Antiviral Therapy :71 81 (doi: /IMP1490)

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1 Antivirl Therpy 21 15:71 81 (doi: /IMP149) Originl rticle Efficcy of peroxisome prolifertor ctivted receptor gonist in the tretment of virus-ssocited hemophgocytic syndrome in rbbit model Wen-Chun Hsieh 1, Bu-Shin Ln 2, Yi-Ling Chen 1, Yo Chng 1,2, Hui-Chi Chung 3, Ih-Jen Su 1,2,3,4 * 1 Division of Infectious Diseses, Ntionl Helth Reserch Institutes, Tinn, Tiwn 2 Grdute Institutes of Microbiology nd Immunology, Ntionl Cheng Kung University Medicl College, Tinn, Tiwn 3 Grdute Institutes of Bsic Medicine, Ntionl Cheng Kung University Medicl College, Tinn, Tiwn 4 Deprtment of Pthology, Ntionl Cheng Kung University Hospitl, Tinn, Tiwn *Corresponding uthor e-mil: suihjen@nhri.org.tw Bckground: Virus-ssocited hemophgocytic syndrome (VAHS) is ftl compliction of virl infections, such s Epstein Brr virus nd H5N1 influenz, tht results from mcrophge ctivtion nd proinflmmtory cytokine injuries. The high comorbidity nd mortlity of current therpy urgently demnds n idel gent bsed on VAHS pthogenesis. Peroxisome prolifertor ctivted receptor (PPAR) gonists, regultors of metbolic syndrome, cn exhibit immunomodultory effects on mcrophge ctivtion nd cytokine secretion. Methods: In this study, we dopted rosiglitzone, PPAR-g gonist, for VAHS control in Herpesvirus ppio ()- infected rbbit model. Vrious doses of rosiglitzone were orlly dministered to rbbits on dy 7 or dy 2 fter intrvenous chllenge with copies of. Results: The rbbits tht received 4 mg/dy rosiglitzone hd significntly incresed survivl when treted t n erly stge of infection (P<1), wheres higher dose (8 mg/dy) ws required t the dvnced stge of the disese (P<5). All rosiglitzone-treted rbbits hd significntly improved lbortory prmeters nd plsm tumour necrosis fctor- levels. Importntly, rosiglitzone could lso inhibit virl repliction in vitro nd in vivo. Conclusions: PPAR gonists could represent potentilly new gent for the therpy of VAHS. Introduction Virus-ssocited hemophgocytic syndrome (VAHS) is ftl compliction ssocited with severe virl infections, such s Epstein Brr virus (EBV) nd H5N1 influenz [1 4]. Previous studies on the pthogenesis of VAHS suggest tht this condition results from n berrnt immune response following virus infection [1,2,5,6]. This virus host interction could led to enhnced proinflmmtory cytokine secretion nd systemic tissue injuries. Clinicl signs of VAHS include fever, heptosplenomegly, cytopeni nd cogulopthy. The disese, if untreted, will progress to the full-blown stge with extensive orgn filure, severe cytopeni nd florid hemophgocytosis in bone mrrow. Hypercytokinemi is consistent finding nd tumour necrosis fctor-α (TNF-α) hs been identified s one of the cndidte cytokines in VAHS pthogenesis [7,8]. The enhnced TNF-α secretion is reportedly medited by the NF-κB/SAP/ERK (nucler fctor-κb/slam-ssocited protein/extrcellulr signl-regulted kinse) signlling pthwy tht is ctivted by EBV ltent membrne protein 1 in EBV-infected T-cells [1,9]. The therpy for VAHS, especilly childhood hemophgocytic lymphohistiocytosis (HLH), hs significntly improved in the pst decde primrily due to the erly recognition nd tretment of the disese [2,1]. However, the high comorbidity ssocited with the current tretment of HLH nd H5N1 influenz urgently demnd novel nd effective gent to be incorported into the current protocol bsed on the VAHS pthogenesis [2 4,6]. Peroxisome prolifertors ctivted receptor (PPAR) gonists, in ddition to their remrkble effects on the tretment of metbolic syndrome [11,12], hve been found to exert immunomodultory effects on T-cell ctivtion, mcrophge ctivtion nd suppression of proinflmmtory cytokine secretion, ll of which re chrcteristic fetures of VAHS nd re primrily 21 Interntionl Medicl Press (print) (online) 71

2 W-C Hsieh et l. medited vi NF-κB signlling [13 16]. Preliminry reports hve lso reveled inhibitory effects of PPAR-g gonist on repliction of HIV nd respirtory syncytil virus (RSV) [17,18]. These combined effects of PPAR gonists suggest their pproprite ppliction in VAHS therpy. A rbbit model of Herpesvirus ppio ()- induced VAHS hs been estblished [19,2]. The virus is n EBV homologue nd -induced VAHS rbbit model mnifested clinicopthologicl fetures similr to VAHS in humns [21]. We therefore dopted this rbbit model to test the efficcy of rosiglitzone, PPAR-g gonist, for VAHS therpy t different stges of the disese. Methods The rbbit model of -induced VAHS The rbbit VAHS model ws previously developed by Hyshi et l. [19] nd estblished in our lbortory using [22]. The viruses were hrvested from culture superntnts of 594S cells, the virus titres djusted to 1 8 copies/ml nd stored t -7 C until use. A totl of copies of were inoculted into rbbits by intrvenous injection. On the bsis of the virus host interction, s previously estblished [22], rbbits were strted to be fed orlly with rosiglitzone (Avndi, GSK, Phildelphi, PA, USA) on dy 7 (group A) or dy 2 (group B) fter inocultion, representing the erly nd lte stge of the disese, respectively. The tretment ws continued until the end of this study. Seril exmintions of clinicl nd lbortory prmeters were recorded. This niml study hs been performed under the pprovl of the Committee of Animl Centre of Ntionl Cheng Kung University Medicl School, Tinn, Tiwn. Histopthologicl exmintions The rbbits were utopsied fter deth or scrificed under euthnsi on dy 45 fter infection with n overdose of pentobrbitl sodium (MTC Phrmceuticls, Cmbridge, Cnd). Multiple orgns including the spleen, lymph nodes, liver, kidneys, lungs nd spinl cord were hrvested, exmined mcroscopiclly nd then fixed with 1% neutrl formlin. The formlin- fixed, prffin-embedded tissue blocks were sectioned t 3 mm thickness nd studied for conventionl histopthology. Detection of in tissues nd quntifiction of virl lods by rel-time PCR The viruses in tissues were detected by in situ hybridiztion of EBV-encoded RNA-1 (EBER-1), s previously described [22]. Virl DNA in plsm or culture superntnt ws extrcted with the QIAmp DNA Mini kit (QIAGEN GmbH, Hilden, Germny) ccording to the mnufcturer s protocol. For virus determintion, the primers used in this ssy were HPNA2-1485S nd HPNA2-1691A, s described previously [22]. The Tqmn probe ws 5 -FAM- AACACCAAGTCCGC CAATACTC-3 TAMRA. Detection of TNF- in rbbit plsm For the determintion of rbbit plsm TNF-, purified ntirbbit TNF- ntibody (BD Bioscience, Sn Diego, CA, USA) ws used s cpture ntibody for ELISA. After blocking, recombinnt rbbit TNF- (BD Bioscience) or rbbit plsm were dded nd incubted for 2 h. The plte ws wshed nd biotin-conjugted ntirbbit TNF- ntibody (BD Bioscience) mixed with strepvidin horserdish peroxidse (BD Bioscience) ws dded. After incubtion for 1 h, the plte ws wshed gin nd tetrmethylbenzidine substrte ws pplied to the plte for 2 min. Finlly, the plte ws mesured t OD 49 nm using n ELISA reder fter dding stop solution. Detection of ntibody-coted red blood cells by flow cytometry Our previous studies hve demonstrted tht ntibodycoted red blood cells (RBCs) ply crucil role in RBC phgocytosis nd cn predict the subsequent development of VAHS [22]. The titre of ntibody-coted RBCs is thus good indictor of VAHS severity. For the detection of ntibody-coted RBCs, blood smples were collected pre- nd post- inocultion nd then incubted with fluorescein isothiocynte (FITC)- lbelled ntirbbit immunoglobulin (DAKO, Glostrup, Denmrk) for 3 min. After setting forwrd sctter nd side sctter detectors to log mode nd gting RBC region, the surfce fluorescence on the rbbit RBC ws mesured by fluorescence-ctivted cell sorting (FACS, Clibur; Becton Dickinson, Frnklin Lkes, NJ, USA). Demonstrtion of in vitro inhibition of virl repliction by rosiglitzone To test whether rosiglitzone cn inhibit virl repliction, the EBV replictive system nd its lytic ntigens were dopted for this study. This pproch ws employed becuse ntibodies ginst the lytic ntigens were not vilble nd becuse EBV nd re close homologues [21]. Immunofluorescence ssy to detect virl ntigens For the detection of EBV virl ntigens, the mrmoset cell line B95.8 (which contins EBV) ws used in this study. B95.8 cells were treted with different dosges of rosiglitzone for 24 h. Cells were hrvested nd then seeded on 12-well slides. Slides were ir-dried nd fixed with cetone for 5 min t 4 C. Anti-EBV erly ntigen D (EAD) nd virl cpsid ntigen (VCA) ntibodies were Interntionl Medicl Press

3 PPAR gonist for hemophgocytic syndrome pplied to the slides for 3 min nd then wshed with phosphte-buffered sline (PBS). The secondry ntibody, Alex Fluor 488 got ntimouse immunoglobulin G (H+L; Invitrogen, Crlsbd, CA, USA) ws then dded to the slides for nother 3 min. After wshing, slides were mounted with glycerol contining 4,6-dimidino- 2-phenylindole (DAPI) for nucler stining control nd then observed under fluorescent microscopy. Western blot nlysis of the inhibition of virl repliction by rosiglitzone in the B95.8 cell line To clrify whether rosiglitzone cn inhibit virl repliction, the EBV-contining mrmoset cell line B95.8 ws used. Phorbol ester (TPA) ws used to induce the lytic cycle of EBV. The EBV lytic proteins VCA nd EAD were detected by immunofluorescence. Western blot nlysis ws lso performed on the induction of EBV lytic protein EAD. t different dosges ws exmined for the inhibitory effect on lytic induction. Sttisticl nlysis Survivl of the rbbits in ech group, s compred with the infected, untreted control rbbits, ws nlysed by the log rnk test. TNF- nd virl lods from plsm mong different groups were nlysed by the Kruskl Wllis test following the Dunn s post test if P<5. All of the tests used the GrphPd Prism 4. softwre. Sttisticl significnce ws defined t P<5. Results Gross observtions nd histopthologicl exmintions Tretment with rosiglitzone ws strted on either dy 7 (erly stge of infection, group A) fter inocultion, when plsm TNF- nd bnorml lbortory prmeters strted to be detected, or on dy 2 fter inocultion (dvnced stge of the disese, group B) when utontibody-coted RBCs ppered nd hemophgocytosis ws observed in tissues, representing dvnced or full-blown disese ctivities (Figure 1A). The lbortory prmeters such s pltelet count usully showed remrkble decrese of beyond 5% t this dvnced stge. A totl of 38 rbbits were used in this study. Among these 38 rbbits, 36 were infected with nd the 2 uninfected rbbits were used s norml control nd drug toxicity control (rosiglitzone, 8 mg/dy). Eight rbbits were infected but received no rosiglitzone tretment (infected nd untreted control). ws dministered to 28 of the -infected rbbits nd these were divided into group A (16 rbbits) nd group B (12 rbbits). The rosiglitzone dosges were further strtified into three subgroups mong group A rbbits: 1 mg/dy (6 rbbits), 2 mg/dy (5 rbbits) nd 4 mg/dy (5 rbbits). Higher rosiglitzone dosges were dministered to group B rbbits nd these were divided into two subgroups: 4 mg/dy (6 rbbits) nd 8 mg/dy (6 rbbits). The -uninfected rbbits for the drug toxicity control remined helthy throughout the course of the experiment. Therefore, high dose of 8 mg/dy rosiglitzone showed no detectble toxicity in rbbits. However, the 8 -infected but untreted rbbits developed clinicl illness, such s loss of ppetite, on dy 22 3 nd ll died before dy 35. Necropsies of the rbbits in the untreted group reveled swollen lymph nodes, heptosplenomegly nd pulmonry congestion. The rbbits fed with lower doses (1 nd 2 mg/dy rosiglitzone) in group A nd the 4 mg/dy subgroup in group B hd necropsy findings similr to those of the untreted group, lbeit to milder degree. By contrst, the rbbits receiving 8 mg/dy of rosiglitzone in group B hd mild to moderte bnorml findings, such s the size of the spleen nd liver t dy 45 post-inocultion (Figure 1B). The 4 mg/dy subgroup rbbits in group A hd reltively norml gross findings. Histopthologicl exmintions from infected but untreted rbbits reveled progressive lymphoid depletion in spleen nd lymph nodes nd extensive necrosis of bone mrrow (Figure 1C). Hemophgocytosis ws frequently observed in lymph nodes nd spleen. Mny infiltrting lymphocytes were observed in portl res of livers (Figure 1C) nd in perivsculr res in kidneys nd lungs (pictures not shown). Similr findings were lso observed in the low-dose subgroups of rbbits (1 nd 2 mg/dy in group A nd 4 mg/dy in group B). However, in the 4 mg/dy rosiglitzone-treted rbbits in group A, the spleen nd lymphoid orgns showed reltively norml findings, with even myeloid hyperplsi in bone mrrow. In the 8 mg/dy treted rbbits in group B, mild to moderte lymphoid hypoplsi, congestion nd occsionl RBC phgocytosis were observed in the spleen nd lymph nodes. The lymphoid infiltrtion in the portl res of the liver ws reltively mild (Figure 1C). The uninfected rbbit receiving 8 mg/ dy rosiglitzone showed norml microscopic findings similr to those of the norml control rbbit (dt not shown), indicting no evidence of drug toxicity t this high dose. therpy improves the survivl rte of -infected rbbits All the rbbits in the untreted group died between dy 25 nd dy 35 with medin survivl of 28 dys. In the treted group A, the rbbits tht received 1 nd 2 mg/dy rosiglitzone hd improved survivl s compred with the untreted group, with medin survivl of 36 dys (1 mg/dy; P=125) nd 37 dys (2 mg/dy; P=24). The rbbits tht received 4 mg/ dy rosiglitzone in group A showed significntly Antivirl Therpy

4 W-C Hsieh et l. Figure 1. Therpeutic design for -induced VAHS nd histopthologicl exmintions of -infected rbbits Virus infection Group A (dy 7) Group B (dy 2) A Time, dys Lbortory prmeters TNF-α present in serum Virus present in serum Anti-RBC ntibody production B Control, dy 26 (ded) Group A, dy 45 Group B, dy 45 C Control, dy 26 (ded) Group A, dy 45 Group B, dy 45 Bone mrrow Spleen Liver (A) According to the virus host interction previously estblished in Herpesvirus ppio ()-infected rbbits [22], the rbbits were fed orlly with rosiglitzone on dy 7 (group A) or dy 2 (group B) fter inocultion. (B) Gross fetures of the spleen in -infected rbbits. The -infected, untreted rbbits hd enlrged spleens. In the treted rbbits, the spleen size ws decresed significntly. (C) Representtive histopthologicl fetures of bone mrrow, spleen nd liver in norml control (control), -infected but without tretment (), 4 mg/dy rosiglitzone-treted rbbits in group A nd 8 mg/dy rosiglitzone-treted rbbits in group B. All sections were stined with hemotoxylin nd eosin (mgnifiction 2). The untreted -infected rbbits hd mrrow necrosis, depletion of the lymphoid system with red blood cell (RBC) phgocytosis (inset, 1, mgnifiction), nd portl infiltrtion of lymphoid cells with mild ftty liver. The rbbits treted with 4 mg/dy rosiglitzone in group A hd much improved results, wheres the rbbits treted with 8 mg/dy rosiglitzone in group B still showed mild to moderte pthology, lthough were much improved s compred with the untreted rbbits. TNF-α, tumour necrosis fctor-α; VAHS, virus-ssocited hemophgocytic syndrome Interntionl Medicl Press

5 PPAR gonist for hemophgocytic syndrome Figure 2. tretment showed significntly improved survivl of -infected rbbits A 1 B Survivl, % mg 2 mg 4 mg Survivl, % mg 8 mg Time, dys Time, dys versus 1 mg rosiglitzone; P=125 versus 2 mg rosiglitzone; P=24 versus 4 mg rosiglitzone; P=36 versus 4 mg rosiglitzone; P=554 versus 8 mg rosiglitzone; P=4 The survivl curves of (A) group A nd (B) group B represent rbbits receiving rosiglitzone tretment on dy 7 or dy 2 fter Herpesvirus ppio () infection. -treted rbbits showed better survivl times thn untreted rbbits in dosge-dependent mnner in both groups. A higher dosge of 8 mg/dy ws required to chieve improved survivl if treted t the dvnced stge of disese. prolonged survivl: t the end of the study (dy 45), 3 of 5 rbbits still survived nd looked helthy, s described bove in the histopthologicl exmintion (P=36; Figure 2, group A). But, if treted t lte or full-blown disese stge (group B), only rbbits treted with the higher dose of 8 mg/dy rosiglitzone showed significntly improved survivl (P=4; Figure 2, group B). Lbortory prmeters nd plsm TNF- levels The rbbits in both therpeutic groups showed improved lbortory prmeters in dose- dependent mnner (Figure 3). Antibody-coted RBC titres, n indictor of RBC phgocytosis [22], hd dosedependent, delyed emergence in plsm (Figure 3A). The pltelet counts were lso more stble nd hd dose-dependent effect in both groups (Figure 3B). However, the pltelet counts in group B rbbits continued to decrese to severe thrombocytopenic levels t dy 45. The results of TNF- production in ech group of rbbits re shown in Figure 3C. Plsm TNF- ws detectble on dy 12 fter inocultion in the untreted group. There ws wide spectrum of TNF- levels: one rbbit hd n unusully high level up to 5 ng/ml. In untreted rbbits, enhnced TNF- levels persisted, but still fluctuted, prior to deth (Figure 3C, dy 26). TNF- production showed delyed increse nd levels were mintined t reltively low levels (<1 ng/ml) in most of the rosiglitzone-treted group A rbbits, except the 1 mg/dy subgroup which hd high levels of TNF- (Figure 3C, upper pnel). On dy 26, the TNF- levels in the 4 mg/dy group A rbbits remined t undetectble levels (P<5, s compred with untreted group). In group B rbbits, the TNF- levels returned from the mildly incresed level before therpy to undetectble levels 3 dys fter therpy (dy 23) in both the 4 mg/dy nd 8 mg/dy subgroups (P<1), indicting therpeutic effect of rosiglitzone. However, TNF- levels incresed gin on dy 26 long with the disese progression. Detection of in tissues nd quntifiction of virl lods in plsm In situ EBER-1 hybridiztion ws performed on formlin- fixed tissues. The EBER-1 signls in the infiltrting lymphocytes of spleen, lymph node nd liver were much lower in the treted groups thn in the untreted rbbits (Figure 4A). There ws dosgedependent effect in both groups, especilly in the 4 mg/dy-treted rbbits in group A, which showed remrkble decrese of the EBER-1 signls. In prllel to the results t the tissue level, plsm virl lods from rosiglitzone- treted groups showed significnt decrese of round hlf log scle s compred with tht in the untreted groups on dy 23 nd dy 26 (P<5); gin, this decrese occurred in dosgedependent mnner (Figure 4B). Antivirl Therpy 15.1

6 W-C Hsieh et l. Figure 3. The PPAR-g gonist improved lbortory prmeters nd TNF- levels of -infected rbbits A Antibody coted RBC, % mg 1 mg 4 mg Group A Antibody coted RBC, % mg 4 mg Group B Time fter inocultion, dys Time fter inocultion, dys B Pltelet, 1 3 /µl mg 1 mg 4 mg Pltelet, 1 3 /µl mg 4 mg Time fter inocultion, dys Timefter inocultion, dys C Dy 12 Dy 23 Dy 26 A 1 mg A 2 mg A 4 mg A 1 mg A 2 mg A 4 mg A 1 mg A 2 mg A 4 mg Dy 12 Dy 23 Dy 26 b b B 4 mg B 8 mg B 4 mg B 8 mg B 4 mg B 8 mg (A) Antibody-coted red blood cells (RBCs) decresed slowly in treted groups in dose-dependent mnner. (B) Pltelet counts lso improved in rosiglitzone-treted groups. All pnel A nd pnel B dt re presented s the men vlues. (C) The plsm tumour necrosis fctor- (TNF-) levels of Herpesvirus ppio ()-infected rbbits decrese fter tretment with the peroxisome prolifertor ctivted receptor-g (PPAR-g) gonist ( P<5, b P<1). All dt were duplicted nd presented s the men vlues Interntionl Medicl Press

7 PPAR gonist for hemophgocytic syndrome Figure 4. Virl lods decresed in both tissues nd plsm smples of rosiglitzone-treted rbbits A, dy 26 (ded) Group A, dy 45 Group B, dy 45 Spleen Lymph nodes Liver Virl lod, copies/ml B Group A, dy 12 Group A, dy 23 Group A, dy 26 A 1 mg A 2 mg A 4 mg Virl lod, copies/ml b A 1 mg A 2 mg A 4 mg Virl lod, copies/ml A 1 mg A 2 mg A 4 mg Virl lod, copies/ml Group B, dy 12 Group A, dy 23 Group A, dy 26 Virl lod, copies/ml B 4 mg B 8 mg b B 4 mg B 8 mg Virl lod, copies/ml B 4 mg B 8 mg (A) Epstein Brr virus-encoded RNA-1 in situ hybridiztion of the spleen, lymph nodes nd liver in untreted Herpesvirus ppio ()-infected nd rosiglitzonetreted (4 mg/dy in group A nd 8 mg/dy in group B) -infected rbbits (originl mgnifictions ). (B) virl lods in plsm of both groups. The treted rbbits showed significnt reductions of virl lod on dy 23 nd dy 26 ( P<5, b P<1) in dosge-dependent mnner. Antivirl Therpy

8 W-C Hsieh et l. inhibits lytic production of EBV in B95.8 cells According to the bove results, rosiglitzone seems to hve n inhibitory effect on repliction in rbbits. To confirm this, we performed in vitro studies to test whether rosiglitzone cn prevent the virus from entering the lytic cycle. As no ntibodies ginst lytic proteins re vilble, the lytic cycle ntigens of EBV, the humn homologue of, were employed in this study. The EBV in B95.8 cells ws induced to enter the lytic cycle nd express lytic proteins such s EAD or VCA following stimultion with TPA. The lytic proteins Figure 5. inhibits EBV lytic protein expression in B95.8 cell line A Untreted TPA TPA+R1 TPA+R1 TPA+R5 TPA+R1 VCA DAPI B Untreted TPA TPA+R1 TPA+R1 TPA+R5 TPA+R1 EAD DAPI C TPA, 5 ng/ml , µm EAD Actin Immunofluorescence studies of (A) Epstein Brr virus (EBV) virl cpsid ntigen (VCA) nd (B) erly ntigen D (EAD) reveled decresed levels of EBV lytic proteins in rosiglitzone-treted, phorbol ester (TPA)-induced B95.8 cells in dosge-dependent mnner. 4,6 -Dimidino-2-phenylindole (DAPI) ws control for nucler stining. (C) Western blotting lso showed tht EBV-EAD expression ws downregulted by rosiglitzone in dose-dependent mnner. TPA: 5 ng/ml; R1, R1, R5 nd R1: 1 mm, 1 mm, 5 mm nd 1 mm rosiglitzone, respectively. Actin ws used s internl control in western blot nlysis Interntionl Medicl Press

9 PPAR gonist for hemophgocytic syndrome EBV-VCA (Figure 5A) nd EBV-EAD (Figure 5B) could be induced by TPA nd were then downregulted in dose-dependent mnner by rosiglitzone tretment, s ssyed by immunofluorescence microscopy. Using western blot nlysis, the sme findings were observed for the expression of EAD (Figure 5C). Tken together, these dt suggest tht rosiglitzone cn inhibit the lytic ctivtion of EBV nd this is probbly lso true for, explining the decrese of virl lods in the rosiglitzone-treted rbbits. Discussion In this study we demonstrted for the first time tht the PPAR-g gonist rosiglitzone could significntly improve the therpy of VAHS in -infected rbbit model. ws found to suppress the secretion of the proinflmmtory cytokine TNF- nd lso to improve lbortory prmeters nd survivl. Interestingly, rosiglitzone could inhibit, lthough not eliminte, virl repliction in vitro nd in vivo. or other PPAR gonists might therefore represent potentil gents for considertion s lterntive or combintion drugs for VAHS therpy. PPAR gonists re prt of current therpies for metbolic syndrome nd relted disorders, such s nonlcoholic ftty liver [23 26], nd might lso exhibit wide spectrum of biologicl effects, including regultion of the immune system nd nti- inflmmtory properties [13,27,28]. PPAR gonists might lso function s nticncer gents [29 31]. The min mechnistic ction of PPAR gonists on metbolic syndrome, on inflmmtion nd on tumours is medited minly through the inhibition of NF-κB signlling, which is one of the key pthwys involved in inflmmtion nd cncer development [32 34]. The pthogenesis of VAHS is reportedly medited vi the NF-κB signl pthwy to ctivte TNF- nd proinflmmtory cytokines [1,9]. In this study, we confirmed tht the plsm TNF- levels showed significnt reduction fter the institution of rosiglitzone on dy 12 of group A rbbits. The rosiglitzone- treted rbbits reveled remrkble effects on meliorting tissue injuries of the lymphoid system, liver nd bone mrrow. Degrees of mcrophge ctivtion nd RBC phgocytosis were lso significntly decresed nd correlted with the delyed emergence nd decresed titres of serum nti-rbc ntibodies. Similr to PPAR gonists, the lipid- lowering sttins re lso known to modulte nd meliorte bcteril sepsis. This effect hs lso been reported in the literture to be medited vi the inhibition of proinflmmtory cytokine secretion nd of NF-κB signlling [35 37]. One importnt, yet unexpected, observtion of this study ws the remrkble decrese of serum virl lods in the rosiglitzone-treted groups of -infected rbbits. Similr observtions were noted in EBER-1 in situ hybridiztion on the spleen, lymph node nd liver, s compred with the untreted group on dy 45. Although the virus in our study persisted t the end of the study period on dy 45, chrcteristic for herpes virus infection, the inhibition of virl repliction by rosiglitzone ws further supported by in vitro studies using the homologue EBV. Previous studies reveled tht PPAR gonists could lso inhibit the repliction of HIV nd RSV [17,18]. The exct mechnism for the inhibition of virl repliction by PPAR gonists remins to be investigted in the future [38 ]. On the bsis of the lbortory nd survivl dt obtined in this study, we showed tht rosiglitzone hs significnt effect on the suppression of TNF- levels nd virl lods, nd could improve the survivl time for rbbits if treted t n erly stge of infection. However, higher dosge of the drug is required to meliorte VAHS if treted t the dvnced stge of the disese nd only suboptiml therpeutic effects could be chieved. Rbbits in the dvnced stge of VAHS usully exhibited severe bone mrrow depletion, severe lymphoid depletion, extensive tissue injuries nd superimposed infections, ll of which mke the prognosis ominous. Lstly, nd importntly, the introduction of PPAR gonists in VAHS therpy will shed light on the therpy of H5N1 influenz, s the high morbidity nd mortlity of H5N1 influenz hs been reported to result from lethl proinflmmtory cytokine injuries in systemic orgns nd tissues in response to H5N1 virus [41]. As mentioned bove, the combined effects of PPAR gonists on the regultion of mcrophge ctivtion, the suppression of proinflmmtory cytokine secretion nd the inhibition of virl repliction mke PPAR gonists new gent for improving the control of severe virl infection nd probbly other cytokine immune disorders. Acknowledgements This study ws supported by grnts from the Ntionl Helth Reserch Institutes nd Ntionl Science Council (IJS). Disclosure sttement The uthors declre no competing interests. References 1. Chung HC, Ly JD, Hsieh WC, Su IJ. Pthogenesis nd mechnism of disese progression from hemophgocytic lymphohistiocytosis to Epstein-Brr virus-ssocited T-cell lymphom: nucler fctor-kpp B pthwy s potentil therpeutic trget. Cncer Sci 27; 98: Antivirl Therpy

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