Angioedema without urticaria: novel findings which must be measured in clinical setting.

PURPOSE OF REVIEW: Angioedema without urticaria is composed of an increasing subtype's variety and presents a challenging diagnosis. This review summarizes the subtypes recently described and subsequent new findings helpful within their classification. RECENT FINDINGS: New methods to measure cleaved high molecular weight kininogen and activated plasma kallikrein have emerged as potential biochemical tests to identify bradykinin-mediated angioedema. Three new subtypes of hereditary angioedema (HAE) with normal C1 inhibitor were described in the past two years: HAE due to mutation in plasminogen gene, in kininogen gene, and in angiopoietin-1 gene; implicating the fibrinolytic and contact systems, and the regulation of vasculature, respectively. The understanding of some mechanisms in angioedema has been improved, compatible to the dominant-negative for some C1 inhibitor variants; furthermore, the increased activation of truncated F12 mutants by plasma kallikrein; and the diminished binding of angiopoietin-1 to its receptor. SUMMARY: The validation of biomarkers for the contact system activation could be beneficial in differentiating bradykinin - from histaminergic-mediated angioedema. Currently, the available laboratorial tests are still somewhat restricted to the evaluation of the complement activation and the mediators of nonhistaminergic and nonbradykinin-mediated angioedema remain to be identified.

Serum complexes between C1INH and C1INH autoantibodies for the diagnosis of acquired angioedema.

Acquired angioedema due to C1-inhibitor (C1INH) deficiency (AAE) is caused by secondary C1INH deficiency leading to bradykinin-mediated angioedema episodes. AAE typically presents in adulthood and is associated with B cell lymphoproliferation. Anti-C1INH autoantibodies (antiC1INHAbs) are detectable in a subset of AAE cases and considered a hallmark of the disease. When free antiC1INHAbs and malignant tumors are not detectable, diagnosis relies on the finding of low C1INH levels and/or function, lack of family history and SERPING1 mutations, age at onset and low or undetectable C1q levels, none of which is specific for AAE. We tested the diagnostic value of a novel enzyme-linked immunosorbent assay (ELISA) for the detection of circulating complexes between C1INH and antiC1INHAbs (C1INH-antiC1INHAb) in the serum of 20 European AAE patients characterized on the basis of their complement levels and function. Free antiC1INHAbs were detected in nine of 20 patients [six of immunoglobulin (Ig)G class, two of IgM class and one simultaneously presenting IgG and IgM classes], whereas C1INH-antiC1INHAb complexes were found in 18 of 20 of the AAE cases, regardless of the presence or absence of detectable free anti-C1INHAbs. Of note, nine of 20 patients showed negative free antiC1INHabs, but positive C1INH-antiC1INHAb complexes in their first measurement. In the cohort presented, IgM-class C1INH-antiC1INHAb are specifically and strongly associated with low C1q serum levels. Detection of C1INH-antiC1-INHAbs provides an added value for AAE diagnosis, especially in those cases in whom no free anti-C1INH antibodies are detected. The link between IgM-class C1INH-antiC1INHAb complexes and C1q consumption could have further implications for the development of autoimmune manifestations in AAE.

THE JEREMIAH METZGER LECTURE NOVEL THERAPEUTIC STRATEGIES OF ALLERGIC AND IMMUNOLOGIC DISORDERS.

Advances in understanding the immunological basis and mechanisms underlying allergic and immunologic disorders have led to effective but costly long-term and repetitive biologic therapies. Gene therapy is a rapidly advancing technology, in which a single administration of an adeno-associated virus encoding the therapeutic protein or monoclonal antibody may provide effective long-term therapy for allergic and immunologic disorders. In this review, we summarize the recent studies from our laboratory developing gene therapy strategies to treat hereditary angioedema and peanut allergy. The unraveling of the pathogenesis of immune-based disorders, including hereditary deficiencies of components of the immune system and allergic disorders, has led to the development of therapies using parenteral administration of recombinant proteins or monoclonal antibodies (1). While many of these therapies are highly effective, they are limited by the half-life of the therapeutic protein or antibody, requiring repetitive administration of days to weeks (2-15). The focus of recent work in our laboratory has been to solve this problem by substituting protein/monoclonal antibody administration with gene therapy, where current technology allows for a single administration of the gene coding for a protein or antibody to provide persistent expression of effective levels of the therapeutic protein or antibody. Gene therapy is a drug delivery platform which uses genetic material, usually in the form of coding exons of the therapeutic gene, to correct, compensate for, or prevent the development of an abnormal phenotype (16). Originally conceptualized as a strategy to treat rare hereditary disorders, gene therapy is being developed for a wide range of human disorders, including common acquired conditions (17-20). In this review, we will describe how we have adopted gene therapy technology to develop therapies for immune-related disorders, using as examples hereditary angioedema, an inherited autosomal dominant disorder, and peanut allergy, a common acquired allergic disorder.

The complex alteration in the network of IL-17-type cytokines in patients with hereditary angioedema.

Hereditary angioedema (HAE) is a rare autosomic-dominant disorder characterized by a deficiency of C1 esterase inhibitor which causes episodic swellings of subcutaneous tissues, bowel walls and upper airways that are disabling and potentially life-threatening. We evaluated n = 17 patients with confirmed HAE diagnosis during attack and remission state and n = 19 healthy subjects. The samples were tested for a panel of IL (Interleukin)-17-type cytokines (IL-1ß, IL-6, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-17, IL-21, IL-22, IL-23) and transforming growth factor-beta (TGF-ß) subtypes. Data indicate that there are variations of cytokine levels in HAE subjects comparing the condition during the crisis respect to the value in the remission phase, in particular type 17 signature cytokines are increased, whereas IL-23 is unmodified and TGF-ß3 is significantly reduced. When comparing healthy and HAE subjects in the remission state, we found a significant difference for IL-17, GM-CSF, IL-21, TGF-ß1 and TGF-ß2 cytokines. These results confirm and extend our previous findings indicating that in HAE there is operating an inflammatory activation process, which involves also T helper 17 (Th17) cytokines and TGF-ß isoforms, associated with localized angioedema attacks and characterized by elevated bradykinin levels.

Diminished capacity of opsonization and immune complex solubilization, and detection of anti-C1q antibodies in sera from patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is an autosomal dominant disease caused by deficiency of C1 esterase inhibitor. Symptoms of HAE include edema, which can potentially cause suffocation. Some patients with HAE exhibit immunological abnormalities, which could prevent an accurate diagnosis. Low levels of complement components are characteristic of HAE and in other settings are thought to reduce elimination of apoptotic cells and immune complex (IC). Thus, we aimed to experimentally clarify the mechanism of immunological abnormalities using sera from HAE patients. METHODS: Serum samples from 18 patients with HAE were collected when free from angioedema attack and compared with normal human pooled sera (NHPS) from 20 healthy volunteers. Opsonization was measured as the rate of phagocytosis of apoptotic Jurkat cells by macrophages differentiated from THP-1 cells incubated with serum. IC solubilization in serum was analyzed by quantifying peroxidase released from a synthetic IC composed of peroxidase and anti-peroxidase antibodies. Anti-C1q antibody levels were detected using an enzyme-linked immunosorbent assay. RESULTS: Serological immunological abnormalities were detected in 12 patients. Opsonization in serum samples from each patient with HAE was lower than that in NHPS (∼20% versus 70%, respectively). The rate of IC solubilization was lower in serum from HAE patients than NHPS. Some patients had high serum anti-C1q antibody levels with increased serum IC levels. CONCLUSIONS: Sera from patients with HAE exhibit anti-C1q antibodies, with a lower capacity for opsonization and IC solubilization. This may be associated with immunological abnormalities and should be investigated further to facilitate accurate diagnosis of HAE.

Contact system revisited: an interface between inflammation, coagulation, and innate immunity.

The contact system is a plasma protease cascade initiated by factor XII (FXII) that activates the proinflammatory kallikrein-kinin system and the procoagulant intrinsic coagulation pathway. Anionic surfaces induce FXII zymogen activation to form proteolytically active FXIIa. Bacterial surfaces also have the ability to activate contact system proteins, indicating an important role for host defense using the cooperation of the inflammatory and coagulation pathways. Recent research has shown that inorganic polyphosphate found in platelets activates FXII in vivo and can induce coagulation in pathological thrombus formation. Experimental studies have shown that interference with FXII provides thromboprotection without a therapy-associated increase in bleeding, renewing interest in the FXIIa-driven intrinsic pathway of coagulation as a therapeutic target. This review summarizes how the contact system acts as the cross-road of inflammation, coagulation, and innate immunity.

The Janus faces of acquired angioedema: C1-inhibitor deficiency, lymphoproliferation and autoimmunity.

Several clinical and biological features of lymphoproliferative diseases have been associated with an increased risk of developing autoimmune manifestations. Acquired deficiency of C1-inhibitor (C1-INH) (AAE) is a rare syndrome clinically similar to hereditary angioedema (HAE) characterized by local increase in vascular permeability (angioedema) of the skin and the gastrointestinal and oro-pharyngo-laryngeal mucosa. Bradykinin, a potent vasoactive peptide, released from high molecular weight kininogen when it is cleaved by plasma kallikrein (a serine protease controlled by C1-INH), is the mediator of symptoms. In total 46% of AAE patients carry an underlying hematological disorder including monoclonal gammopathy of uncertain significance (MGUS) or B cell malignancies. However, 74% of AAE patients have anti-C1-INH autoantibodies without hematological, clinical or instrumental evidence of lymphoproliferative disease. Unlike HAE patients, AAE patients usually have late-onset symptoms, do not have a family history of angioedema and present variable response to treatment due to the hypercatabolism of C1-INH. Experiments show that C1-INH and/or the classical complement pathway were consumed by the neoplastic lymphatic tissues and/or anti-C1-INH neutralizing autoantibodies. Therapy of AAE follows two directions: 1) prevention/reversal of the symptoms of angioedema; and 2) treatment of the associated disease. Different forms of B cell disorders coexist and/or evolve into each other in AAE and seem to be dominated by an altered control of B cell proliferation, thus AAE represents an example of the strict link between autoimmunity and lymphoproliferation.

C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay.

BACKGROUND: Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements. METHODS: The contact phase was reconstituted using the purified components, with C1Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro-Phe-Arg-pNA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics (ROC) were used to calculate the assay's diagnostic performance. RESULTS: The calibration curve was built using C1Inh standard (threshold limit 0.10 × 10(-3) U, i.e., 0.2 pmol), and C1Inh function was quantified in the sample, with a reference interval established based on healthy individuals (n = 281; men: 0.61-1.10 U/ml, median: 0.85 U/ml; women: 0.42-1.08 U/ml, median: 0.74 U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36 U/ml (area under curve [AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61 U/ml (AUC: 1; sensitivity: 100.0%; specificity: 100.0%). CONCLUSION: The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target.

Bradykinin-mediated diseases.

Diseases which have been demonstrated to be caused by increased plasma levels of bradykinin all have angioedema as the common major clinical manifestation. Angioedema due to therapy with angiotensin-converting enzyme (ACE) inhibitors is caused by suppressed bradykinin degradation so that it accumulates. This occurs because ACE metabolizes bradykinin by removal of Phe-Arg from the C-terminus, which inactivates it. By contrast, angioedema due to C1 inhibitor deficiency (either hereditary types I and II, or acquired) is caused by bradykinin overproduction. C1 inhibitor inhibits factor XIIa, kallikrein and activity associated with the prekallikrein-HK (high-molecular-weight kininogen) complex. In its absence, uncontrolled activation of the plasma bradykinin cascade is seen once there has been an initiating stimulus. C4 levels are low in all types of C1 inhibitor deficiency due to the instability of C1 (C1r, in particular) such that some activated C1 always circulates and depletes C4. In the hereditary disorder, formation of factor XIIf (factor XII fragment) during attacks of swelling causes C4 levels to drop toward zero, and C2 levels decline. A kinin-like molecule, once thought to be a cleavage product derived from C2 that contributes to the increased vascular permeability seen in hereditary angioedema (HAE), is now thought to be an artifact, i.e. no such molecule is demonstrable. The acquired C1 inhibitor deficiency is associated with clonal disorders of B cell hyperreactivity, including lymphoma and monoclonal gammopathy. Most cases have an IgG autoantibody to C1 inhibitor which inactivates it so that the presentation is strikingly similar to type I HAE. New therapies for types I and II HAE include C1 inhibitor replacement therapy, ecallantide, a kallikrein antagonist, and icatibant, a B2 receptor antagonist. A newly described type III HAE has normal C1 inhibitor, although it is thought to be mediated by bradykinin, as is an antihistamine-resistant subpopulation of patients with 'idiopathic' angioedema. The mechanism(s) for the formation of bradykinin in these disorders is unknown.

Escalating doses of C1 esterase inhibitor (CINRYZE) for prophylaxis in patients with hereditary angioedema.

BACKGROUND: Nanofiltered C1 inhibitor (human) is approved in the United States for routine prophylaxis of angioedema attacks in patients with hereditary angioedema, a rare disease caused by a deficiency of functional C1 inhibitor. OBJECTIVE: To assess the safety of escalating doses of nanofiltered C1 inhibitor (human) in patients who were not adequately controlled on the indicated dose (1000 U every 3 or 4 days). METHODS: Eligible patients had >1 attack/month over the 3 months before the trial. Doses were escalated to 1500 U every 3 or 4 days for 12 weeks, at which point, the patients were evaluated. If treatment was successful (≤1 attack/mo) or at the investigator's discretion, the patients entered a 3-month follow-up period. The patients with an average of >1 attack/month were eligible for further escalation to 2000 U and then 2500 U. RESULTS: Twenty patients started at 1500 U; 13 were escalated to 2000 U, and 12 were escalated to 2500 U. Eighteen patients reported adverse events. Two patients reported 4 serious adverse events (cerebral cystic hygroma, laryngeal angioedema attack, anemia, and bile duct stone) that were considered by investigators to be unrelated to treatment. Notably, there were no systemic thrombotic events or discontinuations due to adverse events. Fourteen patients were treated successfully (70%), continued to the follow-up period at the investigator's discretion, or experienced a reduction in attacks of >1.0/month. CONCLUSIONS: Dose escalation of nanofiltered C1 inhibitor (human) up to 2500 U was well tolerated and reduced attack frequency in the majority of patients.

The procoagulant and proinflammatory plasma contact system.

The contact system is a plasma protease cascade that is initiated by coagulation factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation or the kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood, however, its in vivo functions are just beginning to emerge. Data obtained in genetically engineered mice have revealed an essential function of the contact system for thrombus formation. Severe deficiency in contact system proteases impairs thrombus formation but does not reduce the hemostatic capacity of affected individuals. The system is activated by an inorganic polymer, polyphosphate that is released from activated platelets. Excessive inherited activation of the contact system causes a life-threatening swelling disorder, hereditary angioedema. Activation of the contact system by pathogens contributes to leakage in bacterial infections. Mast-cell-derived heparin triggers contact-system-mediated edema formation with implications for allergic disease states. Here we present an overview about the plasma contact system in occlusive and inflammatory disease and its contribution to health and pathology.

Strong correlation of high EBNA-1-IgG levels with edematous attacks involving upper airway mucosa in hereditary angioedema due to C1-inhibitor deficiency.

Elevated level of IgG-type antibodies against Type 1 nuclear antigen (anti-EBNA-1-IgG) of the Epstein-Barr virus is a strong risk factor for certain autoimmune diseases. We measured anti-EBNA-1 IgG titers in 107 patients with hereditary angioedema due to C1-inhibitor deficiency (HAE-C1-INH). In the sera from 33 longitudinally tested patients, we found a very strong correlation (R>0.75, p=0.0005) between anti-EBNA-1-IgG titers measured in 2004 and 2010, respectively. High (>200 U/ml) anti-EBNA-1 levels were strongly correlated with the frequency of upper airway attacks (p=0.003) and the dose requirement of C1-inhibitor concentrate (p=0.008), while no significant association with the frequency of subcutaneous and abdominal attacks was found. These novel findings indicate that the underlying/triggering mechanisms of upper airway attacks in HAE-C1-INH may differ from that of other types of attacks and measurement of the anti-EBNA-1 IgG levels may be suitable for the prediction of upper airway attacks and C1-inhibitor concentrate requirement in HAE-C1-INH patients.

C1 inhibitor, a multi-functional serine protease inhibitor.

C1 inhibitor (C1INH) is a serpin that regulates both complement and contact (kallikrein-kinin) system activation. It consists of a serpin domain that is highly homologous to other serpins and an amino terminal non-serpin mucin-like domain. Deficiency of C1INH results in hereditary angioedema, a disease characterised by episodes of angioedema of the skin or the mucosa of the gastrointestinal tract or the oropharynx. Although early data suggested that angioedema was mediated via complement system activation, the preponderance of the data indicate that bradykinin is the mediator. In the past few years, it has become apparent that C1INH has additional anti-inflammatory functions independent of protease inhibition. These include interactions with leukocytes that may result in enhanced phagocytosis, with endothelial cells via E- and P-selectins that interfere with leukocyte rolling and in turn results in suppression of transmigration of leukocytes across the endothelium, and interactions with extracellular matrix components that may serve to concentrate C1INH at sites of inflammation. In addition, C1INH suppresses gram negative sepsis and endotoxin shock, partly via direct interaction with endotoxin that interferes with its interaction with macrophages, thereby suppressing tumour necrosis factor-a and other inflammatory mediators. C1INH treatment improves outcome in a number of disease models, including sepsis and other bacterial infections, possibly malaria, ischaemia-reperfusion injury (intestinal, hepatic, muscle, cardiac, brain), hyper-acute transplant rejection, and other inflammatory disease models. Recent data suggest that this effectiveness is the result of mechanisms that do not require protease inhibition, in addition to both complement and contact system activation.

Complement disorders and hereditary angioedema.

The term complement was introduced more than 100 years ago to refer to a group of plasma factors important in host defense and in the destruction of microorganisms. We now know that there are 3 separate activation pathways that appeared at different times in evolution: the classical, alternative, and lectin pathways. Two of these appear before the evolution of the adaptive immune system and do not require antibody for initiation. All pathways come together to activate C3, the principle opsonic protein of the complement cascade, and all continue together to the generation of biologically active factors, such as C5a, and to lysis of cells and microbes. In general, complete deficiencies of complement proteins are rare, although partial or complete deficiencies of one of the proteins that initiates the lectin pathway, mannose-binding lectin, are far more common. Although genetically controlled complement defects are rare, defects in the proteins in the circulation and on cell membranes that downregulate complement so as to limit uncontrolled inflammation are more common. A number of these are discussed, and because new methods of treatment are currently being introduced, one of these defects, CI inhibitor deficiency associated with hereditary angioedema, is discussed in some detail.

Acquired angioedema associated with hereditary angioedema due to C1 inhibitor deficiency.

Angioedema caused by C1 inhibitor deficiency is a rare disorder that may be either hereditary or acquired, the latter being mainly associated with lymphoproliferative disorders. A 51-year-old woman who had suffered from episodes of acute peripheral edema since she was 12 was diagnosed with hereditary angioedema at the age of 40 and remained stable with stanozolol. Due to a worsening of her symptoms she was reassessed and low levels of C1q and an abnormal lymphocyte count were detected. Immunophenotyping of peripheral blood revealed 9% monoclonal lambda B cells with a follicular center phenotype. The histopathology was consistent with a grade II follicular lymphoma stage IV-A.With chemotherapy, the hematologic disease was controlled and C1q levels returned to normal values. This represents a rare case of a patient with hereditary angioedema who developed acquired angioedema due to a lymphoma that was associated with a reduction in the levels of C1q as her symptoms worsened.

Appraisal of danazol prophylaxis for hereditary angioedema.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by painful, recurrent attacks of inflammation affecting the hands, feet, face, abdomen, urogenital tract, and the larynx. The inflammation can be disfiguring, debilitating, quite painful, and, in the case of laryngeal attacks, life-threatening. Attacks are frequently the source of unnecessary exploratory abdominal procedures, extended hospital stays affecting a patient's ability to retain employment, and severe compromise of the patient's quality of life. HAE is estimated to affect 10,000 people in the US and is caused by deficient or dysfunctional C1-inhibitor, a naturally occurring molecule that is known to inhibit kallikrein, bradykinin, and other serine proteases in plasma. The treatment and management of HAE have been hampered by the dearth of safe and effective therapies. In the United States, there are currently no approved therapies for the treatment of acute HAE attacks. Although prophylactic HAE therapies do exist, they are often viewed as suboptimal due to moderate degrees of efficacy and the existence of adverse effects associated with therapy. Danazol, an attenuated androgen, is the most commonly prescribed prophylaxis treatment for HAE in the United States. Although it has demonstrated moderate efficacy in the prevention of HAE attacks, danazol's side-effect profile can be problematic because there is a correlation between frequency and severity of adverse events and dosage and duration of therapy.