World Journal of Virology

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1 ISSN (online) World Journl of Virology World J Virol 2017 August 12; 6(3): Published by Bishideng Publishing Group Inc

2 W J Contents V World Journl of Virology Qurterly Volume 6 Number 3 August 12, 2017 FIELD OF VISION 49 Additionl ttention to combintion ntiretrovirl therpy-relted lipodystrophy Kobyshi N, Nkhr M, Ok M, Seki K ORIGINAL ARTICLE Bsic Study 53 Highly ctive ntiretrovirl therpy dysregultes prolifertion nd differentition of humn pre-dipocytes Jones E, Mzirk P, McNurln MA, Drrs F, Gelto MC, Cso G I August 12, 2017 Volume 6 Issue 3

3 Contents World Journl of Virology Volume 6 Number 3 August 12, 2017 ABOUT COVER Editoril Bord Member of World Journl of Virology, Kun-Long Ben, PhD, Professor, Kunming Kngto Biotechnology Compny Ltd, Kunming , Yunnn Province, Chin AIM AND SCOPE World Journl of Virology (World J Virol, WJV, online ISSN , DOI: ) is peer-reviewed open ccess cdemic journl tht ims to guide clinicl prctice nd improve dignostic nd therpeutic skills of clinicins. WJV covers topics concerning rbovirl infections, bronchiolitis, centrl nervous system virl diseses, DNA virus infections, encephlitis, eye infections, ftigue syndrome, heptitis, meningitis, opportunistic infections, pneumoni, RNA virus infections, sexully trnsmitted diseses, skin diseses, slow virus diseses, tumor virus infections, viremi, zoonoses, nd virology-relted trditionl medicine, nd integrted Chinese nd Western medicine. Priority publiction will be given to rticles concerning dignosis nd tretment of virl diseses. The following spects re covered: Clinicl dignosis, lbortory dignosis, differentil dignosis, imging tests, pthologicl dignosis, moleculr biologicl dignosis, immunologicl dignosis, genetic dignosis, functionl dignostics, nd physicl dignosis; nd comprehensive therpy, drug therpy, surgicl therpy, interventionl tretment, minimlly invsive therpy, nd robot-ssisted therpy. We encourge uthors to submit their mnuscripts to WJV. We will give priority to mnuscripts tht re supported by mjor ntionl nd interntionl foundtions nd those tht re of gret bsic nd clinicl significnce. Indexing/Abstrcting World Journl of Virology is now indexed in PubMed, PubMed Centrl. FLYLEAF I-IV Editoril Bord EDITORS FOR THIS ISSUE Responsible Assistnt Editor: Xing Li Responsible Electronic Editor: Y-Jing Lu Proofing Editor-in-Chief: Lin-Sheng M Responsible Science Editor: Fng-Fng Ji Proofing Editoril Office Director: Ze-Mo Gong NAME OF JOURNAL World Journl of Virology ISSN ISSN (online) LAUNCH DATE Februry 12, 2012 FREQUENCY Qurterly EDITOR-IN-CHIEF Ling Lu, MD, PhD, Deprtment of Pthology nd Lbortory Medicine, University of Knss Medicl Center, Knss City, 3901 Rinbow Blvd, WHE 3020, KS 66160, United Sttes EDITORIAL BOARD MEMBERS All editoril bord members resources online t EDITORIAL OFFICE Xiu-Xi Song, Director World Journl of Virology Bishideng Publishing Group Inc 7901 Stoneridge Drive, Suite 501, Plesnton, CA 94588, USA Telephone: Fx: E-mil: editoriloffice@wjgnet.com Help Desk: PUBLISHER Bishideng Publishing Group Inc 7901 Stoneridge Drive, Suite 501, Plesnton, CA 94588, USA Telephone: Fx: E-mil: editoriloffice@wjgnet.com Help Desk: PUBLICATION DATE August 12, 2017 COPYRIGHT 2017 Bishideng Publishing Group Inc. Articles published by this Open-Access journl re distributed under the terms of the Cretive Commons Attribution Non-commercil License, which permits use, distribution, nd reproduction in ny medium, provided the originl work is properly cited, the use is non-commercil nd is otherwise in complince with the license. SPECIAL STATEMENT All rticles published in journls owned by the Bishideng Publishing Group (BPG) represent the views nd opinions of their uthors, nd not the views, opinions or policies of the BPG, except where otherwise explicitly indicted. INSTRUCTIONS TO AUTHORS ONLINE SUBMISSION II August 12, 2017 Volume 6 Issue 3

4 W J V World Journl of Virology Submit Mnuscript: DOI: /wjv.v6.i3.53 World J Virol 2017 August 12; 6(3): ISSN (online) Bsic Study ORIGINAL ARTICLE Highly ctive ntiretrovirl therpy dysregultes prolifertion nd differentition of humn pre-dipocytes Eyone Jones, Pvel Mzirk, Mrgret A McNurln, Frnk Drrs, Mrie C Gelto, Giuseppe Cso Eyone Jones, Deprtment of Surgery, Rutgers, Robert Wood Johnson Medicl School, New Brunswick, NJ 08901, United Sttes Pvel Mzirk, Mrgret A McNurln, Giuseppe Cso, Deprtment of Surgery, Stony Brook University Medicl Center, Stony Brook, NY 11794, United Sttes Frnk Drrs, Deprtment of Urology, Stony Brook University Medicl Center, Stony Brook, NY 11794, United Sttes Mrie C Gelto, Deprtment of Medicine, Stony Brook University Medicl Center, Stony Brook, NY 11794, United Sttes Author contributions: Jones E contributed to dt collection; McNurln MA contributed to study design; Drrs F recruited study subjects, collected surgicl smples, nd contributed to drfting the mnuscript; Gelto MC contributed to overll study design; Cso G contributed to study design, performed the experiments; Jones E, Mzirk P, McNurln MA nd Cso G contributed to dt nlysis; Jones E, Mzirk P, McNurln MA, Gelto MC nd Cso G writing of the mnuscript; Jones E, McNurln MA, Gelto MC nd Cso G contributed to dt interprettion; ll uthors pproved the finl version of the mnuscript. Institutionl review bord sttement: The study ws reviewed nd pproved by the Stony Brook University Institutionl Review Bord. All specimens were cquired from ptients fter informed consent nd ethicl permission ws obtined for prticiption in the study. Conflict-of-interest sttement: The uthors hve no conflict of interest to disclose. Dt shring sttement: No dditionl dt re vilble. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.0) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4.0/ Mnuscript source: Invited mnuscript Correspondence to: Giuseppe Cso, MD, PhD, Deprtment of Surgery, Stony Brook University Medicl Center, 100 Nicolls Rd, Stony Brook, NY 11794, United Sttes. giuseppe.cso@stonybrook.edu Telephone: Fx: Received: Jnury 28, 2017 Peer-review strted: Februry 7, 2017 First decision: My 8, 2017 Revised: April 28, 2017 Accepted: June 6, 2017 Article in press: June 7, 2017 Published online: August 12, 2017 Abstrct AIM To investigte the mechnism(s) by which potentil effects of multi-drug highly-ctive ntiretrovirl therpy contributes to lipodystrophy syndrome. METHODS Predipocytes from helthy donors were ssessed for prolifertion nd differentition in the presence of nucleoside reverse trnscriptse inhibitors (NRTIs), nonnucleoside reverse trnscriptse inhibitors (NNRTIs), nd protese inhibitors (PIs) individully nd in combintion. Effects on prolifertion were ssessed with 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyl tetrzolium bromide ssy nd effects on differentition were ssessed from glycerol-3-phosphte dehydrogense (GP DH) ctivity nd quntittion of Oil Red O stining for intrcellulr lipid. Dt were nlyzed with rndomized block ANOVA with post-hoc Fisher s Lest Significnt 53 August 12, 2017 Volume 6 Issue 3

5 Jones E et l. HAART dysregultes humn dipogenesis Difference test. RESULTS Predipocyte prolifertion ws inhibited by combintion of NNRTI + NRTI (14% t 48 h, P < 0.001) nd PI + NRTI (19% t 48 h, P < 0.001) with dditionl suppression when ritonvir (RTV) ws dded (26% t 48 h). The drug combintion of tznvir (ATV) + RTV + emtricitbine (FTC) + tenofovir (TDF) hd the gretest inhibitory effect on prolifertion t 48 h. Predipocyte differentition ws most significntly reduced by the efvirenz + FTC + TDF ssessed either by GPDH ctivity (64%) or lipid ccumultion (39%), P < Combining NRTIs with PI (ATV + FTC + TDF) significntly suppressed differentition (GPDH ctivity reduced 29%, lipid ccumultion reduced by 19%, P < 0.01). This effect ws slightly greter when boosting mount of RTV ws dded (ATV + FTC + TDF + RTV, P < 0.001). CONCLUSION Although combintion ntiretrovirl therpy is cliniclly more efficcious thn single drug regimens, it lso hs much greter inhibitory effect on predipocyte prolifertion nd differentition. Key words: Nucleoside reverse trnscriptse inhibitors; Non-nucleoside reverse trnscriptse inhibitors; Protese inhibitors; Pre-dipocytes; Highly ctive ntiretrovirl therpy; Lipodystrophy The Author(s) Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: We demonstrted n in vitro system for evluting potentil ntiretrovirl regimens for dipose tissue toxicity. In generl, combintion regimens resulted in greter predipocyte prolifertion nd differentition inhibition thn single therpies. The drug combintion of tznvir + emtricitbine + tenofovir hd inhibitory effects on predipocytes nd dding ritonvir t levels equivlent to clinicl boosting, incresed toxicity still further. Jones E, Mzirk P, McNurln MA, Drrs F, Gelto MC, Cso G. Highly ctive ntiretrovirl therpy dysregultes prolifertion nd differentition of humn pre-dipocytes. World J Virol 2017; 6(3): Avilble from: URL: full/v6/i3/53.htm DOI: INTRODUCTION A link between highly ctive ntiretrovirl therpy (HAART) nd HAART-ssocited lipodystrophy (HALS) hs been recognized for well over decde. HALS is ssocited with bnorml chnges in ft distribution throughout the body, insulin resistnce nd ltered levels of triglycerides, cholesterol nd lipoproteins [1]. These chnges impct the helth of n individul s well s their qulity of life nd hve reduced the impct of nti-hiv therpy development [2,3]. HAART regimens with vrious combintions of protese inhibitors (PI), nucleoside reverse trnscriptse inhibitors (NRTI) nd nonnucleoside reverse trnscriptse inhibitors (NNRTI) hve been ssocited with HALS (e.g. [4-6] ). Previously we reported on the in-vitro effects of two PIs, ritonvir (RTV) nd tznvir (ATV) on predipocyte prolifertion nd dipogenesis [7]. In the present study, we report the effects of common first-line combintion regimens used in HIV tretment; efvirenz (EFV) + emtricitbine (FTC) + tenofovir (TDF), ATV + FTC + TDF nd ATV + RTV + FTC + TDF, s well s their individul components, on in-vitro predipocyte prolifertion nd differentition. MATERIALS AND METHODS Ptients nd study design Predipocytes were obtined from bdominl subcutneous ft tissue from helthy kidney donors undergoing nephrectomy. The smples were collected from 10 kidney donors (6 femles, 4 mles) who gve written informed consent. All prticipnts were HIVseronegtive, hd n verge ge of 37 ± 4 yers nd BMI of 29 ± 1 kg/m 2. Subjects were plced under stndrd generl nesthesi nd subcutneous ft tissue ws removed from the peri-umbilicl re during nephrectomy. The specimens were then immeditely plced in sterile Hnk s Buffered Slt Solution (HBSS) t ph 7.4 contining ntibiotics nd mphotericin. All ft smples were processed within one hour. Once isolted, predipocytes were tested in vitro for their bility to replicte nd differentite in the presence of different clsses of ntiretrovirl drugs, which were pplied individully or in combintion. Ft smples from ech donor were processed individully nd ech of the test conditions (drug combintions) were repeted with ech donor smple. The selected drug combintions re recommended ntiretrovirl regimens for (nïve) HIV ptients [8]. These included NNRTI-bsed regimen consisting of NNRTI (EFV) nd 2 NRTIs (TDF nd FTC) (i.e., EFV + TDF + FTC); nd PI-bsed regimen consisting of PI (ATV) nd 2 NRTIs (TDF nd FTC) (i.e., ATV + TDF + FTC). The PI-bsed combintion ws tested with or without the ddition of nother PI, RTV (i.e., ATV + RTV+ TDF + FTC), since this regimen is often recommended to boost the effects of other protese inhibitors [9]. The following drug concentrtions were used in ll the experiments: EFV, 20 µmol/l; FTC, 15 µmol/l; TDF, 1 µmol/l; ATV, 10 µmol/l; RTV, 2 µmol/l. These concentrtions re in the rnge of those observed in the plsm of ptients treted with the specific ntiretrovirl combintion regimens [10-12]. The effects of ntiretrovirl medictions were compred with control smples in which predipocytes were cultured nd stimulted to differentite in the bsence of ntiretrovirl medictions. 54 August 12, 2017 Volume 6 Issue 3

6 Jones E et l. HAART dysregultes humn dipogenesis Pre-dipocyte isoltion nd culture Predipocyte isoltion nd culture from subcutneous ft biopsies were previously described [7]. In brief, ft tissue ws digested with collgense (3 mg/ml, type Ⅱ, Worthington, Lkewood, NJ) to obtin stroml cells tht were then seprted from mture dipocytes by centrifugtion nd incubted in erythrocyte lysing buffer (154 mmol/l NH4Cl, 10 mmol/l K2HPO4, 1 mmol/l EDTA, ph 7.4) for 10 min t room temperture to eliminte red cells. Remining debris ws then removed by filtering cell suspension through 70 µm nylon filter nd then centrifuged. Pelleted predipocytes were plted in bsl medium consisting of DMEM/F-12 (Gibco, Crlsbd, CA) supplemented with 10% Fetl Clf Serum (FCS), 2 mmol/l glutmine, 100 IU/mL penicillin nd 100 µg/ml streptomycin (Gibco) nd incubted for h. After incubtion, ttched cells were extensively wshed with wrm PBS, removed from the pltes with trypsin, suspended nd counted. Predipocytes were then seeded t density of /cm 2. To ssess cell repliction, cultures were incubted in untreted growth medium for 48 h fter which the medium ws exchnged for fresh medium (control) or medium contining drugs. Cell numbers were ssessed over 72 h period. For the ssessment of differentition, cultures were grown to confluence nd differentition ws induced with serum-free medium (control) or the sme medium contining ntiretrovirl drugs s previously described [7,13]. Since some drugs (i.e., EFV) re tightly bound to plsm lbumin, 2 g/l bovine serum lbumin (Sigm, St. Loius, MO) ws dded to differentition medium in ll control nd treted groups. Medium ws replced every 72 h nd differentition ssessed fter 12 d. Assessment of predipocyte prolifertion The effect of ntiretrovirl drugs on predipocyte prolifertion ws ssessed by mesuring the cell number in cultures exposed to the drugs for 48 nd 72 h. Vible cell number ws ssessed with 3-[4,5-dimethylthizol- 2-yl]-2,5-diphenyl tetrzolium bromide bsed ssy, s previously described [7,14]. Since ll cultures were plted t the sme initil cell number, n increse or decrese in vible cells t 48 nd 72 h ws ssumed to represents chnge in the potentil of cells to increse in number, i.e., proliferte. Assessment of predipocyte differentition The differentition of predipocytes into mture dipocytes ws estimted fter 12 d by mesuring the ctivity of the lipogenic mrker enzyme glycerol-3- phosphte dehydrogense (GPDH) nd by quntifying the intrcellulr lipid ccumultion fter stining with Oil Red O. GPDH ssy: Determintion of GPDH ctivity ws bsed on the oxidtion of NADH nd reported s mu where 1 mu corresponded to the mount of enzyme needed to oxidize 1 nmol of NADH/min s reported previously [7]. Oil Red O stining: Cells fixed in 10% formldehyde solution were stined with Oil Red O, extrcted with isopropnol nd ssessed for bsorbnce t 500 nm [7]. Sttisticl nlysis Results re expressed s mens ± SEM. Differences between control nd treted groups nd mong the treted groups were nlyzed with rndomized block ANOVA with post-hoc Fisher s Lest Significnt Difference (LSD) test. P vlues < 0.05 were considered sttisticlly significnt. RESULTS The effect of nti-retrovirl drugs, individully nd in combintion, ws ssessed for two distinct spects of predipocyte metbolism; nmely the prolifertion of predipocytes nd the bility of predipocytes to differentite into dipocytes. Predipocyte prolifertion in the presence of NRTIs, NNRTIs nd PIs All individul ntiretrovirl drugs inhibited the prolifertion of predipocytes incubted in concentrtions of the drugs comprble to the levels seen in the plsm of treted ptients compred with untreted cells (control). This effect ws sttisticlly significnt t 72 h (Figure 1; P < 0.02). The inhibition of prolifertion in FTC-treted cells ws pprent even t 48 h of incubtion (P < 0.01). All drug combintions tested significntly suppressed predipocyte prolifertion. In the presence of EFV + FTC + TDF (NNRTI + NRTIs) predipocyte prolifertion ws inhibited by 14% nd 26% respectively fter 48 nd 72 h compred to controls. Similrly, therpeutic combintions with ATV + FTC + TDF (PI + NRTIs) showed reduction in predipocyte growth of 19 % t 48 h, nd of 30% t 72 h (Figure 1, P < 0.001). When RTV ws dded to ATV + FTC + TDF, s it is cliniclly known to boost other PIs, the inhibitory effect ws more noticeble, with suppression in prolifertion of 26% nd 37% respectively fter 48 nd 72 h compred to controls (Figure 1, P < 0.001). The inhibition of prolifertive ctivity of predipocytes in multi-drug combintions ws more severe thn the inhibition of prolifertion observed with the individul component drugs in some cses, but not ll. In the combintion EFV + FTC + TDF, suppression of prolifertion ws greter thn the suppression observed when EFV nd TDF were used individully (P < 0.02), but there ws not n dditionl effect when EFV + FTC + TDF combintion ws compred to FTC tretment lone (P = NS). Combining ATV + FTC + TDF or ATV + RTV + FTC + TDF incresed the inhibition of dipocyte prolifertion to greter extent thn tretment with the sme concentrtion of the individul drugs both t 48 (P < 0.05) nd 72 h (P < 0.01). 55 August 12, 2017 Volume 6 Issue 3

7 Jones E et l. HAART dysregultes humn dipogenesis A Prolifertion (% of control) B Prolifertion (% of control) Control EFV FTC TDF ATV RTV EFV ATV ATV FTC FTC RTV TDF TDF FTC TDF 0 Control EFV FTC TDF ATV RTV EFV ATV ATV FTC FTC RTV TDF TDF FTC TDF Figure 1 Pre-dipocyte prolifertion in the presence of individul drugs or drug combintions for 48 h (A) nd 72 h (B) (MTT test). The results re expressed s percent vlues of control cultures. Men ± SEM, n = 7. Significntly different from control, P EFV: Efvirenz; FTC: Emtricitbine; TDF: Tenofovir; ATV: Atznvir. A 100 B 0.30 GPDH ctivity (% of control) Oil Red O stining (bsorbnce) Control EFV FTC TDF ATV RTV EFV ATV ATV FTC FTC RTV TDF TDF FTC TDF 0.00 Control EFV FTC TDF ATV RTV EFV ATV ATV FTC FTC RTV TDF TDF FTC TDF Figure 2 Effect of individul drugs or drug combintions on differentition of humn predipocytes. Differentition ws ssessed fter 12 d by mesuring the glycerol-3-phosphte dehydrogense ctivity (A), nd Oil Red O stining (B). All results re expressed s percent vlues of control cultures. Men ± SEM, n = 9. Significntly different from control, P EFV: Efvirenz; FTC: Emtricitbine; TDF: Tenofovir; ATV: Atznvir. Of the three multi-drug regimens, the drug combintion ATV + RTV + FTC + TDF hd more suppressive effect on prolifertion of predipocytes thn the other multi-drug regimens t 48 h (P < 0.02). At 72 h, the combintion, ATV + RTV + FTC + TDF, ws not sttisticlly significntly different from ATV + FTC + TDF but ws significntly different from EFV + FTC + TDF (P = 0.05). Predipocyte differentition in the presence of NRTIs, NNRTIs nd PIs Predipocyte differentition, in the presence of ntiretrovirl drugs, ws ssessed by 2 different techniques, one involved mesuring the enzymtic ctivity of GPDH nd the other involved quntifying intrcellulr lipid ccumultion fter stining with Oil Red O. The two techniques produced comprble results (Figure 2). EFV hd profound inhibitory effect on predipocyte differentition (Figure 2). Both GPDH ctivity nd lipid ccumultion were gretly reduced in cells treted with EFV compred to controls (Figure 2, P < 0.001). Of the other nti-retrovirl drugs tested, only TDF ppered to hve n effect on intrcellulr lipid ccumultion, which tended to be lower when cells were treted with TDF (P = 0.06). Predipocyte differentition in the presence of remining individul drugs did not differ from controls (Figure 2). Figure 2 demonstrtes tht, predipocyte differentition ws significntly reduced when the ntiretrovirl drugs were used in combintion compred to untreted cultures. Compred to control cells, the EFV + FTC + TDF (NNRTI + NRTIs) combintion showed the most suppressive effect on differentition with GPDH ctivity nd lipid ccumultion 64% nd 39% lower respectively (Figure 2, P < 0.001). Combining NRTIs with PI (ATV + FTC + TDF) inhibited GPDH ctivity by 29% nd lipid ccumultion by 19% compred to controls (Figure 2, P < 0.01). This effect ws slightly 56 August 12, 2017 Volume 6 Issue 3

8 Jones E et l. HAART dysregultes humn dipogenesis greter when boosting mount of RTV ws dded (ATV + FTC + TDF + RTV, Figure 2, P < 0.001). The inhibitory effect of multi-drug combintion NNRTI nd NRTI (EFV + FTC + TDF) ws lso compred to the suppression in differentition observed with the nti-retrovirl medictions individully. Suppression with the combintion resulted in greter reduction in predipocyte differentition thn either FTC or TDF lone (P < 0.001). However, suppression of differentition with the combintion of EFV + FTC + TDF ws comprble in mgnitude to tretment with EFV lone suggesting EFV ws ccountble for most of the reduction in differentition observed with the combintion regimen (Figure 2). The two multi-drug regimens contining PI + NRTI were lso exmined reltive to the incubtions with the individul medictions. The multi-drug combintions contining PIs hd greter inhibitory effects on differentition thn tretment with the sme concentrtion of ech individul drug (Figure 2, P < 0.003), with the exception of TDF. The multi-drug regimens hve lso been compred with ech other. The combintion with NNRT nd NRTI (EFV, FTC, TDF) reduced differentition to greter extent thn either of the two regimens with PI + NRTI, whether ssessed s either GPDH ctivity (P < 0.001) or intrcellulr lipid ccumultion (P < 0.02). DISCUSSION There is no doubt tht the etiology of HIV/HAARTssocited lipodystrophy syndrome is multi-fctoril, but ntiretrovirl medictions contribute to the condition. This in vitro technique, with primry cultures of predipocytes isolted from helthy subjects, provides wy of ssessing effects of single nd combintion drug regimens on predipocyte prolifertion nd differentition; nd consequently, on the potentil of drug regimens to contribute to HALS. The ntiretrovirl medictions currently in use hve profound effects on both predipocyte prolifertion nd differentition. All of the ntiretrovirl gents tested inhibited predipocyte prolifertion. Individully, the NRTI, FTC, hd more pronounced effect on predipocyte prolifertion thn the NRTI, TDF; the NNRTI, EFV; or the PIs ATV or RTV (Figure 1). While in generl combintions of ntiretrovirl drugs were more toxic thn the individul drugs, this ws not true for combintions contining FTC. The ddition of TDF (nother NRTI) nd EFV ( NNRTI) to emtricitbine did not produce ny greter toxicity thn ws observed with EFV lone. However, multidrug regimens contining PIs in combintion with NRTI (ATV + FTC + TDF nd ATV + RTV + FTC + TDF) resulted in further suppression of prolifertion. Hving previously demonstrted tht RTV does not suppress predipocyte prolifertion t levels comprble to those used for boosting [7], this study indictes tht dding RTV to combintion of ATV, TDF nd FTC does increse toxicity (Figure 1). Clerly, the toxicity of individul ntiretrovirls cn be ffected by concurrent ntiretrovirl dministrtion. In contrst to predipocyte prolifertion, it is the NNRT EFV tht hs the most profound effect on predipocyte differentition, n effect tht hs been reported previously [15,16]. Combining EFV with NRTIs (EFV + FTC + TDF) does not result in ny greter suppression. Regimens contining PIs nd NRTIs (ATV + FTC + TDF nd ATV + RTV + FTC + TDF) re not s toxic s those contining EFV. However, the multi-drug combintions contining PIs suppressed differentition to greter extent thn the use of ny drug individully. This study highlights the importnce of ssessing both the effects on the prolifertion of predipocytes nd the differentition of predipocytes into mture dipocytes since multi-drug regimens ffect them differently. In conclusion, ntiretrovirl medictions ffect not only the differentition of predipocyte into mture dipocytes, these drugs lso ffect the prolifertion of predipocytes nd cn, therefore, impct on the number of predipocytes tht re vilble. While FTC hs the most profound effect on predipocyte prolifertion, it is EFV tht hs the gretest impct on differentition. Combintions of ntiretrovirl medictions, which hve no impct when used individully, increse the toxicity for predipocytes. COMMENTS Bckground Highly ctive ntiretrovirl therpy (HAART) regimens with vrious combintions of protese inhibitors (PI), nucleoside reverse trnscriptse inhibitors (NRTI) nd nonnucleoside reverse trnscriptse inhibitors (NNRTI) hve long been linked to HAART-ssocited lipodystrophy syndrome (HALS). Once HALS is mnifest, it is difficult to reverse. There is need to develop wys of ssessing drug combintions for the potentil to contribute to HALS. Reserch frontiers The effect of individul nti-retrovirl drugs hve been studied in vitro, but this is the first report of the effect of drug combintions ssessed t cliniclly relevnt concentrtions. The differentil effects on predipocyte prolifertion nd differentition were lso ssessed, providing importnt mechnistic informtion. Innovtions nd brekthroughs The study demonstrtes tht replcing ritonvir-bsed regimens with the cliniclly more cceptble protese inhibitor, tznvir, does not eliminte the potentil for toxic effects on dipose tissue. In ddition, dding ritonvir t boosting levels to regimens contining nucleoside reverse trnscriptse inhibitors nd non-reverse trnscriptse inhibitors lso increses the lipo-toxic potentil of these ntiretrovirl combintions. Applictions Although combintion ntiretrovirl therpy is cliniclly more efficcious thn single drug regimens, the combintion regimens lso hve the potentil to contribute to dipose tissue toxicity through effects of predipocyte repliction nd differentition. The study lso illustrtes the vlue of n in vitro system for screening drug combintions for potentil dipose tissue toxicity. Terminology HALS is condition tht is chrcterized by loss of subcutneous ft, prticulrly in the fce, buttocks, rms nd legs. Antiretrovirl therpy typiclly includes combintion of drugs with different mechnisms of ction including NRTIs, NNRTIs, nd PIs. These drug clsses hve the potentil for differentil effects on dipose tissue nd the effect of combintion therpy my be greter thn the individul drugs lone. This study lso investigted the mechnisms by which ntiretrovirl drugs cn contribute to the loss of dipose tissue; 57 August 12, 2017 Volume 6 Issue 3

9 Jones E et l. HAART dysregultes humn dipogenesis n inbility of predipocytes to proliferte reduces the potentil number of precursor cells for the formtion of dipose tissue, the inbility of pre-dipocytes to differentition reduces the formtion of dipose tissue by rresting precursor cells in n undifferentited stte. Peer-review This mnuscript is worth publishing, reporting the synergistic or coopertive effects of nti-hiv gents on inhibiting dipogenesis by performing in vitro experiments using humn mterils. It will help us to recognize the importnce to tke extr cution in executing HAART. REFERENCES 1 Gurldi G, Stentrelli C, Zon S, Sntoro A. HIV-ssocited lipodystrophy: impct of ntiretrovirl therpy. Drugs 2013; 73: [PMID: DOI: /s ] 2 Lke JE, Wohl D, Scherzer R, Grunfeld C, Tien PC, Sidney S, Currier JS. Regionl ft deposition nd crdiovsculr risk in HIV infection: the FRAM study. AIDS Cre 2011; 23: [PMID: DOI: / ] 3 Quints RC, de Frnç ER, de Petribú KC, Ximenes RA, Quints LF, Cvlcnti EL, Kitmur MA, Mglhães KA, Piv KC, Filho DB. Tretment of fcil lipotrophy with polymethylmethcrylte mong ptients with humn immunodeficiency virus/cquired immunodeficiency syndrome (HIV/AIDS): impct on the qulity of life. Int J Dermtol 2014; 53: [PMID: DOI: /ijd.12400] 4 Domingo P, Estrd V, López-Aldeguer J, Villroy F, Mrtínez E. Ft redistribution syndromes ssocited with HIV-1 infection nd combintion ntiretrovirl therpy. AIDS Rev 2012; 14: [PMID: ] 5 Mrtin A, Moore CL, Mllon PW, Hoy JF, Emery S, Belloso WH, Phnuphk P, Ferret S, Cooper DA, Boyd MA; Second-Line Study Tem. HIV lipodystrophy in prticipnts rndomised to lopinvir/ ritonvir (LPV/r) +2-3 nucleoside/nucleotide reverse trnscriptse inhibitors (N(t)RTI) or LPV/r + rltegrvir s second-line ntiretrovirl therpy. PLoS One 2013; 8: e77138 [PMID: DOI: / journl.pone ] 6 Minmi R, Ymmoto M, Tkhm S, Ando H, Miymur T, Suemtsu E. Comprison of the influence of four clsses of HIV ntiretrovirls on dipogenic differentition: the miniml effect of rltegrvir nd tznvir. J Infect Chemother 2011; 17: [PMID: DOI: /s ] 7 Cso G, Milev I, McNurln MA, Mynrcik DC, Drrs F, Gelto MC. Effect of ritonvir nd tznvir on humn subcutneous predipocyte prolifertion nd differentition. Antivirl Res 2010; 86: [PMID: ] 8 Pnel on Antiretrovirl Guidelines for Adults nd Adolescents. Guidelines for the use of ntiretrovirl gents in HIV-1-infected dults nd dolescents. Deprtment of Helth nd Humn Services. Section ccessed on Mrch 18, 2015 (Tble 6). Avilble from: URL: idsinfo.nih.gov/contentfiles/lvguidelines/adultndadolescentgl. pdf 9 Zeldin RK, Petruschke RA. Phrmcologicl nd therpeutic properties of ritonvir-boosted protese inhibitor therpy in HIVinfected ptients. J Antimicrob Chemother 2004; 53: 4-9 [PMID: DOI: /jc/dkh029] 10 Cly PG, Tylor TA, Glros AG, McRe M, Willims C, McCndless D, Oelklus M. One pill, once dily : wht clinicins need to know bout Atripltrde mrk. Ther Clin Risk Mng 2008; 4: [PMID: ] 11 Kiser JJ, Fletcher CV, Flynn PM, Cunninghm CK, Wilson CM, Kpoginnis BG, Mjor-Wilson H, Vini RM, Liu NX, Muenz LR, Hrris DR, Hvens PL; Adolescent Trils Network for HIV/AIDS Interventions. Phrmcokinetics of ntiretrovirl regimens contining tenofovir disoproxil fumrte nd tznvir-ritonvir in dolescents nd young dults with humn immunodeficiency virus infection. Antimicrob Agents Chemother 2008; 52: [PMID: DOI: /AAC ] 12 von Hentig N, Duer B, Hberl A, Kluke S, Lutz T, Stszewski S, Hrder S. Tenofovir comediction does not impir the stedy-stte phrmcokinetics of ritonvir-boosted tznvir in HIV-1-infected dults. Eur J Clin Phrmcol 2007; 63: [PMID: DOI: /s y] 13 Tchkoni T, Giorgdze N, Pirtskhlv T, Tchouklov Y, Krginnides I, Forse RA, DePonte M, Stevenson M, Guo W, Hn J, Wlog G, Lsh TL, Jensen MD, Kirklnd JL. Ft depot origin ffects dipogenesis in primry cultured nd cloned humn predipocytes. Am J Physiol Regul Integr Comp Physiol 2002; 282: R1286-R1296 [PMID: ] 14 Cso G, McNurln MA, McMilln ND, Eremin O, Grlick PJ. Tumour cell growth in culture: dependence on rginine. Clin Sci (Lond) 2004; 107: [PMID: ] 15 El Hdri K, Glorin M, Monsempes C, Dieudonné MN, Pecquery R, Giudicelli Y, Andreni M, Dugil I, Fève B. In vitro suppression of the lipogenic pthwy by the nonnucleoside reverse trnscriptse inhibitor efvirenz in 3T3 nd humn predipocytes or dipocytes. J Biol Chem 2004; 279: [PMID: DOI: /jbc. M ] 16 Gllego-Escuredo JM, Del Mr Gutierrez M, Diz-Delfin J, Domingo JC, Mteo MG, Domingo P, Girlt M, Villrroy F. Differentil effects of efvirenz nd lopinvir/ritonvir on humn dipocyte differentition, gene expression nd relese of dipokines nd pro-inflmmtory cytokines. Curr HIV Res 2010; 8: [PMID: ] P- Reviewer: Seki K, Shen WJ S- Editor: Song XX L- Editor: A E- Editor: Lu YJ 58 August 12, 2017 Volume 6 Issue 3

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