1

11.. FFRRAAMMEEWWOORRKK OOFF TTHHEE CCLLUUSSTTEERR

Taking into consideration the high sociomedical and socioeconomic importance of thediseases of the musculoskeletal system, the Cluster for Rheumatology, Balneology andRehabilitation was established by the Ludwig Boltzmann Society in 2007. Currently, theCluster consists of the following Ludwig Boltzmann institutes :

Institute for Rheumatology and Balneology, Vienna OberlaaIInnssttiittuutteeffoorrRRhheeuummaattoollooggyyaannddBBaallnneeoollooggyy,,VViieennnnaa OObbeerrllaaaa

Institute for Rehabilitation of Internal Diseases, SaalfeldenIInnssttiittuutteeffoorrRReehhaabbiilliittaattiioonnooff IInntteerrnnaallDDiisseeaasseess,,SSaaaallffeellddeenn

Research Unit for Epidemiology of Rheumatic Diseases, BadenRReesseeaarrcchhUUnniitt ffoorrEEppiiddeemmiioollooggyyooffRRhheeuummaattiiccDDiisseeaasseess,,BBaaddeenn

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The basic and translational research is primarily aimed at investigating pathogeneticmechanisms of inflammatory rheumatic diseases and elucidating the molecular and cellularmechanisms of balneological treatments both in patients and in animal models of arthritis.

The clinical research is focused on developing novel therapeutic tools for rehabilitationprogrammes and on analysing the effectiveness and sustainability of cure and rehabilitationprogrammes.

This unique multi disciplinary combination of basic and clinical research performed in theCluster allows to addresss simultaneously and in a coordinated manner several majorquestions in the fields of rheumatology, balneology and rehabilitation.

2

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Revenues

Funds from previous years 327.785,

Cash in hand 1.455,

Boltzmann Society 326.913,

Third Party 174.506,

Third Party (project related) 68.275,

Other revenues 24.098,

Total 923.032,

Expenses

Investments 48.688,

Research costs 127.720

Personnel costs 443.489,

Administrative expenses 23.829,

Other expenses 1.093,

Total 644.819,

Reserve 278.213,

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Major partners areRheumazentrumWien–Oberlaa GmbH (Center for Rheumatic Diseases, Vienna Oberlaa)Pensionsversicherungsanstalt der Angestellten (Austrian Pension Insurance Organization)Medizinische Universität Wien (Medical Universit y of Vienna)

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In 2010 the advisory board of the Cluster was established which consists of of five members:

Univ. Prof. Dr. Steffen Gay, Universität Zürich, ChairmanUniv. Prof. Dr. Josef, Smolen, Medizinische Universität WienUniv. Doz. Dr. Tanja Stamm, Medizinische Universität WienPrim. Univ. Prof. Dr. Hans Bröll, Rheuma ZentrumWien OberlaaPrim. Dr. Reinhold Hawel, Sonderkrankenanstalt für Erkrankungen des Stütz undBewegungsapparates der PVA, Bad Hofgastein

4

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Ao. Univ.Prof. Dr. Günter Steiner: Cluster coordinator and leader of the ResearchProgrammes RHEUMA and BAL. Major affiliation: Medical University of Vienna, InternalMedicine III, Division of RheumatologyUniv.Doz. Dr. Werner Kullich: Head of the Ludwig Boltzmann Institute for Rehabilitation ofInternal Diseases Saalfelden (LBI Rehab) and leader of the Research Programme REHABPrim. Dr. Anton Ulreich:Head of the LBI Rehab branch in GröbmingDr. Michael Ausserwinkler: Head of the LBI Rehab branch in AlthofenPrim. Dr. Ernst Wagner: Head of the Ludwig Boltzmann Research Unit for Epidemiology ofRheumatic Diseases; place of work: BadenAo. Univ.Prof. Dr.Cem Ekmekcioglu: physiologist; performing studies on clock geneexpression and function in patients with rheumatic diseases; employed via contract forwork; place of work: Institute of Physiology, Medical University of ViennaDr. Makyieh Tohidast Akrad: research associate since 1986, regular employment contract,40 hours per week; responsible for the immunohistochemistry laboratory of the LBI forRheumatology and Balneology branch locatedat the 2nd Department of Medicine, HietzingHospital; place of work: Hietzing Hospital, ViennaMag. Dr. Burkhard Klösch: key researcher at the Ludwig Boltzmann Institute forRheumatology and Balneology; full time employed; major research area: investigations onthe mode of action of hydrogen sulphide ans sulphur bath therapy; place of work: ViennaOberlaaMag. Dr. Bibiane Steinecker: research associate with regular employment status sinceOctober 2009, 16 hours a week; involved in outcome research projects as well as in a basicresearch studies on cellular and molecular mechanisms of NMR therapy; place of work: LBIbranch GröbmingMag. Nicola Fagerer: research associate since October 2005 with regular employment statussince August 2007, 30 hours a week; participation in several projects, such as outcomemeasurement and radical research; place of employment: SaalfeldenMag. Daniela Krehan: PhD student, topic of her thesis: Effects of hydrogen sulphide on theinflammatory state of fibroblast like synoviocytes and T lymphocytes from patients withrheumatoid arthritis or osteoarthritis; employed 30 hours a week; place of work: ViennaOberlaaMag. Eva Luise Hobl: research associate with employment contract from May 2008February 2010, 12 hours a week; place of work: Institute of Physiology, Medical University ofVienna.Mag. Gabriele Nebel: assistant to Prim. Univ.Prof. Dr. Hans Bröll (advisory board member);regular employment contract, 5 hours a week; place of work: Vienna OberlaaDipl.Ing. Norbert Klammer: chemist; study administration and data analysis; employed on asmall basis of less than 10 hours a month; place of work: Institute of Physiology, MedicalUniversity of ViennaMelissa Liszt: biomedical analyst; regular employment since May 2008, 40 hours a week;assistant to Dr. Burkhard Klösch; place of work: Vienna Oberlaa

5

Angela Schwaiger: secretary, assistant to Doz. Kullich; regular employment contract since1995, 30 hours a week; place of employment: SaalfeldenBrigitta Schweiger: biomedical analyst; regular employment contract since 1981, 20 hours aweek; place of work: SaalfeldenMarkus Reiff: technician, 20 hours per week; assistant to Dr. Tohidast Akrad; place of work:Hospital HietzingJohanna Leibl: secretary, assistant to Prof. Steiner and Prof. Dr. Josef Smolen (member ofthe advisory board); 8 hours per week, place of work: Medical University of Vienna,Department of Internal Medicine III.Susanne Humpeler: biomedical analyst; administration of studies; employed on a small basisof less than 10 hours a month; place of work: Institute of Physiology, Medical University ofViennaBrigitte Schweiger: biomedical analyst; organization, accounting; employed on a small basisof less than 10 hours a month; place of work: Vienna

6

22.. RREESSEEAARRCCHH PPRROOGGRRAAMMMMEESS

The three research programmes of the Cluster – RHEUMA, REHAB and BAL –have beenapproved by independent reviewers as well as by the Cluster´s advisory board and aredescribed in the following sections.

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Background

Sulfur bath therapy has long been in use for the therapy of patients suffering fromrheumatic disorders and is still considered helpful for the treatment of diseases such asrheumatoid arthritis (RA) or osteoarthritis (OA). In spa resorts all over the world, such as inVienna Oberlaa hundreds of patients are treated every year. However, scientificinvestigations dealing with the beneficial as well as adverse effects of this kind of treatmentare rare and have sometimes led to controversial results (1 3). Moreover, the underlyingmolecular mechanisms are poorly understood (4, 5). H2S exerts a host of effects on variousbiological targets, resulting in responses that range from cytotoxicity (6 10) tocytoprotective effects (11). Several studies have demonstrated cytoprotective effects ofH2S at micromolar concentrations, which may be related to its ability to neutralize a varietyof reactive species including oxyradicals (12), peroxynitrite (13), hypochlorous acid (14) andhomocysteine (15). Higher (millimolar) H2S exposure tends to be cytotoxic due to freeradical and oxidant generation (8), calcium mobilization (16), glutathione depletion (9), aswell as the induction of mitochondrial cell death pathways (7, 17).Our recently published data suggest that H2S may exert anti inflammatory properties ondifferent cell types (18, 19). At low concentrations and by short term incubation H2Spartially blocked constitutive as well as IL 1 induced IL 6 and IL 8 expression in fibroblastlike synoviocytes (FLS) derived from RA patients (18) and in a human chondrocyte cell line(19). In contrast, previously obtained data showed that H2Smay also have pro inflammatoryproperties.

Results

To further explore the effects of H2S four FLS lines (2 from patients with RA and 2 frompatients with OA) were incubated for 20 min with different concentrations of theexogeneous H2S donor sodium hydrogen sulfide (NaHS). After changing the culturemedium, incubation was continued for a total time of 12 h. At different time points cellculture supernatants were collected and IL 6 secretion was quantified by ELISA.Interestingly, IL 6 expression of 2 FLS lines (one from a patient with RA and one from apatient with OA) was transiently increased by H2S treatment (Fig. 1) while the 2 other linesremained unaffected.

7

RA 26

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A B

Fig. 1. H2S transiently upregulates IL 6 expression in RA and OA FLS. FLS remaineduntreated or were incubated for 20 min with 0.125 mM or 1.0 mM NaHS. After 1, 3, 6 and 12h, IL 6 secretion was quantified by ELISA.

To elucidate the mechanism leading to induction of IL 6 expression, phosphorylation ofextracellular signal regulated kinase (ERK1/2) was analysed by Western blotting at varioustime points post NaHS treatment. The data obtained showed that already 15 min afterinitial H2S exposure ERK1/2 was activated in both cell populations (Fig. 2).

0 15 30 45 60i

0 15 30 45 60 min

ERK1/2Phospho ERK1/2Tubulin

Fig. 2. Western blot analysis of ERK1/2 activation in RA FLS (left) and OA FLS (right)

Further experiments demonstrated that inhibitors of the mitogen activated protein kinases(MAPKs) p38 and MEK1/2 blocked H2S induced IL 6 expression. RA FLS were incubated for20 min with 1.0 mM NaHS only or were treated for 30 min with an inhibitor of p38 MAPK oran inhibitor of MEK1/2 before being stimulated with NaHS. As shown in Fig. 3, both MAPKinhibitors blocked H2S induced IL 6 expression completely.

RA 26

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Fig. 3. The MAPK inhibitors SB203580 andU0126 block H2S induced IL 6 expression. RAFLS were incubated for 20 min with NaHSonly or were treated for 30 min with SB203580or U0126 before stimulated with NaHS. After6 h, IL 6 secretion was quantified by ELISA.

These results demonstrate that H2S at high concentration (1.0 mM) stimulated theexpression of IL 6 and other pro inflammatory genes such as IL 8 and COX 2 (not shown) inFLS derived from patients with RA or OA. This effect was caused through activation of p38and ERK1/2 MAPK. Taken together, H2S is a potent gaseous molecule which can upregulate

8

the expression of pro inflammatory genes in FLS. Therefore, in patients with active RAsulfur bath therapy should be used with great caution.

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Background

Dimethyl sulfoxide (DMSO) is a powerful water miscible solvent that dissolves most waterinsoluble drugs (20). DMSO possesses anti inflammatory properties (21), as well as theability to act as a free radical scavenger (22). Thus, its properties have been exploited in thetreatment of dermatological, rheumatic, and renal manifestations of amyloidosis. DMSO iscapable of inducing or inhibiting cell proliferation, apoptosis and/or differentiation (23).However, limited data are available on the underlying molecular mechanism.Besides the development of new therapeutic drugs for the treatment of RA and OA, there isalso a strong interest in finding new anti inflammatory agents derived from naturalproducts. Dimethly sulfone (DMS) is found in small amounts in many foods, includingunpasteurised milk, grains, meat, eggs, and fish (24 26). It is also present in popular dietarysupplements (27). Reported effects claimed to be associated with DMS include relief ofpain, reduction of inflammation, arthritis, allergies and asthma (28 30).

Results

We were therefore interested to study the effects of DMSO and DMS on the cytokine (IL 6and IL 8) expression in C 28/I2 cells, a human chondrocyte cell line, originally derived from ayoung patient with OA (31, 32). To investigate potential anti inflammmatory properties ofDMSO and DMS, C 28/I2 cells were left untreated or were incubated either with DMSO orDMS over a period of 12 h. At different time points the cell culture supernatants werecollected and IL 6 levels were quantified by ELISA. As shown in Fig. 1A, only the higherDMSO concentration effectively blocked IL 6 expression. Similar inhibitory effects wereobserved when the cells were treated with DMS. Only the higher DMS concentration wasefficiently blocking the secretion of IL 6 (Fig. 1B): after 12 h almost 70 % inhibition wasobtained with both substances. Similar results were obtained when cell culturesupernatants were analysed for IL 8 secretion (data not shown).

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Fig. 4. Long term treatment of C 28/I2 cells with DMSO or DMS results in a reduced IL 6secretion. The cells were left untreated or were incubated for 12 h either with DMSO (0.5 and 1.0%) (A) or DMS (10 and 100 mM) (B). IL 6 levels in cell culture supernatants were quantified byELISA.

9

Furthermore, both DMSO and DMS blocked also IL 1 induced IL 6 and IL 8 expression. Forthis experiment, C 28/I2 cells were either treated for 60 min with DMSO or DMS beforebeing stimulated with IL 1 or were incubated with IL 1 and both of the substancesconcurrently. Blockage of IL 6 and IL 8 secretion was very effective when the cells wereincubated with IL 1 in the presence of either DMSO or DMS.Taken together, these studies show that the antioxidants DMSO and DMS are potentinhibitors of constitutive as well as IL 1 induced cytokine expression in a humanchondrocyte cell line. Identification of the components of the signal transduction pathwaysthat are sensitive to antioxidants may eventually open a new territory to more selectivetreatment of inflammatory disorders.

References

1. Verhagen AP, de Vet HC, de Bie RA, Kessels AG, Boers M, Knipschild PG (1997) Takingbaths: the efficacy of balneotherapy in patients with arthritis. A systematic review. JRheumatol 24:1964 71.2. Verhagen AP, de Vet HC, de Bie RA, Kessels AG, Boers M, Knipschild PG (2000)Balneotherapy for rheumatoid arthritis and osteoarthritis. Cochrane Database Syst RevCD000518.3. Verhagen AP, Bierma Zeinstra SM, Cardoso JR, de Bie RA, Boers M, de Vet HC (2003)Balneotherapy for rheumatoid arthritis. Cochrane Database Syst Rev CD000518.4. Stuhlmeier KM, Bröll J, Iliev B (2009) NF kappaB independent activation of a series ofpro inflammatory genes by hydrogen sulfide. Exp Biol Med (Maywood) 234(11):1327 38.5. Whiteman M, Li L, Rose P, Tan CH, Parkinson DB, Moore PK (2010) The effect ofhydrogen sulfide donors on LPS induced formation of inflammatory mediators inmacrophages. Antioxid Redox Signal Feb 3.6. Attene Ramos MS, Wagner ED, Plewa MJ, Gaskins HR (2006) Evidence that Hydrogensulfide is a genotoxic agent. Mol Cancer Res 4(1):9 14.7. Baskar R, Li L, Moore PK (2007) Hydrogen sulfide induces DNA damage and changes inapoptotic gene expression in human lung fibroblast cells. FASEB J 21:247–55.8. Dorman DC, Moulin FJM, McManus BE, Mahle KC, James RA, Struve MF (2002)Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation: Correlationwith tissue sulfide concentrations in the rat brain, liver, lung, and nasal epithelium. ToxicolSci 65:18 25.9. Truong DH, Eghbal MA, HindmarshW, Roth SH, O’Brien PJ (2006) Molecular mechanismsof hydrogen sulfide toxicity. DrugMetab Rev 38:733–44.10. Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL (2006) Hydrogensulfide is an endogenous modulator of leukocyte mediated inflammation. FASEB J 20:211820.11. Szabó C, Kiss L, Pankotai E (2007) Cytoprotective and anti inflammatory effects ofhydrogen sulfide in macrophages and in mice. Crit Care 11(Suppl.2),P2.12. Kimura Y, Dargusch R, Schubert D, Kimura H (2006) Hydrogen sulfide protects HT22neuronal cells from oxidative stress. Antioxid Redox Signal 8:661–70.13. Whiteman M et al. (2004) The novel neuromodulator hydrogen sulfide: an endogenousperoxynitrite ‘scavenger’? J Neurochem 90:765–68.

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14. Whiteman M et al. (2005) Hydrogen sulfide: a novel inhibitor of hypochlorous acidmediated oxidative damage in the brain? Biochem Biophys Res Commun 326:794–98.15. Yan SK et al. (2006) Effects of hydrogen sulfide on homocysteine induced oxidativestress in vascular smooth muscle cells. Biochem Biophys Res Commun 361:485–91.16. Nagai Y, Tsugane M, Oka J, Kimura H (2004) Hydrogen sulfide induces calcium waves inastrocytes. FASEB J 18:557–59.17. Yang G, Wu L, Wang R (2006) Pro apoptotic effect of endogenous H2S on human aortasmooth muscle cells. FASEB J 20:553–55.18. Kloesch B, Liszt M, Broell J (2010) H2S transiently blocks IL 6 expression in rheumatoidarthritic fibroblast like synoviocytes and deactivates p44/42 mitogen activated proteinkinase. Cell Biol Int 34(5):477 84.19. Kloesch B, Liszt M, Steiner G, Broell J (2010) Inhibitors of p38 and ERK1/2 MAPkinaseand hydrogen sulphide block constitutive and IL 1 induced IL 6 and IL 8 expression in thehuman chondrocyte cell line C 28/I2. Rheumatol Int Dec 14. [Epub ahead of print]20. Santos NC, Figueira Coelho J, Martins Silva J, Saldanha C (2003) Multidisciplinaryutilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects. BiochemPharmacol 65:1035 41.21. Melchior D, Packer CS, Johnson TC, Kaefer M (2003) Dimethyl sulfoxide: does it changethe functional properties of the bladder wall? J Urol 170:253 58.22. Xing L, Remick DG (2005) Mechanisms of dimethyl sulfoxide augmentation of IL 1 betaproduction. J Immunol 174:6195 202.23. Bolduc L, Labrecque B, Cordeau M, Blanchette M, Chabot B (2001) Dimethyl sulfoxideaffects the selection of splice sites. J Biol Chem 276:17597 602.24. Pearson TW, Dawson HJ, Lackey HB (1981) Natural occurring levels of dimethylsulfoxide in selected fruits, vegetables, grains, and beverages. J Agric Food Chem 29:108991.25. Steely JS. Sulphur Compounds in Foods (1994) American Chemical Society, WashingtonD.C.26. Silva Ferreira AC, Rodrigues P, Hogg T, Guedes DP (2003) Influence of sometechnological parameters on the formation of dimethyl sulfide, 2 mercaptoethanol,methionol, and dimethyl sulfone in port wines. J Agric Food Chem 51:727 32.27. Jacobs S, Lawrence RM, Siegel M (1999) The Miracle MSM: the Natural Solution forPain. Putnam GP, New York.28. Barrager E, Schauss AG (2003) Methylsulfonylmethane as a treatment for seasonalallergic rhinitis: additional data on pollen counts and symptom questionnaire. J AlternComplement Med9:15 16.29. Hasegawa T, Kumamoto S, Ueno S, Yoshikai Y (2004) Jpn J Pharmacol Ther 32:420 27.30. Kim LS, Axelrod LJ, Howard P, Buratovich N, Waters RF (2006) Efficacy ofmethylsulfonylmethane (MSM) in osteoarthritis pain of the knee: a pilot clinical trial.Osteoarthritis Cartilage 14:286 94.31. Goldring MB (2004) Immortalization of human articular chondrocytes for generation ofstable, differentiated cell lines. Methods Mol Med 100:23 36.32. Goldring MB (2004) Culture of immortalized chondrocytes and their use as models ofchondrocyte function. Methods Mol Med 100:37 52.

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Background

Nearly every physiological and biochemical process in mammals, when not provenotherwise, is influenced by the master clock and shows time dependent, especiallycircadian, variations. This also includes the immune system. For example, the expression ofcytolytic factors and cytokines in NK cells from rats follows the oscillation of clock genesand should be under circadian control (Arjona et al, 2006). Additionally, it has beendescribed that disruption of the circadian rhythm in humans by shift work or a modulatedexpression of clock genes, leads to increased secretion of inflammatory cytokines (Guess etal, 2009; Castanon Cervantes et al., 2010). Furthermore, in mouse models of arthritis clockgene expression was shown to be disturbed (Hashiramoto et al., 2010). Finally, TNF alpha,one major proinflammatory cytokine in RA, has been shown to inhibit the expression of Ebox regulated clock genes (Per1, Per2) in mouse fibroblasts (Cavadini et al., 2007).Taken together, these findings sugest a connection between the circadian system, clockgene expression, inflammatory cytokines and RA. It is therefore our goal (1) to examine andcompare the expression of the major clock genes in synovial tissues from RA patientscompared to synovial tissues from OA patients, which show a lower grade of inflammation;(2) to investigate differences in the induction and possibly synchronization (over 24h) ofclock gene expression by serum shock and dexamethasone between fibroblast likesynoviocytes (FLS) isolated from patients with RA compared to those from patients withOA or normal juvenile fibroblasts; (3) to study the influences of TNF on clock gene and IL6 expression in FLS from RA patients compared to FLS from OA patients or juvenilefibroblasts; (4) to proof a direct connection between proinflammatory markers and clockgenes RNA silencing experiments will be performed to study the influences of Per1 blockageon IL 6 expression. Per1 is one of the most important clock genes and its expression ishighly affected by stimulants, like horse serum or dexamethasone.

Results

The expression of clock genes was analysed by real time qPCR in synovial tissues (n = 3 foreach group) from patients with RA or OA. The preliminary results show that in 2 RA patientsthe clock genes Bmal1 and Clock were upregulated compared to samples from OA patients(Fig. 1). The results will be confirmed by analysing additonal patient samples.

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Figure 1. Clock gene expression in synovial tissues from patients with RA compared to

patients with OA

Furthermore, the effect of serum shock, dexamethasone or TNF on clock gene expressionwas studied in FLS derived from RA (n=3) or OA (n =3) patients and in juvenile fibroblastsfrom 1 one individual. The results especially show (Table 1):As expected, a higher expression of IL 6 after TNF treatment in RA > OA > JFAs expected, an induction of Per1 after serum shock and DXMAn inhibition of the E box regulatory Per1 and Per2 by TNF after 4hA higher induction of the Per1 2 and Cry 1 genes by serum shock in juvenile (dermal)fibroblasts compared to those from RA and OA patients

Table 1. Expression of IL 6 and different clock genes in RA or OA patients and in juvenilefibroblasts after serum shock, dexamethasone and TNF treatment.

t1 = 2h IL-6 Per 1 Per 2 Bmal 1 Cry 1 Clock

JF OA RA JF OA RA JF OA RA JF OA RA JF OA RA JF OA RA

UT 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

HS 48,20 377,15 1035,80 4,78 3,92 2,52 4,79 2,24 2,16 1,49 1,52 1,18 3,33 1,95 2,09 1,40 0,93 0,87

DXM 0,31 1,24 0,20 7,66 3,92 4,35 0,93 1,14 1,19 1,15 0,96 0,90 0,74 0,86 0,87 0,95 0,99 0,89

TNF 43,37 89,18 154,17 2,04 1,53 0,97 0,84 1,02 0,93 1,17 1,06 1,32 0,96 1,01 1,06 1,09 1,02 0,94

t2 = 4h IL-6 Per 1 Per 2 Bmal 1 Cry 1 Clock

JF OA RA JF OA RA JF OA RA JF OA RA JF OA RA JF OA RA

UT 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

HS 9,36 21,51 32,88 0,63 0,44 0,13 2,29 1,73 1,09 1,38 3,25 2,99 3,82 2,34 2,20 1,29 0,79 0,89

DXM 0,48 0,23 0,88 5,69 5,50 5,07 0,92 1,41 1,00 1,09 0,98 1,01 1,02 1,01 0,91 0,95 0,75 0,80

TNF 14,03 115,80 185,34 0,23 0,11 0,16 0,72 0,71 0,52 1,21 1,45 2,13 1,11 1,29 1,25 1,03 0,96 1,26

12

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Thus, the expression of some clock genes in synovial tissues from RA patients seem to bemodified compared to OA patients. However, induction of clock genes between RA and OApatients seem to be not significantly different with few exceptions, like Per1. TNF is ableto inhibit clock gene expression in human cells.

References

Arjona A, Sarkar DK. Evidence supporting a circadian control of natural killer cell function.Brain Behav Immun. 2006 Sep;20(5):469 76.Castanon Cervantes O, et al.. Dysregulation of inflammatory responses by chronic circadiandisruption. J Immunol. 2010 Nov 15;185(10)Cavadini G, et al..TNF alpha suppresses the expression of clock genes by interfering with Ebox mediated transcription. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12843Guess J, et al.. Circadian disruption, Per3, and human cytokine secretion. Integr CancerTher. 2009 Dec;8(4):329 36.Hashiramoto et al. Mammalian clock gene Cryptochrome regulates arthritis viaproinflammatory cytokine TNF alpha. J Immunol. 2010 Feb 1;184(3):1560 5. .Tai CC, Ding ST. N 3 polyunsaturated fatty acids regulate lipid metabolism through severalinflammation mediators: mechanisms and implications for obesity prevention. J NutrBiochem.2010 May;21(5):357 63Zainal Z, et al.. Relative efficacies of omega 3 polyunsaturated fatty acids in reducingexpression of key proteins in a model system for studying osteoarthritis. OsteoarthritisCartilage 2009 Jul;17(7):896 905

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The scientific projects of the Ludwig Boltzmann Institute for Rehabilitation of InternalDiseases in Saalfelden and the Institute’s Branch for Clinical Rheumatology and PhysicalMedicine in Gröbming on the field of rehabilitation research were continued during the year2010.

In the planned project “REMERI” the sustainability and modification of risk factors ofinpatient rehabilitation programmes for patients suffering from metabolic syndrome will beinvestigated. By the end of 2010 the proposed study gained a positive vote by the ethicscommittee. Unfortunately, the permission by the PVA (pension insurance institution) forthe participation of the involved rehabilitation centres in Saalfelden, Bad Hofgastein,Gröbming, and Bad Tatzmannsdorf had not yet been obtained. Thus, the recruiting ofpatients was delayed and will presumably be started in spring 2011. Neverthelss,preliminary studies to this project involving patients with metabolic syndrome additionallysuffering from RA have been perfomed and addressed the impact of adipocytokines. Asexpected, increased values of the Body Mass Index (BMI) were associated with elevatedlevels of the pro inflammatory adipokine leptin with significant differences between normalweight and obese patients. Adiponectin a protein of fat cells that burns fatty acids and canprevent endothelial inflammation and expression of adhesion molecules decreasedcontemporaneously with increasing BMI. The sCD40 ligand – a marker for activation of Tcells and inflammation – correlated with BMI. Remarkably, normal weight RA patientswithout cardiovascular diseases showed significantly higher levels of adiponectin and lowerlevels of leptin compared to obese RA patients who had an increased risk for cardiovasculardiseases.

A study performed in collaboration with the health resort Bad Hofgastein and the researchinstitute Gastein of the Paracelsus Medical University Salzburg on the OutcomeMeasurement of the effectivity of cure in Bad Hofgastein including radon therapy wascompleted by the end of the year and the data obtained from more than 200 patients arecurrently being analysed.

At the beginning of the year the recruiting of rehabilitation patients for the trial concerningthe influence of a new extension device “GammaSwing” generating controlled harmonicvibrations on the spine of low back pain patients has started. The majority of the proposednumber of patients (n = 60) could already be included in 2010. The controlled, randomisedstudy compares the effect of 6 applications of the GammaSwing extension treatment withthe effect of 6 back massages of the rehabilitation programme of low back pain patients.The study design includes a postal follow up survey after 3 and 6 months.

An outcome measurement of various rehabilitation methods involving more than 700patients is performed in collaboration with the SKA of the PVA Weyer over a period of oneyear, the total data analyses is scheduled for 2011.

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In April 2010 a new project was started, approved by the ethics committee and supportedby The Austrian Research Promotion Agency FFG, together with the University Salzburg,Workgroup for Neurodynamics and Neurosignalling. In the course of this study agedprobands with handicaps should improve the limitation of cognitive functions and sleepdisorders using a special music therapy device that is currently in developmental stage.Prototypes were constructed after a usability inquiry in summer 2010.

For some time past nuclear magnetic resonance (NMR) is therapeutically applied fortreatment of rheumatic and orthopaedic diseases. Previous studies of the LBI Saalfeldenhave proved NMR to be an efficient treatment with sustainable effects on osteoarthritis ofthe knee or finger joints and low back pain. Since there are only a few reports on the cellularand molecular basis of this therapy cell culture experiments were performed to investigatepossible effects on repair mechanisms and analgesia. The experiments showed aremarkable expression pattern of components of the NFAT pathways in CAL72 bone andCAL78 cartilage cells following 10 hours NMR application (especially NFATc1, NFATc3 andNFAT5). Furthermore, bone and cartilage cells were stimulated with interleukin 1 to studythe cell reactions of NMR treatment on immunoactivation and/or activation ofinflammation. In future experiments C28 I2 chondrocytes will be included in theinvestigations (kindly provided by Prof. Mary Goldring, Weill Cornell Medical College, NewYork).

Investigations on inflammatory processes of rheumatic diseases, especially RA, included theanalyses of the effects of chemokine expression on pteridine metabolism. Neopterin levelsin serum correlated significantly with all tested chemokines (CCL2/MCP 1; CXCL13/BCA 1;CXCL1/fractalkine). These chemokines play an important role in immune response andlymphocyte migration concerning the induction of inflammation, angiogenesis, andchemotaxis of inflammatory cells. The results were presented at an international pteridinecongress in Tyrol, and a manuscript has been submitted.

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The focus of research in 2010 was the evaluation of costs of soft tissue rheumatism(eriarthropathia of the shoulder = impingement syndromes). The study has been approvedby the local ethical review committee and patient recruitment has been started. The costevaluation by questionnaire will comprise approximately 70 patients. The questionnairehas already been applied in the studies on costs of osteoarthritis of hip and knee and lowback pain (ARTHROSE), and was adapted according to the particular pain problems andactivity limitations of patients with shoulder diseases. Cost domains that will beinvestigated are: medications, physical therapy, aids, physician`s visits, diagnosticprocedures, hospitalization, in patient rehabilitation, costs of transportation, limitations ofactivity and participation (not only vocational, but also leisure activities), and sick leaves.Additionally co morbidities willbee documented.

The studies on risk factors and course of low back pain following a back school programmewill be continued and finished by Sept 2011.

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Since the Ludwig Boltzmann Institute for Physiological Rhythm Research in BadTatzmannsdorf was closed the planned study „Vergleichende Untersuchung des Effekts vonMoorbädern und Kohlendioxidbädern bei PatientInnen mit Kniegelenksarthrose” (studieson the effectiveness of mud bath therapy in comparison to carbondioxide bath therapy inpatients with osteoarthritis of the knee) had to be cancelled. A new baneological study onthe effectiveness of sulphur bath therapy in patients with osteoarthritis is in preparation.This study will be performed at the Center for Rheumatic Diseases, Vienna Oberlaa.

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The established cooperations with hospitals and research instutions inside and outsideAustria have been described in detail in the Annual Report 2009. A new collaboration hasbeen initiated with Univ.Doz. Hans Peter Kiener from the Medical University of Vienna,Department of Internal Medicine III. This collaboration will be focused on therapeuticinterventions targeted at synovial fibroblasts. Of particular importance is the collaborationwith Siemens and the Austrian Institute of Technology in the context of the FFG fundedresearch project PORACCS (Pathogenesis of Rheumatoid Arthritis on a Cell on a ChipSystem) where a microanalytical computer controlled cell analysis platform is beingdeveloped that allows to monitor living cells in a non invasive manner. In a first applicationpatient derived synovial fibroblasts that have been treated with hydrogen sulphide orsulphur containing drugs will be analysed. Subsequently, the system will be adapted forother cell types.

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The Institute for Rehabilitation of Internal Diseases organized the 29th “RheumatologicalProfessional Development Symposium” in collaboration with the “ÖsterreichischeGesellschaft für Rheumatologie” and the Austrian Pension Insurance Institution (PVA) inSaalfelden on June 26st 2010. The Symposium was well attended, lecturers and participantscame from Austria and Germany.

In collaboration with the PVA the Institute also organised a professional developmentsymposium about supply with protheses in Gröbming. The LBI Saalfelden organized furthermeetings with oral presentations of invited speakers on “Update Rheumatology – Diagnosisand modern therapy of RA, case reports”, “GammaSwing Extension in the Therapy of LowBack Pain”, and “A new ergotherapeutical and practical shoulder report form based on ICF.”

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Steine G.r:

The role of T cells, nucleic acids and genes in the pathogenesis of autoimmune arthritis inrats and menInternational Conference on AutoimmunityLjubljana, Slowenien, 5. 9.5. 2010

Steiner G.:

Anti RA33 and arthritisAutocure Workshop on Specific immunity and immune memory in RASigtuna, Schweden, 26. 28.9. 2010

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Steiner G:

Autoimmundiagnostik Was bringt die ZukunftJahrestagung der ÖGKMCSalzburg 27. 30.10. 2010

Steiner G.:

Autoantibodies as Diagnostic and Prognostic Markers in Early ArthritisAnnual Scientific Meeting of the American College of RheumatologyAtlanta, 8. 11.11. 2010

Fagerer N., Kullich W.:

„Effects of Rheumatoid Arthritis and Coronary Artery Disease on the Expression ofChemokines and Neopterin“29th International Winter Workshop “Clinical, Chemical, and Biochemical Aspects ofPteridines”St. Christoph/Arlberg, 28. Februar – 7. März 2010

Bernatzky G., Presch M., Kullich W.:

„Musik im Alter“1.Jahrestagung der Österreichischen Gesellschaft für Musik und MedizinWien, 19. März 2010

Lichtenschopf A.:

„Welche Erfolge sind in der „every day“ Situation der stationären Rehabilitation möglich?Outcomes in der pneumologischen Rehabilitation“10. wissenschaftliche Sitzung des Arbeitskreises der ÖGPWien, 30. April 2010

Kullich W.:

„Entzündungsmechanismen und Therapie: Moderne Entwicklungen“Salzburger Schmerzkurs für Ärzte „Aus der Praxis – für die Praxis“Leogang, 18. Juni 2010

Kullich W.:

„Ernährung bei Schmerzen: Möglichkeiten zur Hilfe“Salzburger Schmerzkurs für Ärzte „Aus der Praxis – für die Praxis“Leogang, 18. Juni 2010

Bernatzky G., Presch M., Kullich W.:

„Music: A non pharmacological intervention in clinical pain patients“11th International Conference on Music Perception and CognitionSeattle, 23. – 27. August 2010

Lichtenschopf A., Kullich W., Müller R.:

“Diminished weight gain with a standardized inpatient smoking cessation program”(Poster)European Respiratory Society Annual CongressBarcelona, 18. – 22. September 2010

Kullich W., Müller R.:

„Inpatient rehabilitation of musculoskeletal diseases in centres of the Austrian Pension

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Insurance Institution – data of outcome measurement“ (Poster)8th Central European Congress of RheumatologySopron, 23. – 25. September 2010

Bernatzky G., Presch M., Kullich W.:

„Musik im Alter“12. Int. MusikmesseRied im Innkreis, 7. – 10. Oktober 2010

Kullich W., Fagerer N.:

“Expression of Neopterin and Chemokines in Patients with Rheumatoid Arthritis andCoronary Disease” (Poster)Jahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

Steinecker Frohnwieser B., Weigl L., Fagerer N., Kullich W., Kress H.G.:

„Modulation of VEGF and Cytokines by Therapeutic Nuclear Magnetic Resonance“ (Poster)Jahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

Wagner E., Falkenbach A., Kullich W., Ammer K., Eberl G., Hawel R., Kirchheimer J., Mayrhofer

F., Mur E., Schwann H., Singer F., Skoumal M., Ulreich A., Wicker A., Zeindler H.:

„Aspekte der Rehabilitation in Österreich“ (Poster)Jahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

Wagner E., Falkenbach A., Kullich W., Ammer K., Eberl G., Hawel R., Kirchheimer J., Mayrhofer

F., Mur E., Schwann H., Singer F., Skoumal M., Ulreich A., Wicker A., Zeindler H.:

„Rehabilitation – Evolution Zukunftsperspektiven“ (Poster)Jahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

Klösch B., Liszt M., Ertl P., Charwat V., Joksch M.PORACCS: Pathogenesis of Rheumatoid Arthritis on a Cell on a Chip System (Poster)Jahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

Krehan D., Liszt M., Klösch B., Bröll H., Steiner G.

Einfluss von H2S auf aktivierte T ZellenJahrestagung der Österreichischen Gesellschaft für Rheumatologie und RehabilitationWien, 25. – 27. November 2010

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Prof. Steiner and Prof. Ekmekcioglu gave lectures, held seminars and organized JournalClubs for students of medicine and PhD students at the Medical University of Vienna.

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The investigations on the biological effects of hydrogen sulphide and sulphur bath therapy,respectively, will on the one hand continue to explore the effects on synovial fibroblasts,and on the other hand address potential effects on cells of the immune system, such as Tcells and monocytes. Furthermore, it is planned to investigate the effects of hydrogensulphide and sulphur containing drugs in collagen induced arthritis, one of the most widelyused experimental models of RA.

Studies on clock gene expression and function in patients with RA or OA will be continuedat the molecular and immune histochemical level in order to confirm and substantiate thedata obtained so far. Furthermore, it is planned to study clock gene expression in animalmodels of arthritis such as collagen induced or pristane induced arthritis.

Furthermore, it is planned to investigate expression of citrullinated proteins in synovialtissue, with a particular focus on citrullinated vimentin which will be done in collaborationwith the diagnostic company Orgentec.

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The project REMERI will be started in spring 2011 where the sustainability and modificationof risk factors of inpatient rehabilitation programmes for patients suffering from metabolicsyndrome will be investigated.

The “Gamma Swing study” will be continued and completed by the end of 2011.

An outcome measurement of various rehabilitation methods performed in collaborationwith the SKA of the PVA Weyer will be completed.

The investigations on the effects of NMR treatment on fibroblasts and chondrocytesperformed ion collaboration with the Medical University of Vienna will be continued

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A new baneological study on the effectiveness of sulphur bath therapy in patients withosteoarthritis is in preparation. This study will be performed at the Center for RheumaticDiseases, Vienna Oberlaa.

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Kloesch B, Liszt M, Broell J. H2S transiently blocks IL 6 expression in rheumatoid arthriticfibroblast like synoviocytes and deactivates p44/42 mitogen activated protein kinase. CellBiol Int 2010;34:477 484.Kloesch B, Liszt M, Steiner G, Broell J. Inhibitors of p38 and ERK1/2 MAPkinase andhydrogen sulphide block constitutive and IL 1 induced IL 6 and IL 8 expression in thehuman chondrocyte cell line C 28/I2. Rheumatol Int 2010;Dec 14. [Epub ahead of print]Nell Duxneuner V, Machold K, Stamm T, Eberl G, Heinzl H, Hoefler E, Smolen JS, Steiner G.Autoantibody profiling in patients with very early rheumatoid arthritis a follow up study.Ann Rheum Dis 2010; 69:169 174.Hoffmann M, Hopf R, Niederreiter B, Heinz Redl H, Smolen JS, Steiner G. Gait changesprecede clinical arthritis and strongly correlate with symptoms and histological features ofpristane induced arthritis. Arthritis Res Ther. 2010; 12(2):R41.Leibetseder V, Humpeler S, Zuckermann A, Svoboda M, Thalhammer T, Marktl W,Ekmekcioglu C. Time dependence of estrogen receptor expression in human hearts. BiomedPharmacother. 2010 ;64(3):154 9.Ekmekcioglu C, Touitou Y. Chronobiological aspects of food intake and metabolism andtheir relevance on energy balance and weight regulation. Obes Rev. 2010.epubFagerer N., Kullich W.Adipozytokine bei Rheumatoider Arthritis und Adipositas. Effects ofRheumatoid Arthritis and Coronary Artery Disease on the Expression of Chemokines andNeopterin. Pteridines 2010; 21, 35 36.Fagerer N., Kullich W.Adipozytokine bei Rheumatoider Arthritis und Adipositas. Wien MedWochenschr 2010; 160(15 16), 391 398.Kullich W., Müller R. Inpatient rehabilitation of musculoskeletal diseases in centres of theAustrian Pension Insurance Institution – data of outcome measurement. HungarianRheumatology 2010; 212 213.

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Steinecker Frohnwieser B., Weigl L.G., Höller C., Sipos E., Kullich W., Kress H.G. Influenceof NMR therapy on metabolism of osteosarcoma and chondrosarcoma cell. Arab HealthShow Daily 2010; 4, 7.Kullich W., Fagerer N. Expression of Neopterin and Chemokines in Patients withRheumatoid Arthritis and Coronary Disease” (Abstr). J Mineralstoffwechsel 2010; 17(4), 151152.Steinecker Frohnwieser B., Weigl L., Fagerer N., Kullich W., Kress H.G. Modulation of VEGFand Cytokines by Therapeutic Nuclear Magnetic Resonance“ (Abstr.) J Mineralstoffwechsel

2010; 17(4), 155 .