Differentiating the cellular and humoral components of neuromuscular blocking agent-induced anaphylactic reactions in patients undergoing anaesthesia.

BACKGROUND: The significance of IgE antibodies to neuromuscular blocking agent (NMBA)-induced anaphylactic reactions during anaesthesia is unclear. We investigated the relevance of IgE to rocuronium using an in vitro technique. METHODS: Serum samples from 61 patients with anaphylactic reactions during anaesthesia were investigated. On the basis of clinical history, allergy to NMBA was considered likely in 48 patients, further assessed using intradermal skin tests for several commonly used NMBAs, including rocuronium, vecuronium, and succinylcholine. To determine the presence of rocuronium IgE in human serum, a rocuronium-human serum albumin (rocHSA) conjugate was coupled to a solid phase and a radioallergosorbent test performed. The biological effects of patient serum NMBA-IgE on histamine release were investigated using in vitro sensitized basophils from healthy blood donors. RESULTS: IgE to rocuronium was found in 23 of 48 serum samples (48%) with NMBA allergy, although only two of these were able to sensitize basophils to release histamine in response to rocHSA. IgE-responsiveness in the basophil test was only observed with conjugated rocHSA and not with unconjugated rocuronium or the other NMBAs evaluated. However, unconjugated rocuronium inhibited the histamine release induced by rocHSA. Correlation between skin-test reactivity to rocuronium and IgE to rocHSA was low (P>0.1). In contrast, striking correlation between IgE to rocuronium and skin-test reactivity to succinylcholine was found (P<0.001). CONCLUSIONS: Our results indicate that NMBA-related anaphylaxis requires not only IgE NMBA reactivity, but also altered cellular reactivity in the patient. The latter may be demonstrable by testing basophils from the patient, a skin test with (steroidal) NMBA, or both.

Clinical response, pharmacokinetics, development of human anti-chimaeric antibodies, and synovial tissue response to rituximab treatment in patients with rheumatoid arthritis.

OBJECTIVES: To analyse whether persistence of synovial B lineage cells and lack of clinical response to rituximab treatment in patients with rheumatoid arthritis (RA) are associated with low rituximab serum levels and anti-rituximab antibody (ARA) formation. METHODS: Fifty-eight patients with RA were treated with rituximab. The clinical response was determined 24 weeks after each treatment course using the Disease Activity Score evaluated in 28 joints (DAS28) and EULAR response criteria. Rituximab serum levels, ARAs and synovial B lineage cell numbers were determined before and after treatment. RESULTS: Four weeks after treatment rituximab serum levels were highly variable. Low rituximab levels were associated with ARA formation (in five patients (8.6%)) and high baseline erythrocyte sedimentation rate. Interestingly, serum rituximab levels were not related to persistence of synovial B lineage cells or clinical response. Furthermore, response to treatment and re-treatment was similar in ARA-positive and ARA-negative patients. CONCLUSION: There is clear variability in serum levels after rituximab treatment, but rituximab levels are not lower in patients with persistence of synovial B lineage cells or lack of clinical response. The current treatment schedule suffices to induce and maintain a clinical response, even when ARAs are formed.

Immunoglobulin G4: an odd antibody.

Despite its well-known association with IgE-mediated allergy, IgG4 antibodies still have several poorly understood characteristics. IgG4 is a very dynamic antibody: the antibody is involved in a continuous process of half-molecules (i.e. a heavy and attached light-chain) exchange. This process, also referred to as 'Fab-arm exchange', results usually in asymmetric antibodies with two different antigen-combining sites. While these antibodies are hetero- bivalent, they will behave as monovalent antibodies in most situations. Another aspect of IgG4, still poorly understood, is its tendency to mimic IgG rheumatoid factor (RF) activity by interacting with IgG on a solid support. In contrast to conventional RF, which binds via its variable domains, the activity of IgG4 is located in its constant domains. This is potentially a source of false positives in IgG4 antibody assay results. Because regulation of IgG4 production is dependent on help by T-helper type 2 (Th2) cells, the IgG4 response is largely restricted to non-microbial antigens. This Th2-dependency associates the IgG4 and IgE responses. Another typical feature in the immune regulation of IgG4 is its tendency to appear only after prolonged immunization. In the context of IgE-mediated allergy, the appearance of IgG4 antibodies is usually associated with a decrease in symptoms. This is likely to be due, at least in part, to an allergen-blocking effect at the mast cell level and/or at the level of the antigen-presenting cell (preventing IgE-facilitated activation of T cells). In addition, the favourable association reflects the enhanced production of IL-10 and other anti-inflammatory cytokines, which drive the production of IgG4. While in general, IgG4 is being associated with non-activating characteristics, in some situations IgG4 antibodies have an association with pathology. Two striking examples are pemphigoid diseases and sclerosing diseases such as autoimmune pancreatitis. The mechanistic basis for the association of IgG4 with these diseases is still enigmatic. However, the association with sclerosing diseases may reflect an excessive production of anti-inflammatory cytokines triggering an overwhelming expansion of IgG4-producing plasma cells. The bottom line for allergy diagnosis: IgG4 by itself is unlikely to be a cause of allergic symptoms. In general, the presence of allergen-specific IgG4 indicates that anti-inflammatory, tolerance-inducing mechanisms have been activated. The existence of the IgG4 subclass, its up-regulation by anti-inflammatory factors and its own anti-inflammatory characteristics may help the immune system to dampen inappropriate inflammatory reactions.

Immunogenicity does not influence treatment with etanercept in patients with ankylosing spondylitis.

BACKGROUND: Immunogenicity, specifically the onset of antibodies against tumour necrosis factor (TNF) blocking agents, seems to play an important role in non-response to treatment with these drugs. OBJECTIVES: To assess the relation of clinical response of ankylosing spondylitis (AS) to etanercept with etanercept levels, and the presence of antibodies to etanercept. METHODS: Patients with AS were treated with etanercept 25 mg twice weekly, according to the international Assessment in Ankylosing Spondylitis (ASAS) working group consensus statement. Sera were collected at baseline and after 3 and 6 months of treatment. Clinical response was defined as a 50% improvement or as an absolute improvement of 2 points on a (0-10 scale) Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Functional etanercept levels were measured by a newly developed ELISA, measuring the binding of etanercept to TNF. Antibodies against etanercept were measured with a two-site assay and antigen binding test. Clinical data were used to correlate disease activity with serum etanercept levels. RESULTS: In all, 53 consecutive patients were included. After 3 months of treatment 40 patients (76%) fulfilled the response criteria. Mean etanercept levels were 2.7 mg/litre and 3.0 mg/litre after 3 and 6 months respectively. Characteristics and etanercept levels of responders and non-responders were similar. No antibodies to etanercept were detected with any of the assays. CONCLUSION: Etanercept levels of responders and non-responders were similar and no antibodies to etanercept were detected with any of the assays. This study indicates that etanercept is much less immunogenic compared with the other TNF-blocking agents.

Induction of interleukin-10 and suppressor of cytokine signalling-3 gene expression following peptide immunotherapy.

BACKGROUND: Allergen-derived (T cell epitope) peptides may be safer for immunotherapy than native allergen, as they do not cross-link immunoglobulin (Ig)E. However, HLA polymorphism results in multiple potential epitopes. Synthetic peptides of phospholipase (PL) A(2) were selected for a peptide vaccine, on the basis of binding affinity for commonly expressed HLA-DR molecules. OBJECTIVE: To evaluate treatment with an HLA-DR-based PLA(2) peptide vaccine in subjects with mild honeybee allergy in an open, controlled study. METHODS: Twelve volunteers with allergy to bee venom received nine intradermal injections of PLA(2) peptides, with six untreated subjects serving as controls. Outcome was assessed by the size of the late-phase cutaneous reaction to allergen, peripheral blood mononuclear cell (PBMC) proliferation, cytokine release, and expression of genes associated with immune regulation. RESULTS: Subjects receiving peptides showed a decrease in the magnitude of the late-phase cutaneous reaction to bee venom compared with controls (P=0.03). The proliferation of venom-stimulated PBMCs decreased in treated subjects compared with controls (P=0.01). Peptide treatment reduced the production of IL-13 by PLA(2)-stimulated PBMCs (P<0.01) and IFN-gamma (P<0.01), and increased the production of IL-10 (P=0.02). Transcription of the suppressor of cytokine signalling (Socs)3 gene was significantly increased following therapy. A transient, but modest, increase in allergen-specific IgG was also observed. CONCLUSION: HLA-DR-based T cell epitopes modify surrogate markers associated with successful immunotherapy and induction of immune regulation, supporting the concept that this form of treatment may be efficacious in human allergic disease.

Immunoglobulin E and G4 antibody responses in occupational airway exposure to bovine and porcine plasma proteins.

BACKGROUND: Production of both antigen-specific immunoglobulin (Ig)E and IgG4 antibodies is dependent on stimulation of B cells by T helper 2 cell-derived cytokines. However, there is controversy as to their interaction. In this study, we investigated the interdependency of IgE and IgG4 antibody responses to a relatively high range of airway exposure to animal-derived proteins in an occupational setting. Moreover, associations with self-reported airway symptoms and bronchial hyperresponsiveness were established. METHODS: In a cross-sectional design, employees of an animal plasma spray-drying factory were questioned about airway symptoms, exposure was measured with personal sampling technique, and serology was performed. In a selection of subjects from this population, serology was repeated 15 months later, and bronchial hyperresponsiveness was measured. RESULTS: IgE and IgG4 antibodies were detected in 17 and 57% of all employees and were both associated with degree of exposure. Only IgE antibodies showed an independent association with self-reported airway symptoms and bronchial hyperresponsiveness. The presence of IgE antibodies was limited to employees with high levels of IgG4. Employees with IgE and symptoms appeared to have less IgG4 than asymptomatic IgE-positive individuals. The level of specific IgG4 antibodies was stable over a 15-month period. CONCLUSIONS: In high-range airway exposure, development of IgE and IgG4 antibodies depended on the level of exposure. The threshold for development of IgG4 antibodies appeared to be less than that for IgE antibodies, and IgG4 antibodies may protect against the development of symptoms.

Do levels of immunoglobulin G antibodies to foods predict the development of immunoglobulin E antibodies to cat, dog and/or mite?

BACKGROUND: In children at high risk of inhalation allergy, food sensitization is associated with an increased risk for sensitization to inhalant allergens. Furthermore, this association was also found in a cross-sectional study. OBJECTIVE: To examine in a prospective study, whether levels of IgG to foods (i.e. mixture of wheat and rice, mixture of soy bean and peanut, egg white, cow's milk, meat, orange and potato) indicate an increased risk for the future development of IgE antibodies to inhalant allergens in a low-risk population and whether they can be used as predictors of the subsequent development of IgE antibodies in young, initially IgE-negative children. METHODS: Coughing children, aged 1-5, visiting their GPs, were tested for IgE antibodies to mite, dog and cat (RAST) and IgG (ELISA) to foods. All IgE-negative children were retested for IgE antibodies after two years. The IgG results (66 percentiles) of the first blood sample were compared to the RAST-scores of the second blood sample. RESULTS: After two years, 51 out of 397 (12.8%) originally IgE-negative children, had become IgE-positive for cat, dog and/or mite. An increased IgG antibody level to wheat-rice (OR = 2.2) and to orange (OR = 2.0) indicated an increased risk of developing IgE to cat, dog or mite allergens. In addition to IgG to a mixture of wheat-rice and orange; total IgE, breastfeeding, eczema as a baby and age were the most important predictors for the subsequent development of IgE to inhalant allergens. DISCUSSION: An increased IgG antibody level to a mixture of wheat-rice or orange, indicates an increased risk of developing IgE to cat, dog or mite allergens. This indicates that excessive activity of the mucosal immune system is present before IgE antibodies to airborne allergens can be demonstrated. Nevertheless, IgG to foods is not very helpful (with a positive predictive value of 16.5%, and negative predictive value of 90.6%) in identifying individual children at risk in clinical practice. However, besides other risk factors, IgG to wheat-rice and to orange could be useful as a screening test for studies in the early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing IgE antibodies in the future.

Inadvertent BSA-induced elution of IgE in the BSA-RAST.

BACKGROUND: Bovine serum albumin (BSA) is widely used to block nonspecific binding in immunochemical assays. Whereas a previous study had indicated that soluble allergen present during the incubation with anti-IgE in the RAST did not affect bound IgE, we reinvestigated this in the current study, using IgE elution from BSA by soluble BSA as a test system. METHODS: Sepharose-coupled BSA (0.08, 0.4, 2, or 10 microg BSA/test) was incubated overnight with serum and washed. The Sepharose was then incubated with different concentrations of soluble BSA (0, 12, 60, 300, or 1500 microg/test), washed again, and incubated with radioactive anti-IgE. The effect on IgE binding was investigated for various incubation periods (t=0, 1, 2, 4, and 20 h). RESULTS: Incubation in buffer without BSA did not change IgE binding. Soluble BSA eluted IgE antibodies from immobilized BSA by up to 85%. If the BSA density on the solid phase was > or =2 microg/test, the elution efficiency was dependent on the levels of both immobilized BSA and soluble BSA. At lower densities, the dissociation was dependent only on the concentration of soluble BSA. The time needed to obtain 50% IgE elution (t(1/2)) was less if the density of immobilized BSA decreased. Below the critical density (0.8 microg BSA/mg solid phase), t(1/2) was independent of the coating density (45 min). Probably all IgE antibodies are monovalently bound below this density. CONCLUSIONS: Dissociation of IgE from immobilized protein in the presence of soluble protein should be taken into account, particularly when IgE to mammalian serum albumin is involved (milk, meat, or animal dander).

Structural aspects of cross-reactivity and its relation to antibody affinity.

In the context of IgE/allergen interactions, affinity is largely determined by the stability of the allergen-IgE complex: a low affinity is usually equated with a rapid dissociation of the complex. Regular solid-phase assays are not well suited for affinity estimates because of multivalency effects, "unstirred layer" effects and "invisible" antibodies. Elution of IgE bound to solid-phase coupled allergen might be a good measure of intrinsic affinity, provided that reassociation of antibodies is prevented by a high concentration of soluble allergen. Allergen-mediated IgE-dependent triggering of a mast cell is presumably a two-step process. During the first step, the allergen is bound to a cell-bound IgE antibody and dragged over the cell surface. The second step is the interaction between this cell-bound allergen and another IgE antibody. The hypothesis is that the affinity requirements for the first step are higher than for the second. The implication is that a mast cell can be triggered by a single high-affinity antibody in combination with one or more low-affinity antibodies.

Relation between IgG antibodies to foods and IgE antibodies to milk, egg, cat, dog and/or mite in a cross-sectional study.

BACKGROUND: Because IgG antibodies to foods can be detected before IgE antibodies to inhalants, increased levels of IgG antibodies to foods might be used as a predictor of IgE-mediated allergy in initially nonatopic children. OBJECTIVE: To examine the cross-sectional relation between IgG to foods (i.e. mixture of wheat and rice, mixture of soybean and peanut, egg white, cow's milk, meat, orange and potato) and specific IgE to cat, dog, mite, milk and egg white in 1-year-old children. METHODS: All atopic children (n = 120; 58 with and 62 without eczema) and a random sample of the nonatopic children (n = 144) of the Bokaal study were tested on their IgG response to foods. The IgG results of the food assays were dichotomized high or low using the 66th centile as a cut-off value. RESULTS: Atopic children more often had high IgG levels to foods than nonatopic children. IgG to egg white (OR = 7.50) and mixture of wheat and rice (OR = 4.79) were most strongly associated with positive specific IgE. In a stepwise logistic regression analysis egg white, mixture of wheat and rice, and orange were selected (OR = 3.76, OR = 2.43, and OR = 2.11, respectively). In children without eczema higher levels of IgG to foods were still significantly associated with atopy, which was most prominent for egg white, orange and cow's milk. CONCLUSION: An increased IgG antibody level to foods, especially to egg white, orange, and mixture of wheat and rice, indicates an increased risk of having IgE to cat, dog, mite, egg and/or milk allergens, even in the noneczematous group. Therefore, in another prospective study we are currently investigating the usefulness of IgG in early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing allergic diseases in the future.

An IL-13 promoter polymorphism associated with increased risk of allergic asthma.

IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position -1055. The IL-13 -1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. We postulate that the presence of this polymorphism predisposes to the development of allergic asthma.

Standardization of in vivo and in vitro diagnostic procedures in food allergy.

For most foods, true standardization is not yet feasible because there is insufficient information on the relative importance of individual allergens and their variants. Standardization without sufficient information may easily be counterproductive because improvements are less likely to be implemented. In the analysis of natural test material, the following principles apply: 1) RAST inhibition is usually inferior to other means of allergen quantitation. 2) Immunoblot is inefficient for some "important" allergens and overefficient for some "unimportant" allergens and may therefore be deceptive. 3) Single-component assays are the only satisfactory way to describe complex mixtures. Improving the actual food-testing procedure is important, but will not alone result in a reliable diagnostic procedure. Tests for measuring "effect modifiers" will have to be developed in order to predict in vivo reactions to foods.

Allergy to bumblebee venom. II. IgE cross-reactivity between bumblebee and honeybee venom.

To obtain more information on IgE cross-reactivity between bumblebee venom and honeybee venom, we tested sera from venom-sensitized patients for specific IgE against venoms from the European bumblebee (Bombus terrestris), the North American bumblebee (Megabombus pennsylvanicus), and the honeybee (Apis mellifera). RAST, RAST-inhibition, and immunoblotting experiments indicate that bumblebee venom and honeybee venom contain venom-specific IgE-binding epitopes. These results suggest that immunotherapy using honeybee venom may not be effective in all bumblebee venom-allergic patients. Our experiments also revealed differences in IgE binding for venom from European and American bumblebees.

The role of IL-13 in IgE synthesis by allergic asthma patients.

IgE antibodies play a crucial role in allergic type I reactions. Only IL-4 and IL-13 are able to induce an immunoglobulin isotype switch to IgE in B cells. A major question is to what extent these cytokines contribute to the production of IgE in allergic patients. To address this question we used an in vitro culture system in which the production of IgE is dependent on endogenously produced IL-4 and IL-13. In cultures of purified T and B cells from allergic asthma patients and non-atopic controls, T cells were polyclonally stimulated to obtain IL-4, IL-13 and subsequently IgE secretion. The absolute amount of IgE produced was not significantly different between patients and controls. When neutralizing IL-4 antibodies were included during culture, the production of IgE was dramatically inhibited in both patients and controls (production of IgE was reduced to 12%). However, neutralization of IL-13 led to a significantly stronger inhibition of IgE production in the patient group: production of IgE was reduced to 23 +/- 3% versus 50 +/- 10% in the control group. Corresponding with these results, we also observed a higher production of IL-13 by the patients, while the production of IL-4 was not significantly different. A more detailed analysis of the production of IL-13 revealed that patients' T cells were less sensitive to a negative signal controlling IL-13 production. Our results indicate that, at least in vitro, IgE production in allergic asthma patients is more dependent on IL-13 than in non-atopics, due to enhanced IL-13 production and to enhanced IgE production in response to IL-13.