Protein C and procollagen III peptide levels in patients with hepatic dysfunction after allogeneic hematopoietic stem cell transplantation

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1 (25) 6, & 25 Nture Pulishing Group All rights reserved /5 $. Protein C nd procollgen III peptide levels in ptients with heptic dysfunction fter llogeneic hemtopoietic stem cell trnsplnttion M Pihusch 1, H Wegner 2, P Goehring 1,2, C Slt 2, V Pihusch 2, R Andreesen 1, H-J Kol 2, EHoller 1,2 nd R Pihusch 2 1 Ateilung fuer Hemtologie und Internistische Onkologie, Klinikum der Universitet Regensurg, Regensurg, Germny; nd 2 Medizinische Klinik III Grosshdern, Klinikum der Ludwig Mximilins-Universitet Muenchen, Muenchen, Germny Summry: Veno-occlusive disese (VOD) is one of the most serious complictions following hemtopoietic stem cell trnsplnttion (HSCT) nd is ssocited with high mortlity. We conducted lrge tril on the clinicl significnce of protein C (PC) nd procollgen III peptide (PNPIII) levels, which hve een descried s possile dignostic mrkers of VOD. In totl, 5 ptients undergoing llogeneic HSCT were included. PC nd PNPIII levels were nlyzed prior to conditioning nd weekly until 8 weeks fter the HSCT. Signs of VOD nd other trnsplnttion-relted complictions (grft-versushost disese (GVHD), toxicity, microngiopthic hemolytic nemi, infection) were recorded weekly throughout the tril. Ptients showed significnt drop of the PC levels in VOD (7. vs 96.%, Po.1) nd with incresing severity of GVHD. Steroids incresed the PC levels (69.% vs 19.%, Po.1). The highest PNPIII levels were registered in ptients with VOD (men 6. IU/ ml). Ptients with GVHD showed n elevtion of PNPIII, especilly ptients with heptic GVHD. PC levels during conditioning do not predict VOD (98.5 vs 76.5%, NS). Although PC nd PNPIII my ply role in the pthogenesis of VOD they cnnot discriminte etween complictions with jundice nd re only of limited help in the differentil dignosis of VOD. (25) 6, doi:1.18/sj.mt.17511; pulished online 1 August 25 Keywords: hemtopoietic stem cell trnsplnttion; protein C; procollgen III peptide; veno-occlusive disese Heptic veno-occlusive disese (VOD) is recognized s one of the most serious complictions fter llogeneic hemtopoietic stem cell trnsplnttion (HSCT). 1 This sinusoidl Correspondence: Dr med. M Pihusch, Ateilung fuer Hemtologie und Internistische Onkologie, Klinikum der Universitet Regensurg, Frnz- Josef-Struss-Allee 11, 95 Regensurg, Germny. E-mil: Mrkus.Pihusch@t-online.de Received 21 Mrch 25; ccepted 21 June 25; pulished online 1 August 25 occlusion syndrome is chrcterized y pinful heptomegly, jundice, scites, fluid retention, nd weight gin. Severe VOD is oserved in out 5% of the recipients 2 nd complicted y portl hypertension, heptorenl syndrome, nd multisystem orgn filure. It remins ftl in the vst mjority of the cses. VOD is elieved to e cused y toxic injury of the sinusoidl endothelil cells nd heptocytes with susequent dmge of centrl veins in zone of the heptic cinus. The endothelil cell dmge my led to exposure of tissue fctor nd deficiencies in the nturl nticogulnts. 5 Chnges in plsm PC levels re mostly relted to redistiution into edem nd scites fluid. The serum minopropeptide of type III procollgen (PNPIII) my e sign of enhnced heptic collgen formtion. 6 The onset of VOD is usully oserved within 1 month fter the HSCT. In this period conditioning effects, infection, cute grft-versus-host disese (GVHD), or microngiopthic hemolytic nemi (MAHA) my mimic the syndrome. Nevertheless prompt detection of VOD, often sed on clinicl criteri, is essentil, since erly ntithromotic therpy represents the only promising tool so fr. 7 Numerous serum nd plsm proteins hve een reported to e norml in ptients with VOD, including endothelil cell mrkers nd cogultion proteins. The plsm PC (protein C) 8 nd the serum PNPIII 9,6 hve een reported s predictive lortory prmeters for the dignosis of VOD. As there is considerle controversy out the clinicl significnce of PC nd PNPIII we conducted lrge tril in llogeneic stem cell recipients to vlidte the dignostic relevnce of these prmeters llowing discrimintion etween this syndrome nd other forms of liver injury following HSCT. Ptients nd methods Study popultion Between Jnury 1998 nd Octoer 21, 5 ptients 12 yers of ge nd older receiving llogeneic hemtopoietic stem cell grfts were included in this prospective nlysis. Ptients were dmitted for vriety of hemtologicl disorders t vrious stges of their disese. A totl of 71 weeks were documented with medin oservtion period of 8 weeks (rnge 2 151). The seline demogrphic of the

2 62 ptients nd the trnsplnt chrcteristics re summrized in Tle 1. Signed informed consent ws otined from ll ptients or their legl representtives llowing nlysis of ll clinicl nd lortory dt mentioned in this pper. Tril design nd procedures This ws single-center (José-Crrers Trnsplnttion Unit, University of Munich, Germny), prospective study evluting the specifity nd sensitivity of PC plsm levels nd PNPIII serum levels in ptients with VOD nd other TRC with jundice following HSCT. No exclusion criteri were defined. Trnsplnttion course Once week t the timepoint of PC nd PNPIII mesurements the clinicl sitution ws documented. Uncomplicted HSCT ws defined s the sence of orgn dysfunction (defined s iliruin 2. mg/dl, cretinine 1.5 mg/dl, PCO 2 o2 mmhg, PCO 2 5 mmhg, systolic lood pressure o9 mmhg), no ppernce of TRC (defined s: GVHD (grde I), VOD, MAHA, infection, regimen relted toxicity), nd no need of plsm exchnge (defined s plsmpheresis with ppliction of fresh frozen plsm, dilysis, nd hemofiltrtion). Grft-versus-host disese prophylxis ws given s shown in Tle 1. Acute grft-versus-host disese (GVHD) ws grded y clinicl nd iochemicl criteri 1 nd confirmed y iopsy in minority of ptients. GVHD ptients with GVHD (grde I) received first line stndrd therpy with prednisolone (mximum mg/kg/dy). Corticosteroid tretment ws reduced (1% of dosge every dys) s soon s GVHD ws controlled. Chronic grft-versus-host disese (cgvhd) ws grded y clinicopthologicl spects. 11 Heptic VOD ws dignosed nd clssified y the clinicl syndrome of liver enlrgement nd pin, fluid retention nd weight gin, nd elevted serum iluruin concentrtion 2 mg/dl. 2 The dignosis ws confirmed y ultrsound, Doppler ultrsound, nd trnsjugulr liver iopsy in ll cses. Ptient chrcteristics re given in Tle 1. MAHA ws defined s the simultneous occurrence of intrvsculr hemolysis chrcterized y the ppernce of 5% schistocytes in peripherl lood smers, de novo thromocytopeni, nd elevtion of LDH levels. 12,1 Only moderte or severe cses of MAHA requiring therpeutic intervention (ppliction of fresh frozen plsm or therpeutic plsmpheresis) were registered. Infectious complictions were defined y clinicl nd lortory prmeters (fever: 8.51C irrespective of underlying microorgnisms; sepsis: fever plus orgn dysfunction s indicted y disseminted intrvsculr cogultion (DIC), positive lctte, new renl insufficiency, or PCO 2 o2 mmhg, no need of vsopressors; septic shock: sepsis nd need of vsopressors). Blood smpling nd ssessments Prior to conditioning nd once week lood smples were otined on the morning vi the centrl vein ctheter. Tle 1 Epidemiologic nd trnsplnttion chrcteristics of the ptients studied Ptients Totl 5 Sex Mle 26 (58.9%) Femle 1 (1.1%) Age (yers (medin (rnge))) (1 65) Underlying disese Chronic myeloid leukemi 118 (.7%) Acute myeloid leukemi 95 (27.1%) Non-Hodgkin s lymphom 6 (17.1%) Acute lympholstic leukemi 6 (1.%) Myelodysplstic syndrome 16 (.6%) Severe plstic nemi 1 (2.9%) Hodgkin s disese 5 (1.%) Others 1 (2.9%) Stem cell source Relted donor 177 (5.6%) Unrelted donor 17 (9.%) Bone mrrow 21 (66%) Peripherl lood 119 (%) Conditioning regimen Full intensity 26 (67.%) Reduced intensity 11 (2.6%) Busulfn contining regimen 6 (18.%) Conventionl chemotherpy 17 (.9%) GVHD prophylxis CyA-MTX 222 (6.%) CyA-MMF 128 (6.6%) Antithymocyte gloulin 291 (8.1%) Cse numers (percentges) re given. Full intensity conditioning (FIC) ¼ conditioning regimen contining dose of 12 Gy ( Gy for dys, dose rte.5 cgy/min) totl ody irrdition or conditioning regimen contining usulfn (orlly dministered mg/kg BW for dys) comined with intrvenous cyclophosphmide ( mg/kg for 2 dys); reduced intensity conditioning (RIC) ¼ conditioning regimen contining intrvenous fludrine ( mg/m 2 for dys) or intrvenous fludrine ( mg/m 2 for dys) in prllel with intrvenous mscrine (1 mg/m 2 for dys) nd intrvenous cytrine (2 mg/m 2 for dys) comined with totl ody irrdition (2 Gy for 1 dy, dose rte.5 cgy/min), nd intrvenous cyclophosphmide ( mg/kg for 2 dys); conventionl chemotherpy ¼ Second stem cell infusion (sme donor) fter conventionl chemotherpy ecuse of relpse or engrftment rejection (cytrine; fludrine-mscrinecytrine; melphlne; ifosfmide-cyclophosphmide-etoposide; fludrinecytrine-idruicine; cytrine-mscrine; BCNU-etoposide-cytrinemelphlne; cyclophosphmide-vincristine-leomycin); ntithymocyte gloulin ¼ intrvenous ATG (1 mg/kg for 5 dys); CyA ¼ continuous infusion of cyclosporine from dy 1 untildy28(1mg/kgcyaondy 1; 5 mg/kg CyAfromdyuntildy;mg/kgCyAfromdy5untildy28)followedy orlly dministered cyclosporine from dy 28 (2 mg/kg CyA; plsm levels 15 to 2 ng/ml in the first months fter HSCT with grdul tpering); MTX ¼ intrvenous methotrexte (15 mg/m 2 on dy 1, 1 mg/m 2 on dy nd 6); MMF ¼ intrvenous mycophenolte mofetil ( 5 mg from dy ). Citrted lood smples (. ml Primvette S s cogultion tues, Ke Lortechnik, Nuemrecht-Elsenroth, Germny;.8% sodium citrte 9:1) were centrifuged fter collection (. g t room temperture for 15 minutes) for complete pltelet removl. Serum tues (1 ml; Ke Lortechnik, Nuemrecht-Elsenroth, Germny) were used for nlyses of serum prmeters. EDTA tues

3 ( ml; Ke Lortechnik, Nuemrecht-Elsenroth, Germny) were otined for routine lood count. All smples were processed loclly (Institute of Clinicl Chemistry, University of Munich, Germny). Dily vlidtion of the interssy precision showed stndrd devition of less thn 5% throughout the study period given y the mnufcturer. Pltelet count (norml rnge: /l) ws nlyzed using H6 nlyser (Coulter, USA). Liver function ws detected y totl iliruin (DPD Method, Roche, norml rnge: o1. mg/dl). PC plsm ctivity (Chromogenic test; Berichrom PC Dde Behring; Germny; norml rnge: 7 1%) nd procollgen III peptide serum levels (Prokollgen III-Peptid RIA; Schering; Germny; norml rnge:..8 U/ml) were determined y commercil tests. Aliquots were frozen nd stored t 71C until ssyed. Concomitnt mediction Concomitnt mediction, plsm exchnge nd trnsfused lood products (erythrocytes, pltelets, fresh frozen plsm) were noted weekly. All ptients received intrvenous heprin (1 IE/h) prophylcticlly during the whole trnsplnttion period nd full prenterl nutrition from dy 1 until engrftment. Nutrients contined ll lipophilic nd hydrophilic vitmins nd trce elements. Gut decontmintion ws performed using orl nysttine, colistin, nd mphotericin B. Herpes simplex virus prophylxis ws conducted y intrveneous dminstrtion of ciclovir (1 mg/kg BW t.i.d.). Antiiotic nd ntimycotic prophylxis ws given s indicted. Sttisticl nlysis Bsed on the Jones criteri nd on recent retrospective dt 1 with the ssumption tht the expected proportion of ptients with VOD recruited to the study ws %, smple size of t lest ptients ws plnned to chieve numer of t lest 1 VOD ptients. Dt re given s percentges or s mens nd 95% confidence intervls for continuous vriles if not otherwise indicted. Ctegoril nd ordinl vriles were given s medin nd rnge. Differences mong the groups were nlyzed y the Mnn Whitney U test for ordinl dt, nd y Person s w 2 test for ctegoril vriles. Continuous vriles were compred with the Student t test if they followed norml distriution or with the Wilcoxon rnk test if they followed skewed distriution. All sttisticl tests were two-tiled, P-vlues elow.5 eing regrded s significnt. Sttisticl nlyses were performed with the Sttisticl Pckge for the Socil Sciences (SPSS for Windows 1., SPSS Inc., Chicgo, IL, USA). Results Uncomplicted HSCT There ws no difference etween full intensity conditioning (FIC) regimens (see Tle 1) nd reduced intensity conditioning (RIC) regimens (see Tle 1) regrding the PC nd PNPIII serum levels, nd the routine lortory prmeters. Protein C: During the conditioning phse PC plsm levels temporrily dropped (see Figure 1). Sunlysis revels tht totl ody irrdition (TBI) hd no impct on the PC plsm levels (dt not shown). Also conditioning regimens with or without usulfn did not impir the PC plsm levels (usulfn: 97.8 vs 98.5%, NS; non usulfn: 98. vs 98.5%, NS). However, ATG lowered the PC levels significntly (86 vs 98%; Po.5), independently from the previous conditioning regimen. During the further course of uncomplicted trnsplnttion the lowest PC plsm levels within the norml rnge were chieved during plsi. After engrftment PC levels recovered (see Figure 1). Procollgen III peptide: Serum levels of PNPIII were elevted efore HSCT, incresed continully nd reched the highest levels weeks fter the HSCT (see Figure 1). The different conditioning regimens, the ATG prophylxis, nd TBI did not impct on the PNPIII levels (dt not shown). Trnsplnt relted complictions The incidence of the different TRC is shown in Tle 2. Ptients, who hd TRC hd lower PC levels until week 2 fter HSCT nd showed higher PC levels in week nd compred to ptients with uncomplicted HSCT (dt not shown). Ptients with ATG prophylxis hd significntly more TRC (infection, GVHD, MAHA, VOD) compred to ptients without ATG prophylxis (71.2 vs 65.2%, P ¼.8). The PNPIII serum levels in ptients with VOD nd GVHD re presented in Figure 2 nd. Veno-occlusive disese: Ptients with VOD hd n medin onset of this TRC t dy 28 (1 12). Ptients with VOD showed significnt lower PC levels compred to ptients without ny TRC (7. vs 96.%, Po.1). The PC plsm levels efore stem cell trnsfusion did not differ in ptients who developed lter VOD from those ptients who hd no lter VOD (week 2: 98.5 vs 76.5, NS; week 1: 8. vs 82.6%, NS; week : 89. vs 79.9%, NS). The highest PNPIII levels re registered in ptients with VOD compred to ptients with other complictions (dt not shown). In ptients with PNPIII serum levels ove 2.2 IU/ml the sensitivity for VOD ws 1%, the specifity for VOD 8.5%. Infection, MAHA, nd GVHD: The PC levels decresed inversely proportionlly to the severity of infection (dt not shown). Ptients with MAHA hd lowered PC plsm levels within the norml rnge compred to ptients without ny TRC (68.6 vs 96.%, Po.1). Ptients showed significnt drop of the PC levels with incresing severity of GVHD independent from the orgn mnifesttion efore ppliction of steroids (dt not shown). Strting steroids ecuse of GVHD incresed the PC plsm levels significntly (68 vs 18%; Po.5). The PNPIII levels strongly correlted to the severity of infection independent from the leukocyte count (see Figure ). An GVHD led to n elevtion of 6

4 6 PNPIII levels depending on the grde of the heptic GVHD, independently from the use of steroids (see Figure ). Tle 2 Trnsplnttion relted complictions Ptients Procollgen-III-peptide[IU/ml] N = * * * * * Grft-versus-host disese Acute 19 (58.1%) Chronic 2 (7.%) Infection 196 (56.%) Fever 1 (1.1%) Sepsis (9.2%) Septic shock 22 (6.7%) Hemosttic complictions Microngiopthy 57 (17.5%) Venoocclusive disese 15 (.%) Deth 16 (6.6%) Infection 85 (2.%) Relpse 17 (5.2%) GVHD nd infection 17 (5.2%) GVHD 1 (.%) Veno-occlusive disese 1 (.%) Bleeding 5 (1.5%) Others 11 (.1%) 11 * * * Numers give cses (percentge). 1 Protein C [%] c N = * * * * Procollgen-III-peptide [IU/ml] Biliruin [mg/dl] N = HSCT-week 1 Figure 1 ( c) Plsm levels of PNPIII, PC, nd iliruin in ptients with uncomplicted stem cell trnsplnttion course. Mrks indicte men vlues, the rs show the 95% CI. Gry ckground shows the norml rnge of PNPIII, PC, nd iliruin. N ¼ oservtion weeks. *Po.5 compred to the initil vlue efore conditioning (week 2). is defined s the week immeditely fter HSCT. 6 Procollgen-III-peptide [IU/ml] Figure 2 ( ) Procollgen-III-levels in ptients with VOD () nd GVHD I IV () Gry ckground shows the norml rnge of PNPIIIlevels.

5 PNPIII serum levels showed wide overlp in ll trnsplnt-relted complictions with jundice (see Figure c). Procollgen-III-peptide[U/ml] Procollgen-III-peptide[U/ml] c Procollgen-III-peptide [U/ml] N = 16 No Compliction N = * * 121 Fever >8.5 C 1 Sepsis 5 Septic shock Grde of cute heptic GVHD 1 C N = Cond. GVHD MAHA infection VOD Toxic * * * Discussion The clinicl dignosis of VOD is difficult nd sed on the clinicl trid of heptomegly, weight gin, nd jundice. 2, However, mny cuses of jundice nd liver dysfunction such s GVHD, MAHA, toxic effects, or infection cn mimic the symptoms. Nevertheless, prediction of VOD in high-risk ptients would e helpful, since erly nd correct therpeutic decisions re crucil. 7 Confirmtion of the dignosis y liver iopsy is cumersome nd poses significnt risk of leeding in the thromocytopenic ptient, prt from possile flsely negtive results. Ultrsound findings re not sufficiently sensitive in the erly phse. 15 Also, the trnsvenous wedged heptic venous pressure mesurements re highly specific ut only modertely sensitive for VOD. 16 Numerous plsm nd serum proteins hve een reported to e norml in VOD ptients including cogultion proteins nd procollgen peptides. 17 In mny retrospective nlyses the nturl nticogulnt PC, 8,5 synthesized nd relesed y heptocytes, nd the serum minopropeptide of type III procollgen, ccumulting in enhnced collgen formtion, were nlyzed to figure out whether they were useful mrkers of heptic VOD fter HSCT. 9,18 Results were controversil though. While some uthors found PC to e useful mrker to predict VOD 19 or confirm its dignosis, 2,5 other studies stted tht this hemosttic prmeter is not helpful to identify VOD ptients. 17 PNP III ws lso shown to predict nd dignose VOD fter HSCT y some uthors 21,6,22 while others did not find significnt chnges of the propeptide of type III procollgen. 17 On the other hnd, there re dt from niml models showing n initition of sinusoidl ostructive syndrome y chnges in sinusoidl endothelil cells followed y emolistion of the siudoid y sinusoidl lining cells. Altertions of hemosttic prmeters re suggested to e n epiphenomenon in this cse. 2 As there is considerle controversy out the dignostic utility of these mrkers we conducted lrge prospective tril to vlidte the sensitvity nd specifity of PC nd PNPIII levels in the dignosis of VOD following HSCT. Our study shows tht low PC levels during conditioning do not predict VOD. Prophylxis with ATG decresed the PC levels. The PNPIII levels continully incresed during the post tnsplnttion course. The highest PNPIII serum Figure ( c) Procollgen-III-levels in trnsplnttion-relted complictions. N ¼ oservtion weeks. Grey ckground shows the norml rnge of PNPIII-levels. ( nd ) Mrks indicte men vlues of PNPIII, the rs show the 95% CI. *Po.5 significnt s compred to cses without infection or GVHD. Only cses with one single trnsplnt-relted compliction re nlyzed. N ¼ oservtion weeks. (c) Crosses represent the mximum PNPIII-levels of ech single ptient with iliruin levels ove 2. mg/dl. Mrks indicte the men of the mximum PNPIII levels. Cond.: jundice only during conditioning period. GVHD: Jundice only during GVHD I. MAHA: Jundice only during microngiopthy. Infection: jundice only during fever, sepsis, nd septic shock irrespective of underlying microorgnisms. VOD: jundice only during episodes of VOD. Toxic: jundice only cused y drugs (cyclosporin, ntiiotics). N ¼ ptients. 65

6 66 levels were registered in ptients with VOD compred to other TRC. However, PNPIII levels re only of limited help for the differentil dignosis of TRC with hyperiliruinemi. Activted PC plys pivotl role in the inhiition of monocytes nd mcrophges 2 with consecutive inhiition of inflmmtory meditors nd of the cogultion cscde. We could not prove predictive vlue of this nturl nticogulnt for occurrence of VOD. In our lrge tril we oserved low PC levels during conditioning. However, this is rther due to the ATG-induced DIC, reflected y incresing D-dimer levels, low pltelets, nd consumption of ntithromin. 25 A further drop in the PC levels ws registered during the first week fter HSCT. As PC is minly synthesized y heptocytes, this might e indictive for n impired liver function fter conditioning or for disppernce into scites or edem fluid, which is seen in other TRC different from VOD, nd is thus not predictive for lter VOD. Our dt indicte incresing PNPIII levels during HSCT. Elevted PNPIII serum levels re descried in multitude of disorders ccompnied y relevnt liver firosis, 26 s seen in liver iopsies of VOD ptients. Firous ltertions in the heptic sinusoids occur during the development of VOD. These chnges re ccompnied y the deposition of type III collgen in the liver tissue. 6 VOD ptients nd ptients with severe heptic GVHD hd the highest levels of PNP III in our tril. Both TRC re ccompnied with significnt firous ltertions nd heptic remodelling processes. In some smller trils serum concentrtes of PIIINP seemed to e of vlue s n erly mrker for VOD 6,18 while in some other studies they did not. 17 PNPIII, precursor peptide of type III collgen, ccumultes in enhnced collgen formtion s possile in VOD nd severe GVHD of the liver. A rod overlp of the PNPIII levels ws seen in ll TRC with hyperiliruinemi. PNPIII is therefore only of very limited help for the differentil dignosis of VOD. However, the smll smple size of 15 ptients with VOD nd the heterogeneity of the studied popultion in terms of underlying disese, conditioning regimens, or dditionl mediction might e responsle for the lck of significnce in this context. Incresing iliruin levels during HSCT, cused y toxic drugs, might e one reson for the elevtion of PNPIII levels, s PNPIII is cleved y the liver. 27 In ddition, tuulr dmge 28 y immunsuppressive drugs such s cyclosporine, lso results in n increse of PNPIII, which is normlly secreted vi the kidneys. 28 On the other hnd, sepsis nd MAHA, which ffect the renl nd heptic clernce function lso led to high serum levels of PNPIII in our tril. The controversy of the dt presented y different uthors might on the on the hnd e due to smll numers of ptients included in the studies. Therefore, we conducted lrge tril with more thn stem cell recipients included over severl yers. On the other hnd the incidence of VOD fter llogeneic HSCT differs lot etween the different studies. Most of the uthors did not use the strict clinicl criteri ccording to Jones et l. 2 This is why mny of their ptients ctully did not meet ll VOD criteri ut were identified s VOD ptients. Correspondingly the PNPIII levels were rther elevted in other TRC with hyperiliruinemi thn in VOD. According to ig retrospective tril of our group 15 nd dt from groups using the criteri ccording to Jones et l 2 we gin could prove low incidence of VOD with very high mortlity. Conclusion PC levels decrese during the development of VOD fter HSCT. PNPIII plys pivotl role in firous ltertions in heptic TRC. However, we conclude from this first lrge prospective study tht PC plsm levels nd PNPIII serum levels re only of limited help in the differentil dignosis of VOD. Acknowledgements This work is prt of the doctorl thesis of Holger Wegner. References 1 Wdleigh M, Ho V, Momtz P, Richrdson P. 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