Birkelund, Svend

Carlsen, Thomas Gelsing

Christiansen, Gunna

Deleuran, Bent

Hjelholt, Astrid

Jurik, Anne Grethe

Schiøttz-Christensen, Berit

English

    Aalborg UniversitetIncreased levels of IgG antibodies aganist human HSP60 in patients withspondyloarthritisHjelholt, Astrid; Carlsen, Thomas Gelsing; Deleuran, Bent; Jurik, Anne Grethe; Schiøttz-Christensen, Berit; Christiansen, Gunna; Birkelund, SvendPublication date:2013Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Hjelholt, A., Carlsen, T. G., Deleuran, B., Jurik, A. G., Schiøttz-Christensen, B., Christiansen, G., & Birkelund, S.(2013). Increased levels of IgG antibodies aganist human HSP60 in patients with spondyloarthritis. Posterpresented at Interdisciplinary Symposium Inflammation at Interfaces, Hamburg, Germany.http://www.nature.com/natureevents/science/events/17091-Interdisciplinary_Inflammation_at_Interfaces_SymposiumGeneral rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: December 26, 2020Increased	  Levels	  of	  IgG	  An2bodies	  against	  Human	  	  HSP60	  in	  Pa2ents	  with	  Spondyloarthri2s	  I	  n	  t	  r	  o	  d	  u	  c	  t	  i	  o	  n	   C	  o	  n	  c	  l	  u	  s	  i	  o	  n	  s	  ①  A	   sta&s&cal	   significant	   associa&on	   between	   the	  SpA	   group	   and	   IgG1	   &	   IgG3	   an&bodies	   against	  human	  HSP60	  (Figure	  1D,	  p<0.001).	  ②  Cross-­‐reac&vity	   to	  bacterial	  HSP60	   could	  not	  be	  supported.	  ③  IgG3	  an&bodies	  against	  human	  HSP60	  correlated	  to	   disease	   ac&vity	   for	   HLA-­‐B27+	   pa&ents	  (Spearman,	  r=0.48,	  p=0.001).	  ④  These	   results	   suggest	   that	   an&bodies	   against	  human	  HSP60	   in	   connec&on	  with	  HLA-­‐B27	  may	  be	  associated	  with	  SpA.	  	  	  R	  e	  s	  u	  l	  t	  s	  M	  e	  t	  h	  o	  d	  s	  Spondy l o a r t h r i & s	   ( S pA )	   comp r i s e s	   a	  heterogeneous	   group	   of	   inflammatory	   diseases,	  with	   strong	   associa&on	   to	   human	   leukocyte	  an&gen	  (HLA)-­‐B27.	  SpA	  is	  suggested	  triggered	  by	  bacterial	   infec&on,	   and	   bacterial	   heat	   shock	  protein	  (HSP)	  seems	  to	  be	  a	  strong	  T	  cell	  an&gen.	  Since	   bacterial	   and	   human	   HSP60,	   also	   named	  HSPD1,	   are	   highly	   homologous,	   cross-­‐reac&vity	  has	   been	   suggested	   in	   disease	   ini&a&on.	   In	   this	  study,	   levels	   of	   an&bodies	   against	   bacterial	   and	  human	  HSP60	  were	  analysed	  in	  SpA	  pa&ents	  and	  healthy	   controls,	   and	   the	   associa&on	   between	  such	   an&bodies	   and	   disease	   severity	   in	   rela&on	  to	  HLA-­‐B27	  was	  evaluated.	  tc@hst.aau.dk	  Astrid	  Hjelholt1,	  Thomas	  Carlsen2,	  Bent	  Deleuran1,3,	  Anne	  Grethe	  Jurik4,	  Berit	  Schiø\z-­‐Christensen5,	  Gunna	  Chris&ansen1,6,	  Svend	  Birkelund2,6	  1)Department	  of	  Biomedicin	  –	  Medical	  Microbiology	  and	  Immunology,	  Aarhus	  University;	  2)Department	  of	  Health	  Science	  and	  Technology,	  Aalborg	  University;	  3)Department	  of	  Rheumatology,	  Aarhus	  University	  Hospital;	  4)Department	  of	  Radiology,	  Aarhus	  University	  Hospital;	  5)Aarhus	  Clinic	  for	  Rheuma&c	  Diseases;	  6)Loke	  Diagnos&cs,	  Risskov,	  Denmark.	  Serum	   samples	   from	   82	   pa&ents	   and	   50	  controls	   were	   analysed	   by	   Enzyme-­‐linked	  immunoso rben t	   a s s ay	   ( E L I SA )	   fo r	  immunoglobulin	  (Ig)G1,	   IgG2,	   IgG3	  and	  IgG4	  an&bodies	  against	  human	  HSP60	  and	  HSP60	  from	   Chlamydia	   trachoma.s,	   Salmonella	   e.	  Enteri&dis	  and	  Campylobacter	  jejuni.	  Disease	  severity	  was	  assessed	  by	  the	  clinical	  scorings	  Bath	   Ankylosing	   Spondyli&s	   Disease	   Ac&vity	  Index	   (BASDAI),	   Bath	   Ankylosing	   Spondyli&s	  Func&onal	  Index	  (BASFI)	  and	  Bath	  Ankylosing	  Spondyli&s	  Metrology	  Index	  (BASMI).	  	  Pa2ents	  Serum	   samples	   was	   collected	   from	   the	  outpa&ent	   clinic	   at	   Aarhus	   University	   Hospital	  afer	   informed	   wri\en	   consent	   was	   given,	  according	  to	  the	  Danish	  Data	  Protec&on	  Agency,	  the	   Local	   Ethics	   Commi\ee	   (project	   number	  20050046)	  and	  the	  Declara&on	  of	  Helsinki.	  	  References	  SpA group (IgG1)Control group (IgG1)SpA group (IgG3)Control group (IgG3)0204060IgG concentration (!g/mL)SpA group (IgG1)Control group (IgG1)SpA group (IgG3)Control group (IgG3)0204060IgG concentration (!g/mL)SpA group (IgG1)Control group (IgG1)SpA group (IgG3)Control group (IgG3)0204060IgG concentration (!g/mL)   A     C. trachomatis HSP60                   B                 C. jejuni HSP60    C            S. enteritidis HSP60             D                 Human HSP60  p<0.001 p<0.001SpA group (IgG1)Control group (IgG1)SpA group (IgG3)Control group (IgG3)0204060IgG concentration (!g/mL)Figure	  3.	  An2body	  levels	  in	  the	  SpA	  &	  Control	  group	  1.	  Oldstone	  MB:	  Molecular	  mimicry	  and	  immune-­‐mediated	  diseases.	  The	  FASEB	  Journal	  1998,	  12:1255-­‐1265	  2.	  McMichael	  A,	  Bowness	  P:	  HLA-­‐B27:	  natural	  func&on	  and	  pathogenic	  role	  in	  spondyloarthri&s.	  Arthri.s	  Res	  2002,	  4	  Suppl	  3:	  153-­‐158.	  3.	  Inman	  RD:	  Mechanisms	  of	  disease:	  Infec&on	  and	  spondyloarthri&s.	  Nat	  Clin	  Rheumatol	  2006,	  2:163-­‐169	  Table	  1.	  Comparison	  of	  IgG1	  &	  IgG3	  an2bodies	  against	  human	  &	  bacterial	  HSP60	  Median (IQR) IgG1 IgG3 Statistical result of the Mann Whitney U test (p-value) Human HSP60 2.91 (1.71;6.53) 3.38 (1.31;21.42) P=0.53 C. trachomatis HSP60 5.50 (3.07;10.81) 1.52 (1.07;3.08) p0.0001 C. jejuni HSP60 2.14 (1.64;3.22) 1.64 (1.41;2.08) P=0.0001 S. enteritidis HSP60 3.22 (2.00;6.03) 1.55 (1.12;2.31) p0.0001 Billede	  y 4.0977xx 1,  d 0.080507,  r 0.995071234&RQFHQWUDWLRQ>ȝJP/@1 2 3 4IgG1 standard: AQPFigure	  2.	  Standard	  curve	  	  Mark 12Human HSP60 C. trachomatis HSP60S. Enteritidis HSP60C. jejuni HSP60200 kDa116,3 kDa 97,4 kDa66,3 kDa55,4 kDa 36,5 kDa÷ IPGT÷ IPGT÷ IPGT÷ IPGT+ IPGT+ IPGT+ IPGT+ IPGTA     B     C     D      E     F       G      H       I      J     K        L      M** p<0.001; * p<0.01Total SpA group                    HLA-B27                    HLA-B27BASDAIBASFIBASMI  IgG1           IgG3                  IgG1          IgG3          IgG1          IgG30.13-0.030.160.050.010.34*0.180.080.050.000.48*0.05-0.27-0.130.02-0.130.050.29+ -Figure	  1.	  SDS	  PAGE	  Size	  and	  purity	  of	  each	  recombinant	  protein	  were	  evaluated	  by	  10%	  SDS-­‐PAGE	  gel.	  	  The	   different	   HSP60	   proteins	   for	   ELISA	   were	   purified	   using	   a	   Ni+	   column	   and	  imidazole	   as	   a	   compe&ng	   reagent.	   Vector	   transforma&on	  was	   confirmed	  by	   IPGT	  induc&on.	  Lane	  A:	  Molecular	  weight	  marker.	  Lane	  B,	  E,	  H	  &	  K:	  purified	  HSP60.	  Lane	  C+D,	  F+G,	   I+J	  &	  L+M:	  Total	  protein	  content	   from	  E.	  coli	   strain	  BL21	  (DE3)	  with	  (+)	  and	  without	  (-­‐)	  IPGT	  induc&on.	  A	  standard	  curve	  made	  with	  Magellan	  data	  sofware	  used	  to	  quan&fy	  the	  A450nm	  values	  from	  ELISA.	  An	  ELISA	  plate	  was	  coated	  with	  a	  known	  serial	  dilu&on	  (0.0625	  -­‐	  4	  µg/mL)	  of	  na&ve	  IgG1	  subclass	  from	  human	  myeloma	  plasma	  before	  incuba&ng	  wells	  with	  HRP-­‐conjugated	  secondary	  an&body.	  Correla&on	  between	  A450nm	  values	  and	  concentra&on	  was	  fi\ed	  by	  the	  regression	  output	  (r	  =	  0.99507).	  Table	  2.	  Spearman	  Rank	  Correla2ons	  coefficient	  (r)	  for	  the	  correa2on	  between	  an2-­‐human	  	  HSP60	  IgG1	  &	  IgG3	  and	  BASDAI,	  BASFI	  &	  BASMI	  

Increased levels of IgG antibodies aganist human HSP60 in patients with spondyloarthritis

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Increased levels of IgG antibodies aganist human HSP60 in patients with spondyloarthritis

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