Conventional suspension delivery versus tattooing pen-assisted suspension delivery in non-cultured epidermal cell suspension procedure for vitiligo: A randomized controlled trial.

Non-cultured epidermal suspension (NCES) is one of the most widely used surgical therapy for stable vitiligo patients in which recipient size preparation plays an important role in the outcome of NCES. The primary objective is to evaluate and compare the efficacy and safety of conventional suspension delivery after manual dermabrasion (CSMD) versus tattooing pen-assisted suspension delivery (TPSD) in NCES. Paired vitiligo units (VU) in 36 patients, matched with respect to size and location were divided into two groups. The VU in Group 1 underwent suspension delivery by CSMD while the VU in Group 2 underwent same by TPSD. All the VU were followed up at regular intervals until 24 weeks. At the end of 24 weeks, 31 VU (86.1%) in Group 1 achieved >75% repigmentation which was significantly higher (p = .02, chi-square test) as compared to 22 VU (61.1%) in Group 2. The color matching in both the groups VU was also comparable (p = .84, chi-square test). The patient global assessment (PGA) was significantly higher in Group 1 VU as compared to Group 2. Treatment response in terms of repigmentation and PGA was significantly better in VU treated with CSMD as compared to TPSD. Recipient site complications were seen more commonly in Group 1 VU as compared to Group 2. Perilesional halo at the recipient site was seen in none of the VU in Group 2 which was significantly lower than 6 VU in Group 1 than (p = .02, chi-square test). Better results may be possible with technical improvisations in tattooing pen needle diameter and depth of penetration.

Transcriptional variations at perilesional site in vitiligo patients: probably a fight for cell-survival.

Exploration of gene expression variations is a potential source to unravel biological pathways involved in pathological changes in body and understand the mechanism underneath. Vitiligo patients were explored for gene expression changes transcriptionally at perilesional site in comparison to normal site of same patients for melanogenesis pathway (TYR, DCT & TYRP1) cell adhesion (MMPs & TIMP1), cell survival (BCL2 & BAX1) as well as proliferation, migration & development (SOX9, SOX10 & MITF) regulatory system, using skin biopsy samples. Results were also compared with changes in gene expression for melanocytes under stress after hydrogen peroxide treatment in-vitro. Gene amplification was carried out via real time PCR. We found increased expression of proliferation, migration & development regulatory genes as well as melanogenesis pathway genes at perilesional site of patients. In-vitro study also supports induced MITF expression and disturbed melanogenesis in melanocytes under stress. Expression level ratio of cell survival regulatory genes' (BCL2/BAX1) as well as cell adhesion regulatory genes (MMPs/TIMP1) was observed upregulated at patient's perilesional site however downregulated in hydrogen peroxide treated melanocytes in-vitro. Observed upregulated gene expression at perilesional site of patients may be via positive feedback loop in response to stress to increase cell tolerance power to survive against adverse conditions. Gene expression analysis suggests better cell survival and proliferation potential at perilesional site in vitiligo patients. It seems in-vivo conditions/growth factors supports cells to fight for survival to accommodate stressed conditions.

Whole-body and targeted narrowband ultraviolet B phototherapy effectively stabilize acral vitiligo with negligible repigmentation beyond wrists and ankles: Results from a split-body randomized controlled trial.

BACKGROUND: Narrowband ultraviolet B (NB-UVB) phototherapy promotes stability and repigmentation in vitiligo. No studies have compared targeted NB-UVB with whole-body NB-UVB in treatment of acral vitiligo. OBJECTIVES: This randomized split-body study compared whole-body NB-UVB with targeted NB-UVB in inducing stability and repigmentation in acral vitiligo. METHODS: Thirty-two patients with bilaterally symmetrical acral vitiligo lesions (distal to elbows and knees) were recruited. Patients received whole-body NB-UVB treatment, with one hand and one foot shielded until elbow and knee, followed by targeted NB-UVB treatment on the shielded side. Patients were assessed at 4-week intervals for 24 weeks using Vitiligo Disease Activity (VIDA) score, Vitiligo Skin Activity Score (VSAS), Vitiligo Area Scoring Index (determined through fingertip method, using the method to calculate facial-VASI) and degree of repigmentation. RESULTS: After 12 weeks, 87.5% of patients achieved a VIDA score of 3, with none having active disease at 24 weeks. Over 50% repigmentation was observed in 42.2% and 37.5% of limbs in whole-body and targeted groups, respectively (p = .95). No improvement in F-VASI scores of hands and feet (distal to wrist and ankles) was noted with either modality over the 24-week period. CONCLUSION: Our study showed comparable repigmentation rates between whole-body and targeted NB-UVB groups. Limited effectiveness of phototherapy in repigmentation of hands and feet underscores an important therapeutic gap.

Covid-19 Vaccination and Chronic Urticaria: A Paradigm Study.

Background: Multiple vaccines were introduced during 2020-2021 to combat Covid-19 pandemic, being one of the successful vaccine programmes in the present era. Very few studies are available on status of chronic urticaria (CU) post Covid-19 vaccination. Aim: The aim of this study was to study effect of Covid-19 vaccination on our urticaria cohort. Materials and Methods: In this retrospective study, case records of CU patients registered in urticaria clinic, who had received any type of Covid-19 vaccine during the interval of March 2021-2022 were retrieved. Patients were classified as 'vaccine induced urticaria' (VIU) when CU developed for first time post-vaccination and 'vaccine exacerbated urticaria' (VEU) when administration of vaccine exacerbated disease activity in previously diagnosed CU. Results: Overall, 353 CU patients registered with us during this period, 265 had received atleast one dose of a Covid-19 vaccine, of which 12 reported VEU (ten of whom had received adenovirus vector vaccine), and three patients were diagnosed with VIU (all had received inactivated virus vaccine). Mean vitamin D3 levels were significantly higher in patients who had VEU as compared to those CU patients without exacerbation (p = 0.003). Significant correlation was observed between level of concern regarding adverse effects of vaccination, pre-vaccination, and post-vaccination urticaria activity score (UAS-7), (Pearson correlation coefficient = 0.66, P = 0.007) in both VEU and VIU. Urticaria symptoms were controlled in 75% and 66.6% patients, respectively, of VEU and VIU, after one month of initiating standard antihistamine treatment. Conclusion: Hence, we conclude that though Covid-19 vaccines can trigger CU, standard treatment protocols control disease activity in most patients.

Altered levels of lymphocyte enhancer-binding factor-1 modulates the pigmentation in acral and non-acral lesions of non-segmental vitiligo patients: a follow-up-based study in North India.

BACKGROUND: Lymphocyte enhancer-binding factor-1 (LEF1) is responsible for melanocyte proliferation, migration and differentiation and its downregulation may result in depigmentation in vitiligo. Narrowband UVB (NB-UVB) phototherapy is known to enhance melanocyte migration from hair follicles to lesional epidermis; hence, it may have a role in the upregulation of LEF1. OBJECTIVES: We intended to assess the expression of LEF1 both before and after NB-UVB therapy and correlate it with the extent of re-pigmentation. MATERIALS AND METHODS: In this prospective cohort study, 30 patients of unstable non-segmental vitiligo were administered NB-UVB phototherapy for 24 weeks. Skin biopsies were obtained from acral and non-acral sites in all patients, both prior to initiation and after completion of phototherapy and LEF1 expression was measured. RESULTS: Amongst the 16 patients who completed the study, at 24 weeks, all patients achieved > 50% re-pigmentation. However, > 75% re-pigmentation was achieved in only 11.1% of acral patches, whereas it was achieved in a significantly higher number of non-acral patches (66.6%) (p = 0.05). A significant increase was observed in the mean fluorescent intensity of the LEF1 gene in both acral as well as non-acral areas at 24 weeks as compared to baseline (p = 0.0078), However, no difference was observed between acral and non-acral lesions in the LEF1 expression at 24 weeks or the change in LEF1 expression from baseline. CONCLUSION: LEF1 expression modulates the re-pigmentation of vitiligo lesions after treatment with NBUVB phototherapy.

A prospective observational study to sequentially determine the dermoscopic features of vitiligo and its association with disease activity in patients on medical treatment: Dermoscopy and disease activity in vitiligo.

Prospective data on correlation between dermoscopic features of vitiligo and disease activity are scarce. This study was conducted to sequentially determine the dermoscopic features of vitiligo and to evaluate their association with disease activity. A cohort of 30 patients with 60 active vitiligo patches on medical therapy was subjected to sequential clinical and dermoscopic examination at four weekly intervals till 16 weeks. Disease activity at each visit was assessed using serial clinical photographs and modified vitiligo activity severity index. The dermoscopic images were merged and analysed for a predefined set of dermoscopic parameters by two blinded dermatologists. Paired analysis of dermoscopic features was done between baseline, and stabilized vitiligo patches at 12 and 16 weeks. Pigment network changes (absent and reduced pigment network, p < .001), perifollicular depigmentation (p = .02), ill-defined margins (p = .04) and satellite lesions and micro-Koebner phenomenon (p < .001) were associated with active vitiligo while perifollicular repigmentation (p < .001) was associated with stabilizing and repigmenting vitiligo. Satellite lesions and micro-Koebner's phenomena were suggestive of unstable disease irrespective of site of target lesion, while perifollicular repigmentation was suggestive of stabilized/repigmenting disease only at non-acral sites. We found sequential dermoscopy to be useful to assess disease activity and potential for repigmentation in localized vitiligo.

Factors Predicting the Outcome of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis: A 5-Year Retrospective Study.

BACKGROUND: Clinicodemographic and laboratory parameters predicting the outcome of Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) may vary among populations owing to genotypic and environmental variations. There is a scarcity of studies evaluating these parameters in Indian population. AIMS: To analyze clinicodemographic and laboratory parameters predicting disease outcome in patients of SJS/TEN. MATERIALS AND METHODS: Clinical records of patients admitted with a diagnosis of SJS/TEN from January 2014 to December 2018 were reviewed retrospectively with respect to data pertaining to clinicodemographic details, laboratory parameters, and disease outcome. RESULTS: Of 51 patients included in the study, 24 (47.06%) were females. Anticonvulsants [phenytoin (19.6%), carbamazepine (13.7%), others (5.88%)] were the most commonly implicated drugs followed by NSAIDs (19.6%). The overall mortality was 21.6% [SJS (0%), SJS-TEN overlap (18.8%), and TEN (28.6%)]. The mean detached body surface area (BSA) (35.4% ± 10.4% vs. 25.7% ± 11.8%; P = 0.02) was significantly higher among patients with mortality. Blood urea nitrogen, serum HCO3 - levels, and random blood sugar were significantly associated with mortality. Presence of sepsis during the disease course was associated with higher mortality (9/12 vs. 2/39; P = 0.001). Other components of SCORTEN like age and heart rate were not significantly associated with poor outcome in our study. None of our patients had associated malignancy. CONCLUSION: A higher detached BSA, presence of sepsis, higher blood urea nitrogen and random blood sugar, and lower serum HCO3 - levels were associated with mortality. Refinement of scoring systems predicting the outcome of SJS-TEN is needed for better disease prognostication.

Vitiligo: Translational research and effective therapeutic strategies.

This is an exciting phase of vitiligo research with the current understanding of vitiligo pathogenesis and its translation to successful treatment. The pathogenetic origin of vitiligo revolves around autoimmunity with supporting role from many other factors like oxidative stress, inherent melanocyte defects, or defective keratinocytes and fibroblasts. Vitiligo can be classified into segmental or non-segmental depending upon the clinical presentation, or it can be classified as progressing or stable based on the activity of the disease. Vitiligo treatments need to be stratified depending upon which type of vitiligo we are treating and at which phase the vitiligo patient presents to us. There are two different aims of treatment of vitiligo. The first involves rescuing the melanocytes from the damage to arrest the depigmentation. The second strategy focuses on replenishing the melanocytes so that successful repigmentation is achieved. It is also important to maintain the disease in a stable phase or prevent relapse. As stability in non-segmental vitiligo is a dynamic process, maintenance of the stability of repigmentation is also an important consideration in the management of vitiligo. In this review, we shall briefly discuss the current options and future insight into the management of vitiligo.

Surgical Interventions for Patients With Vitiligo: A Systematic Review and Meta-analysis.

Importance: Surgical interventions are a key part of the therapeutic arsenal, especially in refractory and stable vitiligo. Comparison of treatment outcomes between the different surgical procedures and their respective adverse effects has not been adequately studied. Objective: To investigate the reported treatment response following different surgical modalities in patients with vitiligo. Data Sources: A comprehensive search of the MEDLINE, Embase, Web of Science, and Cochrane Library databases from the date of database inception to April 18, 2020, was conducted. The key search terms used were vitiligo, surgery, autologous, transplantation, punch, suction blister, and graft. Study Selection: Of 1365 studies initially identified, the full texts of 358 articles were assessed for eligibility. A total of 117 studies were identified in which punch grafting (n = 19), thin skin grafting (n = 10), suction blister grafting (n = 29), noncultured epidermal cell suspension (n = 45), follicular cell suspension (n = 9), and cultured epidermal cell suspension (n = 17) were used. Data Extraction and Synthesis: Three reviewers independently extracted data on study design, patients, intervention characteristics, and outcomes. Random effects meta-analyses using generic inverse-variance weighting were performed. Main Outcomes and Measures: The primary outcomes were the rates of greater than 90%, 75%, and 50% repigmentation response. These rates were calculated by dividing the number of participants in an individual study who showed the corresponding repigmentation by the total number of participants who completed the study. The secondary outcomes were the factors associated with treatment response to the surgical intervention. Results: Among the 117 unique studies and 8776 unique patients included in the analysis, rate of repigmentation of greater than 90% for surgical interventions was 52.69% (95% CI, 46.87%-58.50%) and 45.76% (95% CI, 30.67%-60.85%) for punch grafting, 72.08% (95% CI, 54.26%-89.89%) for thin skin grafting, 61.68% (95% CI, 47.44%-75.92%) for suction blister grafting, 47.51% (95% CI, 37.00%-58.03%) for noncultured epidermal cell suspension, 36.24% (95% CI, 18.92%-53.57%) for noncultured follicular cell suspension, and 56.82% (95% CI, 48.93%-64.71%) for cultured epidermal cell suspension. The rate of repigmentation of greater than 50% after any surgical intervention was 81.01% (95% CI, 78.18%-83.84%). In meta-regression analyses, the treatment response was associated with patient age (estimated slope, -1.1418), subtype of vitiligo (estimated slope, 0.3047), and anatomical sites (estimated slope, -0.4050). Conclusions and Relevance: The findings of this systematic review and meta-analysis suggest that surgical intervention can be an effective option for refractory stable vitiligo. An appropriate procedure should be recommended based on patient age, site and size of the lesion, and costs.

Immune-modulatory effects of lenalidomide inhibited the progression of lesions in a vitiligo mouse model.

Vitiligo, an autoimmune disorder, is associated with altered cytokine levels and T lymphocytes. Lenalidomide modulates immune system components by altering cytokine production and regulating T-cell stimulation. In this study, effect of lenalidomide was checked on the development of vitiligo lesions, level of various cytokines, and T lymphocytes in the mouse model. The vitiligo mouse model was developed by immunizing C57BL/6 mouse with anti-mouse tyrosine-related protein 2. Lenalidomide was orally given to mice daily, and the effect was observed on the development of vitiligo lesions. The level of T lymphocytes in blood was checked by flow cytometry. Serum cytokine levels were checked by enzyme-linked immunosorbent assay. Vitiligo lesions were found significantly smaller in lenalidomide-treated mice models. It significantly decreased the serum levels of IFN-γ, TNF-α, IL-1ß, and IL-6 but elevated the levels of IL-4 and IL-10. It non-significantly elevated CD4+ /CD8+ T-cell ratio. Lenalidomide had an inhibitory effect on the development of vitiligo lesions in mice models by suppressing the serum level of pro-inflammatory cytokines and increasing anti-inflammatory cytokine levels. It modulated the immune response in vitiligo mice models toward an anti-inflammatory profile suggesting its use in the management of vitiligo.

Dendritic cells sub-sets are associated with inflammatory cytokine production in progressive vitiligo disease.

In autoimmune onset of vitiligo, perilesional area shows inflammatory cells including T cytotoxic, helper cells and macrophages. Dendritic Cells (DCs) regulate immune activities by antigen presentation to T cells or cytokine production. It is evident that pro- and anti-inflammatory DCs are responsible for their respective cytokines release. However, role of DCs in vitiligo is enigmatic. In the present study, we assessed DCs markers (CD11b and CD11c) along with pro- and anti-inflammatory cytokines (IL-17A, IL-10 and IL-12p70) in stable and active vitiligo patients. Our results revealed a significant augmented expression of CD11b+CD11c+ (pro-inflammatory DC) in peripheral blood mononuclear cells (PBMCs) and skin tissues of active vitiligo patients versus control and stable vitiligo group. Unlikely, CD11b+ (anti-inflammatory DC) levels were significantly impeded in active vitiligo group as compared to another two groups. CD11c (T helper 1 stimulating DC) presented no significant alterations in any group. Furthermore, we perceived significantly up-regulated IL-17A (pro-inflammatory cytokine) and down-regulated IL-10 (anti-inflammatory cytokine) expressions in active vitiligo group as compared to control and stable group (in sera, PBMCs and skin tissue). Also, a significant positive correlation was observed between CD11b+CD11c+ and IL-17A; and CD11b+ and IL-10. Contrarily, CD11b+CD11c+ and CD11b+ were negatively correlated with IL-10 and IL-17A, respectively. In conclusion, modulation of pro- and anti-inflammatory DCs in active vitiligo patients may affect cytokines production and thereby, lead to further depigmentation of skin.

A prospective, randomized clinical study to compare the efficacy of recipient site preparation using dermabrasion, cryoblister, and dermaroller in autologous noncultured epidermal cell suspension in stable vitiligo.

Noncultured epidermal cell suspension (NCES) is a well-established surgical treatment modality for stable vitiligo. The outcome of this procedure significantly depends on the method of recipient site preparation, a critical step to achieve cosmetically acceptable repigmentation. To compare the efficacy of recipient site preparation using three methods namely, dermabrasion, cryoblister, and dermaroller followed by NCES in stable vitiligo. In this single-center, prospective, intra-patient, randomized clinical trial; 36 participants having at least three vitiligo patches in same anatomic region with minimum lesional stability of 1 year were randomized 1:1:1 for recipient site preparation using manual dermabrasion, cryoblister, and dermaroller followed by NCES. Patients were followed up at 4, 8, and 12 weeks and assessment of extent and pattern of repigmentation, color match and patient satisfaction were done. Among 36 patients, 22 (61.1%) were females; mean (SD) age was 28.33 (9.4) years. Dermabrasion and cryoblister techniques showed equal efficacy with respect to extent of repigmentation (>75% repigmentation; 55.6% vs 47.2%; P = .63) and patient satisfaction score (20.2 ± 9.6 vs 19.9 ± 7.9, P = .194). However, dermabrasion was superior to cryoblister in terms of rapidity (65% vs 32.5% at 4 weeks, P = .04) and color match (47.2% vs 19.4%, P = .004). Dermaroller had poor repigmentation outcomes compared to both dermabrasion and cryoblister. Cryoblister as a method of recipient site preparation is equally effective as manual dermabrasion in NCES for attaining good to excellent repigmentation, but with risk of hyperpigmentation. However, dermaroller is inferior to both dermabrasion and cryoblister.

Immune modulatory effects of lenalidomide on the cultured peripheral blood mononuclear cells from vitiligo patients.

Vitiligo is a depigmentary disease in which epidermal melanocytes are lost. It is considered to be an autoimmune disease. Lenalidomide, an immunomodulatory drug is being employed in the treatment of various autoimmune and inflammatory disorders. In the present manuscript, the effect of lenalidomide on T cells and major cytokines in the cultured peripheral blood mononuclear cells (PBMCs) derived from vitiligo patients was checked. Eight patients with a clinical diagnosis of active vitiligo volunteered for the study. Blood was collected from them and PBMCs were isolated, cultured, and treated with lenalidomide. After 72 hours, PBMCs were harvested and checked for CD8+ and CD4+ T cells by flow cytometry. Further supernatant was collected and the levels of cytokines namely tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-10 (IL-10), and interleukin-4 (IL-4) were checked using ELISA kits. Lenalidomide nonsignificantly decreased the level of CD8+ T cells but increased CD4+ T cells leading to increased CD4+ /CD8+ T cell ratio. It declined the level of pro-inflammatory cytokines, that is, TNF-α and IFN-γ whereas elevated anti-inflammatory cytokines, that is, IL-10 and IL-4, thus ultimately decreasing the ratio of pro-inflammatory to anti-inflammatory cytokines. Lenalidomide suppressed the proliferation of T lymphocytes and modulated the cytokines secretion toward an anti-inflammatory profile.

Chemical induced pathognomonic features observed in human vitiligo are mediated through miR-2909 RNomics pathway.

BACKGROUND: Chemicals like Monobenzyl Ether of Hydroquinone (MBEH) and 4-Tertiary Butyl Phenol (4-TBP) have been widely recognized to induce clinical lesions that resemble vitiligo, but exact molecular pathway through which these chemicals initiate vitiligo is still far from clear. OBJECTIVES: Since vitiligo is widely considered as an autoimmune disease, this study was an attempt to understand miR-2909 RNomics in vitiligo pathogenesis using MBEH treated primary melanocytes as an archetype cellular model because MBEH causes pathological features indistinguishable from clinical vitiligo. METHODS: Primary melanocytes were treated with MBEH and 4-TBP and the role of miR-2909 RNomics at transcriptional and translational level was explored through qRT-PCR, western blot analysis, flow cytometry, immunocytochemistry, immunohistochemistry and in silico binding affinities. 4 mm punch biopsies were also obtained from lesional sites of vitiligo patients to validate the results observed in cell culture experiments. RESULTS: MBEH induced miR-2909 RNomics led to downregulation of MITF, TYR, TYRP1, and TYRP2 leading to decreased melanin synthesis which in turn is a characteristic trait of vitiligo. On the other hand, 4-TBP increased TGF-ß which also has the intrinsic capacity to downregulate MITF leading to decreased melanin synthesis and thereby initiation of vitiligo. CONCLUSION: Based upon our results we propose a molecular pathway which has the inherent capacity to resolve the mechanism through which these chemicals may induce vitiligo. This mechanism was also found to be involved in the lesional biopsies of vitiligo patients. These results could be exploited in better understanding the pathogenesis as well as in treatment of vitiligo.

Aberrant ETS-1 signalling impedes the expression of cell adhesion molecules and matrix metalloproteinases in non-segmental vitiligo.

Cell adhesion is a complex process that involves multiple molecules on the cell surface (ie cell adhesion molecules [CAMs]), surrounding cells and extracellular matrix (ECM). Repigmentation in vitiligo is dependent on the ECM remodelling and cellular migration, primarily attributed to the transcriptional activation of matrix metalloproteinases (MMPs). In this study, we aimed to demonstrate the role of ETS-1 signalling in the regulation of MMPs and CAMs. Therefore, we studied the expression of ETS-1, MMPs (MMP-2, MMP-9) and CAMs including E-cadherin, ITGA-1 and ICAM-1 in vitiligo (both active and stable) ex vivo. Further, we compared melanocyte morphology and their adhesion towards collagen IV and laminin between control and vitiligo groups in vitro. Also, we silenced ETS-1 in melanocytes cultured from control skin and observed post-silencing effect on above-mentioned MMPs and CAMs. We perceived absent ETS-1 and significantly reduced CAMs and MMPs in vitiligo compared with normal skin. Scanning electron microscopy (SEM) revealed a translucent material surrounding individual melanocytes in stable vitiligo and controls, whereas active vitiligo melanocytes demonstrated loss of this extracellular substance. Adhesion assays revealed significantly decreased binding of cultured melanocytes to collagen IV and laminin V plates in both stable and active vitiligo. Importantly, ETS-1 silencing resulted in significantly reduced expression of CAMs and MMPs. In conclusion, absent ETS-1 expression in both stable and active non-segmental vitiligo seems to impede the expression of CAMs, apart from MMPs, probably leading to progressive depigmentation in active disease and absence of spontaneous repigmentation in stable disease.

Effect of Different Methods of Trypsinization on Cell Viability and Clinical Outcome in Vitiligo Patients Undergoing Noncultured Epidermal Cellular Suspension.

BACKGROUND: Noncultured Epidermal Cell Suspension (NCECS) is a surgical modality used in treating stable vitiligo. Trypsinization of the epidermis may be done either at 4°C overnight (cold) or at 37°C for 30 to 50 minutes (warm). Recently, trypsinization was done at room temperature (25°C) in an in vitro trial. OBJECTIVE: To compare different trypsinization techniques in NCECS regarding cell viability and clinical outcome. METHODS: This comparative multicenter study was conducted on 20 patients with stable nonsegmental vitiligo. In each patient, 3, nonacral vitiligo lesions were randomly assigned for treatment by NCECS prepared by warm, room temperature, and cold trypsinization techniques, respectively. A perilesional biopsy was taken from each of the 3 treated lesions as an objective measure of disease stability. After transplantation, all patients received narrow-band ultraviolet B twice weekly for 6 months. Cell viability was assessed in each technique, as well as clinical outcome in all treated lesions. RESULTS: Warm and room temperature trypsinization techniques were comparable with each other. Both were significantly better than the cold technique regarding viability and repigmentation. CONCLUSION: Room temperature trypsinization can be used as a convenient substitute to warm trypsinization. Cold trypsinization is not recommended because of its poor results and poor patient satisfaction.

Expression of Th17- and Treg-specific transcription factors in vitiligo patients.

BACKGROUND: Vitiligo is mainly considered an autoimmune skin disease as the number of IL-17 producing Th17 cells, involved in the development of autoimmune and inflammatory pathologies, increased in vitiligo skin. T regulatory cells (Tregs) seem to be altered during the disease. Thus, there must be some upstream molecular factors that regulate the cellular response to apoptotic and inflammatory stimuli. OBJECTIVES: To investigate the expression of Th17- and Treg-specific transcription factors in PBMCs and to evaluate the correlation between these transcription factors and cytokines in vitiligo patients. METHODS: We investigated 30 active NSV patients for Th17- and Treg-specific transcription factors RORγt (retinoic acid-related orphan receptor gamma t), FOXP3 (forkhead/winged helix), HELIOS, EOS, and IRF4 (Interferon Regulatory Factor 4) as well as apoptotic marker NALP1 (NACHT-leucine-rich-repeat protein 1) in PBMCs with RT-qPCR. Immunostaining was done for transcription factors and cytokines on skin sections. RESULTS: The mRNA level of FOXP3 was significantly lower in patients (0.76 fold, P < 0.001), whereas RORγt was slight but not significantly increased (0.76 fold, P = 0.06). Furthermore, NALP1 in lymphocytes was found to be increased in patients (0.69 fold, P < 0.01). The immunostaining results revealed increased expression of RORγ, IL-17A, NALP1, and IL-1ß in vitiligo skin when compared to normal healthy skin. CONCLUSION: Reduced FOXP3/RORγt mRNA ratio suggests thriving of the Th17 cell population in PBMCs of vitiligo patients. Increased NALP1 levels indicate the existence of an apoptotic phenomenon which correlates with the increased expression of IL-1ß in vitiligo pathogenesis.