Abstracts from the 10th C1-inhibitor deficiency workshop

s from the 10th C1-inhibitor deficiency workshop Budapest, Hungary. 18–21 May 2017 © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Preface The 10th C1-inhibitor deficiency workshop will be held between 18 and 21 May 2017 in Budapest (2017.haenetworkshop.hu), among the picturesque surroundings of Margaret Island. As indicated by the name of this event, most of the interest focused on angioedema due to C1-inhibitor deficiency in 1999, when it was first organized. The name is unchanged, but the range of angioedemas has expanded since to include all known varieties of hereditary and acquired angioedemas with a bradykinin-mediated pathomechanism. Looking back to the agenda of this biennial conference, many questions remained unanswered and new issues have arisen despite the enormous scientific progress made. On this occasion, 318 participants have registered from 42 countries— this is the greatest attendance in the history of the Workshop since the start of the series. Eighty-six presentations have been submitted for this 4-day long scientific forum. The scientific program sounds interesting—it comprises novel achievements by leading scientific teams in basic research into bradykinin-mediated angioedema, the new findings of diagnostics and genetics, promising therapeutic solutions awaiting introduction, and the experience accumulated with the latest therapeutic procedures. Several presentations discuss the efforts related to improving the patients’ quality of life. This time, we have invited five prominent experts—namely, Alvin Schmaier (Cleveland, OH, USA), Marco Cicardi (Milan, Italy), Avner Reshef (Tel-Hashomer, Israel), Dumitru Moldovan (Tirgu-Mures, Romania) and Attila Mócsai (Budapest, Hungary). Alvin Schmaier will show us that our knowledge about the underlying mechanisms of bradykinin-mediated angioedemas is still limited—this may be remedied by extending our interest to other forms of angioedema with different pathophysiological backgrounds. Marco Cicardi will expose the similarities and the differences between bradykinin-mediated edema formation, and the idiopathic systemic capillary leak syndrome. Avner Reshef will explore a similar issue in his presentation titled ‘Angioedema–Histamine or Bradykinin?’. The lecture on neutrophil granulocytes by Attila Mócsai will take us closer to understanding the pathomechanism of angioedema. The agenda also contains the traditional roundtable session, an opportunity to develop consensus and international guidelines—this year, genetics will be in the limelight. In this session, the keynote lectures will be read by Margarita-Lopez Trascasa (Madrid, Spain) on the extended diagnostic approach integrating serological and genetic methodology; by Anastasios Germenis (Larissa, Greece) on the latest techniques for studying the SERPING1 gene; and by Nancy Brown (Nashville, TN, USA) on the pharmacogenetics of angiotensin-converting enzyme inhibitor-associated angioedema. Notwithstanding the remarkable progress made in South-America and in the former Soviet-bloc countries of Europe, state-of-the-art diagnostic and therapeutic modalities are still not available in many regions of the World. Dumitru Moldovan will review the stages along the way to making these accessible, and the experience accumulated in the effort to achieve high levels of patient care. The conference will be attended both by researchers and by clinicians—medical professionals and nurses, by the representatives of patient organizations, and by pharmaceutical industry experts involved in drug development, in order to assist the efforts of each other through joint thinking. Within the framework of this fruitful cooperation, the pharmaceutical companies also lent financial support to the conference—in addition to their scientific contribution. The travel grants, make it possible for an increasing number of professionals involved in the research or the management of patients with angioedema to attend the Workshop. The generous support by our Sponsors enabled us again to present the “For HAE Patients” award, as well as the “Grant for Young Investigators”. The major donors to this event are CSL Behring and Shire. Pharming Group NV, Swedish Orphan Biovitrum, BioCryst Phamaceuticals, KininX SAS also contributed the sponsorship of the Workshop. On occasion of this tenth, jubilee event, the “For HAE Patients” award goes to Bruce Zuraw (San Diego, USA), who will present his lecture ‘Let the Treatment Fit the Disease’ on the festive session of the scientific section on Day 1. His achievements in bettering the management of patients will be recalled by Antony Castaldo, the chair of the International Patient Organization for C1 Inhibitor Deficiencies. The concluding event of the conference will be the awarding of the ‘Grant for Young Investigators’ to the top four young presenters. The support referred to above made it possible to publish the submitted abstracts of the Workshop in the journal Allergy, Asthma, and Clinical Immunology, in order to make them available to an even broader range of professionals interested in this subject.

The underlying physiology of angioedema (AE) involves swelling of the skin or submucosal surfaces, caused by leakage of plasma into the tissues, owing to increased vascular endothelial hyperpermeability. This is the final common pathway of all AE syndromes, which should be differentiated from edema formation due to increased hydrostatic venous pressure, reduced oncotic pressure or decreased lymphatic drainage. At the vascular level, endothelial integrity is maintained by adherens junctions, consisting of homotypic dimers of VE-cadherin bridges that allow normal paracellular transport of solutes. These bridges are stabilized and protected by adjacent catenin molecules. Two chemical mediators: histamine (H) and bradykinin (BK) are playing a central role in most AE syndromes. Both can induce phosphorylation of adherens junctions, and catenin-mediated coupling of VE-cadherins with cytoskeletal actomyosin, resulting in opening of cell-cell junctions. This process was shown to be activated in hereditary AE by signalling through BK receptors (BKRB 1&2). AE syndromes are clinically heterogeneous and therefore present a considerable diagnostic challenge. Urticaria is characterized by the appearance of wheals which involves superficial cutaneous layers, surrounded by a reflex erythema and associated with intense pruritus. Angioedema (hereditary and acquired) involves the deep dermis, subcutaneous tissue and mucous membranes, and is associated with circumscribed swelling and dull pain. Both conditions are chronic/ relapsing, with attacks and remissions. Interestingly, attacks mediated by BK are often preceded by premonitory signs and symptoms (prodromes), but not H-mediated AE. Despite the apparent dichotomy between these two entities, there is recent evidence for a possible interaction between H-mediated AE and BK-mediated AE syndromes. Rarely, C1-INH deficient patients may present with prodromal pruritic rash that may evolve into a typical edematous HAE attack, and there are few reports of urticaria in HAE patients. Conversely, BK is involved in allergic reactions. In experimental shock/edema induced by insect stings increased FXIIa-C1-INH and kallikrein-C1-INH complexes were demonstrated, and total plasma high-molecular kininogens (HK) were cleaved within minutes. Cleavage of HK plasma pools was also demonstrated in patients with foodinduced anaphylaxis and mast cell (MC)-derived tryptase can induce vascular permeability by releasing BK. Additionally, IgE-mediated anaphylaxis was blocked in FXII-deficient, BKB2R knock-out and HK/ PreKallikrein-deficient mice. Recently, ablation of FXII, BKRB2 receptors and depletion of C1-INH protected mice from activated MC heparin induced vascular leakage. Additionally, negatively charged heparin from activated MC was shown to induce excessive vascular leakage in C1-INH deficient mice, and to trigger FXII auto-activation, while pretreatment with BKB2R antagonist (icatibant) inhibit vascular hyperpermeability and hypotension. In summary, both H and BK are attributable to vascular endothelial hyperpermeability in AE syndromes. Recent data indicate that there might be a 'cross-talk' between MC-mediated 'allergic' events, and activation of BK-generating contact cascade. Allergy Asthma Clin Immunol 2017, 13(Suppl 2):29 After the Second World War Eastern Europe was left to the Communist USSR zone, which resulted in a widening gap between East and West. It was only in 1989 when a new era began in this part of the world. Romania (as well as other former communist countries) was going to join EU and NATO. The West-East exchange programs and freedom of travel greatly improved the condition of Romanian physicians. It was in 2005 when I was invited the first time to join a C1-inhibitor workshop in Budapest. Despite the big post-communist handicaps of the Romanian health system I decided to dedicate the next few years to developing an HAE comprehensive program in my country. Surrounded by a handful of enthusiastic colleagues, I started to look for sporadic cases in some personal databases of colleagues from immunology labs, and also asked for the addresses of those with low C1 inhibitor results. Since 2005 we have organized workshops related to the annual conferences of the Romanian Society of Allergology and Clinical Immunology and later meetings centered on HAE only. From the beginning the support of HAE experts from Europe and later from Israel and USA was significant. In these past 12 years, we have made presentations at the conferences of the national meetings of paediatrics, dermatology, internal medicine, emergency medicine and published papers in order to increase the awareness about this life-threatening disease. In the past 10 years, we have participated in 12 multicentre clinical trials. In 2006 we founded the Romanian Network for Hereditary Angioedema, a professional non-profit organization who allowed us to have our own website, to develop a Romanian HAE guideline and to put together a national registry of HAE patients, which currently includes 99 patients. With our support, the Association of the Romanian HAE patients was founded in 2012. Until 2013 no modern treatments were available. Recombinant C1-inhibitor and a C1-inhibitor concentrate were offered in ED in a small amount from 2015 and it was promised that the B2 receptor antagonist (icatibant) will be reimbursed through a national program that will start soon. Hopefully, the quality of life of these patients will improve considerably. There is still a lot to do to raise the awareness and expand the education of both physicians and patients about this disorder; however, our aim is above all to address the challenge of finding an estimated 250 patients that are missing from our database. Neutrophils are double-edged swords playing a critical role in innate immune defense but also contributing to tissue damage during excessive inflammation. Their exact role in those processes and the molecular details of their function are still poorly understood. We and others have recently shown that neutrophils are critically involved in various in vivo models of innate immunity and inflammation in experimental mice. Neutrophils are also able to release a number of pro-inflammatory mediators including chemokines, cytokines and lipid mediators such as LTB4. Many of the mediators released by neutrophils promote the recruitment or activation of other neutrophils, therefore establishing positive feedback amplification loops during neutrophil-mediated inflammation. Immunoreceptor-induced neutrophil responses required receptor-proximal Src-family kinases, Syk and PLCγ2 which were also critical for neutrophil-mediated in vivo inflammatory reactions. Those molecules were involved in the release of proinflammatory mediators from neutrophils but not in the intrinsic ability of the cells to migrate to the site of inflammation. Interestingly, the downstream gene expression regulator CARD9 was required for the release of chemokines and cytokines but not for LTB4 release or other classical neutrophil responses, providing a unique opportunity to test the in vivo role of neutrophil gene expression changes and neutrophilderived chemokines/cytokines. Neutrophil-specific deletion of CARD9 attenuated various different in vivo inflammation models, providing the first direct evidence for the role of neutrophil gene expression changes during an in vivo inflammatory reaction.

RT-1 Molecular analysis of SERPING1 gene: to whom, when and how?
Margarita López-Trascasa, Alberto López Lera Unidad de Inmunología, Hospital Universitario La Paz, IdiPAZ, CIBERER U754, Madrid, Spain Correspondence: Margarita López-Trascasa (mltrascasa@salud.madrid.org) Allergy, Asthma & Clinical Immunology 2017, 13(Suppl 2):RT-1 Hereditary angioedema due to C1 inhibitor deficiency (HAE-C1INH) can be diagnosed by two specific complement parameters: C4 levels and C1 inhibitor function. In patients with a family history, these measurements are generally sufficient to establish or discard the presence of the disease. As a general rule, genetic studies represent an additional tool for reaching a conclusive diagnosis; especially in three situations which are relatively common in HAE-C1INH diagnosis: de novo cases, relatives of affected patients without clear evidences of HAE symptoms and those samples with inconclusive results following complement testing. Nowadays, mutational studies are easier and cheaper. In addition to traditional Sanger sequencing, the development of integrated analysis platforms including RT-PCR (splicing mutations), RT-qPCR (RNA expression studies), MLPA and XL-PCR (large insertions/deletions) allows a complete, rapid and efficient identification of most of the disease-causing mutations. In our lab, these techniques are paralleled by the study of complement levels and function and integrated as a trademark (Complementest ® ). We systematically perform a full HAE-C1INH study to every new patient referred due to suspicion of the disease. Upon compatible C4 levels and C1Inh function, we first attempt to characterize the mutation in the propositus and then to establish segregation of the mutation in their first degree relatives. In the case of newborns with an affected parent, a simple Sanger sequencing of the altered exon is enough for a straightforward diagnosis of point mutations. When HAE is not associated with C1 inhibitor dysfunction or low C4 levels, HAE with normal C1 inhibitor (HAE-nC1INH) should be considered. In such cases, mutations in the ninth exon of coagulation F12 are present in less than 40% of cases, so that the clinical presentation, lack of response to allergic treatments and familial history should all be considered in support of a HAE diagnosis. In addition to that, and although acquired angioedema with conserved C1q levels is very unusual and presents different features, genetic studies can unambiguously discriminate hereditary and acquired situations. common in women than in men. We have found that the −2399A allele is associated with decreased plasma APP activity in men and women, but that the variant allele is associated with ACE inhibitor-associated angioedema only in men [2]. Neprilysin also degrades bradykinin and substance P. Our group has observed an association between a polymorphism in the gene encoding neprilysin (MME) and ACE inhibitorassociated angioedema of African American ancestry in a case-control study in Tennessee as well as in the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET) study [3]. In a genome-wide association study in 175 individuals with ACE inhibitor-associated angioedema and 489 ACE inhibitor-exposed controls without angioedema from Nashville (Tennessee) and Marshfield (Wisconsin), we found no associations of genome-wide significance [3]. Of the SNPs that were associated modestly with ACE inhibitor-associated angioedema in Nashville/Marshfield analysis, two were also significantly associated with ACE inhibitor-associated angioedema (rs500766 and rs2724635) in ONTARGET cases versus controls. Rs500766 is a polymorphism in the gene encoding for protein kinase C θ (PRKCQ). In both the Nashville/Marshfield sample and in ONTARGET, the T allele was significantly associated with a reduced risk of ACE inhibitor-associated angioedema. Rs2724635 is a polymorphism in ETS variant gene 6 (ETV6), also known as TEL (or translocation ets leukemia), and the G allele was associated with an increased risk of ACE inhibitor-associated angioedema in both African Americans in the Nashville/Marshfield sample and in the ONTARGET sample. Both of these genes are involved in immune regulation and their association with ACE inhibitor-associated angioedema is intriguing as the risk of ACE inhibitor-associated angioedema is increased in patients with seasonal allergies and with immunosuppressant use [4,5].
With every passing day, genotyping of subjects who may suffer from hereditary angioedema becomes more indispensable in the clinical practice. However, the pronounced allelic heterogeneity of SERPING1 gene as well as the fact that approximately 20-25% of all unrelated patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) represent sporadic cases carrying de novo SERPING1 mutations with the same mutational spectrum as the familial ones, makes SERPING1 a prime example of mutagenic lability. Over 450 different SERPING1 mutations related with C1-INH-HAE have been detected up to now, scattered over all exons and exon/intron boundaries of the gene. The majority of C1-INH-HAE-related SERPING1 mutations are missense mutations (34%) followed by frameshift alterations and small indels (31%), large gene rearrangements (17%), splice-site defects (10%), nonsense mutations (7%), and regulatory mutations (1%). Currently, the molecular analysis of SERPING1 initiates with the prioritized amplification of all exons and the exon/intron boundaries by PCR and the detection of mutations by direct sequencing. If no mutation were found, further analysis for the identification of large gene rearrangements is taking place, usually performed by two different techniques, namely the long-range PCR and the multiplex ligation-dependent probe amplification (MLPA). This cumbersome and time-consuming approach is fraught with pitfalls as it is highly dependent on the users' experience and knowledge. Sometimes, for example, a not yet reported missense mutation could be considered as the causal genetic defect because its detection during the early steps of the analysis has prevented the discovery of an indel leading to premature truncation of protein synthesis. Moreover, recently recognized damaging intronic alterations located in the exon/intron boundaries-and not only-may escape the conventional approach, as they require the design of specific primers. On the other hand, however, modern genotyping methods, like the next-generation sequencing (NGS), present many advantages towards a more meticulous SERP-ING1 analysis. Therefore, the increasingly frequent use of genotyping angioedema patients in the clinical practice and research imposes the standardization of the methods and workflows in use for SERPING1 analysis. Special attention should be paid in regard with the interpretation of the detected sequence variants according to the recently developed recommendations.
Background: Few anti-bradykinin (BK) B 1 and B 2 receptor (B 1 R, B 2 R) antibodies are validated, especially for work in intact cells. Since kinins bind their receptor through their C-terminal region and that their N-terminal region is close to the extracellular fluid, fusion protein of the type H 2 N-Protein-Spacer-Agonist-COOH were generated and evaluated for the detection of each receptor type. We recently reported the fusion of the enhanced green fluorescent protein (EGFP) to the C-terminal of each spacer-agonist module (the amphibian sequence maximakinin (MK) for B 2 Rs, (Asn-Gly) n -Lys-des-Arg 9 -BK for B 1 Rs, n = 5 or 15), yielded bright imaging tools of nanomolar potency that do not bind to peptidases. The specificity and design of ligand fusion proteins was further explored. Methods: Using various molecular biology techniques, vectors encoding for different fusion proteins were generated. These vectors were then expressed in producers cells which were ultimately lysed to extract the fusion proteins. The cell lysate was cleared of cellular debris and used on recipient cells. Results: Further studies showed that MK (the 19-mer DLPKINRKG-PRPPGFSPFR possessing the BK sequence at its C-terminus) is largely a species-specific spacer-agonist module that does not bind well to human B 2 Rs, but supports the detection of rat and rabbit B 2 Rs. Moreover, the B 2 R putative agonist (Asn-Gly) 5 -BK has very little affinity for either human or rat B 2 Rs, proving that each spacer-agonist module has a critical structure-activity relationship for each receptor subtype. The fusion of the engineered soybean peroxidase APEX2 to appropriate spacer-agonist modules led to BK receptors ligands compatible with widely available luminescent or imaging peroxidase co-substrates and supported the detection of physiological levels of BK receptors. APEX2-(Asn-Gly) n -Lys-des-Arg 9 -BK constructions (n = 15, 30, 45 or 60) all bind human B 1 Rs. APEX2-(Asn-Gly) 15 -MK was developed as a B 2 R ligand and supports the detection of rat B 2 Rs in a specific manner. The fusion protein human serum albumin-MK did not bind B 2 Rs, showing that some proteins are incompatible with this strategy, possibly due to steric hindrance between the fused protein and the receptor. Other functional proteins, such as β-galactosidase and the Fc region of IgG, are being investigated for the generation of BK receptor ligands. Conclusions: The fusion proteins of the type H 2 N-Protein-Spacer-Agonist-COOH represent the basis for the generation of better tools for the detection of BK receptors in intact cells. Such probes could have diagnostic applications and support physiological and pathological investigations especially in HAE. Background: Multiple pathways have been proposed to generate bradykinin (BK)-related peptides from blood, including some dependent of secretory products from neutrophils or platelets. We applied various forms of activation to fresh blood obtained from healthy subjects to compare kinin formation with reference to mechanisms, kinin concentrations, time course and validation of biological activity at both BK receptor subtypes. Materials and methods: An enzyme immunoassay for BK (Phoenix Pharmaceuticals) was applied to reconstituted extracts of citrated blood incubated at 37 °C under gentle agitation for 0-2 h in the presence of activators and/or inhibitory drugs. Validation of the presence of biologically active kinin is necessary because the immunoassay is reactive with a limited BK C-terminal sequence only. The applied bioassays were the contractility of the human umbilical vein and signaling (c-Fos accumulation) in HEK 293a cells that express recombinant human B 2 or B 1 receptors (B 2 R, B 1 R). Results: The ACE inhibitor enalaprilat did not induce immunoreactive BK (iBK) formation in human blood, but considerably potentiated the effect of the stimuli that were effective. Rapidly (5 min) and intensely (>200 ng/ml iBK) acting stimulants were recombinant tissue kallikrein (KLK-1) and Kontact-APTT, a particulate material that recruits the contact system. Corresponding extracts contracted the umbilical vein via the BK B 2 R and stimulated recombinant B 2 Rs; B 1 Rs were also stimulated by KLK-1-treated blood extracts. Recombinant tissue plasminogen activator was a slowly (≥1 h) acting stimulus inhibited by corn trypsin inhibitor and PKSI-527. Stimulating neutrophil secretion using cytochalasin B + f-Met-Leu-Phe or NETosis with interleukin-8 did not generate iBK. ADP (≥50 μM) was also inactive in this respect, even if the blood was submitted to shear stress in an aggregometer. Conclusions: Comparing the proposed kinin generation pathways in whole normal blood, where endogenous inhibitors abound, put some of them in perspective, e.g. lack of effect of secreted neutrophil PR3 and of platelet activation, indirect and slow effect of fibrinolysis via the contact system. Both KLK-1 and the contact system ultimately recruit kinin receptors. Whether atypical kinin formation/potentiation participate to hereditary or acquired/sporadic angioedema attacks is plausible. Allergy Asthma Clin Immunol 2017, 13(Suppl 2):29 Background: Hereditary angioedema resulting from the deficiency of C1 inhibitor (C1-INH-HAE) is characterized by increased endothelial permeability. Mannan-binding lectin-associated serine protease-1 (MASP-1)-one of the enzymes blocked by C1-inhibitor-is the key serine protease of complement lectin pathway. We previously showed that (1) MASP-1 can activate endothelial cells via protease activated receptor (PAR) cleavage and (2) endothelial cells become activated during C1-INH-HAE attacks. These findings led us to ask whether MASP-1 can increase endothelial permeability and thus take part in the triggering of edematous attacks. To answer this question it was necessary to develop a new and simple permeability test. Using human umbilical vein endothelial cell (HUVEC) culture as an in vitro model we also aimed to explore the mechanisms by which MASP-1 exerts its cellular effects. Results: We have successfully optimized a new, simple, high-throughput and cheap method for permeability measurements, which is based on the reaction of biotinylated gelatin and a streptavidin-conjugated fluorescent dye. In this test, MASP-1 could significantly and dose-dependently increase endothelial permeability 20 min after treatment. This effect was completely blocked by C1-inhibitor and a highly specific artificial MASP-1 inhibitor, SGMI-1. We demonstrated that the permeability increasing effect of MASP-1 is mediated mostly by PAR-1. We also found that MASP-1 induced endothelial permeability requires the activity of p38 mitogen activated protein kinase (p38 MAPK) and p160 Rho associated coiled-coil containing protein kinase (p160 ROCK). In fluorescence microscopy experiments we demonstrated that MASP-1 induces diphosphorylation of myosin light chains resulting in cytoskeletal actin rearrangement, which was also dependent on p160 ROCK activity. Visualization of key molecules involved in cell adhesion showed that MASP-1 changes the molecular pattern of PECAM-1, VE-cadherin and ZO-1, and induces paracellular gap formation. To analyze the long-term effects of MASP-1 on the gene expression profile of HUVECs we carried out an Agilent microarray. MASP-1 significantly changed the expression of 12 permeability-related genes; among others it down-regulated genes involved in cell junctions and actin cytoskeleton regulation, while it up-regulated B2 bradykinin receptor. Conclusion: In our experiments MASP-1 was found to be a potent short-term permeability increasing agonist, but according to our microarray results it may also have a long-term effect on endothelial permeability. These findings raise the possibility that MASP-1 could play an important role in the pathomechanism of C1-INH-HAE, which makes SGMI-1 a potential target of future drug development. Proteases are generally synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or autoactivation. The lectin pathway of the complement system is initiated by mannose binding lectin (MBL)associated serine proteases, MASP-1, and MASP-2, which are known to be present as proenzymes. The third serine protease of the lectin pathway, MASP-3, was shown to be the major activator, and the exclusive "resting blood" activator of pro-factor D (pro-FD), producing factor D (FD), the initiator protease of the alternative pathway. Because only activated MASP-3 is capable to carry out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood.
We demonstrated the presence of active MASP-3 in blood by a more direct technique. First MASPs were partially purified by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool only the zymogen form of MASP-1 was detected by Western blot, whereas MASP-3 was over 70% active. The active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not capable to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood.
In an attempt to explain this phenomenon we modeled the fluidphase activation and subsequent inactivation by C1 inhibitor of lectin pathway proteases using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation if we assume efficient intercomplex activation of zymogen MASP-3 by zymogen MASP-1, however the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and part of them is present in the form inactive serpin-protease complexes. Our approach can be useful to model protease-C1 inhibitor reactions in C1 inhibitor sufficient and deficient conditions in the future.
Background: Angioedema (AE) is characterized by localized swelling of subcutaneous tissues or mucosa of the upper respiratory or gastrointestinal tract. It accounts for as many as 80,000-112,000 Emergency Department (ED) visits annually with 18% resulting in hospitalisation [1]. There are two distinct subtypes of AE, caused by different pathological processes involving either mast-cell mediators, including histamine or bradykinin. The resulting AE often cannot be distinguished on clinical grounds and misdiagnosis may lead to inappropriate or delayed management which can be fatal particularly in patients with swelling of the upper respiratory tract. In a real-life setting, most ED visits for AE, unless the patient is known to suffer from hereditary AE (HAE), will involve allergic or idiopathic AE, with or without concomitant urticaria or evidence of anaphylaxis. These forms of AE are typically mediated by histamine. Their management, usually familiar to ED staff, is made of adrenalin, antihistamines and corticosteroids [referred to as standard therapy (ST), Figure 1]. The key challenge in the ED, however, is recognizing and treating potential nonhistaminergic (bradykinin-mediated) AE. We suggest the distinction to be based on response to Standard Therapy. Results: A satisfactory response to ST will support the diagnosis of histamine-induced AE and the patient will eventually be evaluated accordingly. In the case of non-optimal response, when inadequate dosing of antihistamines or potential beta-blockade have been addressed, bradykinin-mediated AE must be considered. Unlike histamine-mediated AE, bradykinin-mediated AE is not associated with urticaria, does not respond to antihistamines or corticosteroids, and is poorly responsive to adrenalin. It tends to be more severe, longer lasting, and much more likely to involve concurrent abdominal symptoms than its counterpart. Angiotensin-converting enzyme (ACE) inhibitor-associated angioedema represent the first cause of bradykinin-induced AE. ACE inhibitors then cause local elevations in plasma bradykinin because of inhibition of its normal breakdown. HAE is associated with increases in bradykinin following some trigger because of a reduced C1 esterase inhibitor (C1-INH) activity while the acquired form is found with lymphoproliferative diseases or autoimmune disorders as a result of the production of neutralizing autoantibodies against C1-INH or increased C1-INH consumption. Bradykinin-targeted first-line therapy [2] includes intravenous infusion of plasma-derived C1-INH (Berinert, 20 U/kg or Cinryze 1000 U) or recombinant C1-INH (Ruconest, 50-100 U/kg) or subcutaneous injection (30 mg) of icatibant, a bradykinin inhibitor (Firazyr) or ecallantide (Kalbitor), a highly specific plasma kallikrein inhibitor. Solvent detergent-treated freshfrozen plasma (2 U) may be used as a second-line agent only in the absence of first-line agents. The selection of the therapeutic agent in individual patient management plans and the threshold for initiating treatment will be influenced by patient preferences and local policy. Conclusions: This consensus algorithm focuses on the presentation of isolated AE disorders to the ED. It should permit a rapid identification of bradykinin-induced AE for specific therapy.
Background: Laboratory assays for AO diagnosis target complement while AO is subsequent to contact phase activation and bradykinin (BK) accumulation. The aim of this study was to compare diagnostic performances between methods targeting complement and contact phase. Materials and methods: Diagnostic of hereditary angioedema with C1 inhibitor (C1Inh) deficiency (C1Inh-HAE) of patients was performed according to recommendations (1) and confirmed by genetic analysis of SERPING1 gene. Patients taking tranexamic acid or danazol were excluded. Patients and blood donors provided informed consent to participate in the investigation. Antigenic C4 was quantified by nephelometry. C1Inh function was measured by chromogenic assays targeting C1s using commercial kits (Technochrom, Technoclone GmbH or Berichrom, Siemens), by an in-house method (2) or by a new method targeting kallikrein (KK) (3). Spontaneous amidase activity was performed as described (4).
Diagnostic performance values and statistical analysis were performed using Xlstat ® software, and Mann-Whitney U tests, with P < 0.05 as statistically significant. Results: Samples (n = 519) were assayed for C1Inh function; assay systems targeting C1s protease or a new assay targeting KK (3) were compared. Fifty eight samples displayed one discordance between methods. All methods presented with a very good specificity, while the sensitivities were different, mainly for intermediate or transient C1Inh deficiency. The in-house method presented a higher sensitivity than commercials kits, while its sensitivity was lower than the assay targeting KK. A comparison between C4 and spontaneous amidase activity as companion test in C1Inh-HAE is described for 99 healthy subjects and 185 patients. Antigenic C4 and spontaneous amidase activity assays showed good specificity and high positive predictive value for C1Inh-HAE diagnosis. Nevertheless, spontaneous amidase activity displayed higher sensitivity and negative predictive values. Conclusion: Assays targeting contact phase (C1Inh using KK or spontaneous amidase activity) displayed higher sensitivity than assay targeting complement (C1Inh using C1s or antigenic C4), in line with its pertinence for AO physiopathology. The higher sensitivity is advantageous for angioedema screening and biological diagnosis.

Background:
The current diagnostic assay for functional C1 inhibitor (C1INH) measurement employs inhibition of activated C1s of the complement cascade by C1INH, utilizing either a chromogenic assay or a complex ELISA method. Since both the methods have limitations, there is a need to develop a more specific, sensitive and reliable method. Results: We have previously reported that C1INH deficiency can be determined by ELISA methodology based on the concept that C1INH inhibits plasma kallikrein as well as activated factor XII. Employing either enzyme as ligand, we demonstrated superiority to the commercial ELISA method that is based on inhibition of activated C1. Our method is more accurate and sensitive. Although the commercial chromogenic assay is sensitive and specific than the commercial ELISA, a direct comparison between chromogenic and our ELISA was not done. We report results of a direct comparison of the chromogenic assay that the commercial laboratories use with our ELISA method employing either factor XIIa or kallikrein as the C1 inhibitor ligand. We assayed 30 normal controls and 57 patients with type I/II HAE. The values for normal controls by chromogenic assay was 146 ± 25; by factor Allergy Asthma Clin Immunol 2017, 13(Suppl 2):29 XIIa ELISA was 148 ± 36; and by kallikrein ELISA was 129 ± 37 whereas the values for type I/II HAE were 37 ± 22 (chromogenic); 23 ± 12 (factor XIIa ELISA) and 19 ± 11 (kallikrein ELISA). The p value for the chromogenic assay comparing normal plasma to HAE plasma was <0.0001. The p value for the factor XIIa ELISA (or Kallikrein ELISA) comparing normal plasma to HAE plasma was also <0.0001. A comparison of HAE patients done by the chromogenic assay vs. our factor XIIa ELISA (or kallikrein ELISA) also had a p value of <0.0001. Conclusion: The chromogenic method had greater uncertainty; one of the HAE plasma was misdiagnosed and 7 were considered equivocal. The factor XIIa ELISA (or kallikrein ELISA) method is rapid, easily scaled up for large number of samples and appears superior to the chromogenic method. Introduction: Phospholipase enzymes (PL) play central role in numerous cellular events including inflammation and immunoregulation by catalysing the lysis of phosphorylated lipids. Four distinct families of PL have been described and are named A, B, C and D. These enzyme family members are categorised based on the stereospecifically numbered sites within phospholipids where they promote cleavage. Secreted PLA 2 (sPLA 2 ) cleaves the sn-2 acyl chain, releasing arachidonic acid and lysophosphatidic acid. Upon downstream modification by cyclooxygenases, arachidonic acid is modified into active compounds called eicosanoids that include prostaglandins and leukotrienes, which are vascular permeability factors. Moreover, sPLA 2 , released in the blood and biological fluids, exert other biological effects relevant to the initiation and regulation of inflammatory and immune responses. Lipoprotein-associated PLA2 (Lp-PLA 2 ) also known as platelet-activating factor acetylhydrolase is a PLA 2 that catalyzes the hydrolysis of acetyl ester at the sn-2 position of PAF. It has been shown that bradykinin modulates vascular permeability through the activation of PL. Therefore we supposed that PLA 2 may have a role in pathogenesis of angioedema. The aim of this study was to analyze the plasma activity of PLA 2 in patients with C1-INH-HAE. Methods: 71 controls and 105 C1-INH-HAE patients were studied. PLA 2 and LP-PLA 2 activity was measured by fluorescent assay. Angiogenic factors [vascular endothelial growth factors (VEGF-A), angiopoietins 1 and 2] were evaluated by ELISA. Results: Plasma activity of sPLA 2 and LP-PLA 2 were higher in C1-INH-HAE patients in remission than in controls. There was no correlation between the activity of sPLA 2 and LP-PLA 2 . Interestingly, LP-PLA 2 activity was higher in C1-INH-HAE patients experiencing more than 12 attacks per year than in those who had less than 12 attacks. sPLA 2 activity was decreased in patients with higher Ang1 and Ang2 levels. Whereas, there was positive correlation between activity of LP-PLA 2 and Ang1 and negative correlation between LP-PLA 2 and Ang2 and Ang2/Ang1 ratio (an index of vascular permeability). Moreover, the activity of these mediators were decreased during attack compared with basal conditions. Conclusions: Plasma activity of PLA 2, that alter vascular permeability through their activation by bradykinin and eicosanoid production, are increased in patients with C1-INH-HAE in remission and decreased during attacks. Based on these observations that support immunomodulator role of PLA 2 , we hypothesize that these enzymes along with an increased release of bradykinin, VEGFs and Angs, induce a state of "vascular pre-conditioning" that may change the threshold for development of angioedema attacks.

Background:
Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is a potentially life-threatening rare disease, characterized by recurring and spontaneously resolving edematous attacks. Previously many studies published on the activation of the plasma enzyme systems during edematous attacks, nevertheless kinetic follow-up has never been performed. For the first time, we aimed to study the kinetics of parameters in the coagulation and fibrinolytic systems in a spontaneously resolved edematous attack of a C1-INH-HAE patient. Furthermore, we aimed to study the kinetics of these parameters in a healthy subject during a 24-h period, served as control. Materials and methods: In a 56-year-old female with C1-INH-HAE we monitored the severity of the symptoms during the entire observation period and altogether twelve blood samples were obtained. Blood samples collected from a healthy volunteer at 5 different times during a 24-h period served as a control. We measured factors XI and XII activities (FXIa, FXIIa), as well as concentration of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT)-complex, D-dimer, fibrinogen. Results: After a 24-h symptom-free period and another 19-h prodromal period, the patient had a 29-h-long edematous attack in multiple skin locations, and was followed up for another day. It is remarkable that during prodromal stage-which was characterized by erythema marginatum-the levels of D-dimer, F1 + 2 and TAT-complex were as constantly low as those levels measured in the healthy control. Levels of F1 + 2 and TAT-complex were significantly elevated at the moment of the onset of edematous symptoms whereas level of D-dimer was elevated after 6 h. Levels of all three parameters reached maximum 12 h after reaching the maximum severity score. Highest level of D-dimer was almost 100-fold higher than the levels measured during prodromal stage. Fibrinogen levels were constantly elevated during prodromal stage whereas during edematous attack, fibrinogen levels were similarly low as levels were measured in the healthy control. In the healthy control subject, all measured parameters were stable during 24-h observational period. Conclusions: This study was a part of a project aimed to the better understanding of the mechanisms leading to the onset and to the resolution of edematous attack. Real-time monitoring of F1 + 2 and TAT complex suggest that thrombin contribute to the development of edema formation. We confirmed that D-dimer is a prominent biomarker of an ongoing edematous attack. Elevated levels of fibrinogen before the onset edematous symptoms raise the possibility of using it as a predictive marker. The prediction revealed higher C trough with 40 IU/kg (40.2%) and with 60 IU/kg (48.0%) compared with 1000 IU IV (~30%) and a lower peakto-trough ratio with a more consistent elevation of the C1-INH(f ) for the 40 and 60 IU/kg SC doses compared to the 1000 IU IV dose. Conclusions: C1-INH(f ) was well described by a one-compartment model with first order absorption. Body weight was a significant covariate that affected CL. The PK profile for SC administration of C1-INH shows higher C trough with a more consistent and evenly distributed plasma level of C1-INH(f ) compared to the profile for IV administration. Allergy Asthma Clin Immunol 2017, 13(Suppl 2):29 Background: The Phase 3 COMPACT trial showed that a high-concentrate subcutaneous (SC) formulation of C1-INH (CSL830) provided dose-dependent, physiologically-relevant increases in trough C1-INH functional activity (C1-INH[f ]), and demonstrated its safety and effectiveness in preventing hereditary angioedema attacks (HAEAs). Data from this trial was used to (1) study an exposure-response (E-R) relationship between C1-INH(f ) and risk of an HAEA and (2) explore the relationship between C1-INH(f ) and levels of complement factor 4 (C4), a marker of activation of the complement system. Materials and methods: Data from the COMPACT program were used to develop a population E-R model. An interval-censored repeated time-to-event (TTE) model was developed that enabled the C1-INH(f ) to be directly related to HAEAs. The final model included two components, a baseline hazard and a non-linear drug effect. The relationship between C1-INH(f ) and C4 antigen was visually inspected in an exploratory manner. Background: Cinryze, a human plasma-derived C1 inhibitor (C1-INH) is approved for routine prophylaxis against hereditary angioedema (HAE type I and II) attacks in adults, adolescents, and children. We present interim efficacy and safety results as well as the pharmacokinetics and pharmacodynamics of two doses of intravenous (IV) C1-INH in patients 6-11 years of age. Methods: This is an ongoing randomized, phase 3, multi-centre, single-blind, crossover study (NCT02052141). We present data from 6 patients who completed the study. Following a 12-week baseline observation period (BOP), patients were randomised to receive 500U or 1000U IV C1-INH every 3-4 days for 12 weeks, and then switched to the other dose for 12 weeks. The primary efficacy endpoint was the number of monthly attacks in a treatment period. Secondary efficacy endpoints included attack severity and the number of attacks requiring acute treatment. Clinical and laboratory safety were evaluated.
Blood samples were collected before and 1 h after doses 1, 12, and 24 to measure functional C1-INH activity, C1-INH antigen, and C4. Results: Six patients (all female with HAE type I and a median age of 10.5 years) had a mean (SD) of 2.26 (1.62) attacks per month during the BOP. The mean (SD) difference in the number of attacks between the BOP and treatment periods was −1.89 (−1.31) and −1.89 (−1.11) for the 500 and 1000 U doses, respectively, i.e. an 84.8% (20.1) and 88.1% (13.4) reduction in the number of attacks. The safety profile of either dose was comparable. No deaths, serious adverse events (AEs), thrombotic or thromboembolic events occurred. Frequently reported treatment-related AEs included fatigue and irritability. Baseline-adjusted C1-INH functional activity (corrected for its pre-first dose level) observed pre-dose 12 (mean [SD] of 0.145 [0.152] U/ml for 500 U and 0.210 [0.098] U/mL for 1000 U; normal range: >0.68 U/ml) was approximately maintained to dose 24. An hour post-dosing, C1-INH functional activity and antigen concentration increased two-fold or more. Mean (SD) baseline-adjusted C1-INH antigen concentrations pre-dose 12 were 0.039 (0.024) g/L for 500 U and 0.044 (0.032) g/L for 1000 U (normal range: 0.21-0.39 g/L). For dose 12, C4 concentration was variable with a mean (SD) of 36.2 (36.7) mg/L for 500 U and 49.0 (42.1) mg/L for 1000 U, (normal range: 160-700 mg/L). Conclusion: In this 24-week study, twice-weekly IV administration of 500 and 1000 U C1-INH in 6 children aged 6-11 years was safe and well-tolerated, and demonstrated efficacy in reducing HAE attack frequency. Background: BCX7353 is a potent once daily oral kallikrein inhibitor. A Phase 2, double-blind, dose-ranging, placebo-controlled, 3-part, parallel-group study (APeX-1) evaluated the efficacy, safety, pharmacokinetics and pharmacodynamics of BCX7353 as a prophylactic treatment in HAE patients. A planned interim analysis of a subset of Part 1 is reported (28 of 36 planned subjects). Materials and methods: In Part 1, patients with HAE Type I or II with a recent history of frequent angioedema attacks were randomized 1:1 to receive 28 days of treatment with BCX7353 350 mg QD or placebo. Parts 2 and 3 will include an analysis of lower doses (125 and 250 mg [Part 2] and 62.5, 125 and 250 mg [Part 3]). Subjects recorded details of HAE attacks in a diary. Efficacy was assessed by the number of attacks over the entire and effective dosing period (EDP, Days 8-29) when BCX7353 steady-state conditions were achieved. Plasma samples were drawn for BCX7353 concentrations and kallikrein inhibition at steady state. Safety was monitored by adverse events (AEs), laboratory assessments, vital signs, physical exam findings and ECGs. Results: Twenty-eight subjects with a mean of 1.0 HAE attacks per week were enrolled and included in the interim analysis. Four subjects were excluded from the per-protocol (PP) population: two subjects on BCX7353 discontinued early (non-treatment emergent liver enzyme elevations and gastroenteritis with liver enzyme elevations) and two subjects (one per treatment) did not have confirmed Type I or II HAE. The least-squares mean HAE attack rates during the EDP in the PP population were 0.92 (placebo) and 0.34 (BCX7353) attacks per week, translating to a reduction in attacks of 63% (p = 0.006). Four of 11 subjects (36%) were attack-free on BCX7353 during the EDP compared to 1 of 13 on placebo (7.7%; PP population). All trough concentrations drawn on BCX7353-treated subjects at steady-state exceeded the predefined minimum therapeutic target (fold range 2.3-7.0, mean 4.7). Mean ex-vivo plasma kallikrein activity was suppressed by an average of 82-89%, and this was sustained over the dosing interval. There were no SAEs and no severe drug-related AEs. BCX7353-treated subjects had a numerically higher number of mild to moderate gastrointestinal AEs; additionally, BCX7353-related gastrointestinal AEs may have been reported as HAE attacks. Conclusions: Once-daily BCX7353 was associated with a clinically meaningful, statistically significant reduction in attacks in subjects with HAE. BCX7353 was generally safe and well tolerated. These findings support the ongoing evaluation of a range of doses of BCX7353 for prophylaxis of angioedema attacks in HAE patients. Background: Lanadelumab (DX-2930/SHP-643) is a long-acting, highly-specific, potent, human monoclonal antibody targeting plasma kallikrein that received fast track and breakthrough therapy designations. Results from a Phase 1b study (NCT02093923) did not identify safety signals and supported efficacy of lanadelumab in preventing hereditary angioedema (HAE) attacks. A pivotal randomized, doubleblind (DB), placebo-controlled, parallel arm study (NCT02586805) is ongoing, and an open-label extension (OLE; NCT02741596) is currently enrolling patients (pts). Here, we describe the design of a Phase 3 OLE study to evaluate the long-term safety and efficacy of lanadelumab for prevention of angioedema attacks in patients with HAE. Materials and methods: This OLE will include pts (≥12 years old; Type 1/2 HAE) rolling over from the DB study and an additional 50-100 pts who did not participate in the DB study (non-rollover). The non-rollover population will include pts switching to lanadelumab from another prophylactic therapy. Rollover pts will initially receive a single 300 mg subcutaneous dose of lanadelumab and will not receive another dose until after their first HAE attack. Thereafter, lanadelumab 300 mg q2 weeks will be administered until Day 350, followed by a 4-week safety follow-up. Non-rollover patients will be dosed q2 weeks regardless of their first attack. Pts may qualify to self-administer lanadelumab. The primary objective of the OLE will be to assess long-term safety. In the phase 1b study, 25% pts had local adverse effects following lanadelumab treatment vs 23.1% following placebo. Secondary objectives include evaluation of efficacy (time to first HAE attack to determine outer bounds of the dosing interval, attack rate, number attacks requiring acute treatment, are moderate/severe, or are associated with high-morbidity). Lanadelumab 300 and 400 mg was associated with a 100 and 88% reduction in attacks, respectively, in the Phase 1b study. Immunogenicity, pharmacokinetics/pharmacodynamics, quality of life, characteristics of breakthrough attacks, selfadministration and safety/efficacy in pts switching to lanadelumab from another prophylactic therapy will be evaluated. Results: Results of the OLE are expected in 2018. Conclusions: Results of this study will provide additional important data on the long-term safety, efficacy and dosing frequency of lanadelumab, a first-in-class subcutaneous therapy for prevention of angioedema attacks in patients with HAE. Background: Hereditary angioedema (HAE) is a rare, potentially lifethreatening disease characterized by acute skin and mucosal oedema. Current treatments of HAE are limited by route of administration and adverse events, since all HAE drugs are administered intravenously or subcutaneously, and may be associated with drug-specific adverse effects. There is a need for safe orally-administered therapies that control plasma kallikrein activity, prevent HAE attacks, and are welltolerated. Approximately 900 compounds were screened on the basis of chemical structure, selectivity for plasma kallikrein, and kallikrein inhibition. This study evaluated the safety of ATN-249, a novel, potent, and selective orally administered plasma kallikrein inhibitor that potentially treats HAE by blocking kallikrein-mediated production of bradykinin. Materials and methods: Genotoxicity was evaluated in bacterial reverse mutation, chromosomal aberration, and micronucleus assays. Good laboratory practice (GLP) repeat dose (28-day) toxicity was evaluated in rats and monkeys. Metabolite profiles were measured by dose recovered in intact rats and bile duct cannulated (BDC) rats. Safety pharmacology was evaluated in the functional observational battery (FOB) assessment in rats and in cardiovascular and respiratory telemetry studies in monkeys. Results:

O-17 Safety study of ATN-249, a new oral kallikrein inhibitor for hereditary angioedema
• Genotoxicity No genotoxicity was noted in bacterial reverse mutation, chromosomal aberration, and micronucleus assays. • Toxicity No noteworthy findings on clinical signs, hematology, coagulations, serum chemistry, urinalysis, and gross necropsy in GLP 28-day rat and monkey toxicity studies at doses up to 100 mg/kg/day. • Metabolism Following a single oral administration: • In intact rats, 100% of ATN-249 was recovered within 0-168 h, with 99% in feces. • In BDC rats, 97% of ATN-249 was recovered within 0-72 h; with 52% recovered in feces and 39% recovered in bile. • Previously, bioavailability was shown to be >50% in rats.
• Safety pharmacology No adverse effect was observed in rat FOB studies or monkey cardiovascular and respiratory studies.

Conclusions:
Studies demonstrating the absence of genotoxicity, absence of adverse events in rat and monkey toxicity, high bioavailability, comprehensive recovery following oral administration, and absence of adverse events in safety pharmacology suggest that ATN-249's safety profile is desirable and appropriate for further clinical evaluation. These results, along with previously published high potency and high selectivity results, suggest ATN-249 has a wide therapeutic window with once-daily dosing potential, and may be a novel, safe, potent, and selective orally-administered plasma kallikrein inhibitor for treatment of hereditary angioedema (HAE). Results: A fit-for-purpose quasi-quantitative assay of FXIIa-mediated kallikrein activity has been developed. Precision both within and between assays was well within 30% CV. The assay is sensitive to spiked concentrations of CSL312 in the range of 1-10 µg/mL. The assay also discriminates HAE and healthy plasma samples. Less than 10 µg/mL of CSL312 added to HAE plasma restored amidolytic activity to levels seen in normal subjects. The CSL312 concentration needed to treat HAE samples to achieve a target kallikrein activity may be used to predict effective CSL312 doses in HAE patients. Data will also be presented showing characterisation of different HAE patient samples using this assay. Conclusions: CSL312 is a potent inhibitor of FXIIa-mediated kallikrein activity in normal and HAE plasma. This assay shows promise to monitor CSL312 effects and inform clinical dosing regimens. Hereditary angioedema (HAE) is an autosomal dominant inherited disease characterized by painful edema episodes with variable localization and severity. Most of diagnosed patients present either mutation on C1 inhibitor gene (SERPING1), with abnormal levels or functionality (HAE type I and II, respectively) or Factor 12 (FXII) gene (HAE-FXII). These mutations leads to exacerbated bradykinin (BK) production, the peptide responsible for edema formation. In addition, there are several HAE patients of unknown molecular etiology (HAE-U), presenting same symptoms but no mutation in both classical genes. Symptoms are widely variable in patients presenting the same mutation, even in the same family. This variability highlights the role of several other modulator genes on HAE pathophysiology. In order to determine these candidates, 15 genes were sequenced in patients with HAE-U using next generation sequencing (NGS) method. From the genes analysed, KLKB1 seems to participate as a causative gene, wherein two missense mutations were identified on the HAU-U patients. Those mutations were submitted to "in silico" analysis. Mutation p.Gln442Pro was classified as benign on Mutation Taster, PolyPhen2 and SIFT, whereas, Ingenuity analysis revels a gain of function for this mutation. The mutation p.Asp619Glu was classified on "in silico" analysis as probably pathogenic by Mutation Taster, possible pathogenic on PolyPhen2 and tolerable on SIFT. In addition, the two variants were visualized on YASARA software, in order to determine possible intramolecular interactions. Gln442 seems to make a hydrogen bound with Val444 residue, but this interaction occurs on the C-terminal carbon. Therefore, substitution on amino-acid side chain might not play a major role on this interaction. In the case of Asp619, it is highly conserved among several species and could interact with Lys507 residue. This pocket is located at helix 5 close to disulfide bound, responsible for maintenance of light and heavy chains linked, after FXIIa cleavage. Changes in this pocket could lead the protein to assume a new conformation, modifying interactions between kallikrein and FXIIa, either favoring or hindering its activation. Nurses will be encouraged to register to the IHNO and receive information through the website.

Conclusions:
The new website will foster better communication between the nurses, increase awareness of health-care providers and other medical professionals and help the IHNO to achieve its proclaimed goals. Background: Prevalence of Hereditary Angioedema is approximately 1:50,000 inhabitants. Latin America population in estimated as 635,000,000, so we would expect 12,700 HAE patients. In 2013, less than 5% of the patients had been identified. Misdiagnosis, difficult access to biochemical tests and no access to therapy have been the routine in developing countries. We organized a meeting, supported by the Latin American Association for HAE (ALAeh), in order to update our situation. Methods: Physicians interested on HAE and its development in their country were invited to present the identified patients. Background: The painful and life-threatening hereditary angioedema (HAE) attacks are associated with oedema leading to disability and impairment in daily activities [1]. This is very stressful for patients and their families and may be associated with anxiety and depression [2]. Here, findings from the subject-reported outcome measures in the Phase 3 COMPACT study are reported. Materials and methods: In this study, 90 subjects with HAE were randomized to 1 of the 4 treatment sequences to receive 40 or 60 IU/ kg C1-INH(SC) twice-weekly for 16 weeks, preceded or followed by placebo for 16 weeks. Analyses were carried out in those subjects who provided at least one subject-reported outcome assessment Background: C1-INH-HAE is a rare disease with impairment in health related quality of life (HRQoL) as measured by generic HRQoL questionnaires (SF-36, E5QD). We aimed to study HRQoL in adult patients with C1-INH-HAE by means of generic (E5QD), symptom specific (AE-QoL) and disease-specific (HAE-QoL) questionnaires. Methods: The study was approved by Hospital La Paz Ethics Committee (PI-2297). Spanish patients ≥18 year. with C1-INH-HAE were included. C1-INH-HAE activity was measured by HAE-AS (range 0-30). HRQoL was measured by E5QD (range 0-1), HAE-QoL (range 25-135) and AE-QoL (range (0-100). Data were entered into an excel database and statistical analysis was performed using SPSS v. 20 for IOS. Results: 58 patients (35 females, 60.3%; 23 males, 39.7%) were included. Mean age was 45.6 ± 14.1 year. Mean HAE-AS was 7.0 ± 4.6. Mean HAE-QoL score was 100.8 ± 25.8, whereas mean adjusted AE-QoL score was 33.6 ± 23.4. Mean E5QD value was 0.927 ± 0.098. There was a significant negative correlation between HAE-QoL and AE-QoL scores (−0.897, p < 0.0001). The most affected dimension in HAE-QoL was "perceived control over disease" and the least affected "treatment difficulties" (with a 39 and 17% mean decrease with respect to dimension maximum score). The most affected dimension in AE-QoL was "fear/shame" (38.9 ± 26.3) and the least affected "functioning" (38.9 ± 28.4). There was a positive weak correlation between age and HAE-QoL score (0.2965) (p = 0.024) and negative weak correlation between age and AE-QoL score (−0.2399) (p = 0.072). HRQoL was more impaired in females than males. HAE-QoL scores (mean ± SD) were higher in males (107.8 ± 20.3) than females (96.2 ± 28.2), whereas AE-QoL scores (mean ± SD) were lower in males (27.5 ± 20.6) vs females (37.5 ± 24.5), but without significant differences. When studying HAE-QoL and AE-QoL domains the only significant differences between males and females were regarding "emotional role/social functioning" (p = 0.0397) and "mental health" (p = 0.0462) dimensions in HAE-QoL. C1-INH-HAE disease activity (HAE-AS score) was negatively correlated with HAE-QoL (−0.704, p < 0.001) and E5QD (−0.622, p < 0.001) scores and positively correlated with AE-QoL score (0.736, p < 0.001). Conclusions: HAE-QoL and AE-QoL scores show a high and significant negative correlation. Both HAE-QoL and AE-QoL had a moderate correlation with C1-INH-HAE activity as measured by HAE-AS. Perceived control over disease was the most affected dimension in HAE-QoL.

O-32 Study of health-related quality of life and disease activity in adults with HAE in Sweden
Patrik Nordenfelt 1,2 , Mats Nilsson 3 , Anders Lindfors 4 , Carl-Fredrik Wahlgren 5,6 , Janne Björkander 1,3 Background: Hereditary angioedema (HAE) is known to affect healthrelated quality of life (HR-QoL) [1,2]. To better understand the impact on HR-QoL of diseases with recurrent angioedema, like HAE, two instruments, Angioedema Quality of Life (AE-QoL) and Angioedema Activity Score (AAS) have been developed [3,4]. Aim: To better understand the impact HAE has on HR-QoL in Swedish adults, we used the new instruments AE-QoL and AAS in combination with the generic HR-QoL instruments EuroQoL 5 Dimension 5 level (EQ-5D-5L) and RAND-36. Methods: All identified allegeable adults with HAE received a questionnaire with RAND-36, EQ-5D-5L, AE-QoL and AAS instruments. AAS was filled in during 4 weeks. A questionnaire about medication and sick leave was completed. RAND-36 gives results in 9 dimensions from 0 to 100, where 100 is the most favourable result [5]. EQ-5D-5L gives a value ranging from sometimes negative values to 1, where 1 is the best [6]. AE-QoL produces results 0-100 in four dimensions, where 0 is the most favourable. The AAS increases with the disease activity. Background: With every passing day, genotyping of subjects who may suffer from hereditary angioedema (HAE) becomes more indispensable in the clinical practice. The conventional approach to the detection of disease-causing SERPING1 variants is time-consuming and fraught with many pitfalls. Materials and methods: A NGS custom platform (NGS-HAE) was designed using the Ion AmpliSeq ThermoFisher Scientific Designer ® , in order to analyze SERPING1 in its full length (all exons, introns, promoter, 5′-and 3′-untranslated regions-UTRs). A 100% coverage of all translated regions and UTRs was achieved with missing areas located only in intronic regions (overall coverage ≈ 83%). An intermediate version of the platform analyzing up to 825 amplicons was also tested aiming to examine its ability to detect copy number variations (CNVs). Annotation of variants and CNVs analysis was performed by variant-Caller v5.04.0 ® and Ion Reporter software v.5.2 ® (Thermo Scientific). Ninety-three conventionally typed DNA samples from HAE patients carrying different SERPING1 variants (52 single nucleotide variants-SNVs, 28 indels, 13 large defects) were blindly analyzed for the forward validation of NGS-HAE, with reverse validation still pending. Results: NGS-HAE results were concordant with conventional SERP-ING1 typing in 88/93 cases (51/52 SNVs, 26/28 indels and 11/13 large defects). Out of the five discrepancies observed, the conventional approach had miss-assigned two substitutions as large defects, while the NGS-HAE analysis algorithm was not able to identify one small deletion and two nucleotide substitutions. Hotspot regions were introduced in the variantCaller analysis for the detection of these three unidentified variants, elevating the concordance rate in regard with SNVs detection to 100%. Moreover, in 12/28 indels nomenclature differences were reported, with Ion Reporter assignment verified by Sanger reanalysis. Loss of heterozygosity of intronic mutations was observed in 6/13 large defects, indicating the presence of large deletions. CNV analysis resulted in the identification of CNVs of altered extend in all samples bearing large defects as well as false positive CNVs in samples bearing SNVs. Conclusions: Our NGS custom platform represents a time-and costefficient screening approach for SERPING1 typing that is valid for the detection of the vast majority of disease-associated SNVs. Given that conventional typing methods and workflows are highly dependent on the users' experience and knowledge, the NGS-HAE could be a useful initial approach for detecting HAE-causing SERPING1 variants. The potential triggering factors for HAE attacks included stress (9.2%), trauma (7.1%) and infection (4.7%). However, in most attacks triggering factor was not identified (71.9%). The most frequent were abdominal attacks (54.7%) followed by peripheral oedema (33.8%). Laryngeal oedema was presented in 10.6% of attacks. 20.9% attacks were combined. Prodromal symptoms (most often erythema marginatum, weakness or nausea) were reported by 13.6% of attacks. 2060 attacks (81.9%) were actively treated (65.8% icatibant, 21.5% recombinant C1-INH, 5.9% plasma derived, highly purified, nanofiltered C1 inhibitor (pnfC1-INH), 0.5% plasma derived, nanofiltered pdC1-INH (nfC1-INH), 0.1% fresh frozen plasma, 4.2% increase in androgens dosage, 1.8% increase in tranexamic acid dosage). Treatment had to be repeated in 312 attacks (15.1%). Hospitalization was necessary in 25 attacks (1.2%). Emergency medical service (EMS) was used in 11 attacks (0.5%). Conclusions: The analysis of HAE attacks gives further insight into their course. Our results show marked clinical variability in HAE patients. The fact that in more than 15% of attacks required repeated treatment of single attack shows that although various therapeutic Background: Our previous studies have shown that, in patients with C1-INH-HAE, SERPING1 mutations and the carriage of the F12-46C/T polymorphism are independently associated with the age of disease onset [1,2]. Plasma kallikrein is involved in the cascade of bradykinin production and activation of factor XII, and the G allele of its S143N (rs3733402) variant has been recently associated with less plasma kallikrein activity [3]. Our aim was to investigate the possible contribution of the KLKB1-Ser143Asn polymorphism (rs3733402) in C1-INH-HAE clinical phenotype. Materials and methods: 218 type I C1-INH-HAE patients (age at analysis 37.03 ± 18 years) from 100 European families (41 Hungarian, 32

P-2 Hereditary angioedema: report from the Czech registry
Greek, 16 German, 11 Romanian) were studied. All patients were previously genotyped for SERPING1 and F12 variants. The KLKB1-Ser143Asn polymorphism was detected by direct sequencing of exon 5. The possible associations of Ser143Asn polymorphism with the clinical features, combined or not with the carriage of SERPING1 mutations and/ or the F12-46C/T polymorphism, were explored by the use of generalized estimating equations (GEE), an extension of the generalized linear model that accounts for the within-subject correlations. Results: 117 patients were genotyped as heterozygous, 57 as homozygous for the A allele and 44 as homozygous for the G allele (allele frequency: G = 47%, A = 53%). In a GEE linear regression model with age at disease onset as dependent variable and adjusted for carriage of the F12-46C/T polymorphism, the presence of the G allele of KLKB1-Ser143Asn polymorphism was significantly associated with a delayed disease onset by 4.4 years compared with patients carrying the A allele (p < 0.011).

Conclusions:
The presence of the G allele of KLKB1-Ser143Asn polymorphism is an independent genetic factor strongly correlated with a delayed disease onset in patients with type I C1-INH-HAE. leads to severe complement C1q consumption, both features being diagnostic landmarks of AAE. Objective: To analyze the clinical and biochemical characteristics of 12 AAE cases and the negative effects of anti-C1INH autoantibodies on C1INH conformation, residual inhibitory activity on KK and C1s, and contact system activation status in plasma. Results: At the time of diagnosis, 10/12 of patients had low C1INH levels, autoantibodies were initially detected in 6/12 and C1q was undetectable in 9/12. Residual C1INH inhibitory activity was extremely variable and did not correlate with the observed complement consumption. Purified IgG from one patient dose-dependently induced C1INH cleavage in the presence of KK. Moreover, among autoantibody-negative patients and by using a novel in-house ELISA assay, we for the first time detected circulating C1INH:IgG or C1INH:IgM in 5/6 otherwise autoantibody-negative and in all positive cases in our series. Conclusion: The detection of plasma C1INH:Ig complexes in autoantibody-negative patients is clinically relevant and indicates that anti-C1INH autoantibodies could be underestimated by conventional screening methods and represent an additional diagnostic tool. Reduced activity of C1-INH may result in an instability of kinin pathway with the generation of bradykinin inducing in increased vascular permeability. Bradykinin increases the release of nitric oxide and Vascular Endothelial Growth Factor (VEGF) from endothelial cells. VEGF, Angiopoietin 1 (Ang1) and Ang2 are released at sites of inflammation and/or angiogenesis regulating vascular permeability.We have previously demonstrated that plasma concentrations of VEGF-A, VEGF-C, Ang1, and Ang2 were higher in patients with C1-INH-HAE in remission than in healthy controls. The level of these mediators was correlated with severity of disease phenotype measured by number of attacks per year [1]. There are no data on the role of vascular permeability factors during angioedema attack and in patients on prophylaxis. In this study we analyzed plasma concentration of VEGF-A, VEGF-C, Ang1 and Ang2 in patients with C1-INH-HAE, both in remission and during acute attacks and we compared angiogenic factors plasma levels in patients taking or not prophylactic treatment. Methods: Ninety-four untreated patients and 29 patients undergoing prophylaxis with attenuated androgens were studied. Then, we analysed 20 C1-INH-HAE patients in remission and during angioedema attack. Concentrations of angiogenic (VEGF-A, Ang1, Ang2) and lymphangiogenic (VEGF-C) factors were evaluated by ELISA. Results: We could not detect differences in the levels of the tested factors between untreated patients and patients undergoing prophylaxis with attenuated androgens. However, these are preliminary data that we plan to expand in a follow-up study.

P-6 Role of vascular permeability factors in patients with hereditary angioedema with C1 inhibitor deficiency
VEGF-A, VEGF-C and Ang2 levels were not modified during attack compared to basal conditions. By contrast, Ang1 levels (a vascular stabilizer) were increased during acute phase and the ratio Ang2/Ang1 (an index of vascular permeability) was decreased.

Conclusions:
The results of this study show that the plasma concentrations of vascular permeability factors during acute attack changes scenario compared to basal conditions. There is an increase of vascular stabilizer and a decrease of index of vascular permeability. Moreover, preliminary experiments suggest that the prophylaxis do not modify vascular permeability factors concentration maybe because vascular preconditioning is not influenced by androgens. Bradykinin (BK) and its receptors (BDKRB1 and BDKRB2 receptors) play important roles in a wide variety of physiological and pathological processes. As mediators of pain and inflammation, they exert a variety of biological effects on endothelium and peripheral circulation. The aim of our study is finding a distribution pattern of the both bradykinin receptors on monocytes and lymphocytes CD4+ and CD8+ in attack of angioedema and remission of the disease. Method and material: Peripheral blood mononuclear cells (PBMC) collected from 10 healthy volunteers (7 women and 3 men in the age 20-62 year) and 10 patients with hereditary angioedema type I in attack and remission of the disease (5 women and 5 men in the age 26-50 year) were isolated. Subsequently, PBMC were stained to distinguish: lymphocytes (anti-CD3, anti-CD4, anti-CD8), monocytes (anti-CD14, anti-CD16, anti HLA-DR) and their expression of B1 (anti-BDKRB1) and B2 (anti-BDKRB2) receptors and flow cytometry was performed. Also the analysis of B1 and B2 gene expression by real time PCR as a preliminary study was performed. Results: We found the higher expression of BDKRB2 mainly on monocytes during attack of angioedema compering to the remission and to the reference group. The expression of BDKRB2 was highly variable in lymphocytes CD4 and CD8 both during the attack and remission. Expression of BDKRB1 was residual on both types of lymphocytes. The expression of BDKRB1 detected on monocytes was at the same level in both groups under the study. As preliminary results of gene expression the presence of mRNA for B1 and B2 receptors in PBMC was observed. Conclusion: In patients with HAE and in the reference group the expression of BDKRB1 and BDKRB2 was detected both on monocytes and lymphocytes. The expression of BDKRB1 was low contrary to the expression of BDKRB2, which was generally high in attack of angioedema. There was a huge variation between individuals in expression of BDKRB2 on

Materials and methods:
We developed ELISA assays using commercially available antibodies specific to FXI, FXII, C1s and C1r, as well as an affinity-purified anti-C1-INH IgG produced by our laboratory. Similarly, commercially available active FXII, and FXI, as well as active recombinant C1s, C1r and commercial plasma-derived C1-INH-further purified by ion-exchange chromatography-were used to generate the complexes required for the standards. We checked the quality of the complexes with SDS PAGE. The ELISA assays were tested on various types of blood samples (serum, and plasma anticoagulated with EDTA, citrate, hirudine, or EDTA+ protease inhibitor cocktail). The samples were collected from healthy volunteers. Results: The complexes were produced from molar equivalent quantities of active enzymes and of C1-INH. In average 85.3% (70-96) of the initial enzymes formed complex with C1-INH, based on SDS-PAGE analyses. In each developed sandwich-ELISA assay the detection threshold was less than 0.01% of the plasma concentration of the enzyme. The accuracy of the recovery of the complexes was 92% (75.17-119) on average. Except C1r/C1-INH complex measurement, the serum levels of the complexes were always higher than plasma values. Intra-and inter-assay variation was 11.88% (7.51-17.55) and 15.18% (9.01-19.3), respectively. Conclusions: We successfully developed a set of ELISAs for the sensitive determination of various enzyme-inhibitor complexes, which makes the simultaneous investigation of C1-INH-regulated activation systems possible in C1-INH-HAE. This could greatly contribute both to a better understanding of the pathomechanism of this disease, and to the exploration of attackkinetics. In addition to C1-INH-HAE, this method might prove suitable for studying other disorders related to the impaired regulation of plasma enzyme systems. Background: Hereditary angioedema (HAE) due to C1 inhibitor deficiency (C1-INH-HAE) is characterized by recurrent episodes of disabling and painful swelling. Ruconest is a recombinant human C1 inhibitor that is approved for treatment of HAE attacks. A treatment registry was established in Europe to review the adverse event profile and efficacy of Ruconest following single and repeated treatment with Ruconest. Methods: Patients with C1-INH-HAE were enrolled following a decision to treat with Ruconest and after providing written informed consent. Medical history and baseline HAE information was collected at a screening visit. Treatment decisions were at the discretion of the health care professionals (HCP) involved in the patients' care according to their standards for the management of C1-INH-HAE and in line with the approved Ruconest summary of product characteristics. Following treatment with Ruconest, the HCP entered data using a web-based questionnaire about the attack, response to therapy, and any adverse events. Background: Idiopathic systemic capillary leak syndrome (ISCLS) presents with recurrent potentially life-threatening episodes of hemoconcentration and hypovolemic shock. Due to the rarity of the disease (about 250 cases described) and to misdiagnosis, evidence based approaches as well as validated protocols are still lacking. Aim: To report our experience on the treatment of acute shock emergency in patients with ISCLS. Materials and methods: Analysis of records from 12 ISCLS patients (9 men), from a cohort of 22, admitted once or several times to hospital for hypovolemic shock. Results: Mean age at symptoms' onset was 51.5 years (range 35-68). ISCLS crises occurred with variable frequency (from 3 to 8), timing of the acute phase (mean duration 3-4 days) and severity. Mean followup was 6 years (1 month to 18 years). Prodromal symptoms (identified in all subjects) consisted of: arterial hypotension (11/12 patients), fatigue (10/12), oliguria (10/12), worsening edema (9/12), weight gain (7/12), presyncopal/syncopal episodes (6/12), abdominal pain (6/12), nausea, vomiting, diarrhea (5/12), arthromyalgia (5/12), sore throat, dysphonia, cough (5/12), dizziness (4/12), high temperature (4/12), thirst/polydipsia (3/12), headache (2/12), diaphoresis (3/12), dyspnea (3/12), altered consciousness (3/12), livedo reticularis (1/12). The acute phase could develop very rapidly, with alteration of vital parameters, mild to moderate alteration of consciousness and finally distributive shock, with marked hypotension, tachycardia, oligoanuria, edema (often with "stone-like" consistency, paresthesia or pain). Blood tests always revealed high hemoglobin (highest recorded value 25.8 g/dl) and hematocrit (up to 72%), hypoproteinemia (minimum serum albumin 9 g/L). IgG monoclonal band was present in all patients without changes outsides and during shock. Treatment was based on crystalloids (11/12 patients), amines (11/12); 10/12 patients were treated with small boluses of high molecular weight plasma expanders (e.g. 250 mL bolus of colloids every 4 h), 7/12 steroids, 5/12 albumin, 5/12 diuretics, 1/12 methylene blue, 1/12 iv Ig. Long-term prophylaxis consisted of: verapamil (11/12), theophylline (8/12), terbutaline (2/12), iv Ig (2/12). Complications recorded were: acute renal failure (10/12), compartment syndrome and neuropathy (7/12), rhabdomyolysis (5/12), myocardial edema (5/12), pericardial effusion (5/12), pleural effusion or abdominal free fluid (4/12), cerebral involvement (2/12), acute pulmonary edema (2/12), DVT (1/12). CVVH was performed in 4/12 cases, oro-tracheal intubation in 1/12, fasciotomy in 1/12, ECMO in 1/12. Conclusions: ISCLS may induce severe shock associated with hemocencentration and hypoproteinemia. Fluid replacement and amines should be minimized, with judicious use of colloids to maintain adequate perfusion. Careful surveillance of potential complications is warranted. Aim of the study: To assess the hypothesis of a bidirectional crosstalk/interplay between autonomic nervous system (ANS) modulation and contact/complement system activation in the development of angioedema attacks. Materials and methods: 23 HAE patients (6 males, mean age 47.5 ± 11.4 years) during remission and 24 healthy volunteers (8 males, mean age 45.3 ± 10.6 years) underwent recording of ECG, beatby-beat blood pressure (BP), respiratory activity during rest (R; 10′) and 75-degrees-head-up tilt (T; 10′), together with blood withdrawals for C1INH, C4 and cleaved high molecular weight kininogen (HK). In a subgroup of patients, plasma catecholamines were also evaluated. Spectral analysis of heart rate variability allowed to extract low (LF) and high (HF) frequency components, markers of sympathetic and vagal modulation respectively. Results: SAP was significantly higher in C1-INH-HAE patients than in controls (134.0 ± 19.0 vs 112.1 ± 17.4 mmHg, p = 0.001 at R, 141.4 ± 28.8 vs 121.7 ± 17.3 mmHg, p = 0.01 during T), while only in controls T induced a significant increase in mean SAP and SAP variance. LF nu increased significantly after orthostatic challenge in both groups (69.7 ± 26.1 after T vs 57.7 ± 24.9, p < 0.05 in patients and 78.0 ± 20.7 vs 51.5 ± 21.2, p < 0.01 in controls), but only in healthy subjects there was a significant increase of LF/HF ratio, index of sympathovagal balance. As expected, plasma C1-INH antigen and function as well as C4 antigen were markedly lower in C1-INH-HAE patients than in controls. Both proteins showed a tendency to increase after tilt. Noradrenaline was higher in patients at R and increased in both groups after tilt test. Cleaved kininogen (cHK), marker of contact system activation, was increased in C1-INH-HAE patients compared to controls. Tilt test induced a significant increase in cHK only in HAE patients (49.5 ± 7.5 after T vs 47.1 ± 7.8% at R, p = 0.01 in patients and 40.0 ± 6.1 vs 38.7 ± 7.2%, p = 0.06 in controls). Conclusions: Our data are consistent with an alteration of ANS modulation in HAE patients, who present increased sympathetic activation at rest and blunted response to orthostatic challenge. Tilt test-induced increased HK cleavage suggests a link between stress and bradykinin production. ) with no gender difference observed between early and late treating groups. Early self-treatment varied across countries, ranging from 69.6% (Germany/Austria) to 11.6% (France). Early treaters vs late treaters treated attacks localized to skin, abdomen and larynx at a similar rate (P = 0.6105, P = 0.3398 and P = 0.8219 respectively). Similarly, no statistically significant difference between early vs later treater groups was observed based on grouped attack severity (very mild/mild/moderate vs severe/very severe; P = 0.164). Comparing early versus late treatment, respectively, a significant reduction (P < 0.001) in median (Q1,Q3) time to resolution [4.5 h (1.0, 11.5) versus 8.0 h (3.0, 24.5)] and attack duration [5.0 h (1.5, 12.0) versus 13.0 h (6.0, 33.0)] was observed (243 patients; 1353 attacks with complete information on time to treatment, time to resolution and duration of attack). Conclusion: Early treaters had shorter time to resolution and attack duration compared to late treaters, possibly indicating the importance of early access to icatibant in the face of HAE attacks. Differences in local practice patterns, icatibant availability, and tendency of early treaters to treat any symptoms without delay may drive prevalence of early use across countries. These and other findings from this analysis are hypothesis generating and should be further evaluated. The aim of this study was to describe the pattern of treatment in acute attacks of angioedema during pregnancy and breastfeeding. Methods: Retrospective analysis of data collected during pregnancies and lactation of patients attended at the Allergy Department of Hospital University La Paz by physicians specialized in the management of HAE. The following variables were collected: number of attacks in the previous six months (T1), pregnancy (T2) and breastfeeding (T3), severity of the attack, time to treatment administration, time to onset of improvement, time to complete improvement, percentage (%) of improvement 4 h after and type of acute treatment received. Results: We reviewed 395 attacks of 13 patients, 18 pregnancies (2 abortions). There were no significant differences in the number of attacks among T1, T2 and T3. The number of attacks in pregnancy and breastfeeding was positively correlated (Pearson = 0.798, p = 0.017). There were no differences in the percentage of acute attacks treated with C1-inhibitor concentrate (pdC1INH) between the three periods. There were more acute attacks treated in T2 (68.9%) than in T1 (54.1%) (p = 0.044). Acute attacks were treated earlier in T2 and T3 compared to T1, although without significant differences (p = 0.327). The time to onset of improvement was significantly different among groups (p < 0.001) (T2 3.61 ± 2.79) (T3 2.70 ± 2.45) (T1 5.54 ± 6.41). There were no differences in the time to complete improvement (p = 0.179). The percentage of improvement after 4 h was higher in T3 (57.0% ± 34.2) than in T2 (40.1% ± 33.5) (p = 0.007). The total duration of attacks was positively correlated with the time to treatment administration (p < 0.001). Mean total duration of attacks was not significantly different among groups (p = 0.980) (T1 29.28 ± 15.42) (T2 29.81 ± 76.17) (T3 28.02 ± 32.30). Conclusions: This is the first study that provides data about treatment outcomes in acute attacks during pregnancy and breastfeeding in patients with hereditary angioedema. Time to onset of improvement and percentage of improvement at 4 h in breastfeeding and in pregnancy was shorter compared to the situation prior to pregnancy, although further studies are needed to confirm these results. Contrary to HAE the late onset of angioedema attacks (>40 years) and negative family history are characteristic for AAE. Materials and methods: The aim of the study was to analyze patients with late onset of angioedema attacks from the group of above 350 persons with angioedema attacks due to C1-INH deficiency registered in our center. Results: The late onset of angioedema attacks due to deficiency of C1-INH was found in the group of 10 patients (2.9%) ( Table 1). In 6 patients we confirmed AAE (group A) but in 4 cases we have no proof of any other disease or abnormality and they are still under observation (group B). Conclusions: Late onset of angioedema attacks due to C1-INH deficiency may appear in patients with negative family history. Precise work up and differential diagnosis with AAE is very important. Clinical course of the disease in the group B is identical to HAE. We also observed efficacy of treatment with C1 INH concentrates. • We find 9 splicing, 6 missense, 7 frameshift and 2 nonsense mutations. 10 of them have been found that were not described in the consulted database (http://www.ensembl.org/index.html), but in general only 6 mutations didn't mention in ensemble.org, because we have duplicates of mutations. • 4 small deletions affecting exon 3 (c.520-524delATCGC, p.Ser173fs254X), exon 8 (c.1293delA, p.Leu430fs449X), exon 7 (c.1106delA, p.Glu368fs396X) and exon 5 (c.744-745delCA, p.247Serfs255X) have been find. All of them are producing frame-shifts leading to premature stop codons. In most cases an on-demand therapy of acute attacks is sufficient, in severe cases, however, a prophylactic therapy is needed. Therefore C1-INH intravenously (IV) was shown to be safe and efficient. Methods: We present the case of a patient with HAE-I who was under prophylactic therapy with C1-INH IV due to a high number of attacks during on-demand therapy. An implanted port guaranteed a periodical and safe apply of the medication until the device had to explanted due to an infection. Because of a bad vein status repeated IV application failed. After stopping the prophylactic therapy he suffered from recurrent and partially severe attacks again. Therefore we tried a subcutaneously off-label use of 1500 IE C1-INH as prophylaxis over more than one year. Results: After a brief training session the self-application was easily managed by the patient. Under the prophylaxis the number of attacks was reduced from 4.33 to <1/month. No severe attack and none of the upper airway was noticed. The quality of life measured by the AE-Qol could be improved. The results were similar to those under the approved IV therapy. Discussion: Subcutaneous use of 1500 IE C1-INH seems to be easy and safe. In our case it showed similar effectiveness compared to the IV therapy. No adverse events could be noticed. The quality of life measured by the AE-Qol could be approved. By learning a self-application the patient gained independence. The results of this case seem promising, however bigger studies are needed to underline our findings. The anamnesis is the crucial first instrument for discerning bradykininmediated from histamine-mediated angioedemas. Patients with onset of angioedemas in late adulthood and no family history should be considered for acquired angioedema (AAE) or late-onset hereditary angioedema (HAE) with spontaneous mutations. Once specific drug intake such as angiotensin-converting-enzyme inhibitors (ACE-inhibitors), angiotensin-II-receptor antagonists (sartans) and dipeptidylpeptidase-4 (DPP-4) inhibitor in some oral antidiabetics is excluded, a blood test for C4, C1-esterase inhibitor antigen (C1-INH-a) and function (C1-INH-f ) should be performed. As levels for these tests are low as well for HAE (type I and II) as for AAE, levels of C1q can help discern between the two pathologies. Two patients with late onset of bradykinin mediated angioedemas presented to our clinics. Both women, aged 53 and 66, showed low levels for C4, C1-INH-a and C1-INH-f, but only the younger had low C1q. AAE is known to be tightly correlated to lymphoproliferative diseases, but also anti-C1-inhibitor-autoantibodies and other malignancies with subsequent consumption of the factors mentioned above [1]. For this woman no malignancy was known, oncological examination including PET-CT didn't reveal any suspicious result, but she will undergo regular oncological visits. About 14% of patients with AAE don't show any correlated disease [2]. The second patient had a history of breast cancer, splenomegaly and a submucosal gastric formation suspicious for MALT-lymphoma, an endoscopic biopsy was unsuccessful. Unexpectedly, levels for C1q were normal and testing for anti-C1-inhibitor-autoantibodies was negative. In literature there are reports about cases of AAE with normal levels of C1q [3]. As the patient mentioned that her daughter started having swellings, we had to re-consider HAE type I as diagnosis. Genetic analysis showed no mutation for SERPING-1 gene and the daughter's blood levels for C4, C1-INH-a and C1-INH-f were normal. Even if HAE could not be excluded for sure, AAE has to be considered as the most probable diagnosis. A new endoscopic biopsy of the suspected MALT-lymphoma is already planned, as therapy of the malignant disease can stop the symptoms of AAE. In the meanwhile, she self-administers bradykinin B2 receptor antagonist for acute attacks. C1q is an important marker to discern between HAE and AAE, especially if the correlation between AAE and lymphoproliferative diseases is considered. Even with rarely normal levels of C1q, the diagnosis of AAE should lead to clarify the presence of a malignant disease in order to detect and cure it by time.

P-18 The importance of C1q in diagnosis of acquired angioedema (AAE)
Consent to publish: Consent to publish was obtained from the patients.
Ukraine is undergoing a medical reform. , and 78% are type I. The mean age of the first attack was 2 years and 6 months, the median age was also 2 years and 6 months old, and the earliest age at initial symptoms was 2 months old. The mean age at diagnosis was 7 years. 86% of patients had cutaneous involvement, 64% had gastrointestinal symptoms at any time since diagnosis and 64% had laryngeal involvement at least once, with the youngest patient at 2 months of age for laryngeal symptoms (confused as a whipping cough reactivation every month until the specific treatment). One patient reported an asthmatic-like attack, which improved rapidly after infusion of plasma derived C1-INH. 50% of our patients have used plasma-derived CI-INH at least once for acute attacks only. Because of the poor availability of this treatment, 43% of patients were treated with nadroparin for acute attacks, with successful improvement of the symptoms. We do not use danazol in children because the adverse effects reported in this population. No deaths have been reported in paediatric patients at our Institution. Conclusions: Misdiagnosis is common in paediatric population, especially in those with initial gastrointestinal symptoms or atypical manifestation as asthmatic-like attack. Subcutaneously administered Nadroparin has been reported to be effective for symptoms resolution [2], however it is not recommended in current guidelines. As there is poor availability of plasma derived C1-INH in our country, further   Family history of HAE related death was reported in 37 patients (53%). Based on the frequency of attacks patients are grouped in 5 categories (n = 53) ( Table 3). The location of attacks data is presented in Table 4. Treatment modalities are presented in Table 5. It presently contains 937 entries from Italy, the number is expected to rapidly grow with the contribution of centers from other countries. The governance of the registry will be in charge to a committee where all centers and patients' supporting groups are represented. The technical handling is provided by the data managing society Cloud-R s.r.l., which developed the new registry on the cloud. Thanks to the application architecture of the registry, patients will be able to directly provide part of the information and to retrieve individual data via web or mobile. Centers will own and will be able use the data they provide. The committee will manage aggregated data and will release periodic outputs. Data will be available for health and regulatory agencies according to the law. Conclusions: The first disease registry for different forms of angioedema has been built. It is open globally, scalable, secure and it will provide prospective data to expand the understanding of the disease and to improve the standard of care.

Introduction:
Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare, oftentimes misdiagnosed disease, with consequent delays in diagnosis and associated morbidity. We analyzed patient data from the Icatibant Outcome Survey (IOS) to evaluate the relationship between patient age (birth date) and both age at diagnosis and delay in diagnosis (time between first symptom onset and diagnosis).

Methods:
The IOS is an international, observational study (NCT01034969). Data were collected from 12 countries between July 2009 and January 2017. Study participants provided written informed consent. Patients with a family history of C1-INH-HAE who were diagnosed before their first symptoms were excluded from analysis. To avoid potential bias inherent in the data collected from the youngest generation (time to diagnosis is limited by their age), only patients born before 1990 and diagnosed before 25 years of age were included. Linear regression analyses were performed.  Table 1). Conclusion: Although delay in diagnosis of patients with C1-INH-HAE has improved over time, there remains a need to raise awareness of HAE.  [1]. Swelling of the intestine may cause symptoms suggestive of an acute abdomen, and this may mislead physicians to consider surgical treatment.
We described a case of a 42 year old man with abdominal pain attacks for the past 8 years. The patient described the attacks of abdominal pain as abrupt and quite intense, sometimes associated with nausea or vomit. He only described two episodes, where in addition of the abdominal pain he experienced local swelling of his forearm and hand, the swelling had no apparent cause and it resolved in less than 3 days. During one of his attacks he underwent open appendicectomy, but the histopathology result was negative for appendicitis. Five years after, he experienced another severe attack of abdominal pain that again brought him to the emergency department. As part of his medical approach an abdominal CT scan was performed, showing peripancreatic and subdiaphragmatic fluid along with mesenteric lymphadenitis. He persisted with acute abdominal pain, so he underwent an exploratory laparotomy from which a biopsy of a mesenteric lymph node was taken and ascitic fluid sent for culture. Under the suspicion of infection, he received antibiotic, but afterwards the histopathology report showed only unspecific inflammatory response, and no bacteria was found in the ascetic fluid culture. To continue studying the cause of his abdominal attacks the patient also had a push enteroscopy showing esophagitis and erosive gastritis, histopathology report from stomach, duodenum, and jejunum biopsies were normal.
In the course of one of his visits to the emergency department, aside from the clinical suspicion of pancreatitis, finally angioedema was considered as a differential diagnosis. Hereditary angioedema with C1-INH deficiency type I was confirmed with laboratory tests (Table 1). presented at least an attack treated with Berinert ® . These patients presented 287 attacks: abdominal (29.0%), facial (22.0%), laryngeal (14.0%), peripheral (10.0%) and multi-location (25%). 60.0% of the attack were rated "severe", 38.0% "moderate" and 2% "low". A trigger event was described in 25.4% of crisis in relation with stress or trauma for 15.0% of the cases. 39.8% (98) of attacks have been treated with 20 UI/kg, 54% (135) with 10-20 UI/kg. On available data, symptoms began to be improved in less than 1 h in 35.0% and in less than 2 h in 27%. Symptoms disappeared in less than 24 h in 68.5%. A treatment failure occurred for 8 out 287 attacks (11.1%): 4 attacks were treated with a low dose (<20 UI/kg), 1 attack was treated 8 h after starting, no precise information are available for the 3 remaining attacks. Patients' satisfaction rate was 94.6%. Conclusion: COBRA registry affords the opportunity to systematically describe type I-II bradykinin mediated AE patients treated with Berinert ® and to monitor its efficacy in attack treatment. Background: Acute angioedema attacks in hereditary angioedema (HAE) are unpredictable; they may cause activity impairment and may be disfiguring, painful or even life-threatening. Specific treatment is administered through subcutaneous or intravenous route. Attending to healthcare facilities cause economic costs, absenteeism from work and delay in treatment. Home-and self-administration programs reduce morbidity, disease burden and potential mortality, improving quality of life. Materials and methods: In this observational retrospective study, HAE patients attending to our third level Hospital, were offered a home self-administration training program. They followed 2-6 sessions lasting for 45 min with a specialist nurse. One partner for each patient was also considered. Injection/infusion skills were annually reviewed.
The number of annual angioedema attacks and their need for emergency department (ED) attendance for the last 5 years was registered. implications were discussed and this common decision was made. The dose used per application: 4200 U/20 ml (2 vials solved in 10 ml solvent, each) was injected intramuscularly in two different sites, either into m. gluteus maximus or m. quadriceps femoris. During the 14-week follow-up no severe breakthrough attacks occurred. The patient reported significant improvement in the quality of life and daily activities were restored.

Results
No side effects at the application site and from the medication were reported from any of the patients. Conclusions: Intramuscular administration of rhC1inh could be an alternative to the intravenous route of application, especially when intravenous administration is compromised or access to medical care facilities is difficult. Intramuscular application of rhC1inh seems to be safe and effective in the presented cases: as on-demand therapy and in prophylaxis. 2100 U rhC1inh can be successfully solved in 10 ml solvent and the intramuscular application shows no adverse effects. Long-term prophylaxis (LTP) with rhC1inh seems to be safe and effective in subjects with severe HAE. Application of two vials (4200 IU) twice weekly seems to be an effective dose for LTP.
Consent to publish: Consent to publish was obtained from the patients.

P-40
Are we ready to propose a pharmacological approach for Hereditary Angioedema with normal C1 Inhibitor (HAEnlC1INH)?
Stephanie Background: Hereditary angioedema with normal C1 inhibitor (HAEnlC1INH) was first described in 2000. It affects both genders with higher prevalence off females due to hormonal influence. Symptoms have been associated with bradykinin and part of the patients present factor 12 mutation. Considering the limited knowledge about the mechanism involved, therapy for HAE with C1INH deficit has been applied to those cases. We evaluated the response to therapy of symptomatic patients with HAEnlC1INH proposing a personal approach. Methods: Patients with suggestive symptoms of HAE, familial history and normal C1-INH were included. DNA samples were evaluated for the presence of mutations on exon 9 of the F12 gene. The protocol was approved by ethical committee. Results: Fourteen families (8 with factor 12 mutation) were included within a total of 38 (33F:5M) patients (median age 34.5; 9-69 years). Initial symptoms appeared at 18.5 years old (median) and 23/29 between 10-30 years old. Three families reported bruising as prodromal symptoms. Edema mainly affected the following: abdomen 27/29; face 19/29; extremities 12/29; upper airways 13/29. Prophylactic therapy was: combined contraceptive exclusion 8/24; tranexamic acid (medium dosage 500-750 mg/day) 20/29; progestins 15/24; danazol 9/24; oxandrolone 3/24. The attacks were treated with higher dosages of tranexamic acid in 9/29 patients; icatibant 8/29 and plasma derived C1INH in 4/29. Conclusions: Although recognition of physiopathology in HAEn-lC1INH is restricted, we face the need of therapy in these patients. Approximately 2/3 of the patients present high risk for upper airway obstruction. We propose a stepwise approach for those patients: combined contraceptive exclusion; low dosages of tranexamic acid; progestins and finally androgens as prophylaxis. High dosage of tranexamic acid was effective for mild symptoms during the attacks but icatibant and plasma derived C1 inhibitor had been used preferentially.