Eur J Pediatr (2005) 164: 486–490 DOI 10.1007/s00431-005-1673-4

O R I GI N A L P A P E R

Gotho Geishofer Æ Alexander Binder Æ Martin Mu¨ller Bettina Zo¨hrer Æ Bernhard Resch Æ Wilhelm Mu¨ller Jo¨rg Faber Æ Adam Finn Æ Georg Endler Christine Mannhalter Æ Werner Zenz Æ the Central European Meningococcal Genetic Study Group

4G/5G promoter polymorphism in the plasminogen-activator-inhibitor-1 gene in children with systemic meningococcaemia Received: 7 January 2005 / Revised: 7 March 2005 / Accepted: 10 March 2005 / Published online: 21 April 2005
Springer-Verlag 2005

Abstract Meningococcal disease may present as sepsis, meningitis or a combination of both. Impaired fibrinolysis and massive elevation of the plasminogen activator inhibitor-1 (PAI-1) is a characteristic feature of meningococcal sepsis. Previously, an association between mortality and the functional 4G/5G promoter polymorphism of the PAI1gene in a cohort of UK and Dutch children with meningococcal sepsis was reported. We carried out a prospective, multicentre study to investigate the association of the 4G/ 5G PAI-1 polymorphism, diagnosis, and outcome in meningococcal disease in a Central European and UK population. Blood samples and clinical information of 347 previously healthy children with meningococcal infection were collected from 95 paediatric hospitals in Germany, Switzerland, Italy, the United Kingdom, and Austria from 2000 until 2002. Mortality was significantly associated with the 4G/4G genotype (12 of 90 (13%) vs. 15 of 240 (6%), P =0.037), resulting in an odds ratio of 2.31. The diagnosis of sepsis (independent of symptoms of meningitis) was significantly more frequent in carriers of the 4G/4G genotype (P =0.01), resulting in an odds ratio of 2.21 to develop sepsis. Meningitis was not associated with the PAI-1 4G/5G polymorphism, and allele frequencies were G. Geishofer Æ A. Binder Æ M. Mu¨ller Æ B. Zo¨hrer Æ W. Zenz (&) the Central European Meningococcal Genetic Study Group Department of General Paediatrics, Medical University of Graz, Auenbruggerplatz 30, 8036 Graz, Austria E-mail: [email protected] Tel.: +43-316-3852605 Fax: +43-316-3852045 B. Resch Æ W. Mu¨ller Department of Neonatology, Medical University of Graz, Graz, Austria J. Faber Department of Paediatrics, Johannes Gutenberg University, Mainz, Germany A. Finn University of Bristol, Institute of Child Life and Health, Department of Clinical Sciences at South Bristol, Bristol, UK G. Endler Æ C. Mannhalter Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Vienna, Austria

similar in patient and control groups. Conclusion: Our data show a correlation between the 4G/4G genotype in the plasminogen activator inhibitor-1 gene and poor outcome in children with meningococcal infection. In addition, 4G homozygous patients were prone to develop sepsis. We found no influence of the plasminogen activator inhibitor-1 polymorphism on the susceptibility to invasive meningococcal infection. Keywords Fibrinolysis Æ Meningococcal disease Æ Plasminogen activator inhibitor-1 Æ Polymorphism Æ Sepsis Abbreviations CEMR: Central European Meningococcal Research Æ HWE: Hardy-Weinberg equilibrium Æ PAI-1: plasminogen-activator-inhibitor-1 Æ UKMR: United Kingdom Meningococcal Research Co-investigators of the Central European Meningococcal Study Group were: Ba¨rbel To¨pke (Ostalb-Klinikum, Aalen), Peter Fucik (Krankenhaus Amstetten, Amstetten), Johann M. Penzien (Zentralklinikum Kinderkliniken, Augsburg), Gedeon Diab (Kreiskrankenhaus Bad Hersfeld, Bad Hersfeld), Robert Miething (DiakonieKrankenhaus, Bad Kreuznach), K.H. Deeg (Klinikum Bamberg, Bamberg), Ju¨rg Hammer (Universita¨ts-Kinderspital beider Basel, Pa¨diatrische Intensivmedizin, Basel), Ulrich Heininger (Universita¨tsKinderspital beider Basel, Abt. fu¨r Infektiologie, Basel), Verena Varnholt (Uni-Klinikum Charite´, Campus Virchow Klinikum, Berlin), Andreas Schmidt (St.-Agnes-Hospital, Bocholt), Lutz Bindl (Rheinische Friedrich-Wilhelms-Universita¨t, Bonn), Ursula Sillaber (Landeskrankenhaus Bregenz, Abt. fu¨r Innere Medizin, Bregenz), Christian Huemer (Landeskrankenhaus Bregenz, Abt. fu¨r Kinderheilkunde, Bregenz), Primrose Meier (Zentralkrankenhaus Links d. Weser, Bremen), G. Simic-Schleicher (Zentralkrankenhaus BremenNord, Bremen), Markus Markart (Ospedale di Bressanone, Brixen), Eberhard Pfau (DRK-Krankenhaus Chemnitz-Rabenstein, Chemnitz), Hans Broede (Klinikum Lippe-Detmold GmbH, Detmold), Bernd Ausserer (Krankenhaus Dornbirn, Dornbirn), Hermann Kalhoff (Sta¨dt. Kliniken Dortmund, Dortmund), Volker Arpe (St. Marien-Hospital Du¨ren-Birkesdorf, Du¨ren), Susanne SchweitzerKrantz (Evangelisches Krankenhaus, Du¨sseldorf), Johannes-Martin Kasper (St. Georg Klinikum Eisenach GmbH, Eisenach), Kathrin Loranth (Krankenhaus der Barmherzigen Bru¨der, Eisenstadt), Hans J. Bittrich (Helios Klinikum Erfurt, Erfurt), Burkhard Simmer (Landeskrankenhaus Feldkirch, Feldkirch), Nicola Weigand (Klinikum Justus-Liebig-Universita¨t, Gießen), Egbert Herting (GeorgAugust Universita¨ts-Kinderklinik, Go¨ttingen), Karl-Heinz Smolle

487 (Medizinische Universita¨tsklinik, Graz), Christoph Fusch (Klinikum d. Ernst-Moritz-Arndt-Universita¨t, Greifswald), Alois Gruber (Krankenhaus der Schulschwestern Grieskirchen, Grieskirchen), Ulf Schimmel (Allg. Krankenhaus Hagen GmbH, Hagen), Suzanne Knaufer-Schiefer (Ohrekreis-Klinikum, Haldensleben), Wolfgang La¨ssig (Sta¨dt. Krankenhaus Martha-Maria Halle-Do¨lau GmbH, Halle), Axel Hennenberger (Kinderkrankenhaus Wilhelmstift, Hamburg), Axel von der Wense (Altonaer Kinderkrankenhaus, Hamburg), Roland Tillmann (Klinikum Kreis Herford, Herford), Ju¨rgen Schwarick (Kreiskrankenhaus Herzberg, Herzberg), F.C. Sitzmann (Univ. Klinik fu¨r Kinder- und Jugendmedizin, Homburg/ Saar), Herbert Mu¨ller (Klinik Robert-Weixler-Strasse, Kempten), Peter Kurnik (Landeskrankenhaus Klagenfurt, Klagenfurt), Peter Groneck (Kinderklinik der Stadt Ko¨ln, Ko¨ln), Helene Gro¨blacherRoth (Landeskrankenhaus Krems a. d. Donau, Krems a.d. Donau), Ju¨rgen Bensch (Vinzentius-Krankenhaus, Landau), Reinhard Moser (Landeskrankenhaus Leoben, Leoben), Rudolf Schwarz (LandesKinderklinik Linz, Linz), Kurt Lenz (Koventhospital der Barmherzigen Bru¨der, Linz), Thomas Hofmann (Evang. Krankenhaus, Lippstadt), Wolfgang Go¨pel (Med. Universita¨t zu Lu¨beck, Lu¨beck), Thomas Berger (Kantonspital Luzern, Luzern), Erwin Hauser (Landeskrankenhaus Mo¨dling, Mo¨dling), Kai Martin Fo¨rster (Sta¨dt. Krankenhaus Harlaching, Mu¨nchen), Jochen Peters (Kinderklinik der TU Mu¨nchen, Mu¨nchen), Thomas Nicolai (Klinikum Innenstadt v. Haunersches Kinderspital, Mu¨nchen), Bjo¨rn Kumlien (Kinderklinik des Dritten Ordens, Mu¨nchen), Regina Beckmann (DietrichBonhoeffer-Klinikum, Neubrandenburg), Christiane Seitz (FEKFriedrich-Ebert-Krankenh. Neumu¨nster GmbH, Neumu¨nster), D. Hu¨seman (Ruppiner Kliniken GmbH, Neuruppin), Roland Schu¨rmann (Sta¨dt. Kliniken Neuss Lukaskrankenhaus GmbH, Neuss), Van Hop Ta (Evang. Krankenhaus, Oberhausen), Eckart Weikmann (Landeskrankenhaus Oberwart, Oberwart), W. Evert (Sta¨dt. Kliniken Offenbach, Offenbach), Ju¨rgen Hautz (Klinikum Offenburg, Offenburg), Ju¨rgen Seidenberg (Elisabeth-Kinderkrankenhaus, Oldenburg), Lucia Wocko (Krankenhaus Oranienburg, Oranienburg), Petra Luigs (St.Vincenz-Krankenhaus Frauen-u. Kinderklinik, Paderborn), Hans-Ludwig Reiter (Klinik fu¨r Kinder und Jugendliche am Sta¨dtischen Klinikum, Pforzheim), J. Quietzach (VogtlandKlinikum Plauen GmbH, Plauen), Michael Ko¨nig (Oberschwaben Klinik gGmbH, Ravensburg), Johanna Herrmann (Kreiskrankenhaus Rendsburg, Rendsburg), Horst Mitter (Krankenhaus der Barmh. Schwestern Ried, Ried i. Innkreis), Ekkehard Seidler (Kreiskrankenhaus Obergo¨ltzsch-Rodewisch, Rodewisch), Bernhard Maak (Thu¨ringen-Klinik ‘‘Georgius Agricola’’ GmbH, Saalfeld), Wolfgang Sperl (Landeskrankenanstalten Salzburg, Salzburg), Manfred Meissl (Landeskrankenhaus Scha¨rding, Scha¨rding), Reinhard Koch (Leopoldina-Krankenhaus, Schweinfurt), Manfred Cremer (DRK-Kinderklinik Siegen, Siegen), H.A. Breuer (Sta¨dt. Klinikum Solingen, Solingen), W. Go¨rke (Johanniter Kinderklinik, Stendal), Robert Nossal (Olga Hospital Stuttgart, Stuttgart), Walter Pernice (Kreiskrankenhaus Torgau, Torgau), Hans R. Salzer (Landeskrankenhaus Tulln, Tulln), Hartmut Koch (St. Marien-Hospital, Vechta), Gerhard Schaller (Landeskrankenhaus Villach, Villach), Franz Paky (Landeskrankenhaus Vo¨cklabruck, Vo¨cklabruck), Friedrich Straßer (Klinikum Weiden, Weiden), Franz Eitelberger (Krankenhaus d. Barmh. Schwestern v. heiligen Kreuz, Wels), D. Sontheimer (Harz-Klinikum Wernigerode GmbH, Wernigerode), Andreas Lischka (Wilhelminenspital Wien, Wien), Alfred Dilch (Gottfried von Preyersches Kinderspital der Stadt Wien, Wien), Christian Scheibenpflug (Donauspital im SMZ-Ost der Stadt Wien, Wien), Robert Bruckner (Krankenhaus Wiener Neustadt, Wiener Neustadt), Klaus Runge (Zentrum fu¨r Kinder- und Jugendmedizin, Wuppertal), Wolfgang Kunze (Krankenhaus Muldentalkreis Wurzen, Wurzen), Peter Schermann (Krankenhaus Zwettl, Zwettl).

patients with severe septic shock and the clinical syndrome of purpura fulminans [26,32]. Derangement of coagulation is seen in virtually all patients with meningococcal disease and septic shock and contributes significantly to the pathogenesis [21]. Concentrations of protein C, protein S and antithrombin III are low in plasma [11], the endothelial production of prostacyclin is impaired [14], thrombomodulin is down-regulated on the endothelial surface [18], while procoagulant molecules such as tissue factor are increased on monocytes [27]. This leads to deposition of fibrin with histologically demonstrable widespread microthromboses [10]. In addition, data have been published showing impairment of fibrinolysis [8], possibly due to endotoxin which induces up-regulation of tissue plasminogen activator and plasminogen-activator-inhibitor-1 (PAI-1) [5,25]. PAI-1 is a 50 kDa glycoprotein, belonging to the serpin protease family [19], and is synthesised and secreted by various cell types including endothelial cells, hepatocytes and platelets [22]. PAI-1 is the primary inhibitor of tissue and urinary type plasminogen activator, the major proteolytic activators of plasminogen [20]. In patients with meningococcal sepsis, concentrations of PAI-1 are extremely elevated and a significant correlation between PAI1 levels and mortality has been shown [13, 15, 30]. The human PAI-1 gene contains a common 4G/5G insertion/deletion polymorphism located within the promoter region, 675 base pairs upstream of the transcription start site. This polymorphism plays a role in the regulation of PAI-1gene expression [7,9]. In vitro experiments have shown that the 4G allele produces six times more PAI-1 mRNA than the 5G allele in response to interleukin 1b [7]. Plasma PAI-1 levels are the result of both environmental and genetic influences, and several studies have indicated that individuals with the 4G/4G genotype have PAI-1 levels approximately 25% higher than individuals carrying the 5G/5G polymorphism [6, 7,17]. Association studies on the PAI-1promoter polymorphism in patients with meningococcal disease have produced conflicting results. In British and Dutch children, Hermans et al. [13] showed that carriers of the 4G/4G genotype produce higher concentrations of PAI-1 and have a higher mortality. No influence of the 4G/5G polymorphism on the clinical manifestation of meningococcal disease (sepsis vs. meningitis) was described. These findings were confirmed in other patients by the same working group [12]. In contrast, in a cohort of Dutch children, Westendorp et al. [29] found no association of the 4G/4G genotype with mortality. However, the authors demonstrated a higher prevalence of the 4G/4G genotype in relatives of patients who developed septic shock compared to a higher frequency of the 5G/5G genotype in relatives of patients with meningitis [29] and concluded that the PAI-1 genotype influenced the clinical manifestation of meningococcal disease. To further elucidate the role of the 4G/5G PAI-1 polymorphism on the pathophysiology of meningococcal disease, we present data from patients included in a prospective multicentre study in Germany, Switzerland, Italy and Austria, complemented with a cohort of UK children.

Introduction Subjects and controls Fulminant meningococcaemia is a life-threatening disease with acute onset, septic shock, and progressive haemorrhagic necrosis of the skin [1]. Mortality rate is highest in

Patients with meningococcal disease ( n =347) were included from two cohorts.

488 Table 3 Mortality of meningococcal disease in children classified by PAI-1promoter genotype in the two patient cohorts

Central European Meningococcal Research (CEMR) cohort

4G/5G Genotype

Died

Total (n)

Between March 2000 and October 2002, blood samples and clinical data were collected from 220 patients from 95 paediatric hospitals in Germany, Switzerland, Southern Tyrol, and Austria (see co-investigators list). In addition, archived blood samples and clinical data from 31 patients from eight centres were included during the planning stage of the study before March 2000. The age at disease onset varied from 1 month to 27 years (median age 34 months); 126 patients were males and 125 females. A total of 246 samples from this cohort were successfully genotyped. Clinical information was collected from each patient according to a defined protocol (demographic data, diagnosis (sepsis, meningitis, or combined disease), Glasgow meningococcal septicaemia prognostic score, surgery and outcome). Meningococcal disease was diagnosed in all patients by the presence of typical clinical symptoms such as fever, purpuric rash and meningitis, septicaemia or both and either by isolation of meningococci from blood or CSF, detection of meningococcal antibodies, or positive PCR amplification of the meningococcal genome in blood or CSF. Patients were classified to have meningococcal meningitis if they showed a white blood cell count >10/ll in CSF and/or had evidence of meningism without the presence of sepsis. Meningococcal sepsis was diagnosed if patients had features of severe sepsis such as organ dysfunction [23], but presented with no evidence of meningism or an abnormal CSF. Patients with symptoms of both septicaemia and meningitis were classified as having combined disease.

Cord blood of healthy, unrelated newborns ( n =320) was collected between March 2002 and August 2002 at the Department of Neonatology of the Medical University of Graz and used for genotyping and determination of prevalences. For all subjects, blood samples were collected after parental consent. The study was approved by the ethics committees of all participating hospitals.

United Kingdom Meningococcal Research (UKMR) cohort

Genotyping

4G/4G 4G/5G or 5G/5G Total

CEMR cohort (n)

UKMR cohort ( n)

9/64 (14.1%) 13/182 (7.1%) 22/246 (8.9%)

3/26 (11.5%) 2/58 (3.4%) 5/84 (6.0%)

90 240 330

Table 4 Outcome of meningococcal disease in children classified by PAI-1promoter genotype

4G/5G Genotype

Outcome

4G/4G 4G/5G or 5G/5G Total

Total (n)

Fatal (n)

Non-fatal (n)

12 (44.4) 15 (55.6) 27

78 (25.7) 225(74.3) 303

90 240 330

Caucasian controls

Blood samples and clinical data from 96 patients who had been admitted to the Sheffield Children’s Hospital between March 1995 and January 2000 were enrolled in this cohort with the same disease definitions as for the CEMR patients. The age of disease onset varied from 3 months to 15 years (median age 40 months) with 54 males and 38 females. A total of 84 samples from this cohort were successfully genotyped.

EDTA blood samples obtained by non-traumatic venepuncture were collected and stored at 20C until further processing. DNA was isolated using standard procedures. For the detection of the insertion/deletion polymorphism, an allele specific PCR was used as described previously with minor modifications [16].

Table 1 PAI-14G/5G promoter genotype frequencies in the combined patients cohorts and Caucasian controls

Frequencies were compared by the v2 test. Calculations were performed with the SPSS statistic software package, version 10.0 (SPSS Inc., Chicago). A likelihood ratio v2 statistic was used to determine the Hardy-Weinberg equilibrium (HWE). v2 statistics are given as two-sided Fisher’s exact test for 2·2 tables or two-tailed Pearson’s likelihood

4G/5G genotype

Patients (n)

Controls (n)

4G/4G 4G/5G 5G/5G Total

90 (27.3%) 166 (50.3%) 74 (22.4%) 330

91 (28.8%) 149 (47.2%) 76 (24.1%) 316

Table 2 PAI-14G/5G promoter genotype frequencies in the two patients cohorts

4G/5G genotype

CEMR cohort (n)

UKMR cohort (n)

4G/4G 4G/5G 5G/5G Total

64 (26.0%) 132 (53.7%) 50 (20.3%) 246

26 (31.0%) 34 (40.5%) 24 (28.6%) 84

Statistical analysis

Table 5 Relationship between PAI-1genotype and clinical symptoms in the CEMR cohort

PAI-1genotype

4G/4G 4G/5G or 5G/5G Total a

Clinical symptom (n) Meningitisa

Sepsisa

51 (26.7%) 140 (73.3%) 191

45 (32.1%) 92 (67.9%) 137

All samples (n)

64 (26.0%) 182 (74.0%) 246

The specified clinical symptom is irrespective of the occurrence of other symptoms

489 Table 6 Relationship between PAI-1genotype and clinical diagnosis in the CEMR cohort

PAI-1genotype

4G/4G 4G/5G 5G/5G Total

Clinical diagnosis (n) Meningitis

Mixed

Sepsis

18 (19.1%) 57 (60.6%) 19 (20.2%) 94

33 (34.0%) 45 (46.4%) 19 (19.6%) 97

12 (30.0%) 22 (55.0%) 6 (15.0%) 40

ratio for other crosstables. Logistic regression was used to estimate an odds ratio to measure the size of effect of each genotype.

Results A total of 330 patients were genotyped. The frequencies of the 4G/4G, 5G/4G, and 5G/5G PAI-1genotypes did not differ significantly between the patient cohorts and the control population (P =0.7, Table 1). The frequencies of the PAI-1 4G/5G promoter genotypes in the Caucasian controls and both patient cohorts (Table 2) were in HWE and allele frequencies were similar to previously published results [7,13]. Comparison of patients collected within CEMR and UKMR cohorts indicated no statistical difference in mortality (Table 3) or allele frequencies. Thus both groups of patients were combined for severity analysis. In the combined cohort we observed a significantly higher proportion of deaths among patients with the 4G/ 4G genotype (12 of 90, 13%) than among those with the 4G/5G or 5G/5G genotypes (15 of 240, 6%; P =0.037), resulting in an odds ratio of 2.31 (95% CI 1.04–5.14, Table 4). Analysis of both groups individually showed the same trend, reaching significance when combined. Comparing patients diagnosed with sepsis to non-sepsis patients, in the CEMR cohort, the diagnosis of sepsis (independent of symptoms of meningitis) was significantly more frequent in carriers of the 4G/4G genotype (v2=6.7, P =0.01), resulting in an odds ratio to develop sepsis of 2.21 (95% CI 1.20–4.08) whereas meningitis (irrespective of septicaemia) was not associated with PAI-14G or 5G genotype (Table 5). When patients were classified according to clinical diagnosis (sepsis, mixed disease, and meningitis) in the CEMR cohort, genotype frequency did not vary significantly with respect to clinical diagnosis (P =0.2, Table 6). However, a trend for higher 4G/4G frequencies in the sepsis subgroups compared to the meningitis subgroup was observed.

Discussion We found an association between the 4G/4G promoter polymorphism of the PAI-1gene and mortality in meningococcal disease in a collection of German, Swiss, and Austrian Caucasian patients and a UK Caucasian patients cohort. The mortality risk for patients carrying the 4G/4G genotype was about twice as high as for the other genotypes. This finding fits nicely to the odds ratios found by Hermans et al. [12] (2.0) and Haralambous et al. [13] (1.9) in English and Dutch patients.

Although cohort sizes varied substantially, mortality rates, allele frequencies for patients and controls and impact of the 4G4G genotype on mortality are similar in these studies. Overall mortality rates largely depend on the ratio of sepsis versus pure meningitis cases. Mortality rates for the subgroups including sepsis symptoms (that is the sepsis and the mixed subgroups) are very similar in all studies (23% in the combined cohorts of Hermans et al. [12], 17% in Haralambous et al. [13] combined cohorts], 17% in the cohorts of this study). UK and Dutch patients come from regions with an established high risk of contracting meningococcal disease. Our study confirms the data in a combined cohort of Central European and UK patients, consisting of more patients from a population with a lower incidence of meningococcal disease. In addition, our data indicate that patients carrying the PAI-14G/4G genotype are at a higher risk to develop sepsis. The finding that a gene controlling the inhibition of fibrinolysis determines outcome suits perfectly the hypothesis that the pathophysiology of meningococcal sepsis is in part related to disseminated intravascular coagulation that leads to impaired organ perfusion and eventually to multiorgan failure [28,31]. This hypothesis is supported by observations that plasma concentrations of PAI-1 are significantly higher in patients with sepsis than with meningitis [13]. We did not find an increased risk to develop meningitis (as an isolated clinical symptom as well as in conjuction with septicaemia) associated with the PAI-14G/5G polymorphism. This is in accordance with the hypothesis that disseminated intravascular microthromboses do not play a major role in the development of bacterial meningitis [3,4]. The gene frequencies of the PAI-1promoter polymorphism in patients with meningococcal infection were similar to the frequencies observed in the general population. This is also in accordance with the observations made by others and suggests that this polymorphism is not involved in the predisposition to invasive meningococcal disease. A further interesting observation is that allele distributions and odds ratios for all clinical subgroups in our cohorts show an exact match to those reported by Hermans et al. [12] as well as Haralambous et al. [13]. These findings endorse the huge influence that genetics may play in the field of infectious diseases. The only adjunctive treatment for septic shock hitherto shown to be effective in reducing mortality rate is activated protein C [2]. This substance not only inhibits procoagulant pathways but also neutralises PAI-1 [24]. Thus our data provide a further explanation for the therapeutic efficacy of activated protein C in sepsis. Another therapeutic option to neutralise the PAI-1 is tissue plasminogen activator which is also able to dissolve intravascular clots. However, in a restrospective multicentre study in patients with severe meningococcal infection, a high risk of intracerebral bleedings has been described with the unrestricted systemic use of this substance [31,32]. In summary our study confirms evidence for an important link between regulation of coagulation and fibrinolysis and manifestation and outcome of meningococcal infection in a patient population with low prevalence for this disease. Acknowledgements This study was supported by the Jubila¨umsfond der O¨sterreichischen Nationalbank, grants 8842 and 10112.

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