evaluated. Signs of immunodeficiency (decrease of T- and B-lymphocytes counts, impaired synthesis of immunoglobulins, defects of phagocytosis, decrease of NK number) were revealed in all of the patients. Majority of them (81.3%) had defects in humoral immunity. Decrease of CD31, CD4+ and CD8+ was detected in 86.7, 35.7 and 91.7% of the patients respectively. Study of serum immunoglobulins performed in 15 patients showed decrease of IgG, IgM and IgA levels in 6 (40%), 1 (6.7%) and 6 (40%) of the patients respectively. Agammaglobulinemia was diagnosed in one patient in which only trace quantities of IgA and IgG were detected and IgM level was well below the normal. Congenital deficiency of IgA was diagnosed in 3 children. Majority of the children (11 from 12) had comorbidities (frequent respiratory infections, dermatitis, changes of intestinal microflora). Thus, immunocompromised condition of a child is a risk factor for VAPP after administration of alive oral poliovaccine.     

Glycosylation of IgG B cell receptor (IgG BCR) in multiple myeloma: relationship between sialylation and the signal activity of IgG BCR

Little is known about the glycosylation of the isotype switched B cell receptor (BCR) in multiple myeloma, and the way it might affect receptor function. In this work IgG BCRs isolated from the individual lysates of peripheral blood lymphocytes (PBL) of 32 patients with IgG multiple myeloma and healthy controls were investigated for the expression of sialic acid (SA), galactose (Gal) and N-acetylglucosamine (GlcNAc), the sugars known to specify the glycoforms of human serum IgG. The degree of glycosylation and signaling status of all 32 isolated myeloma IgG BCRs were correlated and compared with the glycosylation of the IgG paraproteins isolated from sera of the same patients. It was shown that BCR IgG in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of IgG BCR is associated with higher levels of tyrosine phosphorylation (signaling activity) of the IgG BCR supramolecular complex and that BCR IgG and serum IgG paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. The results suggest that the development of the malignant clone in MM from post-switch B cells expressing IgG BCR at their surfaces to plasma cells secreting IgG paraprotein may be followed by permanent glycosylation changes in the IgG molecules.     

Mixed IgA-IgG aggregates as a model of immune complexes in IgA nephropathy

Patients with IgA nephropathy have circulating immune complexes containing IgA, IgG, and C3. We have mixed human IgG and IgA1 and heated them to form mixed aggregate. On sucrose density gradients IgG aggregates were 11 to 19S whereas IgA aggregates were either 11S or greater than 19S. Mixed aggregates had both an 19 and 11 S peak. The isoelectric point of aggregates with only IgG was 7 to 9 and of only IgA 4.5 to 5.5. The isoelectric point of mixed aggregates decreased as the percent IgA increased. IgG aggregates mixed with normal human serum caused 30% C3 activation (20 min, 37 degrees C) whereas IgA aggregates causes no activation. There was a linear decrease in C3 activation as the percent IgA increased. Mixed aggregates that contained either radiolabeled IgG or IgA were mixed with normal human serum (1 h, 37 degrees C) and then solubilized, reduced, and separated by 10% SDS-PAGE. Heavy m.w. bands, consistent with covalent bonding of C3b and C3bi to Ig H chain were only seen in lanes with labeled IgG. This was confirmed by Western blot analysis. A human dimeric IgA1 myeloma protein with rheumatoid factor activity was also studied. It caused 15% alternative pathway C3 activation but did not fix C3 to its H chain. Binding of aggregates (+/- C3) to E was tested. Aggregates with IgG C3 bound but IgA (+/- C3) did not. Addition of greater than 10% IgA to an IgG-C3 aggregate inhibited E binding. We conclude that IgG in mixed aggregates is the site of C3 fixation. In contrast, IgA does not fix C3 but instead lowers the isoelectric point, increases the size and inhibits binding to E. These properties would inhibit clearance and promote mesangial deposition and local C activation.     

Characterization of anti-Cryptosporidium IgA antibodies in sera from immunocompetent individuals and HIV-infected patients.

Human antibody response to Cryptosporidium parvum has been previously shown as involving immunoglobulin (Ig)M and IgG isotypes. The interest in anti-cryptosporidial IgA antibody response has been recently stimulated by studies on the therapeutic effects of secretory IgA antibodies to Cryptosporidium in animal models and in patients. In the present study, isotypes of serum anti-Cryptosporidium antibodies have been characterized in donors of the following categories: (a) healthy adults, (b) healthy children, (c) immunocompetent children with transient cryptosporidial diarrhea, (d) HIV-infected patients without clinical and parasitological evidence of Cryptosporidium infection and (e) AIDS patients with cryptosporidial diarrhea. Antibodies were detected using C. parvum oocysts purified by density gradient centrifugation from bovine faeces. The IgA antibodies were revealed using alpha-chain specific antibodies. Indirect immunofluorescence analysis with oocysts was used as control. Although high levels of serum antibodies of the IgA class were detected in some donors in the group of healthy adults, elevated values were consistently found in HIV-infected patients. Higher values were found in HIV patients with clinical cryptosporidiosis. The presence of a secretory component in serum IgA antibodies in these patients has been documented. Data indicate that IgA serum antibodies are produced as well as IgM and IgG antibodies upon contact with the parasite, and suggest that elevated IgA serum antibodies to Cryptosporidium are not associated with protection in HIV patients.     

白癜风患儿抗核抗体、免疫球蛋白及补体检测的临床分析

目的:探讨白癜风患儿中抗核抗体(ANA)、免疫球蛋白(IgG、IgA、IgM)以及补体(C3、C4)的表达及临床意义。方法:收集2014年10月至2016年5月我院收治的30例白癜风患儿为病例组,并于同期随机选取30例健康体检儿童为对照组,患儿的血清ANA、12种自身抗体谱分别采用间接免疫荧光法及免疫印迹法进行检测,IgG、IgA、IgM,C3、C4水平采用免疫透射比浊法进行检测。结果:病例组中ANA阳性率为13.33%(4/30),与对照组的3.33%(1/30)比较,差异无统计学意义(P0.05)。12种自身抗体谱中,分别有1例(3.33%)患儿dsDNA、SmDNA、SS-A/Ro60KD、SS-B/La、CENP-B阳性,与对照组比较,差异均无统计学意义(P0.05)。病例组患儿血清IgG、IgA、C4水平低于对照组,差异有统计学意义(P0.05)。ANA阳性患儿血清IgG、IgA、IgM水平高于ANA阴性患儿,而补体C3、C4水平低于ANA阴性患儿,差异有统计学意义(P0.05)。结论:白癜风患儿ANA阳性表达与健康儿童无明显区别,体液免疫功能有明显异常,临床有重要的参考价值。     

IgD Plasma Cell Neoplasia: Clinical Manifestations and Characteristic Features

Three patients, one with plasma cell leukemia and clinically asymptomatic hypernephroma and meningioma, and two others with multiple myeloma, had M-components of IgD/λ type. In the first case, IgD globulin was found in the serum, ascitic and pleural fluids. Including our patients, 50 cases of IgD myeloma have been reported in the literature. A review of this group showed some significant differences from the other classes of multiple myeloma. IgD myeloma seems to involve a larger proportion of younger people, 66% being less than 59 years of age. The involvement of internal organs and renal damage were more frequent in IgD myeloma than in other classes. Serum total protein was frequently not increased, the relative concentration of M-component was often low and in 12% there was no spike in electrophoresis. The diagnosis therefore was sometimes difficult. In a quarter of the cases Bence Jones proteinemia was found and in 15% there were multiple spikes, both these manifestations being rare in IgG or IgA classes of myeloma. In 89%, IgD globulin had lambda type light chain, clearly contrasting with the figure of approximately 30% in other classes. Bence Jones protein was found in the urine in 91%. The survival time seemed to be shorter than in other myelomas.     

Case report: Immunoglobulin A deficiency in patients with juvenile rheumatoid arthritis treated with aspirin

In three patients with juvenile rheumatoid arthritis, serum IgA concentrations were within the normal limit at the onset of disease and before aspirin administration. After aspirin administration, their serum IgA levels gradually decreased. After discontinuation of aspirin, their serum IgA levels gradually increased. These results suggest that IgA deficiency may be due to aspirin administration in such patients. The IgA production in vitro of peripheral blood mononuclear cells from a patient taken 3 months of discontinuation of aspirin was markedly inhibited by preincubation with aspirin. Since the patients' serum IgG and IgM levels hardly changed the heavy chain class switching may be influenced by aspirin through some undefined mechanisms.Abbreviations CH heavy chain constant region - ³H-TdR ³H-thymidine - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - JRA juvenile rheumatoid arthritis - MEM minimum essential medium - PBMCs peripheral blood mononuclear cells - PBS phosphate buffered saline - PFCs plaque forming cells - PWM pokeweed mitogen - SI stimulation index