Marys Medicine

Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo

See discussions, stats, and author profiles for this publication at: ARTICLE in JOURNAL OF INVESTIGATIVE DERMATOLOGY · DECEMBER 2014
Impact Factor: 6.37 · DOI: 10.1038/jid.2014.529 · Source: PubMed
Available from: Hamidreza Pazoki-Toroudi Retrieved on: 28 July 2015 Simvastatin Prevents and Reverses Depigmentation ina Mouse Model of VitiligoPriti Agarwal1, Mehdi Rashighi1, Kingsley I. Essien1, Jillian M. Richmond1, Louise Randall2,Hamidreza Pazoki-Toroudi3, Christopher A. Hunter4 and John E. Harris1 Vitiligo is a common autoimmune disease of the skin that results in disfiguring white spots. There are no Foodand Drug Administration (FDA)-approved treatments, and current treatments are time-consuming, expensive, andof low efficacy. We sought to identify new treatments for vitiligo, and first considered repurposed medicationsbecause of the availability of safety data and expedited regulatory approval. We previously reported that the IFN-g-induced chemokine CXCL10 is expressed in lesional skin from vitiligo patients, and that it is critical for theprogression and maintenance of depigmentation in our mouse model of vitiligo. We hypothesized that targetingIFN-g signaling might be an effective new treatment strategy. Activation of signal transducer and activator oftranscription 1 (STAT1) is required for IFN-g signaling and recent studies revealed that simvastatin, an FDA-approved cholesterol-lowering medication, inhibited STAT1 activation in vitro. Therefore, we hypothesized thatsimvastatin may be an effective treatment for vitiligo. We found that simvastatin both prevented and reverseddepigmentation in our mouse model of vitiligo, and reduced the number of infiltrating autoreactive CD8 þ T cellsin the skin. Treatment of melanocyte-specific, CD8 þ T cells in vitro decreased proliferation and IFN-g production,suggesting additional effects of simvastatin directly on T cells. Based on these data, simvastatin may be a safe,targeted treatment option for patients with vitiligo.
Journal of Investigative Dermatology advance online publication, 22 January 2015; doi: and therefore unknown, although general, nontargeted Vitiligo is a common, disfiguring autoimmune disease of immunosuppression is likely, and no treatments are effective the skin. Psychological consequences are severe, leading to for all patients. An orally available systemic treatment for depression, anxiety, sleep disturbances, sexual dysfunction, vitiligo would be a helpful addition to currently limited feelings of discrimination, and even suicidal thoughts/ treatment options.
attempts. These emotional disturbances are comparable to Vitiligo pathogenesis incorporates both intrinsic defects those suffering from psoriasis and eczema ( within melanocytes that activate the cellular stress response . The estimated pre- and autoimmune mechanisms that target these cells valence of disease is 0.5–1% of the total population , or 1.5–3 million people in the United States and 35–70 million in the world. The estimated direct health-care cost burden of vitiligo in the United States is $175 million each year ), a particularly high cost ). Patients with vitiligo have increased numbers of considering that there are few effective, and no systemic, autoreactive, melanocyte-specific CD8 þ T cells in the skin treatments. The mechanisms of current treatments are untested and blood, and functional studies using human skin ex vivosupport CD8 þ T cells as critical for depigmentation . Recent advances in Division of Dermatology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA; 2Department understanding the key cytokines that promote psoriasis and of Medicine, The University of Melbourne, Melbourne, Victoria, Australia; related autoimmune diseases have resulted in treatments with 3Department of Physiology, Iran University of Medical Sciences, Tehran, Iran excellent efficacy and safety profiles, significantly improving and 4Department of Pathobiology, University of Pennsylvania School of patients' quality of life . However, the Veterinary Medicine, Philadelphia, Pennsylvania, USA cytokines that drive vitiligo pathogenesis are not shared with Correspondence: John E. Harris, Division of Dermatology, Department ofMedicine, University of Massachusetts Medical School, LRB 225, 364 these diseases , and cytokine-targeted Plantation Street, Worcester, Massachusetts 01605, USA.
treatments for psoriasis have been ineffective for vitiligo Abbreviations: FDA, Food and Drug Administration; HMG-CoA, 3-hydroxy-3- We recently discovered that the IFN-g-chemokine axis is methylglutaryl-coenzyme A; i.p., intraperitoneal; RT–PCR, reverse active in human vitiligo, and functional studies in a vitiligo transcriptase–PCR; STAT1, signal transducer and activator of transcription 1 mouse model that we developed revealed that it is critical for Received 26 September 2014; revised 30 October 2014; accepted 17November 2014; accepted article preview online 18 December 2014 both progression and maintenance of vitiligo, implicating this & 2015 The Society for Investigative Dermatology P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo pathway as a potential target for new treatments described previously . Briefly, KRT14- . IFN-g signaling requires the Kitl*4XTG2Bjl (Krt14-Kitl*) mice express a noncleavable, activation of signal transducer and activator of transcription membrane-bound form of Kit ligand under the keratin 14 1 (STAT1) to induce the transcription of downstream gene promoter, and therefore expression is limited to the epidermis.
targets , and humans deficient As a consequence, these mice have black skin and black hair, in STAT1 have severely impaired IFN-driven responses without other sequelae (. Sublethally irradiated (500 rad) Krt14-Kitl* mice were used as hosts with selective STAT1 deficiency have impaired IFN-induced for the adoptive transfer of premelanosome protein (PMEL)- signaling and target gene expression , specific TCR-transgenic CD8 þ T cells (PMELs) that recognize although some IFN-induced genes may be STAT1 -inde- both mouse and human PMEL (also known as gp100), a pendent . We reasoned that inhibitors melanocyte-specific differentiation antigen ( of STAT1 activation might also interfere with IFN-g signaling, ). PMELs were isolated from the spleen of donors, and may prove to be effective treatments for vitiligo.
column-purified using negative selection, and 106 cells were A recent study reported that simvastatin, an HMG-CoA transferred to hosts intravenously. Mice were then infected (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitor intraperitoneally (i.p.) with 106 plaque-forming units of recom- approved by the Food and Drug Administration (FDA) for the binant vaccinia virus that expresses human PMEL (rVV- treatment of hypercholesterolemia, inhibited IFN-g-induced hPMEL), a potent antigenic stimulus for PMELs that results in STAT1 activation in vitro. This effect was specific for the their activation and expansion in vivo .
HMG-CoA reductase pathway rather than an off-target effect, We have found that this treatment results in depigmentation of as it was rescued by the addition of mevalonate, a pathway the skin 5–7 weeks later, and mice are assigned a Vitiligo intermediate downstream of HMG-CoA reductase but up- Score based on the extent of depigmentation on their ears, stream of cholesterol (Interestingly, high- nose, feet, and tail .
dose simvastatin treatment of a patient with both vitiligo and To identify the optimal treatment dose of simvastatin we hypercholesterolemia resulted in rapid repigmentation of his performed a dose response in our model, testing three skin, supporting simvastatin treatment as a potential therapy for increasing doses (0.2, 0.4, and 0.8 mg), with the highest dose vitiligo . Therefore, we tested simvastatin as consistent with treatment in a mouse model of rheumatoid a treatment in our mouse model of vitiligo. We found that arthritis (). Beginning 1 day after vitiligo simvastatin both prevented vitiligo and reversed depigmen- induction, we began treatment of hosts with simvastatin (i.p.
tation in mice with established, widespread disease. It had 3  weekly) or vehicle control until scoring them 5 weeks multiple effects on melanocyte-specific T cells, not only later. We found that simvastatin treatment reduced depig- reducing their numbers in the skin, but also reducing their mentation compared with vehicle-treated controls. There was proliferation and production of IFN-g, suggesting that multiple a strong correlation between the dose and clinical response, pleiotropic effects of simvastatin may contribute to its positive with 0.8 mg having the most significant response .
Flow cytometry analysis revealed a similar correlationbetween the dose and the reduction of total number of PMELs present in the ear skin ), and we selected 0.8 mg as Simvastatin treatment reduces the extent of depigmentation in the optimal dose for repeat experiments (.
Next, we analyzed the ear skin, tail skin, skin-draining In order to test simvastatin as a potential treatment for vitiligo, lymph nodes, spleen, and blood by flow cytometry to quantify we induced vitiligo in mice with black skin and black hair as the total number of PMELs present at those locations in Med dose High dose Low dose Med dose High dose Low dose Med dose High dose Figure 1. Simvastatin dose correlates with clinical response and reduction of melanocyte-specific CD8 þ T cells in the ear skin. Vitiligo was induced and micewere treated with 0.2, 0.4, or 0.8 mg of simvastatin or vehicle control (No Tx) three times weekly for 5 weeks. (a) There is a strong correlation between thesimvastatin dose and clinical response, with 0.8 mg having the most significant effect. P-value calculated by analysis of variance (ANOVA) is shown. Post test forlinear trend was also significant (P ¼ 0.0014). Med, medium. (b) A similar correlation was found between simvastatin dose and reduction of melanocyte-specificCD8 þ T cells (PMELs) in the ear skin, but not skin-draining lymph nodes (LNs). P-value calculated by ANOVA is shown. Post test for linear trend was alsosignificant (P ¼ 0.0003). NS, not significant; *Po0.05; **Po0.01; and ***Po0.001.
Journal of Investigative Dermatology (2015), Volume 00

P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo simvastatin-treated versus vehicle-treated controls. We found toward reduction, but PMEL numbers at other sites were not significantly reduced numbers of PMELs in the skin in simvas- affected, including the dermis . Antibody tatin-treated hosts compared with controls, but no change in neutralization of IFN-g had a similar effect ). These the number of PMELs at other sites These results suggest that simvastatin acutely affects PMEL homing or observations are consistent with our previous studies blocking retention directly within the skin, and may inhibit IFN-g IFN-g in this model , and suggest that simvastatin could be interfering with IFN-g signaling.
Our previous observations revealed that IFN-g is required for PMEL migration to the skin and progression of disease, as Acute treatment with simvastatin reduces autoreactive T cells in well as the maintenance of depigmentation. In addition, it the skin, but does not affect CXCL10 expression appeared that IFN-g-induced CXCL10 may specifically promote Long-term, preventative treatment with simvastatin as above PMEL migration within the skin to the epidermis could affect PMELs in multiple ways, including reducing the In order to determine whether CXCL10 expression activation or proliferation of PMELs early during priming, is directly inhibited by simvastatin, we analyzed CXCL10 migration of PMELs into the skin, or altering their function expression in the skin by reverse transcriptase–PCR (RT-PCR) after their recruitment to the skin. In order to determine in mice treated with vehicle, one dose of simvastatin, or three whether simvastatin affects T cells directly within the skin, daily doses of simvastatin. We found that despite a reduction we induced vitiligo in hosts as before and allowed them to of PMELs in the epidermis following treatment with simvasta- develop depigmentation for 5 weeks. Next, we treated the tin, CXCL10 expression was unchanged .
hosts with 1 dose of simvastatin, 3 daily doses of simvastatin,or vehicle control and analyzed the numbers of PMELs within Simvastatin reduces T-cell proliferation and effector function the skin by flow cytometry. We found that three doses of simvastatin significantly reduced the number of PMELs within Because simvastatin did not affect CXCL10 expression in the the ear epidermis, and even a single dose suggested a trend skin, we tested whether it altered the activation or effector Figure 2. Simvastatin prevents depigmentation and melanocyte-specific CD8 þ T-cell accumulation in the skin despite no global effect on T-cell frequency.
Vitiligo was induced and mice were treated with simvastatin (0.8 mg) or vehicle control three times weekly for 5 weeks. (a) Representative mouse ears, noses,footpads, and tails from each group are shown. (b) The effects of simvastatin on vitiligo score and (c) the total numbers of melanocyte-specific CD8 þ T cells(PMELs) in the ear skin, tail skin, lymph nodes (LNs), spleen and blood. NS, not significant. (d) Representative flow cytometry plots are shown.

P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo Vehicle 1 Dose3 Doses Vehicle 1 Dose3 Doses Vehicle 1 Dose3 Doses Relative fold expression Vehicle 1 Dose 3 Doses Vehicle 1 Dose3 Doses Vehicle 1 Dose3 Doses Vehicle 1 Dose 3 Doses Figure 3. Acute treatment with simvastatin reduces T-cell numbers in the epidermis. Vitiligo was induced and mice were treated with one dose or threedaily doses of simvastatin (0.8 mg) or vehicle control 5 weeks after vitiligo induction. (a) Representative flow plots show a reduced number of melanocyte-specificCD8 þ T cells (PMELs) in the ear epidermis of the mice that received one or three daily doses of simvastatin when compared with vehicle. (b) The effectsof acute treatment with simvastatin on the total number of PMELs on ear skin, tail skin, lymph nodes (LNs), and spleen. NS, not significant. (c) Neutralization ofIFN-g has a similar effect on ear skin. (d) The relative expression of CXCL10 was unaffected in the ear skin of mice treated with one or three daily doses ofsimvastatin.
function of T cells directly. PMELs were isolated from the We next determined whether these direct effects of simvas- spleen of donors and column-purified for CD8 T cells using tatin on T cells were through inhibition of the HMG-CoA negative selection. In order to track proliferation after activa- reductase pathway or through an off-target effect. HMG-CoA tion, purified PMELs were labeled with carboxyfluorescein reductase catalyzes the formation of mevalonate, an inter- succinimidyl ester (CFSE). PMELs were then activated for 3 mediate in the cholesterol synthesis pathway days in vitro using anti-CD3 and anti-CD8 antibodies, and ). In order to test whether mevalonate supplementation treated with either 1, 5, 10, or 100 mM simvastatin or vehicle could rescue proliferation and IFN-g production by PMELs in control. We found that treatment with simvastatin reduced the presence of simvastatin, we supplemented simvastatin- both the proliferation and IFN-g production by PMELs treated PMEL cultures with 1 mM mevalonate or vehicle , suggesting that simvastatin may directly control. We found that mevalonate rescued both prolife- affect T-cell function in vitiligo, rather than indirectly by ration and IFN-g production by PMELs, suggesting that direct decreasing CXCL10.
inhibition of the HMG-CoA reductase pathway, rather than an Journal of Investigative Dermatology (2015), Volume 00 P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo Cells with CFSE dilution No Mevalonate1 mM Mevalonate No Simvastatin μM No Simvastatin μM 5 μM Simvastatin 10 μM Simvastatin 5 μM Simvastatin 10 μM Simvastatin Figure 4. Simvastatin inhibits both the proliferation and IFN-c production of melanocyte-specific CD8 þ T cells in vitro through inhibition of the HMG-CoA(3-hydroxy-3-methylglutaryl-coenzyme A) reductase pathway. (a, b) Melanocyte-specific CD8 þ T cells (PMELs) were isolated, labeled with carboxyfluoresceinsuccinimidyl ester (CFSE), and activated for 3 days in vitro using anti-CD3 and anti-CD8 antibodies in the presence of 1, 5, 10, or 100 mM simvastatin or vehiclecontrol. PMELs were then stained for intracellular IFN-g and analyzed for (a) proliferation or (b) IFN-g production by flow cytometry. (c, d) Simvastatin-treatedPMEL cultures were supplemented with 1 mM mevalonate or vehicle control and CD8 þ T-cell (c) proliferation and (d) IFN-g production were analyzed by flowcytometry. Representative (e) histograms for CFSE dilution and (f) dot plots for IFN-g. Blue represents control unstimulated samples.
off-target effect, was responsible for reduced proliferation and their tails were randomized into two treatment groups, either function of PMELs in the presence of simvastatin with simvastatin 0.8 mg three times weekly or vehicle controlfor a total of 4 to 6 weeks. Photographs of the tails before and Simvastatin reverses established vitiligo after treatment were analyzed using ImageJ to calculate the Because simvastatin prevented depigmentation in our mouse percent pigmentation of the tail, and therefore the degree of model and directly reduced the numbers of PMELs in the skin repigmentation, after treatment. Despite no effect on the total in vivo as well as the function of PMELs in vitro, we tested number of melanocyte-specific CD8 þ T cells present in the whether simvastatin could also reverse established depigmen- skin, we found that there was a significant increase in tation in our mouse model of vitiligo. Mice were induced with pigmentation in the group treated with simvastatin compared vitiligo as above and allowed to depigment and then stabilize with the control group, as evidenced by the newly appearing for 10 to 12 weeks. Those with 450% depigmentation on macules of pigment around the hair follicles (, P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo ), that STAT1 activation is required for IFN-g signaling (), that simvastatin inhibits IFN-g-induced STAT1 activation in vitro ; , and that simvastatin reversed depigmentation in a patient with vitiligo .
We hypothesized that the mechanism of action of simvastatin in vitiligo was to inhibit IFN-g-induced CXCL10 production in the skin, and therefore interfere with proper homing of % Tail pigment change autoreactive T cells to the epidermis. However, despite itsclear efficacy in our mouse model of vitiligo, its mechanism of action does not appear to be straightforward. This is consistent with multiple previous studies showing pleiotropic effects of statins on various T cell–mediated autoimmune diseases Simvastatin did indeed prevent the migration of melano- cyte-specific CD8 þ T cells to the skin during vitiligo progres- sion while not decreasing their viability, as their numbers were unaffected in skin-draining lymph nodes, spleen, and blood.
These effects are similar to what we observed in our model % Tail pigment 20 when we neutralized IFN-g ). Short-term treatment with simvastatin reduced the number of PMELs within the ear epidermis but not tail, and this may be because of the difference in their thickness. The epidermis of the tail is thick, whereas the epidermis of the ear is thinner. CD8 þ T cells, which drive melanocyte destruction in humans and ourmouse model, have been reported to express adhesionmolecules upon entry into the epidermis that helps to retain them there. One possibility is that the thicker epidermis of thetail with more adhesion molecules is better able to retain thePMELs than that of the thinner ear epidermis. Alternatively, simvastatin may have better penetration of the thinnerepidermis of the ear compared with the tail, although highlipophilicity of simvastatin makes this less likely.
Despite the ability of simvastatin to reduce the number of PMELs in the epidermis, we found that CXCL10 expressionwas not reduced in the skin after short-term treatment, Figure 5. Simvastatin reverses established vitiligo. Mice with extensive suggesting that this effect of simvastatin was independent of depigmentation on the tail (450%) were treated with simvastatin (0.8 mg) CXCL10. We then found that simvastatin reduced T-cell or vehicle control three times weekly beginning 12 weeks after vitiligo proliferation and IFN-g production in vitro. This is consistent induction for a total of 4 to 6 weeks. Photographs of each tail before and aftertreatment were analyzed using ImageJ software to calculate the amount of with a study that reported decreased superantigen-induced repigmentation. (a) Simvastatin treatment in established vitiligo did not IFN-g production by human CD4 þ T cells ex vivo isolated significantly affect total number of melanocyte-specific CD8 þ T cells (PMELs) from subjects treated with simvastatin compared with their in skin. (b) The mean percent change in pigmentation from baseline was responses before treatment ), suggesting  3.1% and 8.4% for the mice treated with phosphate-buffered saline (PBS) or that it has additional effects on T cells. STAT1 is also simvastatin, respectively. (c) Paired t-test showed a significant increase in phosphorylated by IL-12 , a cytokine percent tail pigmentation only in mice treated with simvastatin. (d) Arepresentative tail from each group before and after treatment is shown.
that induces IFN-g production, and certain STAT1 mutationsin humans result in decreased IFN-g production after IL-12stimulation (, suggesting thatSTAT1 inhibition by simvastatin may have effects upstream of providing a rationale for clinical studies in humans to test IFN-g production as well. Alternatively, inhibition of HMG- simvastatin as a potential treatment for vitiligo.
CoA reductase may affect signaling mediators other thanSTAT1 that are required for T-cell proliferation and/or IFN-g production. Interestingly, despite reversing vitiligo, simvastatin Here we report the ability of simvastatin to both prevent and did not affect the number of PMELs in the skin in reverse established depigmentation in a mouse model of established disease, suggesting that simvastatin applies this vitiligo. This is consistent with previous observations that the effect primarily by mechanisms other than recruitment of IFN-g signaling pathway is critical for both the progression and autoreactive T cells. Previous studies have shown that statins maintenance of depigmentation in our mouse model could have multiple additional effects on T cells, in addition to Journal of Investigative Dermatology (2015), Volume 00 P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo their ability to influence T-cell recruitment and proliferation.
procedures were approved by the University of Massachusetts Consistent with this, we found a direct effect on IFN-g Medical School Institutional Animal Care and Use Committee.
production after stimulation in vitro, and thus hypothesizethat simvastatin inhibits the activation or effector status of Simvastatin preparation PMELs that remain in the skin to promote repigmentation.
Simvastatin (Sigma-Aldrich, St Louis, MO) was activated before use as Further experiments are needed to elucidate the exact described previously ). Briefly, 12 mg of mechanisms by which simvastatin acts to reverse vitiligo in simvastatin was dissolved in 300 ml of ethanol and then 450 ml of our mouse model.
0.1 N NaOH was added to the solution and subsequently incubated The dose of simvastatin used for our in vivo studies in mice at 50 1C for 2 hours. The pH was then brought to 7.0 by adding HCl, (up to 40 mg kg  1) is much higher than what is used in and the final concentration of the stock solution was adjusted to humans (up to 80 mg day  1, B1 mg kg 1). However, high 4 mg ml  1 and kept at 4 1C.
doses are required for treatment of rodents because of theirrapid upregulation of HMG-CoA reductase in response to Induction of vitiligo and treatment of mice treatment with statins ). The optimal dose we Vitiligo was induced through adoptive transfer of PMEL CD8 þ T cells identified in our mouse model is consistent with established as described previously (. Briefly, PMEL CD8 þ active doses in rodents T cells were isolated from the spleens of PMEL TCR-transgenic mice , as well as doses tested in a mouse through negative selection on microbeads (Miltenyi Biotec, Auburn, model of rheumatoid arthritis (. Notably, CA) according to the manufacturer's instructions. Purified CD8 þ the one vitiligo patient who reportedly responded to sim- T cells (1  106) were injected intravenously into sublethally vastatin clinically was on the highest FDA-approved dose of irradiated (500 rad 1 day before transfer) Krt14-Kitl* hosts (12 to simvastatin, 80 mg per day .
16 weeks of age). Recipient mice also received intraperitoneal Statins are metabolized and concentrate in the liver, result- injection of 1  106 plaque-forming units of rVV-hPMEL (N Restifo, ing in low levels circulating in the plasma and through tissues National Cancer Institute, NIH, Bethesda, MD) on the same day of Therefore, high doses may be transfer. Treatment with simvastatin to prevent vitiligo was performed required for efficacy in humans if the target tissue is the skin or by i.p. injection of the drug three times weekly for the duration of 5 blood. However, the 80 mg dose of simvastatin increases the weeks. Control mice received either no treatment or were treated risk of myopathy and rhabdomyolysis that led to removal of with an equal volume of phosphate-buffered saline. Vitiligo score was this dose by the FDA (. Whether simvastatin objectively quantified by an observer blinded to the experimental will be required for successful treatment of vitiligo patients, or groups, using a point scale based on the extent of depigmentation at whether other statins with fewer side effects could be effective, four easily visible locations, including the ears, nose, rear footpads, is unknown. Simvastatin is highly lipophilic ( and tails as described previously (. Each location , and this may promote its accumulation in was examined, and the extent of depigmentation was estimated as a multiple tissues other than the liver, including the skin. This percentage of the anatomic site; both left and right ears and may contribute to its therapeutic effects in vitiligo, and may left and right rear footpads were estimated together and therefore also explain why it has more side effects. Future studies in evaluated as single sites. Points were awarded as follows: humans may consider testing simvastatin in a topical formu- no evidence of depigmentation (0%) received a score of 0, 40 to lation, as this should result in fewer side effects, and topical 10% ¼ 1 point, 410 to 25% ¼ 2 points, 425 to 75% ¼ 3 points, 475 simvastatin was reported to reduce inflammation in a mouse to o100% ¼ 4 points, and 100% ¼ 5 points. The ‘‘vitiligo score'' was model of irritant dermatitis .
the sum of the scores at all four sites, with a maximum score of Simvastatin is an inexpensive, relatively safe FDA-approved 20 points. Acute treatment with simvastatin was performed by i.p.
medication that prevents and reverses depigmentation in our injection of either 1 dose or 3 daily doses of simvastatin (0.8 mg) vitiligo mouse model, and it reversed depigmentation in one 5 weeks after vitiligo induction, or vehicle control. Acute IFN-g reported vitiligo patient (. Its use may be blockade was through i.p. injection of 3 daily doses of IFN-g– repurposed to provide the first systemic treatment option for neutralizing antibody (500 mg, XMG-6) or vehicle control.
patients with vitiligo, and could also be reformulated as a To induce repigmentation, 10 to 12 weeks after induction of topical treatment option. Based on these observations, a vitiligo, mice with at least 50% tail depigmentation were randomly clinical trial in a small number of patients with high-dose assigned to receive either 0.8 mg of simvastatin as before or vehicle simvastatin would be useful to determine whether larger control for a total of 4 to 6 weeks. Treatment efficacy was objectively studies to prove efficacy would be a worthwhile investment.
quantified by comparison of the tail photographs before and aftertreatment with ImageJ software (NIH, Bethesda, MD). Briefly, all MATERIALS AND METHODS images were converted to 8-bit black and white, and the brightness threshold adjusted to 100, converting all pigmented areas to black KRT14-Kitl*4XTG2Bjl (Krt14-Kitl*) mice were a gift from BJ Longley and all depigmented areas to white. The outline of the tail was (University of Wisconsin, Madison, WI). Thy1.1 þ PMEL TCR-trans- selected, and the mean area over threshold calculated, representing genic mice were obtained from The Jackson Laboratory (Bar Harbor, the fraction of pigmented tail skin. This was multiplied by 100, ME; stock no. 005023, B6.Cg Thy1a/CyTg(TcraTcrb)8Rest/J). All mice converting the fraction to percent pigmentation of the tail. Percent were on a C57BL/6J background, were maintained in pathogen-free change over baseline reflects the percent pigmentation after treatment facilities at the University of Massachusetts Medical School, and minus the percent pigmentation before treatment and, therefore, a P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo positive number reflects percent repigmentation whereas a negative was generated with iScript complimentary DNA synthesis kit number reflects further depigmentation.
(Bio-Rad, Hercules, CA). Real-time PCR was conducted with com-plimentary DNA and iQ SYBR Green (Bio-Rad) in a Bio-Rad iCycler In vitro T-cell proliferation and cytokine production assays iQ according to the manufacturer recommendations. Mouse primer TCR-transgenic CD8 þ T cells that recognize premelanosome protein sequences are as follows: Cxcl10 50-AGGGGAGTGATGGAG (PMEL) were isolated from the spleens of transgenic mice using a AGAGG-30 (sense) and 50-TGAAAGCGTTTAGCCAAAAAAGG-30 MACS CD8-negative isolation kit (Miltenyi Biotec, Auburn, CA) as (antisense); actin-b (Actb): 50-GGCTGTATTCCCCTCCATCG-30 (sense) mentioned above. Isolated CD8 þ T cells were suspended at (antisense). CXCL10 1.0  107 cells per ml in 2 mM carboxyfluorescein succinimidyl ester expression is reported after normalization to expression of ACTB.
(Invitrogen, Carlsbad, CA) in phosphate-buffered saline with 0.1% Data were pooled from three separate experiments and gene expres- fetal bovine serum and incubated for 10 minutes at 37 1C. Subse- sion is reported relative to the lowest expression in untreated mice quently, cold fetal bovine serum was added at an equal volume, the within each experiment after normalization to expression of b-actin.
cells were centrifuged at 350 g, and resuspended in T-cell media(RPMI-1640 Gibco (Life Technologies, Grand Island, NY), 10% fetal Statistical analysis bovine serum, 2 mM glutamax, 1 mM sodium pyruvate, 10 mM HEPES, Statistical analysis was performed using Prism software (GraphPad 0.5  nonessential amino acids, and 50 mM b-Mercaptoethanol).
Software, La Jolla, CA). Dual comparisons were made using 5.0  104 cells per well were incubated in a 96-well plate for the paired or unpaired Student's t-test when applicable. Groups of 72 hours at 37 1C. Wells in a 96-well plate were previously coated Z3 were analyzed by analysis of variance with Dunnett's post-tests.
overnight with 3 mg ml  1 CD3 antibody in phosphate-buffered saline P-values of o0.05 were considered significant.
at 4 1C. Stimulated cells were incubated in the presence of 2 mg ml  1of soluble CD28 antibody and unstimulated cells were incubated in CONFLICT OF INTEREST uncoated wells. Cells were also incubated with simvastatin or both The authors state no conflict of interest.
simvastatin and 1 mM (S)-Mevalonic Acid Lithium Salt (Sigma-Aldrich).
Surface staining for flow cytometry was then performed for CD45 We thank BJ Longley for Krt14-Kitl* mice and N Restifo for recombinant (clone 30-F11; BioLegend, San Diego, CA), CD8b (BioLegend clone vaccinia virus. This project was supported by the National Institute of Arthritis YTS1560707), Thy1.1 (BioLegend clone OX-7), and intracellular and Musculoskeletal and Skin Diseases, part of the NIH, under award number staining was performed for IFN-g (clone XMG1.2; ebioscience, San AR061437, and research grants from the Vitiligo Research Foundation, KawajaFamily Vitiligo Research Initiative Award, and Dermatology Foundation Stiefel Diego, CA). Data were pooled from three separate experiments, and Scholar Award (to JEH).
the average numbers from untreated groups (neither simvastatin normevalonate) were used for normalization.
Aaronson DS, Horvath CM (2002) A road map for those who don't know JAK-STAT. Science 296:1653–5 Ears, tails, spleens, and skin-draining lymph nodes were harvested at Alghamdi KM, Khurrum H, Rikabi A (2011) Worsening of vitiligo and onset of the indicated times. Spleens were disrupted, and the red blood cells new psoriasiform dermatitis following treatment with infliximab. J Cutan were lysed with red blood cell lysis buffer (Life Technologies). Ear and Med Surg 15:280–4 tail skin were incubated in skin digest medium (RPMI containing Alghamdi KM, Khurrum H, Taieb A et al. (2012) Treatment of generalized 0.5% deoxyribonuclease (DNase) I (Sigma-Aldrich) and liberase TL vitiligo with anti-TNF-alpha agents. J Drugs Dermatol 11:534–9 enzyme blend (0.5 mg ml  1) (Roche, Indianapolis, IN)) and pro- Alikhan A, Felsten LM, Daly M et al. (2011) Vitiligo: a comprehensive cessed with a medimachine (BD Biosciences, San Jose, CA) as overview Part I. Introduction, epidemiology, quality of life, diagnosis,differential diagnosis, associations, histopathology, etiology, and work-up.
described previously ). For separation of the J Am Acad Dermatol 65:473–91 dermis and epidermis, tail skin samples were incubated with Baker EL, Coleman CI, Reinhart KM et al. (2012) Effect of biologic agents on dispase (2.4 U ml  1; Roche) for 1 hour at 37 1C. Epidermis was non-PASI outcomes in moderate-to-severe plaque psoriasis: systematic removed and mechanically disrupted with 70 mm cell strainers, and review and meta-analyses. Dermatol Ther 2:9 dermis samples were incubated with collagenase IV (1 mg ml  1) with Bickers DR, Lim HW, Margolis D et al. (2006) The burden of skin diseases: DNase I (0.5 mg ml  1; Sigma-Aldrich) for 1 hour at 37 2004 a joint project of the American Academy of Dermatology Associa- 1C on a shaker.
tion and the Society for Investigative Dermatology. J Am Acad Dermatol Cells were filtered through a 70-mm mesh before analysis. The following antibodies were obtained from BioLegend: mouse (CD8b, Chapgier A, Boisson-Dupuis S, Jouanguy E et al. (2006a) Novel STAT1 alleles in CD45.2, CD90.1, and Fc block). The data were collected and otherwise healthy patients with mycobacterial disease. PLoS Genet 2:e131 analyzed with a BD LSR II flow cytometer (BD Biosciences) and Chapgier A, Wynn RF, Jouanguy E et al. (2006b) Human complete Stat-1 FlowJo (Tree Star, Ashland, OR).
deficiency is associated with defective type I and II IFN responses in vitrobut immunity to some low virulence viruses in vivo. J Immunol Quantitative real-time RT–PCR The expression of CXCL10 in mouse tissues was performed using Dayel SB, Alghamdi K (2013) Failure of alefacept in the treatment of vitiligo.
J Drugs Dermatol 12:159–61 quantitative real-time RT–PCR. Ear and tail skin from three control Desai CS, Martin SS, Blumenthal RS (2014) Non-cardiovascular effects and three simvastatin-treated mice were analyzed in each experi- associated with statins. BMJ 349:g3743 ment. The tissues obtained were stored at  80 1C until RNA was Dimmeler S, Aicher A, Vasa M et al. (2001) HMG-CoA reductase inhibitors extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt according to the manufacturer's instructions. Complimentary DNA pathway. J Clin Invest 108:391–7 Journal of Investigative Dermatology (2015), Volume 00 P Agarwal et al.
Simvastatin Prevents and Reverses Vitiligo Dupuis S, Dargemont C, Fieschi C et al. (2001) Impairment of mycobacterial Ogg GS, Rod Dunbar P, Romero P et al. (1998) High frequency of skin-homing but not viral immunity by a germline human STAT1 mutation. Science melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.
J Exp Med 188:1203–8 Dupuis S, Jouanguy E, Al-Hajjar S et al. (2003) Impaired response to interferon- Ongenae K, Beelaert L, van Geel N et al. (2006) Psychosocial effects of vitiligo.
alpha/beta and lethal viral disease in human STAT1 deficiency. Nat Genet J Eur Acad Dermatol Venereol 20:1–8 Otuki MF, Pietrovski EF, Cabrini DA (2006) Topical simvastatin: preclinical Fehr T, Kahlert C, Fierz W et al. (2004) Statin-induced immunomodulatory evidence for a treatment of skin inflammatory conditions. J Dermatol Sci effects on human T cells in vivo. Atherosclerosis 175:83–90 Glassman SJ (2011) Vitiligo, reactive oxygen species and T-cells. Clin Sci Overwijk WW, Theoret MR, Finkelstein SE et al. (2003) Tumor regression and (Lond) 120:99–120 autoimmunity after reversal of a functionally tolerant state of self-reactive Greenwood J, Steinman L, Zamvil SS (2006) Statin therapy and autoimmune CD8 þ T cells. J Exp Med 198:569–80 disease: from protein prenylation to immunomodulation. Nat Rev Immu- Overwijk WW, Tsung A, Irvine KR et al. (1998) gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells Harris JE, Harris TH, Weninger W et al. (2012) A mouse model of vitiligo with using high-affinity, altered peptide ligand. J Exp Med 188:277–86 focused epidermal depigmentation requires IFN-gamma for autoreactive Passeron T, Ortonne JP (2012) Activation of the unfolded protein response in CD8( þ ) T-cell accumulation in the skin. J Invest Dermatol 132:1869–76 vitiligo: the missing link? J Invest Dermatol 132:2502–4 Kita T, Brown MS, Goldstein JL (1980) Feedback regulation of 3-hydroxy-3- Ramana CV, Gil MP, Han Y et al. (2001) Stat1-independent regulation of gene methylglutaryl coenzyme A reductase in livers of mice treated with expression in response to IFN-gamma. Proc Natl Acad Sci USA 98:6674–9 mevinolin, a competitive inhibitor of the reductase. J Clin Invest 66: Rashighi M, Agarwal P, Richmond JM et al. (2014) CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of Kunisada T, Lu SZ, Yoshida H et al. (1998) Murine cutaneous mastocytosis and vitiligo. Sci Transl Med 6:223ra23 epidermal melanocytosis induced by keratinocyte expression of trans- Sadeghi MM, Collinge M, Pardi R et al. (2000) Simvastatin modulates cytokine- genic stem cell factor. J Exp Med 187:1565–73 mediated endothelial cell adhesion molecule induction: involvement of Laddha NC, Dwivedi M, Mansuri MS et al. (2013) Vitiligo: interplay between an inhibitory G protein. J Immunol 165:2712–8 oxidative stress and immune system. Exp Dermatol 22:245–50 Schallreuter KU, Bahadoran P, Picardo M et al. (2008) Vitiligo pathogenesis: Leung BP, Sattar N, Crilly A et al. (2003) A novel anti-inflammatory role for autoimmune disease, genetic defect, excessive reactive oxygen species, simvastatin in inflammatory arthritis. J Immunol 170:1524–30 calcium imbalance, or what else? Exp Dermatol 17:139–40 Li N, Salter RC, Ramji DP (2011) Molecular mechanisms underlying the Schroder K, Hertzog PJ, Ravasi T et al. (2004) Interferon-gamma: an overview inhibition of IFN-gamma-induced, STAT1-mediated gene transcription in of signals, mechanisms and functions. J Leukoc Biol 75:163–89 human macrophages by simvastatin and agonists of PPARs and LXRs.
Shah AA, Sinha AA (2013) Oxidative stress and autoimmune skin disease. Eur J J Cell Biochem 112:675–83 Dermatol 23:5–13 Linthorst Homan MW, Spuls PI, de Korte J et al. (2009) The burden of vitiligo: Spritz RA (2012) Six decades of vitiligo genetics: genome-wide studies provide patient characteristics associated with quality of life. J Am Acad Dermatol insights into autoimmune pathogenesis. J Invest Dermatol 132:268–73 Stamm JA, Ornstein DL (2005) The role of statins in cancer prevention and Meraz MA, White JM, Sheehan KC et al. (1996) Targeted disruption of the Stat1 treatment. Oncology (Williston Park) 19:739–50 gene in mice reveals unexpected physiologic specificity in the JAK-STAT Toosi S, Orlow SJ, Manga P (2012) Vitiligo-inducing phenols activate the signaling pathway. Cell 84:431–42 unfolded protein response in melanocytes resulting in upregulation of IL6 Mosenson JA, Zloza A, Klarquist J et al. (2012) HSP70i is a critical component and IL8. J Invest Dermatol 132:2601–9 of the immune response leading to vitiligo. Pigment Cell Melanoma Res van de Veerdonk FL, Plantinga TS, Hoischen A et al. (2011) STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis. N Engl J Med Mosenson JA, Zloza A, Nieland JD et al. (2013) Mutant HSP70 reverses autoimmune depigmentation in vitiligo. Sci Transl Med 5: van den Boorn JG, Konijnenberg D, Dellemijn TA et al. (2009) Autoimmune destruction of skin melanocytes by perilesional T cells from vitiligo Mundy G, Garrett R, Harris S et al. (1999) Stimulation of bone formation patients. J Invest Dermatol 129:2220–32 in vitro and in rodents by statins. Science 286:1946–9 van den Boorn JG, Melief CJ, Luiten RM (2011) Monobenzone-induced Ni W, Egashira K, Kataoka C et al. (2001) Antiinflammatory and antiarterio- depigmentation: from enzymatic blockade to autoimmunity. Pigment sclerotic actions of HMG-CoA reductase inhibitors in a rat model of Cell Melanoma Res 24:673–9 chronic inhibition of nitric oxide synthesis. Circ Res 89:415–21 Zhao Y, Gartner U, Smith FJ et al. (2011) Statins downregulate K6a promoter Noel M, Gagne C, Bergeron J et al. (2004) Positive pleiotropic effects of activity: a possible therapeutic avenue for pachyonychia congenita.
HMG-CoA reductase inhibitor on vitiligo. Lipids Health Dis 3:7 J Invest Dermatol 131:1045–52


Judentum Glaube - Brauchtum - Ethik - Politik Inhalt Vorwort . 3 Im Anfang . 4 Was heißt Judentum? . 5 Grundlagen jüdischen Glaubens . 8 - Gott (jüdische "Theologie") . 8 - Mensch (jüdische Anthropologie) . 10 - Schriften (Tora, Talmud, Midrasch) . 11

Nombre: Lola Bravo Ballester y Laura López Flores Curso: 1º Bachillerato 2. Justificación de proyecto…………………………….…….……. Página 4 3. Objetivos………………………………………………………… Página 4 4. Metodología…………………………………….……………… Página 5