A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

乙型肝炎IgM和IgG—补体双特异性循环免疫复合物的意义

对不同临床类型的乙型肝炎患者,采用捕捉法ＥＬＩＳＡ,以ＩｇＭ和ＩｇＧ类抗体排除抗原性异物的免疫反应进行比较研究。结果发现,两种反应能力在慢性ＨＢＣ感染中基本相同,表现出明显的病型差异,而在急性ＨＢＶ感染中则不同,前者反应强度显著主于后者;二者阳性率在慢性ＨＢＶ感染的临床类型中虽均随其肝损害加重而显著上升,但ＩｇＧ／Ｃ３双特异性循环免疫复合物与ＡＬＴ有关,而ＩｇＭ／Ｃ３－ＴＣＩＣ与ＡＬＴ无关;二者阳     

病毒唑对肾综合征出血热患者特异性抗体应答的影响

在用病毒唑(Ribavirin)治疗肾综合征出血热(Hemorrhagic fever with renal syndrome,HFRS)(以安慰剂作对照)的双盲法临床试验中,用微量酶联免疫吸附技术对确诊的64例患者血清和尿液中特异性IgG及IgM抗体水平进行动态观察与分析,结果发现,病毒唑治疗组两种血清抗体水平均较安慰剂对照组低(IgG,P<0.001;IgM,P<0.05),而两组患者尿液中两种抗体水平与阳性率均无明显差异,表明病毒唑对HFRS患者抗体的生成具有抑制作作用,本文就该作用的原因与后果进行了分析评价。     

Are Patients with Erythema Migrans Who Have Leukopenia and/or Thrombocytopenia Coinfected with Anaplasma phagocytophilum or Tick-Borne Encephalitis Virus?

Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.     

Testing for antibodies to tick-borne encephalitis virus using blood dried on filter paper

Possibility to use blood dried on filter paper for serological testing on antibodies to tick-borne encephalitis (TBE). Sensitivity and specificity of specific IgM detection in dry stains of blood by lanthanide fluorescence immunoassay was 94.9% (86.9-100%) and 97.5% (94-100%) respectively, compared with results obtained in tests of sera. Agreement in positive and negative results of tests for IgM against TBE in 562 serum samples and dry blood stains was 95.3%. During analysis of both types of biomaterial high degree of correlation was observed between intensity of fluorescence when testing for both IgM (r=0.86700; p=0.05; n=562), and IgG (r=0.83883; p=0.05; n=337) toTBE virus. Use of this mildly invasive technique of blood draw is reasonable during conduction of large-scale population studies for seroepidemiologic monitoring, investigation of disease outbreaks, control of effectiveness of vaccination against TBE, assessing of level of specific immunity in population of endemic regions, control of treatment, and serologic diagnostics of acute TBE in hospitalized patients, in which blood draw is difficult to perform.     

Specific antigens of the causative agents and their antibodies in the circulating immune complexes in acute dysentery

The level of circulating immune complexes has been determined in 53 patients in the dynamics of the disease. For the first time circulating immune complexes have been found to contain Shigella sonnei K-antigen and Shigella flexneri O-antigen, as well as IgA, IgG and IgM to Shigella. Shigella antigens can be detected from the first week of the disease, and their occurrence does not depend on the level of circulating complexes in patients blood serum.     

Rapid diagnosis of tick - borne encephalitis by immunofluorescence assay of specific serum IgM antibodies.

The indirect IF technique, using suspensions of TBE virus infected and uninfected PS cells as antigen-containing substrate, furnishes a rapid and practical test making possible the detection of specific IgM class serum antibodies in the initial stage of clinically manifest TBE. It enables early confirmation of diagnosis already in the acute phase of the disease and thus it can be instrumental in differential diagnosis and rational therapy, e.g., the administration of specific hyperimmune gamma-globulin.     

Autogenous Immunity to Endogenous RNA Tumor Virus: Differential Reactivities of Immunoglobulins M and G to Virus Envelope Antigens

The autogenous humoral immune response of mice to their endogenous leukemia virus has been examined in terms of the reactivities of individual classes of antibody present in normal B6C3F(1) serum. Whole serum and the immunoglobulin (Ig) M and IgG fractions of serum from animals of different age groups were compared by radioimmune precipitation assays and viral infectivity neutralization assays. Both IgM and IgG fractions were able to precipitate virus, although not as effectively as whole serum. Virus-specific antibody levels, as well as total antibody concentrations in whole serum, appeared to increase with age. Sodium dodecyl sulfate gel electrophoresis analysis was performed with immune precipitates obtained when whole serum or 19 or 7S fractions from animals of different age groups were reacted with disrupted virus. The 19S antibody fraction reacted with three antigenic determinants on the viral envelope. These antigens have apparent molecular weights of 17,000, 43,000, and 68,000. The last two appear to be glycoproteins and may correspond to the M(2) and M(1) antigens. In contrast, the 7S component reacted only with the 17,000-molecular-weight protein. Neutralization assays against BALB:virus-2, a xenotropic endogenous mouse type C virus, revealed that 19S and whole serum but not the 7S fraction possessed neutralizing activity. These findings indicate that there are differential reactivities of IgM and IgG antibodies in normal serum of B6C3F(1) mice, with respect to both recognition of viral envelope antigens and neutralization of endogenous MuLV. These results are consistent with the hypothesis that the autogenous humoral immune response is a systemic host function that may be important in the regulation of endogenous type C virus expression in vivo.     

The characteristics of detecting the tick-borne encephalitis virus antigen in the ELISA and indirect hemagglutination reaction by means of scanning electron microscopy

The surface of polystyrene plates was studied at different stages of the enzyme immunoassay (EIA) and the passive hemagglutination (PHA) test by the method of scanning electron microscopy in the detection of tick-borne encephalitis (TBE) virus antigen. The study revealed that in the process of EIA larger antigens were washed away from the plate surface. The objects detected on the polystyrene surface were identified as conglomerations of the virions of TBE virus, but whole virions were shown to play no decisive role in EIA. The conclusion was made that, due to some specific features of this method, EIA was more sensitive in reaction with small antigens (individual glycoproteids, their small complexes). And, respectively, the PHA test was more sensitive in reaction with large antigenic complexes (whole virions, their conglomerations, immune complexes).     

Lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs

Amid growing evidence that numerous viral infections can produce immunopathology, including nonspecific polyclonal lymphocyte activation, the need to test the direct impact of an infecting virus on the immune system of the host is crucial. This can best be tested in the isolator piglet model in which maternal and other extrinsic influences can be excluded. Therefore, neonatal isolator piglets were colonized with a benign Escherichia coli, or kept germfree, and then inoculated with wild-type porcine reproductive and respiratory syndrome virus (PRRSV) or sham medium. Two weeks after inoculation, serum IgM, IgG, and IgA levels were 30- to 50-, 20- to 80-, and 10- to 20-fold higher, respectively, in animals receiving virus vs sham controls, although <1% was virus specific. PRRSV-infected piglets also had bronchial tree-associated lymph nodes and submandibular lymph nodes that were 5-10 times larger than colonized, sham-inoculated animals. Size-exclusion fast performance liquid chromatography revealed that PRRSV-infected sera contained high-molecular-mass fractions that contained IgG, suggesting the presence of immune complexes. Lesions, inflammatory cell infiltration, glomerular deposits of IgG, IgM, and IgA, and Abs of all three isotypes to basement membrane and vascular endothelium were observed in the kidneys of PRRSV-infected piglets. Furthermore, autoantibodies specific for Golgi Ags and dsDNA could be detected 3-4 wk after viral inoculation. These data demonstrate that PRRSV induces B cell hyperplasia in isolator piglets that leads to immunologic injury and suggests that the isolator piglet model could serve as a useful model to determine the mechanisms of virus-induced immunopathology in this species.     

A calcium-dependent cryoglobulin IgM kappa/polyclonal IgG.

The mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood. A mixed cryoglobulin IgM kappa/IgG (type II) of a patient with Sj?gren's syndrome was studied because of its unique properties. This cryoglobulin precipitated in serum but not in serum containing 10 mM EDTA. The cryoprecipitation was shown to require calcium (Ca) and was optimal at 1 mM of free Ca. Cryoprecipitation was also induced by Ba, Mn, and Sr, but not by Mg and Co. Purified IgM kappa/IgG complexes precipitated in the presence of Ca, but not IgM kappa alone. There was no significant binding of 45Ca to the purified IgM kappa, IgM kappa/IgG complexes formed with purified components, and the cryoprecipitate. The relative affinity of the radiolabeled [125I]IgM kappa for IgG was 3.6 x 10(3) liters/mol at 37 degrees C as assessed by sucrose density gradient ultracentrifugation, and increased to 1.7 x 10(4) liters/mol at 4 degrees C. The addition of Ca produced no change in the affinity at 37 degrees C and 4 degrees C. The absence of a direct effect of Ca on the Ag/antibody reaction was confirmed in experiments using polyethylene glycol as precipitating agent. In conclusion, two independent steps were responsible for the precipitation of this cryoglobulin. The first step was an efficient formation of soluble immune complexes as produced by a drop in temperature. The second step was caused by a change in the physicochemical conditions--the presence of Ca--which induced polymerization of the IgM kappa/IgG complexes.     

Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination

A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.     

Blood levels of immunoglobulins G,A,M, circulating immune complexes, complement and proteins in patients on intermittent peritoneal dialysis

The study involved 17 patients on IPD. Blood serum levels of IgG, IgA, IgM, circulating immune complexes, complement and proteins were determinated at the beginning of therapy, after 3, 6, 12 months on IPD and after 1 year on hemodialysis. The frequency of peritonitis was noted during this time. Peritonitis was the most frequent during first 3 months on IPD. No differences in blood serum levels of IgG, IgA, IgM, in the specific periods of IPD were noted. A significant increase in blood serum circulating immune complexes in patients on IPD and hemodialysis compared to the control group was found. A significant decrease in blood serum of C3 complement in patients on IPD and hemodialysis in comparison with the controls were found. A significant decrease in blood serum proteins at the beginning of IPD and after 3 months IPD in comparison with proteins concentration in patients on hemodialysis was observed.     

Antigen-induced rheumatoid factors. Protein and carbohydrate antigens induce different rheumatoid factor responses

Rheumatoid factors, autologous IgM anti-IgG, were produced after immunization with protein or carbohydrate antigens. After immunization with either type of antigen, the kinetics of the rheumatoid factor response reflected the kinetics of the dominant IgG isotype in the anti-antigen response. Secondary immunization with protein antigens induced an IgM rheumatoid factor response which was consistently greater than that seen after carbohydrate immunization, and almost exclusively specific for the IgG1 isotype. In contrast, primary or hyperimmunization with carbohydrate antigens gave rise to a more heterogeneous response dominated by IgM anti-IgG3, with lesser amounts of IgM anti-IgG2b and anti-IgG1. Direct immunization with immune complexes gave similar results, as complexes composed of IgG1 induced exclusively IgM anti-IgG1, whereas those complexes made up of IgG3 gave rise to IgM rheumatoid factors binding IgG3 and IgG2b. Rheumatoid factor production, with isotypic specificity defined by the immunizing antigen, appears to be a natural consequence of immunization with a variety of protein and carbohydrate antigens.     

Trypanosoma cruzi: Role of different antibody classes in protection against infection in the mouse

Immune serum obtained from mice with a chronic infection of Trypanosoma cruzi was fractionated on Sephadex G-200 or on protein ASepharose 4B. Mice were infected with a standard infective dose of T. cruzi 24 hr after injection with either IgM, IgG, IgG1, or IgG2 + IgG3 fractions. Mice were also pretreated with immune serum depleted by affinity chromatography of either IgG2a, IgG2b, or both subclasses before infection with T. cruzi. Control mice were pretreated with normal mouse serum or immune serum depleted of IgG. The parasitemia and survival of the animals were determined and used as parameters of protection. The results of these experiments demonstrated that the protective antibodies were mostly IgG2 and seem to be preferentially located in IgG2b subclass. IgM and IgG1 fractions were very little, if any, protection.     

Increased immune complexes of hypocretin autoantibodies in narcolepsy

Background

Hypocretin peptides participate in the regulation of sleep-wake cycle while deficiency in hypocretin signaling and loss of hypocretin neurons are causative for narcolepsy-cataplexy. However, the mechanism responsible for alteration of the hypocretin system in narcolepsy-cataplexy and its relevance to other central hypersomnias remain unknown. Here we studied whether central hypersomnias can be associated with autoantibodies reacting with hypocretin-1 peptide present as immune complexes.

Methodology

Serum levels of free and dissociated (total) autoantibodies reacting with hypocretin-1 peptide were measured by enzyme-linked immunosorbent assay and analyzed with regard to clinical parameters in 82 subjects with narcolepsy-cataplexy, narcolepsy without cataplexy or idiopathic hypersomnia and were compared to 25 healthy controls.

Principal Findings

Serum levels of total but not free IgG autoantibodies against hypocretin-1 were increased in narcolepsy-cataplexy. Increased levels of complexed IgG autoantibodies against hypocretin-1 were found in all patients groups with a further increase in narcolepsy-cataplexy. Levels of total IgM hypocretin-1 autoantibodies were also elevated in all groups of patients. Increased levels of anti-idiotypic IgM autoantibodies reacting with hypocretin-1 IgG autoantibodies affinity purified from sera of subjects with narcolepsy-cataplexy were found in all three groups of patients. Disease duration correlated negatively with serum levels of hypocretin-1 IgG and IgM autoantibodies and with anti-idiotypic IgM autoantibodies.

Conclusion

Central hypersomnias and particularly narcolepsy-cataplexy are characterized by higher serum levels of autoantibodies directed against hypocretin-1 which are present as immune complexes most likely with anti-idiotypic autoantibodies suggesting their relevance to the mechanism of sleep-wake cycle regulation.     

Serodiagnosis of microbiosis in mice with a quantitative assay of immunoglobulins by a microcomputer-introduced ELISA system. 1. Anti-Sendai virus antibodies in naturally infected mouse sera

An enzyme-linked immunosorbent assay system combined with microcomputer data analysis was established as a quantitative assay method of immunoglobulins. The assay system was applied to measure IgG and IgM levels of anti-microbe antibodies in animals, especially mouse and rat. And now the measurement of IgG and IgM levels (ng/ml) of anti-Sendai virus (HVJ) antibodies in naturally infected mice is available. The assay system could improve serodiagnosis in the specificity and sensitivity and in the rapid treatment of many serum samples. The operation of this system was performed by a microcomputer, FM 8 connected Titertek Multiskan MC. The limited sensitivity of this assay for IgG and IgM was 10 ng/ml and 30 ng/ml, respectively. Ninety-one of serum samples were positive for IgG and/or IgM (45 samples for IgG and IgM, 44 samples for IgG, 2 samples for IgM) to Sendai virus in the tested 279 mouse sera, and serum titers were ranged from 1: 10 to 1: 12,800 in the IgG, and from 1: 20 to 1: 160 in the IgM. In these titers, serum IgG and IgM amounts were estimated to be 0.1 to 154 micrograms/ml and 0.5 to 4.8 micrograms/ml, respectively. Relationships of serum titers and antibody amounts were almost consisted, being judged like that approximately 10 micrograms/ml is 1: 400, 30 micrograms/ml is 1: 1,600 in IgG, and 2.4 micrograms/ml is 1: 80, 4 micrograms/ml is 1: 160 in IgM.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.