Mechanisms of action that contribute to efficacy of omalizumab in chronic spontaneous urticaria.

The monoclonal anti-immunoglobulin E (IgE) antibody, omalizumab, was the first drug approved for use in patients with chronic idiopathic/spontaneous urticaria (CIU/CSU) who remain symptomatic despite H1 -antihistamine treatment. Omalizumab binds to free IgE, which lowers free IgE levels and causes FcεRI receptors on basophils and mast cells to be downregulated. It has been shown to improve symptoms of CIU/CSU, but its mechanism of action is not currently understood. Potential mechanisms in CIU/CSU include reducing mast cell releasability, reversing basopenia and improving basophil IgE receptor function, reducing activity of IgG autoantibodies against FcεRI and IgE, reducing activity of IgE autoantibodies against an antigen or autoantigen that has yet to be definitively identified, reducing the activity of intrinsically 'abnormal' IgE, and decreasing in vitro coagulation abnormalities associated with disease activity. However, none of these theories alone or in combination fully account for the pattern of symptom improvement seen with omalizumab therapy, and therefore, no one mechanism is likely to be the definitive mechanism of action. Additional research is needed to further clarify the involvement of omalizumab in relieving symptoms associated with the complex, multifactorial pathogenesis of CIU/CSU.

Omalizumab substantially improves dermatology-related quality of life in patients with chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous/idiopathic urticaria (CSU/CIU) has substantial detrimental effects on health-related quality of life (HRQoL) with an effect comparable to or worse than many other skin diseases. OBJECTIVE: To assess the effect of omalizumab on CSU patients' HRQoL, measured by the Dermatology Life Quality Index (DLQI) in three phase III studies ASTERIA I, ASTERIA II and GLACIAL. METHODS: A post hoc analysis examined changes in DLQI scores, distribution of patients across DLQI bands and the proportion reaching minimal clinically important difference (MCID) following omalizumab vs. placebo. RESULTS: Omalizumab 300 mg significantly improved total DLQI scores vs. placebo, with a mean decrease from baseline to week 12 of -10.3 vs. -6.1 (P < 0.0001) in ASTERIA I, -10.2 vs. -6.1 (P = 0.0004) in ASTERIA II and -9.7 vs. -5.1 (P < 0.0001) in GLACIAL. A significant shift from high disease impact on life at baseline towards less impact at week 12 was seen with omalizumab 300 mg vs. placebo (P < 0.001; all studies). The proportion of patients where change in mean total DLQI score from baseline to week 12 reached an MCID of ≥4 was 74.1%, 76.0% and 77.2% in ASTERIA I, II and GLACIAL, respectively (P < 0.01; all studies). LIMITATIONS: Maximum duration of omalizumab treatment was 24 weeks. CONCLUSION: This additional analysis assessed the impact of CSU and benefit of treatment with omalizumab by exploring different facets of DLQI data by treatment arm at multiple assessment points. The original aspects of analysis included applying the concept of the recently validated score for the MCID of the DLQI, changes in DLQI domain scores and in the distribution of subjects based on validated total DLQI score bands. It showed consistently that omalizumab provides significant and clinically relevant improvements in many aspects of HRQoL that are important to patients with CSU. These results contribute to a better understanding of the impact of CSU and its treatment on patients and can support clinical decision-making in routine medical practice.

A novel assay to diagnose hereditary angioedema utilizing inhibition of bradykinin-forming enzymes.

BACKGROUND: Hereditary angioedema types I and II are caused by a functional deficiency of C1 inhibitor (C1-INH), leading to overproduction of bradykinin. The current functional diagnostic assays employ inhibition of activated C1s; however, an alternative, more physiologic method is desirable. METHODS: ELISAs were developed using biotinylated activated factor XII (factor XIIa) or biotinylated kallikrein bound to avidin-coated plates. Incubation with plasma was followed by detection of bound C1-INH. RESULTS: After standard curves were developed for quantification of C1-INH, serial dilutions of normal plasma were employed to validate the ability to detect known concentration of C1-INH in the plasma as a percent of normal. Hereditary angioedema (HAE) types I and II were then tested. The level of functional C1-INH in all HAE types I and II plasma tested was less than 40% of our normal control. This was evident regardless of whether we measured factor XIIa-C1-INH or kallikrein-C1-INH complexes, and the two assays were in close agreement. By contrast, testing the same samples utilizing the commercial method (complex ELISA, Quidel Corp.) revealed the levels of C1-INH between 0 and 57% of normal (mean, 38%), and 42 samples were considered equivocal (four controls and 38 patients). CONCLUSIONS: Diagnosis of HAE types I and II can be ascertained by inhibition of enzymes of the bradykinin-forming cascade, namely factor XIIa and kallikrein. Either method yields functional C1-INH levels in patients with HAE (types I and II) that are clearly abnormal with less variance or uncertainty than the commercial method.

Classification, diagnosis, and approach to treatment for angioedema: consensus report from the Hereditary Angioedema International Working Group.

Angioedema is defined as localized and self-limiting edema of the subcutaneous and submucosal tissue, due to a temporary increase in vascular permeability caused by the release of vasoactive mediator(s). When angioedema recurs without significant wheals, the patient should be diagnosed to have angioedema as a distinct disease. In the absence of accepted classification, different types of angioedema are not uniquely identified. For this reason, the European Academy of Allergy and Clinical Immunology gave its patronage to a consensus conference aimed at classifying angioedema. Four types of acquired and three types of hereditary angioedema were identified as separate forms from the analysis of the literature and were presented in detail at the meeting. Here, we summarize the analysis of the data and the resulting classification of angioedema.

The maddening itch: an approach to chronic urticaria.

Chronic spontaneous urticaria (CSU) is defined as the presence of urticaria with daily or almost daily symptoms for 6 weeks or more. CSU affects 0.1%-0.8% of the population. Its pathogenesis involves autoimmunity, abnormalities in signal transduction, and the action of histamine on H1 receptors. Investigation of CSU should be guided by a thorough history and physical examination. A concise laboratory evaluation, including the CU index, is recommended. This index can provide useful data on severity and response to therapy. Initial treatment should involve increasing doses of nonsedating antihistamines until the intended effect is achieved. Only when a patient is unresponsive to high-dose nonsedating antihistamines (or sedating antihistamines) can we consider CSU refractory and consider immunomodulatory therapy. The most promising drugs are cyclosporine and, more recently, omalizumab.

Unmet clinical needs in chronic spontaneous urticaria. A GA²LEN task force report.

Chronic spontaneous urticaria, formerly also known as chronic idiopathic urticaria and chronic urticaria (CU), is more common than previously thought. At any time, 0.5-1% of the population suffers from the disease (point prevalence). Although all age groups can be affected, the peak incidence is seen between 20 and 40 years of age. The duration of the disease is generally 1-5 years but is likely to be longer in more severe cases, cases with concurrent angioedema, in combination with physical urticaria or with a positive autologous serum skin test (autoreactivity). Chronic spontaneous urticaria has major detrimental effects on quality of life, with sleep deprivation and psychiatric comorbidity being frequent. It also has a large impact on society in terms of direct and indirect health care costs as well as reduced performance at work and in private life. In the majority of patients, an underlying cause cannot be identified making a causal and/or curative treatment difficult. Nonsedating H1-antihistamines are the mainstay of symptomatic therapy, but treatment with licensed doses relieves symptoms effectively in < 50% of patients. Although guideline-recommended updosing up to fourfold increases symptom control in many patients, a substantial number of patients have only little benefit from H1 -antihistamines. Consequently, there is a great need for new therapeutic strategies.

Characterization of human high molecular weight kininogen. Procoagulant activity associated with the light chain of kinin-free high molecular weight kininogen.

Human high molecular weight (HMW) kininogen has been isolated and was found to be a single chain protein of approximately equal to 120,000 daltons. Upon digestion with plasma kallikrein bradykinin is generated, and SDS gel electrophoresis of the kinin-free protein reveals an apparent loss in size of 15,000 daltons. The kinin-free kininogen retains full activity as a coagulation factor and consists of two chains: a heavy chain of approximately equal to 66,000 daltons disulfide-linked to a light chain of 37,000 daltons. The heavy chain of HMW kininogen shares antigenic determinants with LMW kininogen and possesses no detectable coagulant activity. The isolated light chain is shown to be responsible for the coagulant activity of HMW kininogen and contains a unique antigenic determinant that distinguishes HMW kininogen from low molecular weight kininogen.

Substrates of Hageman factor. I. Isolation and characterization of human factor XI (PTA) and inhibition of the activated enzyme by alpha 1-antitrypsin.

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8-9.4 (peak 9.0-9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt alpha-globulin which was isolated free of alpha(1)-chymotrypsin inhibitor, inter alpha-trypsin inhibitor, alpha(2)-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to alpha(1)-antitrypsin, and it was absent in alpha(1)-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as alpha(1)-antitrypsin.

Activation of the classical pathway of complement by Hageman factor fragment.

A fragment of activated Hageman factor (HFf) has been demonstrated to activate the classical pathway of complement in a manner that is analogous to complement activation by antigen-antibody complexes or aggregated IgG. Thus C1, C4, C2, C3, and C5 were found to be depleted on addition of HFf to serum. The reduction of serum hemolytic activity was maximal upon addition of 5 micrograms HFf and an incubation time of 60 min at 37 degrees C. Consumption of the total complement activity and of the individual components proceeded in a dose-dependent fashion. No comparable activity was observed when equimolar concentrations of either the native Hageman factor (HF) or two-chain activated form of Hageman factor (HFa) were incubated with serum. Further, the ability of HFf to convert serum C3 and C4 was similar to that of aggregated IgG as assessed by immunoelectrophoresis. This function of HFf appeared to be independent of plasminogen (or plasmin) since plasminogen-free serum was indistinguishable from normal serum. Radial double immunodiffusion experiments using antiserum to C1q, C1r, and C1s on HFf-treated serum demonstrated the dissociation of the C1 trimolecular complex, with concomitant reduction of C1r antigenicity that is indicative of C1 activation. Thus, HFf appears to lead to C1 activation upon incubation with serum or when incubated with partially purified C1. This may represent a control link between activation of the intrinsic coagulation-kinin pathway and the initiation of the classical complement cascade.

A prealbumin activator of prekallikrein. II. Derivation of activators of prekallikrein from active Hageman factor by digestion with plasmin.

Activation of a plasma fraction containing unactivated Hageman factor and prekallikrein followed by chromatography of this fraction on DEAE-cellulose revealed four peaks having bradykinin-generating activity. Peak 1 contained kallikrein; peaks 2-3, 4, and 5 each contained prekallikrein-activating activity. Elution of peaks 2-3, 4, and 5 from disc gels after electrophoresis at pH 9.3 revealed peaks of prekallikrein-activating activity located at 5-8, 11-12, 15-16, and 20-26 mm, each of which was associated with a peak of clot-promoting activity which specifically corrected Hageman factor deficiency. Conversion of peak 2 to peaks 3, 4, and 5 was associated with a progressive decrease in size, increase in net negative charge, increased prekallikrein-activating activity, and decreased ability to correct Hageman factor deficiency. Plasminogen and plasmin were found on a DEAE-cellulose chromatogram of serum overlapping peaks 2 and 3. Incubation of active Hageman factor with streptokinase-activated plasminogen resulted in enhanced ability of the mixture to activate prekallikrein. Assessment of the products of this reaction by disc gel electrophoresis demonstrated the formation of the prealbumin prekallikrein activator corresponding to the major prekallikrein activator generated by contact activation of human plasma. The conversion of plasminogen to plasmin and the subsequent cleavage of Hageman factor by plasmin to form activators of prekallikrein represents one pathway in which coagulation, fibrinolysis, and inflammation are linked.

Modulation of function of the activated first component of complement by a fragment derived from serum. I. Effect on early components of complement.

An activity designated Kf can be separated from human serum and shown to give a 100-300% enhancement in the hemolytic activity of fully activated, fractionated C1. The enhancement of C1 activity is not because of activation of precursor C1 and it is not attributable to an effect on C1 binding. EAC42 or EAC4 intermediates interacted with C1Kf exhibit a greater T(max) and shorter Z(max) than when such intermediates are reacted with the same number of hemolytic units of C1. C3 consumption by the EAC1Kf42 intermediate greatly exceeds that of the EAC142 intermediate produced from the same EAC4 cells by comparable inputs of the other two complement components. Taken together, these findings suggest that Kf-treated C1 achieves more efficient utilization of C4 and C2 to create a larger number of 42 sites as appreciated on the intermediates by shorter T(max) and a greater Z(max), and an increased capacity to utilize C3. The capacity of Kf to enhance C1 upon introduction into whole serum of a patient with hereditary angioedema (HAE) in a manner comparable to its effect on fractionated C1 suggests that the effect of Kf may be pertinent to certain pathophysiologic conditions of man.

The fibrinolytic pathway of human plasma. Isolation and characterization of the plasminogen proactivator.

The conversion of the plasminogen proactivator to plasminogen activator by activated Hageman factor or its fragments has been recognized as an essential step in the conversion of plasminogen to plasmin. The plasminogen proactivator has been completely separated from prekallikrein and pre-PTA, two other proenzyme substrates of activated Hageman factor or its fragments. Plasminogen proactivator, free of any contaminating proteins as assessed by disc gel electrophoresis or isoelectric focusing, revealed a single band with an isoelectric point of 8.9 corresponding in position to the Hageman factor activatable material eluted from replicate unstained gels. After conversion of plasminogen proactivator by Hageman factor fragments to the plasminogen activator, the active site of the plasminogen activator is not inhibited by C1INH and is thus readily distinguished from that of kallikrein or PTA. The plasminogen activator is susceptible to inactivation by DFP while the plasminogen proactivator is not, as has been the case for esterases having a serine in the active site. Its interaction with plasminogen is inhibited by epsilon-aminocaproic acid.

The selective eosinophil chemotactic activity of histamine.

Histamine diphosphate was shown to selectively attract human eosinophils from mixed granulocyte populations when over 20% eosinophils were used in a modified Boyden chamber chemotactic assay system. This effect of histamine is abolished by incubation with diamine oxidase (histaminase) and was generated by decarboxylation of L-histidine. A linear dose dependent increase in eosinophil migration was observed between 3 X 10(-7) M and 1.25 X 10(-6) M, while higher concentrations of histamine inhibited the migration of eosinophils. The attractant activity of histamine was not inhibited by H-1 or H-2 receptor antagonists, however, the inhibition of migration observed at higher histamine concentrations was reversed by metiamine, an H-2 receptor antagonist. The effects of histamine upon eosinophil migration were demonstrable using three different assays: (a) counting cells that had traversed 5-mum pore, 12-mum thick polycarbonate filters, (b) counting cells that had migrated various distances into a 3-mum pore, 145-mum cellulose nitrate filters, or (c) measuring the number of cells that had traversed an upper polycarbonate filter and migrated into a lower cellulose nitrate filter using 15Cr-labeled cells. The ability of histamine to enhance eosinophil migration was shown to be dependent upon the presence of a concentration gradient; histamine did not cause a dose-dependent increase in random motility. Furthermore, preincubation of the eosinophils with histamine deactivate the cells to further stimulation by histamine or by C5a. It is concluded that in low doses histamine is a chemoattractant for human eosinophils, while in higher doses histamine inhibits eosinophil migration. These observations may relate to the influx and localization of eosinophils in immediate hypersensitivity reactions.

A prealbumin activator of prekallikrein. 3. Appearance of chemotactic activity for human neutrophils by the conversion of human prekallikrein to kallikrein.

Human plasma kallikrein has been shown to directly and selectively attract human neutrophils from a mixed leukocyte population. The capacity of plasma kallikrein to be chemotactic and to generate the nonapeptide bradykinin was maintained during progressive purification. While neither highly purified prekallikrein nor the prealbumin Hageman factor fragments were chemotactic alone, their interaction so as to convert prekallikrein to kallikrein yielded both chemotactic and kinin-generating activity. Both functions of kallikrein were inhibited by treatment with diisopropyl fluorophosphate, indicating an essential role for the active site of the enzyme in the expression of its chemotactic activity.

Monocyte chemotactic and activating factor is a potent histamine-releasing factor for human basophils.

Recombinant monocyte chemotactic-activating factor (MCAF) has been shown to induce histamine release from human basophils with a dose response between 10(-9) and 10(-6) M. The peak of activity was reached at 10(-7) M. Histamine release by MCAF was rapid with an initial rate comparable with histamine release by an optimal dose of anti-IgE. MCAF led to peak histamine release within 1 min. 80% of the subjects tested were responsive to MCAF or anti-IgE, while all were responsive to FMLP. The percentage histamine release by MCAF was, however, less than that seen with anti-IgE or FMLP, but this was attributable to a lesser percent release in nonatopic subjects; atopic subjects responded similarly to all three agonists. MCAF was also shown to activate highly purified human basophils more readily than mixed leukocytes, and its activity was inhibited by a polyclonal rabbit antibody. At a suboptimal concentration (2.5 x 10(-9) M), MCAF was unable to prime the basophil to histamine release by other secretagogues. However, interleukin 3 (IL-3) and IL-5 could each prime basophils for MCAF-induced secretion. Therefore, our results suggest that MCAF may be a major contributor to the histamine-releasing activity seen in peripheral blood mononuclear cell supernatants that has been designated histamine releasing factor(s).

Pathogenesis of chronic urticaria.

Chronic urticaria is defined as the presence of urticaria (hives) for at least 6 weeks with the assumption that it occurs daily or close to it. If we eliminate physical urticarias and urticarial vasculitis from consideration, the remainder can be divided into autoimmune chronic urticaria (45%) and idiopathic chronic urticaria (55%). The autoimmune subgroup is associated with the IgG anti-IgE receptor alpha subunit in 35-40% of patients and IgG anti-IgE in an additional 5-10%. These autoantibodies have been shown to activate blood basophils and cutaneous mast cells in vitro with augmentation of basophil activation by complement and release of C5a, in particular. Binding methods (immunoblot and ELISA) yield positives in many autoimmune diseases as well as occasional normal subjects or patients with other forms of urticaria but most such sera are non-functional. Activation of basophils or mast cells causing histamine release is quite specific for chronic urticaria and defines the autoimmune subgroup. Although pathogenicity is not formally proven, the antibodies cause wealing upon intradermal injection, and removal of the autoantibody leads to remission. A cellular infiltrate is seen to be characterized by mast cell degranulation and infiltration of CD4+ T lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The intensity of the infiltrate and clinical severity of the disease (including accompanying angio-oedema) is more severe in the autoimmune subpopulation. This latter group also has a higher evidence of human leucocyte antigen DR alleles associated with autoimmunity and a 25% incidence of antithyroid antibodies with diagnosed hypothyroidism in some. Hypo-responsiveness of patients' basophils to anti-IgE and hyperresponsiveness to serum defines another subpopulation (at least 50%) that overlaps the idiopathic and autoimmune subgroups. Hypo-responsiveness to anti-IgE has been shown to be associated with elevated levels of cytoplasmic phosphatases that inhibit degranulation. Reversal of the abnormality is seen with disease remission. Further work will be needed to distinguish whether this is a cause or a consequence of persistent urticaria and to further assess the relationship (or lack thereof) of altered responsiveness (decreased or increased) with the presence or absence of activating autoantibodies.