Hereditary and acquired angioedema: Problems and progress: Proceedings of the third C1 esterase inhibitor deficiency workshop and beyond  Angelo Agostoni, MD, Emel Aygören-Pürsün, MD, Karen E. Binkley, MD, FRCPC, Alvaro Blanch, Konrad Bork, MD, Laurence Bouillet, MD, CCA, Christoph Bucher, MD, Anthony J Castaldo, MPA, Marco Cicardi, MD, Alvin E Davis, MD, Caterina De Carolis, MD, Christian Drouet, PhD, Christiane Duponchel, Henriette Farkas, MD, PhD, Kálmán Fáy, MD, Béla Fekete, MD, PhD, DSc, Bettina Fischer, MD, Luigi Fontana, MD, George Füst, MD, PhD, DSc, Roberto Giacomelli, MD, Albrecht Gröner, PhD, C. Erik Hack, MD, PhD, George Harmat, MD, PhD, John Jakenfelds, MD, Mathias Juers, MD, Lajos Kalmár, Pál N. Kaposi, MD, PhD, István Karádi, MD, PhD, DSc, Arianna Kitzinger, Tímea Kollár, MD, Wolfhart Kreuz, MD, PhD, Peter Lakatos, MD, PhD, DSc, Hilary J. Longhurst, MA, MRCP, PhD, MRCPath, Margarita Lopez-Trascasa, MD, PhD, Inmaculada Martinez-Saguer, MD, Nicole Monnier, PhD, István Nagy, Éva Németh, Erik Waage Nielsen, MD, PhD, Jan H. Nuijens, MD, PhD, Caroline O'Grady, RGN, Emanuela Pappalardo, PhD, Vincenzo Penna, Carlo Perricone, Roberto Perricone, MD, Ursula Rauch, Olga Roche, Eva Rusicke, Peter J Späth, PhD, FAMH, George Szendei, MD, Edit Takács, MD, Attila Tordai, MD, PhD, Lennart Truedsson, MD, PhD, Lilian Varga, PhD, Beáta Visy, MD, Kayla Williams, BS, MA, MFA, Andrea Zanichelli, Lorenza Zingale, MD  Journal of Allergy and Clinical Immunology  Volume 114, Issue 3, Pages S51-S131 (September 2004) DOI: 10.1016/j.jaci.2004.06.047 Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 1 Facial edema. Photo: Dermatologische Klinik, Universitätsspital Zurich, Switzerland. Brunello Wüthrich, MD. Reprinted with permission from Swiss Medical Weekly.43a Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 2 Penile edema. Photo credit: Dr. Martin Ludovic. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 3 Various erythematous rashes preceding or accompanying angioedema episodes. A1, Facial erythema marginatum; A2, close view. Photo credit: Brunello Wüthrich, MD. Reprinted with permission from Swiss Medical Weekly.43a B1, Mottling on chest; B2, close view. Photo credit: George Harmat, MD. Reprinted with permission from Acta Dermato-Venereologica.46 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 4 Sagittal sonogram during an abdominal HAE attack. A significant amount of fluid can be seen in the pouch of Douglas, with a swollen intestinal loop visible (arrow) floating in the free fluid. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 5 Transverse sonogram during an abdominal HAE attack, showing bowel and pancreas. Longitudinal section of a swollen bowel: the intestinal wall is edematously thickened (arrows); in addition, the reflectivity of the pancreas is increased. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 6 Sonogram during an abdominal attack of HAE, showing kidney and spleen. Section of a thickened intestinal wall (large arrow) and a small amount of fluid (small arrow) between the left kidney and the spleen. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 7 Sagittal and transverse sonograms during an HAE attack before and after treatment. Sagittal sections are shown above, transverse below. A, A large amount of free peritoneal fluid has accumulated in the pouch of Douglas and, in the sagittal section, a floating intestinal loop is visible. The urinary bladder appears below. B, Soon after treatment with C1-INH concentrate, the amount of peritoneal fluid is somewhat decreased. C, Only a minimal amount of fluid is present in the pouch of Douglas 24 hours after C1-INH treatment. Several sonograms have previously been published in slightly different format.78,81 Transverse panels B and C reprinted with permission from Acta Paediatrica.81 Sagittal panels A-C reprinted with permission from the European Journal of Gastroenterology and Hepatology.78 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 8 Transverse sonogram during an abdominal HAE attack: liver and pancreas. Increased hepatic reflection (starry sky liver) and thickened, echogenic portal veins (arrow); the pancreatic region is also hyperechoic (double arrows). Reprinted with permission from Acta Paediatrica.81 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 9 Transverse sonogram during an abdominal HAE attack: liver and cholecyst. Because of edematous swelling, the wall of the cholecyst is hyperechoic (large arrow) and the small portal veins are also more echogenic (small arrows) in contrast with the liver's overall decrease in echogenicity. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 10 Structure of C1NH gene.85,153 Arrowheads represent Alu elements.150 The positions of the 4 polymorphic sites are indicated by elongated arrows. Nucleotide numbers refer to the C1NH gene, with number 1 denoting the first nucleotide of exon 1. Accession number X54486. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 11 Mutation analysis of the C1NH gene by DHPLC showing data with insertion, point mutation, and deletion. Red and black profiles correspond to patient and control samples, respectively. A, A 10-nucleotide insertion in exon 3 causes the presence of heteroduplexes before and after normal homoduplex. B, A point mutation (A → G) in exon 4 causes the presence of heteroduplexes in close vicinity to a normal homoduplex. C, A 5-nucleotide deletion in exon 5 causes the presence of heteroduplexes before normal homoduplex. Drouet et al, Unpublished data, August 2003. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 12 Analysis of C1NH gene mutations by SSCA. A, SSCA of fragment 2144-2360 including part of exon 3; lanes 1, 2, 4 HAE kindreds without mutations in this fragment; lane 3, mutation 2264-5delAG [R18fsX33]. B, SSCA of fragment 2482-2761 including part of exon 3; lane 1, healthy control; lanes 3, 4, HAE kindreds without mutations in this fragment; lane 2, mutation in exon 3: T2650C [F147S]. C, SSCA of fragment 8690-8895 including exon 6; lanes 1, 3, 4, HAE kindreds without mutations in exon 6; lane 2, exon 6 mutation: C8823G [Y308Stop]. Lopez-Trascasa et al, Unpublished data, June 2003. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 13 Southern blot analysis of patients with HAE-I carrying rearrangements in the C1NH gene. The outer lanes (N) contain control DNA of unaffected individuals and flank different heterozygous deletions/duplications. BclI-digested DNA was hybridized with a full-length cDNA probe. The 20-kb BclI fragment contains the 8 exons of the C1NH gene. Data from Stoppa-Lyonnet et al.150 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 14 Quantitative fluorescence multiplex analysis: exon-specific multiplex PCR results for exons 3, 4, 5, 7, and 8 from C1NH and a control exon from BRCA1. Red and blue profiles correspond to the patient and control samples, respectively. Deletions of exons 3 and 4 (A), duplication of exon 4 (B), deletion of exon 8 (C), and deletion of exon 4 (D) are clearly visible. ex, Exon. Modified from Duponchel et al.182 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 15 Decision algorithm for mutation analyses of the C1NH gene. This algorithm takes into account the importance of mutations in the coding sequence and in the introns, and the abundance of rearrangements. Depending on the methods used, a preliminary screening of rearrangements may be preferred. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 16 α-1-Antitrypsin tertiary structure conserved among the serpin family α-1-antitrypsin is composed of 3 β-sheets (A in red, B in green, and C in yellow) and 9 α-helices (A-I in gray). The blue strand is the reactive center loop (RCL) with the P1 active amino acid. Reprinted with permission from Silverman et al.197 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 17 Different pathways of kinin generation in C1-inhibitor deficiency state. Low C1-inhibitor leads to uncontrolled activation of factor XII, which generates kallikrein and plasmin. Kallikrein liberates bradykinin from HK, whereas plasmin cleaves off C2-kinin from activated C2. Activated C2 is continuously produced during baseline complement activation, which is increased as a result of insufficient control of autoactivation of C1 caused by C1-INH deficiency. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 18 Analysis of vascular permeability. Extravasation of Evans blue dye at 15 to 30 minutes was much more extensive in C1-INH–deficient mice (B) than in wild-type mice (A), particularly after the application of mustard oil to the left ears of the mice. (C) The difference in the amount of extravasation was clearly demonstrated by the rear footpads of mice of each genotype. Administration of human C1-INH resulted in reduced vascular permeability, as did the combination of Bk2R deficiency together with C1-INH deficiency. Reprinted with permission of the American Society for Clinical Investigation.9 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 19 Spectrophotometric analysis of vascular permeability in the small intestine. Quantitation of extravasated Evans blue dye in C1INH−/− mice treated with an ACE inhibitor (captopril), a Bk1R antagonist (des-Arg9,[Leu8]-bradykinin; Bk1RA), and a Bk2R antagonist (Hoe140). Reprinted with permission of the American Society for Clinical Investigation.9 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 20 Spectrophotometric analysis of vascular permeability in footpads. Quantitation of Evans blue dye extracted from the paws of mice of the indicated genotypes, either treated or not treated with intravenous C1-INH (C1I), DX-88 (DX), or Hoe140 (Hoe). Reprinted with permission of the American Society for Clinical Investigation.9 Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 21 Pathways for bradykinin generation and metabolism. On the endothelium, bradykinin generation likely initiates independent of factor XII; however, once kallikrein is generated, it soon catalyzes factor XII activation, thus amplifying the response. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 22 Correlation in female patients with HAE between serum progesterone (A) and SHBG concentrations (B) and the number of edematous attacks in the year after blood sampling. Spearman correlation coefficients and their significance (P values) are indicated. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 23 Underlying angioedema disease in 44 women who had induction or exacerbation of recurrent angioedema caused by OCs or HRT. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 24 Assessment of C4 antigen concentration by polyclonal anti-C4 antibodies. Assessment of C4 antigen levels by polyclonal anti-C4 antibodies might serve as a simple follow up parameter for therapy. Upper left and right panels depict C1-INH versus C4 antigen concentrations in 111 plasma samples from 21 patients with HAE-I. In the left panel, the individual C1-INH concentrations are plotted against C4 concentrations in plasma samples from patients with various therapeutic managements. Open square, no therapy; inversed open triangle, therapy with tranexamic acid; closed circle, therapy with danazol; closed triangle, therapy with stanozolol. Open squares and triangles represent samples from patients with current or recent bacterial or viral infections and therapy with danazol or stanozolol. Open squares with a crossing line indicate samples from an asymptomatic woman, a member of a family with concomitant heterozygous C1-INH deficiency and factor I protein gene mutation, herself heterozygous for C1-INH only. In the right panel, the means (±1 SD) of concentration ranges are depicted. The lower left panel indicates clinical manifestation of the C1-INH deficiency in 111 patients with HAE-I: closed circles give C1-INH concentration in plasma collected after an attack, and open circles indicate mean C1-INH concentrations of 2 assessments between which an attack occurred. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 25 Work-up flow sheet for patients with a clinical history of idiopathic or unknown origin abdominal symptoms or angioedema. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 26 Adverse events attributable to HAE or AAE in patients with and without a supply of C1 inhibitor at home. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 27 Effect of long-term danazol treatment on serum concentrations of C4 (A) and C1-INH (B) in children with HAE. Baseline values are compared with those measured in the first serum sample obtained after treatment had been initiated. C4 levels were determined in 8 children. Patients with high concentrations of nonfunctional C1-INH (233, 214, and 297%) were excluded from the comparison. Danazol treatment significantly increased both C4 and C1-INH levels (P=.002 and P=.0156, respectively; Wilcoxon signed-rank test). Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 28 Flow of data in the European HAE Register. PC, Personal computer. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 29 Window for accessing the European HAE Register. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 30 European HAE Register visit form. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 31 Hungarian HAE Center patient accrual. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

Fig 32 Hungarian HAE Center: flow of responsibilities. Journal of Allergy and Clinical Immunology 2004 114, S51-S131DOI: (10.1016/j.jaci.2004.06.047) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions