Treatment of hereditary angioedema with nanofiltered C1-esterase inhibitor concentrate (Cetor®): multi-center phase II and III studies to assess pharmacokinetics, clinical efficacy and safety.

From 1997, plasma-derived C1-inhibitor concentrate (Cetor®) has been available to HAE and AAE patients. Recently, a virus reducing 15 nm nanofiltration step has been introduced in the production process. A randomized, double-blind controlled cross-over study was performed to compare the pharmacokinetics (PK) of nanofiltered (C1-INH-NF) with conventional C1-inhibitor (C1-INH). Efficacy and safety were investigated in an open-label, on-demand and a prophylactic study. No differences in pharmacokinetic parameters between C1-INH and C1-INH-NF were found (13 non-symptomatic HAE patients). Both C1-inhibitor products equally increased plasma C4 levels. In the on-demand study, 14 acute angioedema attacks in 8 patients were analyzed. In the prophylactic study, 1 AAE and 5 HAE patients experienced in total 31 attacks during 748 observation days. In total 180,000 units of C1-INH-NF were administered. No product-related adverse events occurred, and no anti-C1-antibodies were induced. Nanofiltration in the production process of C1-inhibitor did not affect the pharmacokinetics, efficacy, and safety.

An ultrapure plasma-derived monoclonal antibody-purified factor IX concentrate (Nonafact®), results of phase III and IV clinical studies.

Nonafact(®), an ultrapure, monoclonal antibody-purified factor IX concentrate (FIX) was developed to minimize risk of thrombotic complications and viral transmission. To investigate the pharmacokinetics, efficacy and safety, phase III/IV studies were performed in the Netherlands and Poland from 1996 to 2007. The mean half-life, in vivo response and recovery of Nonafact(®) were 18.7 (SD 2.0) h, 1.1 (SD 0.2) IU dL(-1) per IU kg(-1) b.w. of FIX infused and 49% (SD 10%), respectively. Eleven surgical procedures were performed in eight patients. During two surgeries, both high-risk, blood loss was observed. No postoperative bleeding occurred. The in vivo recovery of FIX was higher than expected. In the phase III follow-up study, 26 previously treated patients (PTP) were included with a median follow-up of 1130 days. From the 1617 minor bleedings, 80.5% was stopped after a single infusion. In the phase IV study thirteen patients were treated for a median study period of 737 days. In the two follow-up studies the investigators rated the effect of Nonafact(®) as excellent/good in 95% of major bleedings. Surgeries for which Nonafact(®) was given prophylactically were without bleeding problems. In total more than 10 million units of Nonafact(®) were used during almost 120 person-years. Only one minor adverse event was reported. No inhibitors, viral transmissions and thrombogenic events occurred. In conclusion, Nonafact(®) is safe and provides excellent haemostasis in haemophilia B patients treated for spontaneous bleeding or undergoing surgical procedures. Due to the excellent in vivo recovery characteristic, treatment with Nonafact(®) is cost saving compared to other FIX products.

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.

Allergenicity assessment of apple cultivars: hurdles in quantifying labile fruit allergens.

BACKGROUND: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes. METHODS: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS). RESULTS: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by approximately 1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen. CONCLUSIONS: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.

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.

The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum.

BACKGROUND: For the detection of allergen-specific IgE in serum, IgE-binding assays such as the radioallergosorbent assay (RAST) are commonly used. In this study, the applicability and sensitivity of the stripped basophil histamine release bioassay was investigated and compared to the RAST. METHODS: Basophils were stripped of their IgE by an acidic buffer, sensitized by human serum and stimulated by allergen with or without interleukin (IL)-3. The histamine release was determined by fluorometric analysis. RESULTS: We showed that for enhancement of the maximal histamine release and the sensitivity of the stripped basophil assay, the priming cytokine IL-3 can be added to the basophils simultaneously to the stimulus. Preincubation of the cells with IL-3, as described in other studies, was not necessary. The bioassay can be used to study the specificity of IgE-mediated reactions. Basophils sensitized by serum absorbed to a particular allergen did not respond to this allergen anymore. This method is very suitable to study cross-reactivity between allergens. The results obtained in the bioassay were comparable to those obtained in the RAST. Using the RAST, lower concentrations of allergen-specific IgE were detected than in the bioassay. However, sera containing IgE against minor allergenic components were negative in the RAST, but strongly positive in the basophil assay. CONCLUSIONS: The stripped basophil histamine release bioassay is useful to complement and extend serological detection of allergen-specific IgE. Especially with sera containing IgE against minor components, this assay is more suitable than the RAST. Furthermore, in this assay, the dependency of IgE and of allergen-specific IgE in reactions can be studied in more detail.