Complement-fixing Ability Research Articles

IgG3 donor–specific antibodies with a proinflammatory glycosylation profile may be associated with the risk of antibody-mediated rejection after kidney transplantation

The pathogenicity of de novo donor-specific antibodies (dnDSA) varies according to their characteristics. While their MFI, complement-fixing ability, and IgG3subclass are associated with ABMR occurrence and graft loss, they are not fully predictive of outcomes. We investigated the role of the Fc glycosylation of IgG3 dnDSA in ABMR occurrence using mass spectrometry after isolation by single HLA antigen beads. Between 2014 and 2018, we enrolled 54 patients who developed dnDSA (ABMR- n=24; ABMR+ n=30) in two French transplant centers. Fucosylation, galactosylation, GlcNAc bisection, and sialylation of IgG3 dnDSA were compared between ABMR+ and ABMR- patients. IgG3 dnDSA from ABMR+ patients exhibited significantly lower sialylation (7.5% vs. 10.5%, p<.001) and higher GlcNAc bisection (20.6% vs. 17.4%, p=.008). Fucosylation and galactosylation were similar in both groups. DSA glycosylation was not correlated with DSA MFI. In a multivariate analysis, low IgG3sialylation, high IgG3%, time from transplantation to kidney biopsy, and tacrolimus-free regimen were independent predictive factors of ABMR. We conclude that a proinflammatory glycosylation profile of IgG3 dnDSA is associated with a risk of ABMR occurrence. Further studies are needed to confirm the clinical interest of DSA glycosylation and to clarify its role in determining the risk of ABMR and graft survival.

OR21 Clinical significance of C3D binding donor specific anti-HLA antibodies detected by single antigen beads after kidney transplantation

Aim Recent interest in characterizing the complement-fixing ability of post-transplant donor specific alloantibodies (DSA) resulted in the enhancement of Luminex solid phase alloantibody assays (SAB) to determine DSA complement-fixing capacity. Previous studies have reported a significantly worse clinical outcome after kidney transplantation for SAB C1q-binding versus non-C1q-binding DSA. A newly available SAB method detects C3d-binding rather than C1q. The purpose of this study was to compare the clinical outcomes in kidney transplant recipients that developed de novo DSA post-transplant stratified by C3d binding by SAB assay (Lifecodes, Immucor). Methods A cohort of 222 recipients transplanted between 2002 and 2013, with previously identified post-transplant de novo DSA, were retested by Luminex SAB IgG and C3d assay using the Immucor LSA Single Antigen Class I and Class II beads. This 222 patient cohort was 45.2 ± 12.1 (19–74) years old, 63% male ( n = 139), 35% African-American ( n = 77), 33% sensitized pre-transplant ( n = 73), and 11% retransplant recipients ( n = 25). The median time to first rejection episode experienced by 112/222 (50.4%) recipients was 18.8 months (range 8 days to 6.6 years). The median time of sera testing was 35.8 months post-transplant (range 8 days to 10 years). Results In 149/222 (67%) recipients, the SAB C3d assay was positive. Analysis of anti-HLA class specificity revealed 72 (32.4%) of recipients were SAB C3d negative, 16 (7%) were anti-HLA Class I only positive, 111 (50%) were anti-HLA Class II only positive, and 23 (10%) were positive for anti-HLA Class I and Class II. Death censored graft survival after sera acquisition for testing was significantly worse for all SAB C3d positive versus C3d negative recipients. The difference in graft survival was most pronounced when anti-HLA Class I SAB C3d binding was detected. Conclusion We conclude that C3d binding in this SAB assay identifies de novo DSA kidney recipients at highest risk for graft loss. I. Balazs: Employee; Company/Organization; Immucor, Inc.

HLA-C antibodies in women with recurrent miscarriage suggests that antibody mediated rejection is one of the mechanisms leading to recurrent miscarriage

HLA-C is the only polymorphic classical HLA I antigen expressed on trophoblast cells. It is known that higher incidence of C4d deposition on trophoblast cells is present in women with recurrent miscarriage. C4d is a footprint of antibody-mediated classical complement activation. Therefore, this study hypothesize that antibodies against HLA-C may play a role in the occurrence of unexplained consecutive recurrent miscarriage.Present case control study compared the incidence of HLA-C specific antibodies in 95 women with at least three consecutive miscarriages and 105 women with uneventful pregnancy. In the first trimester of the next pregnancy, presence and specificity of HLA antibodies were determined and their complement fixing ability. The incidence of HLA antibodies was compared with uni- and multivariate logistic regression models adjusting for possible confounders.Although in general a higher incidence of HLA antibodies was found in women with recurrent miscarriage 31.6% vs. in control subjects 9.5% (adjusted OR 4.3, 95% CI 2.0–9.5), the contribution of antibodies against HLA-C was significantly higher in women with recurrent miscarriage (9.5%) compared to women with uneventful pregnancy (1%) (adjusted OR 11.0, 95% CI 1.3–89.0). In contrast to the control group, HLA-C antibodies in the recurrent miscarriage group were more often able to bind complement.The higher incidence of antibodies specific for HLA-C in women with recurrent miscarriage suggests that HLA-C antibodies may be involved in the aetiology of unexplained consecutive recurrent miscarriage.

New insights in antibody-mediated rejection.

In the present review, we aim to describe the state of knowledge concerning antibody-mediated rejection (ABMR) spectrum and diagnosis criteria before analyzing the present and future promising leads regarding ABMR prognosis markers and treatment. Recent studies regarding complement-binding donor-specific antibodies and the molecular approach highlighted the unmet need for stratification tools for prognosis and treatment inside ABMR disease. ABMR is the leading cause of kidney allograft failure. The recent expansion of its spectrum is related to the paradigm of a continuous process, leading insidiously to a chronic form of ABMR and to the progressive acknowledgement of new entities (such as vascular ABMR, subclinical ABMR, C4d-negative ABMR). Considering the global picture of ABMR, the Banff classification gradually refined the diagnosis criteria so that it now describes a clinically relevant and coherent entity. Nevertheless, if the diagnosis mainly relies on conventional assessment, such as histological findings and circulating donor-specific antibodies, these criteria face serious limitations in terms of stratification of patients at risk of graft loss inside ABMR disease. Recently, new promising tools have emerged in order to identify long-term outcomes at the time of the diagnosis of rejection. In this regard, donor-specific antibodies' complement-fixing ability and the molecular approach contributed significantly. Currently, however, no clinically relevant surrogate marker of treatment efficiency is currently available.

HLA Specificity and Titer Rather Than Complement Fixing Ability of DSA Would Influence the Incidence of Chronic Antibody Mediated Rejection.

HLA Specificity and Titer Rather Than Complement Fixing Ability of DSA Would Influence the Incidence of Chronic Antibody Mediated Rejection.

Liver transplantation with a strongly positive crossmatch: Case study and literature review

A positive crossmatch has been associated with increased risk in liver transplantation. To study the clinical significance of preformed donor-specific human leukocyte antigen antibodies (DSAs) in liver transplantation, we reviewed patients who underwent liver transplantation with a strongly positive flow cytometry crossmatch. DSAs were evaluated with a Luminex solid phase assay. The complement-fixing ability of DSAs was tested with a complement component 1q (C1q) assay. Using an assay correlation between complement-dependent cytotoxicity crossmatch, flow cytometry crossmatch, and DSA results, we reviewed the effects of DSAs on the outcomes of our patients as well as reported cases in the literature. Five of 69 liver recipients had a strongly positive crossmatch: 4 had a positive T cell crossmatch [median channel shift (MCS) = 383.5 ± 38.9], and 5 had a positive B cell crossmatch (MCS = 408.8 ± 52.3). The DSAs were class I only in 1 patient, class I and II in 3 patients, and class II only in 1 patient. Cholestasis, acute rejection, or both were observed in 3 of the 4 patients with a positive T cell crossmatch with an MCS approximately greater than 300. The C1q assay was positive for 3 patients. Two had either persistent cholestasis or early acute rejection. One patient who was treated with preemptive intravenous immunoglobulin had an unremarkable outcome despite a positive C1q result. One of the 2 patients with a negative C1q assay experienced persistent cholestasis and early and recurrent acute rejection; the other had an unremarkable outcome. None of the patients died or lost a graft within the first year of transplantation. Our study suggests that human leukocyte antigen antibody screening, flow cytometry crossmatch MCS levels, DSA mean fluorescent intensity levels, and C1q assays may be useful in assessing the risk of antibody-mediated rejection and timely interventions in liver transplantation.

Post-Transplant Complement-Binding Donor-Specific Anti-HLA Antibodies Are Strong Predictors of Kidney Allograft Outcome

Background: The key role of anti-HLA antibodies in kidney allograft has become better appreciated. Our aim is to characterize the properties of post-transplant HLA-DSA in term of quantification, ability to fix complement, histologic impact and their association with graft outcome. Methods: This is a population-based study of 321 consecutive kidney transplant patients (01/2005 to 01/2009). All patients were systematically screened for the presence of HLA-DSA by Luminex at day-0, at 1-year post-transplant and at the time of rejection. The sera of patients with evidence of post-transplant DSA were screened for complement-fixing ability by Luminex-based assay that detects C1q binding to DSA (DSA-C1q pos). We recorded for each patient rejection and protocol biopsies performed at 1-year post-transplant. The mean follow-up time was 57±15mo. Results: A total of 110 patients (34.3%) had DSA detected in the first year post-transplant: 60 (18.7%) patients presented pre-existing DSA and 59 (18.4%) de novo DSA. Of these, 28 (25.5%) patients had evidence of post-transplant C1q fixing DSA: 12 against HLA class I, 11 against HLA class II and 5 against HLA class I and class II. The mean MFI of post-transplant DSA-C1q pos was 8500±4711, as compared to 2404±2363 of DSA-C1q neg (p< .001). In patients with post-transplant DSA, the ability to fix C1q was associated with a higher ABMR rate in the first year post-transplant (64.3% vs 13.4%, p< .001), higher microcirculation inflammation score on biopsies (2.4±1.4 vs 0.65±0.9, p< .001) and lower GFR at the end of follow-up (38±19 vs 21.57±19mL/min, p< .001) as compared to patients with DSA-C1q neg. Patients with post-transplant DSA, whether C1q neg or C1q pos had significant decreased graft survival as compared to patients without post-transplant DSA (HR=3.03 and 21.8 respectively, p=.04 and p< .0001). The multivariate Cox model revealed that, when adjusted for clinical, histological Banff parameters and day-0 and post-transplant DSA MFI, the presence of post-transplant DSA-C1q pos was independently associated with higher graft loss (HR= 6.9;CI=1.5-32.6, p=.01). Conclusion: Using an unselected population of well phenotyped patients, we determined that the presence of post-transplant complement-fixing HLA-DSA discriminates a group of patients with increased risk of ABMR and the highest risk of graft loss. The identification of these antibodies allows risk stratification, which should help guide monitoring and therapeutic strategies in sensitized patients.

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Aim Clinical management of renal transplant patients is often a clinical dilemma once they have developed donor specific antibodies (DSA). One of the mechanisms of graft injury by DSA is considered complement-mediated. We evaluated complement-fixing ability of DSA during post-transplant monitoring. Methods Frozen serum samples (N = 17) were selected from 6 patients in whom DSA was detected after kidney transplant, including collections from rising and stable creatinine periods. HLA antibody (IgG) was tested by LABScreen PRA and LABScreen Single Antigen beads (SAB, One Lambda). Complement fixing ability was tested by C1q assay (One Lambda). Donor/recipient HLA typing was tested by serology or SSO/SSP. DSA (IgG), DSA (C1q), creatinine, and biopsy results were correlated. Results From 6 patients (3 pediatrics and 3 adults), 17 serum samples are from 7 points during increasing creatinine and 10 points during stable creatinine. In these patients, the first detection of DSA was from 1.2 to 6 years ago. Specificity was class II only in 4 patients and both class I/II in 2 patients. All 17 data points detected maximum DSA (IgG) at >7000 MFI by SAB, and were also equally positive by C1q assay, regardless the creatinine trend or biopsy results. Corresponding biopsy results were available in 12 samples; C4d and T-cell rejection (N = 3), C4d only (N = 5), T-cell rejection only (N = 4), and neither (N = 0). Thus, 8 of 12 serum samples correlated C1q positivity and C4d positivity (66%). Among the 6 patients with C1q positive DSAs, 4 patients had stable creatinine, chronic antibody rejection was recognized 18 months after C1q detection, and one developed rising creatinine due to T-cell rejection. Conclusions All 17 DSA (IgG) positive samples were also positive by C1q assay, regardless of the creatinine or C4d biopsy status. In vivo responses and consequences of DSA are regulated by complex immune mechanisms, which may not correlate to an in vitro complement fixing assay.

Why human pemphigoid autoantibodies do not trigger disease by the passive transfer into mice?

The recapitulation of disease features in animals by the transfer of patient autoantibodies has been used to demonstrate the autoimmune nature of several diseases. Failure of disease induction by the passive transfer of autoantibodies has been assigned to a limited cross-reactivity of the autoantibodies with the murine tissue. However, the possibility that the passively transferred “inflammatory” patient autoantibodies may not be able to unfold their pathogenic potential due to restricted Fc-dependent effector functions has not yet been systematically explored. In this study we analyze the interaction of patients’ autoantibodies with murine complement and granulocytes. Bullous pemphigoid is a blistering disease associated with autoantibodies, which are thought to induce subepidermal blistering by activating complement and granulocytes. The passive transfer of patients autoantibodies failed to induce skin blistering in wild type mice. The cross-reactivity of pemphigoid autoantibodies with murine antigens was analyzed in silico, ex vivo and by the passive transfer of IgG in vivo. Complement-fixing ability of patients’ autoantibodies was evaluated by complement-binding test. Granulocyte activation was assessed by reactive oxygen species production assay and the cryosection model. We have found that although pemphigoid autoantibodies bound to murine skin in vitro and in vivo, they showed a lower capacity to fix murine complement and a reduced ability to activate murine granulocytes when compared with human complement and cells, respectively. These results indicate that for disease models using the passive transfer of patient autoantibodies, their interaction with the innate factors of the host should be optimized to match the human situation.

Prevalence and qualitative properties of circulating anti–human leukocyte antigen alloantibodies after pregnancy: No association with unexplained recurrent miscarriage

In pregnant women, circulating alloantibodies, triggered by exposure to paternal HLA antigens, are frequently detectable. The finding of lower alloantibody levels in women who experience spontaneous abortion (miscarriage) has led to the speculation that antipaternal antibodies could favor maintenance of pregnancy, whereas their lack poses a risk of miscarriage. Postulating a role of alloantibodies in the pathogenesis of unexplained abortion, we examined whether different categories of recurrent miscarriage (RM) can be distinguished according to prevalence or distinct qualitative properties of anti–human leukocyte antigen (HLA) antibody patterns. Sera obtained from 167 women with RM were assessed for complement- and non–complement-fixing anti-HLA alloreactivity using Luminex-based bead array technology. Women with RM had less often detectable anti-HLA class I and/or II reactivity (19%) compared with a control group of 96 multiparous women without a history of miscarriage (49%). However, analysis of different categories of RM (unknown [ n = 112] versus known cause [ n = 55]; primary [ n = 125] versus secondary RM [ n = 42]) did not reveal any differences regarding antibody prevalence, number of targeted HLA single antigens, antigen specificity, binding density, or complement-fixing ability of detected alloantibodies. Our results do not support a link between anti-HLA antibody formation and RM, and argue against a diagnostic value of alloantibody detection in the diagnostic work-up of women with RM.

Antibodies, isotypes and complement in allograft rejection

Classical complement activation is a key step in the process of antibody-mediated rejection. Emphasizing novel diagnostic strategies, this study will discuss recent studies highlighting the particular relevance of alloantibodies with complement-fixing ability. Reinforcing the pivotal role of complement, numerous studies have shown tight associations of capillary C4d deposition, a 'footprint' of alloantibody-triggered complement activation, with the occurrence of allograft injury. Distribution patterns of immunoglobulin isotypes or subclasses, which strongly differ in their ability to activate complement, may not adequately reflect the actual pathogenetic relevance of detected allosensitization. This fact may be explained by the finding that other variables, such as antibody-binding density or a synergism of antibodies against different epitopes of the same antigen, may contribute to complement activation. An attractive approach to distinguish between complement-fixing and presumably less harmful noncomplement-fixing alloreactivities could be the detection of C4d deposition in vitro. Applying such techniques, recent studies have shown that human leukocyte antigen reactivity with C4d-fixing ability, in contrast to noncomplement-fixing sensitization, may strongly predict antibody-mediated rejection and inferior graft survival. Considering the pivotal role of complement, technologies that uncover the complement-fixing ability of alloantibodies may be of particular interest for the selective detection of deleterious sensitization.

Autoantibodies from mixed cryoglobulinaemia patients bind glomerular antigens.

Mixed cryoglobulinaemia (MC) is a disorder characterized by the presence of large amounts of cryoprecipitating IgM-IgG complexes. An immune complex glomerulonephritis develops in one third of all patients, but its occurrence does not seem related to the amount of cryoglobulins in the sera, nor to their complement-fixing ability. In this study we investigated the presence of IgG antibodies reactive with kidney antigens in 33 MC patients (11 with glomerulonephritis, 22 without renal involvement). A total glomerular extract was run on a 10% acrylamide gel, blotted to nitrocellulose and probed with the patients' sera. Sera from half of the patients without renal involvement reacted with several glomerular antigens whose molecular weight ranged between 200 and 29 kD. In the group with renal involvement, sera from 7/11 patients reacted with an antigen of 50 kD, which is also expressed in thymus, but not in the heart or liver. In a follow-up study of four patients with renal involvement, the amount of serum antibody specific for the 50-kD antigen fluctuated, either spontaneously or in response to therapy. These results show that antibodies specific for glomerular antigens are detectable in MC sera. The immune response against a 50-kD antigen expressed in the kidney and thymus seems to be restricted to a subset of MC patients with renal involvement. Circulating autoantibodies specific for glomerular antigens might contribute to the induction of glomerulonephritis in MC forming immune complexes in situ.

Determinants of the Complement-Fixing Ability of Recipient Presensitization Against HLA Antigens

The presence of preformed alloantibodies with the ability to activate complement may pose a particular risk for kidney allograft rejection. The aim of this study was to evaluate variables that determine the complement-fixing capability of human leukocyte antigen (HLA) sensitization. Sixty-five sensitized patients with > or =10% pretransplant panel-reactive antibody (PRA) levels uncovered by immunoglobulin G [IgG]FlowPRA HLA class I and/or class II screening were included. Applying modified FlowPRA screening, sera were evaluated for patterns of alloreactive IgG subclasses and IgM, and, in parallel, for their complement-activating ability assessed by flow cytometric detection of human complement split product deposition ([C4d]FlowPRA). Approximately two-thirds (68%) of tested sera were found to contain complement-fixing alloreactivity (> or =10%[C4d]FlowPRA). IgG1 type panel reactivity was predominant (detectable HLA class I and II reactivity in 93% and 91% of IgG-positive sera), followed by IgG3 (49%/44%), IgG2 (44%/27%), and IgG4 (19%/11%). Applying partial correlation we found an independent correlation of both %[IgG1]FlowPRA and %[IgG3]FlowPRA with %[C4d]FlowPRA reactivities (P< or =0.01). In addition, for IgG1 a contribution of the amount of bound alloantibody to complement-fixation was observed. Complement-fixation was also favored by the simultaneous presence of alloreactive IgG1, IgG3, and IgM. Previous grafting, but not pregnancy and transfusion, was independently associated with complement-fixing sensitization (P<0.05), presumably due to increased IgG1 type reactivity. Anti-HLA antibody-triggered complement activation is dependent on both the pattern of Ig reactivities and the amount of bound antibody. Previous transplantation represents a major risk factor for the development of complement-fixing sensitization.

Purification and Partial Characterization of an Acidic Polysaccharide with Complement Fixing Ability from the Stems of Avicennia Marina

An acidic polysaccharide fraction that had high anticomplementary activity was isolated from the stems of Grey Mangrove in 0.15% yield. The final fractions was designated HAM-3-IIb-II. The polysaccharide fraction appeared to be homogenous by high performance size exclusion chromatography with an estimated molecular weight of 105 kDa. The isolated polysaccharide is more effective than polysaccharide K (PSK) in its anticomplementary activity at 58 microg/ml of PSK and 23 microg/ml of HAM-3-IIb-II that inhibit 50% of complement activity in the complement fixation assay. Structural studies indicated that HAM-3-IIb-II was rich in galacturonic acid along with arabinose, galactose and rhamnose, characterizing a pectin-type polysaccharide, which was also confirmed by FT-IR spectrum. The presence of rich neutral sugar side chains of arabinogalactans may have contributed to the expression of high activity. Traditionally, this mangrove plant is used for medicinal purposes and it appears to have some scientific applications.

Antibodies against Tumor Cell Glycolipids and Proteins, but Not Mucins, Mediate Complement-Dependent Cytotoxicity

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.

Isolation, partial characterisation and immunomodulating activities of polysaccharides from Vernonia kotschyana Sch. Bip. ex Walp

The roots from Vernonia kotschyana Sch. Bip. ex Walp. ( Baccharoides adoensis var. kotschyana (Sch. Bip. ex Walp.) M.A. Isawumi, G.El-Ghazaly & B. Nordenstam) (Asteraceae) are used in Malian folk medicine for the treatment of gastritis, gastro duodenal ulcers, as an aid to ameliorate digestion and as a wound healing remedy. Since a common feature among these conditions is related to immune responses, immunomodulating activities of fractions isolated from both the 50 °C and the 100 °C water extracts from Vernonia kotschyana were investigated in this study. The active principles were identified as acidic polysaccharide fractions, containing pectic arabinogalactan type II structures, which showed both complement fixing ability and T-cell independent induction of B-cell proliferation in vitro. Some activity was also observed on macrophages. The present study may provide additional support for the popular use of this plant to improve intestinal health.

The malian medicinal plant Trichiliaemetica; studies on polysaccharides with complement fixing ability

Trichilia emetica is a tree or bush that has many different traditional uses in Mali, amongst others the leaves have been used as a woundhealing remedy. As polysaccharides have been isolated from other plants that have a long tradition as woundhealing remedies, it was of interest to study the polysaccharides of T. emetica as well. The polysaccharides were extracted at 50 and 100 °C, and the polymeric material was separated into neutral and acidic polymers by anion exchange chromatography. The complement fixation ability of the different fractions was determined. The Acidic fraction 4 of the 100 °C extract was the most active, but most of the other fractions were also quite active. Structural studies showed that the most active fraction was a pectin of the rhamnogalacturonan type 1 with side chains of the arabinogalactan type II. Removal of terminal arabinofuranosides lead to a drop of the activity indicating that this structural unit may be involved in the bioactive site of the molecule.

Origin of bovine IgM structural variants

The structure of IgM determined from two cDNAs isolated from a Holstein (BLV7G1) and an Angus×Hereford cross-bred (B5D8) cow reveals high sequence similarity both at nucleotide (98.7%) and amino acid (97.9%) level and is closest to sheep (89.4%). Three bovine IgM allotypes, designated as IgMa, IgMb and IgMc, are classified based on nucleotide substitutions in all the Cμ exons resulting in amino acid replacements. Further, insertion of three in-frame codons at Cμl and Cμ2 junction of B5D8 IgM from the intervening intron, via cleavage of pre-mRNA at an alternate cryptic 5′ splice donor site, leads to generation of additional bovine IgM variants. The C1q-binding site, involved in classical complement pathway, is identified in bovine IgM where ten amino acids are conserved across species. Interestingly, bovine IgM has the lowest number of proline residues (5) in the Cμ2 domain in comparison to other species (7–9) and this is likely to impose structural constraints on mobility of Fab arms of the bovine IgM during antigen recognition. The rigidity in the bovine IgM Cμ2 domain may, however, facilitate exposure of C1q-binding site subsequent to antigen binding and enhance its complement fixing ability. The restricted mobility of bovine IgM Fab arms may possibly favor generation of an antigen-combining site requiring an unusually long third complementarity determining region of the heavy chain (CDR3H), apart from antigen selection of variable domains. This is consistent with the fact that an exceptionally long CDR3H has not been observed in bovine IgG which bears a long and more flexible hinge region. Additional hydrophilic threonine and serine residues in the Cμ2 domain of bovine IgM, as compared to other species, however, enhance its ability to extend into the solvent. Finally, restricted fragment length polymorphism analysis of genomic DNA from four cattle breeds reveals the presence of; at least, four allelic variants of bovine Cμ gene.

IgG antibody subclass responses determined by immunoblot in infants’ sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine

The introduction of meningococcal serogroup C conjugate vaccines into the UK immunisation schedule has led to the decline of serogroup C disease in those vaccinated but there is no imminent vaccine solution for serogroup B disease. The PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate and an outer membrane vesicle (OMV) vaccine containing six different PorA OMPs (each representing a different serosubtype) has been evaluated in phase II trials with encouraging results. Little is known about the IgG subclass response to the various antigens contained within this vaccine. These responses are important due to the different half-lives and complement fixing abilities of these antibodies. In this study, immunoblotting was undertaken with infants’ sera following either three or four doses of vaccine, and OMVs from six isogenic meningococcal strains differing only in their PorA serosubtype. Following either three or four doses of the vaccine, IgG 3 and IgG 1 subclass antibodies were induced to all six of the isogenic strains, although sera collected after four doses of vaccine showed stronger antibody levels. IgG 3 was found in more sera than IgG 1. For both sets of sera, the two isogenic strains expressing P1.5,2 and P1.5 c,10 induced stronger IgG subclass antibody responses than the other four meningococcal strains. The recombinant hexavalent PorA OMV vaccine stimulates both IgG 1 and IgG 3 subclass antibodies, the subclasses that are most effective in activating the complement system.

Detection of Complement-fixing Antiphospholipid Antibodies in Association with Thrombosis

Antiphospholipid antibody (aPL) is a hallmark of antiphospholipid syndrome (APS), characterized by thrombosis and recurrent fetal loss. We developed a novel ELISA system to detect complement-fixing ability of anticardiolipin antibody (aCL), and evaluated its clinical usefulness through studying the prevalence of the antibodies in rheumatic diseases, especially in association with thrombosis and recurrent abortion. Among 189 patients with rheumatic diseases, the complement-fixing aCL was positive in 26 (83.9%) of 31 patients with APS and 2 (1.3%) of 158 with other disease categories, whereas it was not positive among 52 normal subjects. Twenty-seven of 28 patients (96.4%), who were positive for complement-fixing aCL, had the episodes or history of thrombosis and/or recurrent abortion, at the time we studied. The remaining one in this group developed APS manifesting pulmonary infarction and occlusion of mesenteric artery 6 months after the evaluation. The sensitivity and specificity of this assay system were 75.0% and 99.3%, respectively, in relation with thrombotic episodes. On the other hand, the IgG aCL were positive in 28 (77.8%) of 36 cases with recent thrombotic episodes and 24 (15.7%) of 153 cases with no recent thrombotic episodes. The sensitivity and specificity of IgG aCL assay system were 77.8% and 84.3%, respectively, in relation with thrombotic episodes. These results indicate that complement-fixing aCL may specifically occur in association with the episodes of thrombosis and/or recurrent abortion in patients with APS compared to IgG-aCL. The method for detecting the complement-fixing aCL is simple, and it provides the useful diagnostic marker for thrombotic manifestations associated with APS.