Anti-citrullinated protein antibodies have a low avidity compared with antibodies against recall antigens.

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) are highly specific for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. Recent ongoing evidence indicates that the ACPA response broadens before precipitation of full-blown RA, as indicated by a more extensive isotype usage and an increased citrullinated epitope recognition profile. Nonetheless, the evolution of the ACPA response is still poorly understood and might intrinsically differ from the protective responses against pathogens. METHODS: The avidity and the avidity maturation of ACPA in relation to the avidity of antibodies against recall antigens were analysed. RESULTS: The avidity of ACPA was significantly lower than the avidity of antibodies to the recall antigens tetanus toxoid and diptheria toxoid. Moreover, ACPA did not show avidity maturation during longitudinal follow-up and ACPA avidity was also relatively low in patients who displayed extensive isotype switching. CONCLUSIONS: These observations indicate that the natural evolution of ACPA differs from the development of antibodies against recall antigens. These data also indicate that ACPA avidity maturation and isotype switching are disconnected, whereby extensive isotype switching occurs in the setting of restricted avidity maturation. Intrinsic differences between the RA-specific autoantibody system and protective antibody responses against pathogens could be of relevance for designing novel B cell-targeted therapies for RA.

B-cell maturation and antibody responses in individuals carrying a mutated CD19 allele.

Homozygous CD19 mutations lead to an antibody deficiency due to disruption of the CD19 complex and consequent impaired signaling by the B-cell antigen receptor. We studied the effects of heterozygous CD19 mutations on peripheral B-cell development and antibody responses in a large family with multiple consanguineous marriages. Sequence analysis of 96 family members revealed 30 carriers of the CD19 mutation. Lymphocyte subset counts were not significantly different between carriers and noncarriers in three different age groups (0-10 years; 11-18 years; adults). B cells of carriers had reduced CD19 and CD21 median expression levels, and had reduced proportions of transitional (0-10 years) and CD5(+) B cells (adults). CD19 carriers did not show clinical signs of immunodeficiency; they were well capable to produce normal serum Ig levels and had normal responses to primary and booster vaccinations. The frequency of mutated Vκ alleles was not affected. Heterozygous loss of CD19 causes some changes in the naive B-cell compartment, but overall in vivo B-cell maturation or humoral immunity is not affected. Many antibody deficiencies are not monogenetic, but likely caused by a combination of multiple genetic variations. Therefore, functional analyses of immune cell function should be carried out to show whether heterozygous mutations contribute to disease.

Clinical fluctuations in MuSK myasthenia gravis are related to antigen-specific IgG4 instead of IgG1.

We studied the longitudinal relation between disease severity and titers of antigen-specific IgG subclasses in sera of patients with myasthenia gravis and antibodies to Muscle Specific Kinase (MuSK MG). Six patients were included of whom 55 samples had been collected during 2.5-13.4 years. Anti-MuSK antibodies were determined by ELISA and with a cell-based immunofluorescence assay. Disease severity was scored on a semi continuous scale. Only antigen-specific IgG4, and not IgG1, titers were significantly associated with disease severity in a linear mixed effect model (p = 0.036). Levels of IgG4 antibodies were above IgG1 in all samples except in one patient who went into clinical remission while switching from IgG4 to IgG1. The results support an important role for IgG4 in the pathogenesis of MuSK MG, in contrast to MG with anti-acetylcholine receptor antibodies.

Adenovirus infection in paediatric stem cell transplant recipients: increased risk in young children with a delayed immune recovery.

Adenovirus (HAdV) infections are a frequent cause of morbidity and mortality after allogeneic human stem cell transplantation (HSCT). We report a retrospective single-centre study on 328 consecutive paediatric recipients of an allogeneic HSCT. During the first 6 months after HSCT, HAdV infection occurred in 37 children (cumulative incidence 12%). The highest incidence was found in young children up to 5 years of age, transplanted after 1994, with >2 log T-cell depletion of a graft of another than an HLA-genotypically identical related donor (actuarial frequency at 6 months 84%). Persistence of HAdV and spreading of the virus over multiple sites showed a trend towards the development of HAdV disease or death, but did not reach significance. Recovery of immunity after HSCT, that is, serum concentrations of IgM and peripheral blood counts of T cells and subsets, was delayed in children with an HAdV infection compared with noninfected children. In seven out of seven patients with HAdV DNA in serum and decreasing lymphocyte counts, the infection had a fatal course. Manipulation of cellular immunity either by tapering of immunosuppression, infusion of donor lymphocytes or immunotherapy using HAdV-specific T cells should be considered in graft recipients at risk for a severe HAdV infection.

Dot-immunobinding assay as an accurate and versatile technique for the quantification of human IgG subclasses.

A dot-immunobinding assay on nitrocellulose membranes has been developed for the quantification of human IgG subclasses using subclass-specific monoclonal antibodies. The advantages of this technique can be summarized as follows: (1) possibility of rapid semi-quantitative evaluation and/or precise quantitation from the same dot-pattern; (2) simple procedure with very good reproducibility; (3) sensitivity for nanogram concentrations of individual subclasses, therefore applicable not only to serum but also to other body fluids with a low content of IgG; (4) very small amounts of test material needed; (5) very good correlation of results with other techniques (ELISA, radial immunodiffusion) but without some of the inherent problems of the latter methods.

Immunoblotting techniques for the detection of low level homogeneous immunoglobulin components in serum.

Because of the increasing demand for simple and reliable techniques for the detection of low concentrations of paraproteins against a highly heterogeneous serum background, two techniques were investigated for their sensitivity: isoelectric focusing (IEF) and Wieme high resolution electrophoresis, each with subsequent blotting by diffusion. The techniques were compared using isolated mouse monoclonal antibodies (mAb) of known concentration and specificity. Wieme electrophoresis in combination with immunoblotting (IBL) or antigen-specific immunoblotting (ABL) has a detection limit of 100 ng/ml and 10 ng/ml, respectively. For IEF in combination with IBL or ABL these limits were 1000 and 30 ng/ml, respectively. For ABL, polyvinylidene difluoride (PVDF) and nylon-supported nitrocellulose (NSNC) membranes gave similar detection limits, although for IBL, PVDF is preferred to NSNC. While IEF is essential for investigating the spectrum of the antibody repertoire. Wieme electrophoresis is the most powerful technique for the detection of homogeneous immunoglobulin components (H-Ig). After separation of the proteins. IBL is fast, simple and sensitive enough for routine detection and characterization of H-Ig. However, when the antibody specificity is known, ABL should be chosen for its superior sensitivity.

IgG antibodies to ATG early after pediatric hematopoietic SCT increase the risk of acute GVHD.

Anti-thymocyte globulin (ATG), raised in rabbits, is frequently used in allogeneic hematopoietic SCT (HSCT), to prevent graft rejection and acute GVHD. In solid organ transplant patients, antibodies to rabbit IgG result in an enhanced clearance of ATG. The occurrence of such antibodies in HSCT recipients and their clinical impact is unknown. Concentrations of ATG and anti-ATG antibodies were measured in 72 pediatric HSCT recipients treated with ATG as part of the conditioning. Anti-ATG antibodies were detected in 20 children (28%), all transplanted with a non-depleted graft. IgG anti-ATG, alone or combined with IgM and/or IgA anti-ATG, appeared in 10 children. Four patients developed IgG anti-ATG antibodies early (before day 22) post-HSCT. They had steep drops in ATG levels and showed rapid T-cell recovery, which was associated with a significantly increased risk of acute GVHD. In six patients IgG anti-ATG responses occurred later (range 28-46 days) after HSCT without an increased risk of GVHD. A total of 10 children only mounted an IgM (and IgA) anti-ATG response, which was without major impact on ATG levels. These results indicate that early development of IgG anti-ATG antibodies has a major impact on acute GVHD. Routine analysis ATG/anti-ATG Ab measurement should be considered.

Impaired avidity maturation after tetanus toxoid booster in children with Down syndrome.

Down syndrome children show a decreased avidity of the antibody response after tetanus toxoid booster vaccination at 9 years of age suggesting impaired memory B cell selection in the germinal center. Clinicians need to be aware of this ongoing subtle immunologic deficit in Down syndrome.

Mannose-binding lectin levels and infections in children after allogeneic hematopoietic SCT.

Mannose-binding lectin (MBL) is an activator of the lectin pathway of the C' system and hence an important component of the innate immune system. Although reports are conflicting, MBL deficiency has been reported to influence the infection susceptibility in hematology/oncology patients or recipients of allogeneic hematopoietic SCT (HSCT). MBL levels and the occurrence of infections were retrospectively analyzed in 98 pediatric HSCT patients. MBL deficiency in recipients was not corrected by HSCT using a donor with normal MBL production. In addition, low serum MBL concentrations were not associated with increased susceptibility to any type of infections post-HSCT in this cohort of pediatric HSCT recipients.

Cellular and humoral responses to tetanus vaccination in Gabonese children.

Protection to tetanus is often not optimal in developing countries due to incomplete vaccination schemes, or decreased efficacy of vaccination. In this study we investigated the immunological response to tetanus booster vaccination in school children living in a semi-urban or in a rural area of Gabon. Tetanus-specific total IgG as well as antibody subclasses of the IgG1, IgG2, IgG3 and IgG4 isotype and the avidity of the dominating IgG1 subclass were determined both before and 1 month after the booster vaccination. In addition, tetanus-specific cytokine responses were determined. We found a polarization towards a T helper 1 (Th1) profile in the semi-urban children, whereas the cytokine responses of the rural children showed a T helper 2 (Th2) skewed response. Furthermore, tetanus-specific antibodies of the different IgG subclasses were all increased upon a tetanus booster vaccination and levels of IgG1 and IgG3 were higher in the rural children. In conclusion, a tetanus booster vaccination induced a stronger Th2 over Th1 cytokine profile to tetanus toxoid (TT) in rural children who showed the highest levels of IgG1 and IgG3 anti-TT antibody responses.

Resetting the adaptive immune system after autologous stem cell transplantation: lessons from responses to vaccines.

Autologous stem cell transplantation (ASCT) to treat autoimmune diseases (AID) is thought to reset immunological memory directed against autoantigens. This hypothesis can only be studied indirectly because the exact nature of the pathogenetic autoantigens is unknown in most AID. Therefore, 19 children with juvenile idiopathic arthritis (JIA) or systemic lupus erythematodes (SLE) and 10 adults with multiple sclerosis (MS) were vaccinated with the T-cell-dependent neoantigen rabies and the recall antigen tetanus toxoid after, respectively before, bone marrow harvest. Both vaccinations were repeated after ASCT. All except two of the responders mounted a primary antibody response to rabies after revaccination, and 44% of the responders mounted a primary antibody response to tetanus boost after ASCT. These data show that immunological memory to a neoantigen is lost in most patients with AID after immunoablative pretreatment; however, memory to a recall antigen boosted before bone marrow harvest is only lost in part of the patients. Disease progression was arrested in all patients with JIA/SLE except one, but only in a minority of MS patients. Clinical outcome on a per case basis was not associated with the profile of the immune response toward the vaccination antigens after ASCT.

Association of smoking with the constitution of the anti-cyclic citrullinated peptide response in the absence of HLA-DRB1 shared epitope alleles.

OBJECTIVE: Smoking is a risk factor for anti-cyclic citrullinated peptide (anti-CCP) antibody-positive rheumatoid arthritis (RA) in patients with HLA-DRB1 shared epitope (SE) alleles. It is unknown whether smoking influences not only the presence of these antibodies, but also other characteristics of the anti-CCP response, such as isotype usage. The aim of this study was to determine the influence of smoking on anti-CCP isotypes in RA patients, and to determine whether this influence is observed in the presence and/or absence of SE alleles. METHODS: IgA, IgM, and IgG subclasses of anti-CCP antibodies were measured by enzyme-linked immunosorbent assay in serum obtained at the first visit to the Leiden Early Arthritis Clinic from 216 patients with anti-CCP-positive RA whose smoking habits were also assessed. HLA genotyping data were available for 202 of these patients. RESULTS: IgA and IgM anti-CCP were more frequent in RA patients who were smokers than in those who were nonsmokers (odds ratio 2.8 and 1.8, respectively). In addition, levels of all isotypes of anti-CCP, except IgG3, were significantly higher (P < 0.05) in smokers. The number of anti-CCP isotypes was higher in smokers compared with nonsmokers, both in SE-negative RA (P = 0.04) and in SE-positive RA (P = 0.07). CONCLUSION: Patients with anti-CCP-positive RA who have a current or former tobacco exposure display a more extensive anti-CCP isotype usage in general, and IgA and IgM in particular, compared with patients with anti-CCP-positive RA who have never smoked. In contrast to its influence on the incidence of anti-CCP positivity, the influence of tobacco exposure on the constitution of the anti-CCP response is significant in SE-negative RA. These findings suggest a differential effect of tobacco exposure on the induction as compared with the propagation of the anti-CCP antibody response.

Isotype distribution of anti-cyclic citrullinated peptide antibodies in undifferentiated arthritis and rheumatoid arthritis reflects an ongoing immune response.

OBJECTIVE: The evolution of the rheumatoid arthritis (RA)-specific anti-cyclic citrullinated peptide (anti-CCP) antibody response, as measured by the isotypes of anti-CCP, has not been described. This study was undertaken to determine anti-CCP isotype usage in patients with undifferentiated arthritis (UA), patients with recent-onset RA, and patients with RA of long duration. METHODS: IgA, IgM, and IgG subclasses of anti-CCP were measured by enzyme-linked immunosorbent assay in serum samples that were obtained from IgG anti-CCP antibody-positive patients with UA (n = 110) and IgG anti-CCP antibody-positive patients with RA (n = 152) early after the onset of arthritis. Patients with UA in whom RA developed within 1 year (UA-->RA) were compared with patients with UA in whom RA did not develop within 1 year (UA-->UA). In addition, baseline serum samples obtained from a subset of patients with RA (n = 64) were compared with sera obtained from the same patients a median of 7 years later. RESULTS: IgM anti-CCP was present in early samples from both patients with UA and patients with RA and in followup samples from patients with RA. Several IgG anti-CCP antibody-positive patients who did not have IgM anti-CCP early after disease onset did display IgM anti-CCP later in the course of the arthritis. A diverse pattern of isotype usage was detected in early samples, with a trend toward lower frequencies of all isotypes of anti-CCP in patients with UA compared with patients with RA and in UA-->UA patients compared with UA-->RA patients. Levels of all isotypes except IgG1 had decreased after 7 years. CONCLUSION: These data indicate development of the anti-CCP isotype repertoire into full usage early in the course of arthritis. The sustained presence of IgM anti-CCP indicates ongoing recruitment of new B cells into the anti-CCP response, reflecting a continuous (re)activation of the RA-specific anti-CCP response during the course of anti-CCP-positive arthritis.

Immunoglobulin G (IgG) subclass distribution and IgG1 avidity of antibodies in human immunodeficiency virus-infected individuals after revaccination with tetanus toxoid.

In human immunodeficiency virus (HIV)-infected individuals the amount of antibodies formed after vaccination with T-cell-dependent recall antigens such as tetanus toxoid is proportional to the peripheral blood CD4(+) T-lymphocyte counts. To investigate whether the immunoglobulin G (IgG) subclass distribution and avidity of the antibodies produced after vaccination are affected as well, we gave 13 HIV-infected adults with low CD4(+) T-lymphocyte counts (<200 x 10(6)/liter; group I), 11 HIV-infected adults with intermediate CD4(+) T-lymphocyte counts (>/=200 x 10(6)/liter; group II), and 5 healthy controls booster immunizations with tetanus toxoid. The prevaccination antibody concentrations against tetanus toxoid were similar in the HIV-infected and healthy adults. After vaccination the total IgG and the IgG1 anti-tetanus toxoid antibody concentrations were significantly lower in group I than in group II and the controls. The avidity of the IgG1 anti-tetanus toxoid antibodies formed by HIV-infected adults was within the range for healthy controls, irrespective of their CD4(+) T-lymphocyte counts.

Quality and quantity of the humoral immune response in healthy elderly and young subjects after annually repeated influenza vaccination.

Doubt about the serologic efficacy of annually repeated influenza vaccination prompted investigations into the course of hemagglutination-inhibiting (HI), IgG, and IgA antibody titers and the IgG and IgA avidity index to influenza A/Taiwan/1/86 and A/Beijing/353/89 after annual vaccination. Fifty-four healthy elderly persons >70 years of age and 24 healthy young adults <30 years of age received standard influenza vaccine during 3 consecutive years. On average, prevaccination HI, IgG, and IgA titers to both influenza virus strains increased >=4 fold between the first and the third vaccination (analysis of variance, P<.001). The postvaccination HI and IgG titers remained unchanged after annual vaccination. The avidity index of IgG and IgA antibodies increased somewhat after annual vaccination, although the increase was statistically significant only in the young subjects. These data indicate that annual influenza vaccination of healthy elderly and young subjects results in an overall increase in protective antibodies.

Immunoglobulin levels and monoclonal gammopathies in children after bone marrow transplantation.

Bone marrow graft recipients suffer profound immunodeficiency during at least 3 months after transplantation. B-cell reconstitution following allogeneic bone marrow transplantation (BMT) in children was studied longitudinally by quantification of Ig (sub)class levels in serum and by investigation of numbers and characteristics of homogeneous Ig components (H-Ig); ie, monoclonal gammopathies (MG). For the latter purpose, a sensitive immunoblotting technique capable of detecting H-Ig of a concentration as low as 0.5 microgram/mL was used. Sera of 40 children grafted for a variety of diseases were investigated and followed up for 5 years. It was found that Ig (sub)classes reached normal levels from 3 months after BMT onward. The sequential increase of the different Ig isotypes was in accordance with that seen in normal ontogeny. This was especially clear following BMT for severe congenital immunodeficiency. H-Ig appeared from as early as 6 weeks after BMT in increasing numbers, beginning within IgM, IgG3, and IgG1, and afterward within other isotypes. After an initial increase of serum Ig levels, "overshooting" occurred accompanied by high frequency of H-Ig. H-Ig were still present at 5 years after BMT, when Ig levels normalized. Our data indicate that B-cell reconstitution after allogeneic BMT recapitulates normal ontogeny but in a clonally dysregulated fashion; that is, with overexpression of some clones and underexpression of others.

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation.

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell-depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B-cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: evidence for donor origin of the antibody producing cells.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.