No / To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4¿-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 ¿M H2O2 increase DHPR activities, whereas levels greater than 30 ¿M H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.

No / Vitiligo is a visible condition that is more noticeable in darker-skinned people. Beliefs about illness have been linked to psychosocial adjustment. There is some evidence that such beliefs may be influenced by cultural factors. Surprisingly little is known about beliefs in relation to vitiligo. Objectives The study sought to explore in depth the ways in which British Asian women manage and adjust psychosocially to vitiligo, and the potential role of ethnicity and culture in this process. Methods In-depth semistructured interviews were conducted with seven British women of South Asian decent and analysed using the qualitative method of template analysis. Results Participants described feeling visibly different and all had experienced stigmatization to some extent. Avoidance and concealment were commonplace. Experiences of stigmatization were often perceived to be associated with cultural values related to appearance, status, and myths linked to the cause of the condition. Conclusions The findings of this study present a unique in-depth analysis of British South Asians living with vitiligo and suggest there is a need for further research to explore cultural associations of disfigurement and of adjustment to chronic skin conditions. Furthermore, they suggest that in addition to individual therapeutic interventions there may be a need for community interventions aimed at dispelling myths and raising awareness of sources of support and treatment.

The Melanocyte Keratinocyte Transplantation Procedure (MKTP) is an effective surgical replacement of lost melanocytes in recalcitrant vitiligo and pigmentary skin disorders. However, it is only effective in stable vitiligo lesions because active autoimmunity destroys the newly transplanted melanocytes. Despite careful selection of candidates based on the reported clinical stability, the success of the procedure is still unpredictable. MKTP candidates with non-segmental, segmental, and mixed vitiligo, as well as hypopigmented scars and Piebaldism patients were enrolled to our studies. Our aim was first, to investigate the possible immunological mechanisms responsible for the unpredictable post- transplantation outcomes, including T cell subsets and inflammatory chemokines, by correlating these biomarkers with clinical phenotypes, duration of stability, and surgical outcomes. We used suction blister biopsy, a minimally invasive technique that we developed to sample human skin. Moreover, we quantified transplanted melanocytes in the suspension using flow cytometry. Following MKTP, we corelated these biomarkers to the repigmentation score. We found that CD8+ T cells remain in some clinically stable vitiligo lesions, correlate negatively with the post-surgical score of repigmentation, and inversely impact the durability of the responses. Interestingly, the number of transplanted melanocytes in the suspension and the duration of stability do not have prognostic roles. Based on our findings and in a second group of patients, we suppressed the activity of T cells to enhance the outcomes of MKTP. We used Ruxolitinib, JAK1/2 inhibitor, in a triple blinded randomized controlled within subject study, in comparison with Tacrolimus (a calcineurin inhibitor and the standard of care treatment in vitiligo) as well as placebo control. We found lower T cell infiltrate, lower chemokines, and better skin repigmentation in lesions treated with MKTP plus Ruxolitinib or Tacrolimus than in lesions treated with MKTP plus placebo. Lastly, we compared two different types of laser in preparation of the recipient skin for MKTP - ablative versus fractional Er:YAG laser. We found that the ablative laser is combined with minimal CD8+ T cell epidermal infiltrate and superior repigmentation score in comparison to more infiltrate and lower repigmentation score with the fractional laser. Taken together, these results from our studies provide novel insight to predict the optimal surgical candidates and will improve surgical outcomes. It advances the treatment of vitiligo by uncovering the impact of autoimmunity on the success of repigmentation and discovering new approaches to optimize the surgical treatment options in patients with vitiligo and pigmentary skin disorders.

Vitiligo is a skin disease where the pigment cells that produce melanin die or stop functioning, which causes white patches to appear on the body. Although vitiligo is not considered a serious disease, there is a risk that something is wrong with a person's immune system. In recent years, the use of medical image processing techniques has grown, and research continues to develop new techniques for analysing and processing medical images. In many medical image classification tasks, deep convolutional neural network technology has proven its effectiveness, which means that it may also perform well in vitiligo classification. Our study uses four deep convolutional neural networks in order to classify images of vitiligo and normal skin. The architectures selected are VGG-19, ResNeXt101, InceptionResNetV2 and Inception V3. ROC and AUC metrics are used to assess each model's performance. In addition, the authors investigate the economic benefits that this technology may provide to the healthcare system and patients. To train and evaluate the CNN models, the authors used a dataset that contains 1341 images in total. Because the dataset is limited, 5-fold cross validation is also employed to improve the model's prediction. The results demonstrate that InceptionV3 achieves the best performance in the classification of vitiligo, with an AUC value of 0.9111, and InceptionResNetV2 has the lowest AUC value of 0.8560. / Vitiligo är en hudsjukdom där pigmentcellerna som producerar melanin dör eller slutar fungera, vilket får vita fläckar att dyka upp på kroppen. Även om Vitiligo inte betraktas som en allvarlig sjukdom, det finns fortfarande risk att något är fel på en persons immun. Under de senaste åren har användningen av medicinska bildbehandlingstekniker vuxit och forskning fortsätter att utveckla nya tekniker för att analysera och bearbeta medicinska bilder. I många medicinska bildklassificeringsuppgifter har djupa konvolutionella neurala nätverk bevisat sin effektivitet, vilket innebär att den också kan fungera bra i Vitiligo klassificering. Vår studie använder fyra djupa konvolutionella neurala nätverk för att klassificera bilder av vitiligo och normal hud. De valda arkitekturerna är VGG-19, RESNEXT101, InceptionResNetV2 och Inception V3. ROC- och AUC mätvärden används för att bedöma varje modells prestanda. Dessutom undersöker författarna de ekonomiska fördelarna som denna teknik kan ge till sjukvårdssystemet och patienterna. För att träna och utvärdera CNN modellerna använder vi ett dataset som innehåller totalt 1341 bilder. Eftersom datasetet är begränsat används också 5-faldigt korsvalidering för att förbättra modellens förutsägelse. Resultaten visar att InceptionV3 uppnår bästa prestanda i klassificeringen av Vitiligo, med ett AUC -värde på 0,9111, och InceptionResNetV2 har det lägsta AUC -värdet på 0,8560.

Vitiligo is an idiopathic, non-contagious and often familial depigmentation disorder affecting both sexes equally. The mostly progressive and patchy loss of the inherited skin colour is not only a cosmetical problem, it has a profound impact on the patient's well-being. Stigmatisation and rejection often causes depression, self-consciousness, sexual problems and an impaired quality of life. To further substantiate earlier investigations and to introduce new aspects, we utilised the Dermatology Life Quality Index (DLQI), the Beck Depression Inventory (BDI) and the Adjustment to Chronic Skin Disorders Questionnaire (ACS) with its sub-scales on Social anxiety/avoidance, Helplessness and Anxious-depressive Mood in 422 patients and 55 healthy controls. We also included 103 children, their parents and 18 controls by using the Children's Dermatology Life Quality Index (CDLQI) and an adapted version of the ACS. We found that patients with vitiligo experience high levels of stigmatisation. They have an impaired quality of life and are more socially anxious/avoidant, helpless and (anxious-) depressive compared to healthy controls. The results correlate with disease severity, avoidant behaviour/hiding of vitiligo and the belief that psychological stress influences the disease. Female patients are generally more affected. Treatment with pseudocatalase PC-KUS improves quality of life and reduces anxious-depressive mood. Children also suffer from stigmatisation and an impaired quality of life. Parents are more socially anxious and helpless compared to the control group.

Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Made available in DSpace on 2016-04-28T20:38:21Z (GMT). No. of bitstreams: 1 Augusta Renata Almeida do Sacramento.pdf: 1086153 bytes, checksum: 96ad2507779fcd3e9a62c030beeafa02 (MD5) Previous issue date: 2012-08-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper was aimed at observing the psychosocial variables resulting from vitiligo and symbolic representations of such a disease. Sixty women, aged between 19 and 77 have participated of this study. Thirty out of all of them had vitiligo (experimental group) and other 30 had different skin diseases (control group). The following instruments were used to collect data: #1 Form to characterization of sample, #2 Dermatology Life Quality Index (DLQI), #3 Semi-structured interview and #4 Theme drawing. The DLQI was applied in the two groups and the other instruments applied only in experimental group. The data collected were analyzed under analytical psychology and psychosomatic Jungian. The interviews speeches were analyzed, then 03 categories and 10 subcategories were determined: emotional factors (factors which caused the disease, aggravating factors of the disease); the disease purpose; living with the disease (social support, prejudice, disturbances derived from the disease, behavior towards the disease, the disease impact, exposition to the symptoms, expectations concerning the treatment and fear of kids having the disease). The results revealed a commitment to the life quality not only to the women having vitiligo but also to the women who had other skin diseases, and there was no significant statistical difference between the two groups. It was observed the association of emotional aspects in the appearance and/or aggravating condition of vitiligo as well as behavioral changes, limitations and prejudice experienced by women having the disease. The drawings pointed out certain symbolic representations of vitiligo, showing conflicts and suffering due to the disease / O presente trabalho objetivou observar as variáveis psicossociais decorrentes do vitiligo e representações simbólicas dessa doença. Participaram deste estudo 60 mulheres, entre 19 e 77 anos. Destas, 30 com vitiligo (grupo experimental) e 30 com demais doenças de pele (grupo controle). Os seguintes instrumentos foram utilizados para coleta de dados: 1) formulário para caracterização da amostra, 2) Índice de Qualidade de Vida em Dermatologia (DLQI-BRA), 3) entrevista semi-estruturada e 4) desenho temático. O DLQI-BRA foi aplicado aos dois grupos e os demais instrumentos aplicados somente ao grupo experimental. Os dados foram analisados à luz da psicologia analítica e psicossomática junguiana. Os discursos das entrevistas foram analisados, sendo levantadas 3 categorias e 10 subcategorias: fatores emocionais (fatores desencadeantes da doença, fatores agravantes da doença); finalidade da doença; convívio com a doença (suporte social, discriminação, alterações decorrentes da doença, comportamentos diante da doença, impacto da doença, a exposição dos sintomas, expectativas diante do tratamento e receio que os filhos tenham a doença). Os resultados revelaram um comprometimento na qualidade de vida tanto nas mulheres com vitiligo quanto nas mulheres com demais doenças de pele, não havendo uma diferença estatística significativa entre os dois grupos. Observou-se a associação de aspectos emocionais no surgimento e/ou agravamento do vitiligo, assim como diversas mudanças comportamentais, limitações e discriminações experienciadas pelas mulheres diante da doença. Os desenhos apontaram certas representações simbólicas do vitiligo, sinalizando conflitos e sofrimento devido à doença

Patients with classical vitiligo lose partially their protecting inherited pigment. The cause of the disease is still unknown. Despite massive epidermal oxidative / nitrative stress and signs for DNA-damage in the skin and in the plasma, these patients have no higher prevalence for sun induced non-melanoma skin cancer and increased photo-damage. Protection and DNA-repair have been attributed to a functioning up-regulated wild type p53 / p21 cascade in association with up-regulated p76 MDM2. As some patients with cutaneous melanoma develop depigmentations away from their primary tumour site post surgical excision, it became of our interest, whether this melanoma associated leucoderma (MAL) is the same as classical vitiligo. The purpose of this thesis was two-fold. In part I, we wanted to further substantiate the reasons behind the constantly up-regulated wild-type functioning p53 / p21 cascade in classical vitiligo utilising a panel of proteins with direct and / or indirect action on p53 regulation, including p21, p76MDM2, MDM4/MDM4phospho, SPARC, VEGF-A and TGF-β1. In part II, we wanted to characterize MAL and compare this peculiar leucoderma with classical vitiligo using the same protein panel and methodologies. To achieve our goals, we used in vivo FT-Raman spectroscopy, in vitro cell cultures, in vitro and in situ immuno-fluorescence labelling, Western blot, dot blot and computer modelling techniques. Our data showed distinct differences between classical vitiligo and MAL. Our results in MAL exhibited a concentration dependent protein expression gradient between the basal / suprabasl layers and the upper layers of the epidermal compartment using catalase, ONOO-, p53, p21, MDM4, p76MDM2, TGF-β1 and VEGF-A expression gradient. Moreover, we document for the first time the presence of a nitrated non-fuctional SPARC protein in classical vitiligo which is absent in MAL. Although we show in vivo considerable ROS / RNS- mediated stress in MAL and classical vitiligo documented by FT-Raman spectroscopy, Western blot and in situ immuno-fluorescence, our results prove that MAL and classical vitiligo are two distinct entities.

Vitiligo is an autoimmune skin disease in which the pigment producing cells of the epidermis, melanocytes, are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. Previous work has identified IFNg as the central cytokine driving disease pathogenesis in both human patients and in our mouse model of vitiligo. IFNg signaling induces production of the chemokines CXCL9 and CXCL10, which trigger autoreactive T cell migration into the epidermis where effector T cells can target and destroy melanocytes. However, both IFNg and type I IFN signaling through activation of STAT1 proteins can induce transcription of the chemokines CXCL9 and CXCL10. Therefore, it seems reasonable that type I IFN signaling may also contribute to disease pathogenesis. The role of type I IFN in vitiligo is still unclear. Genome wide association studies identified multiple genes within the type I IFN pathway including TICAM1 and IFIH1 as susceptibility loci in vitiligo. One additional study reported increased epidermal staining of CD123, a marker expressed by pDCs, and the type I IFN induced gene MX1 in vitiligo patient skin. However, this study did not show any functional data to support the role of type I IFN signaling in vitiligo pathogenesis. Since the role of type I IFN in vitiligo is ill-defined, we used two different mouse models of vitiligo to functionally determine the role of type I IFN in disease by inducing vitiligo in hosts which lack the type I IFN receptor (IFNaR). In the first model, we induced vitiligo by adoptive transfer of melanocyte-specific CD8 T cells, which are activated in vivo by infection with recombinant vaccinia virus (VACV) expressing their cognate antigen. Vitiligo induction in IFNaR-deficient mice led to the development of severe disease compared to wild type mice. Acceleration and severity of disease was characterized by increased early recruitment of melanocyte-specific CD8 T cells to the skin, increased production of effector cytokines TNFa and IFNg, and reduced PD-1 expression. Increased production of IFNg by CD8 T cells in the skin of IFNaR-deficient mice led to increased expression of the chemokines CXCL9 and CXCL10 driving disease progression. IFNaR-deficient mice also displayed significantly increased VACV titters compared to wild type hosts. This data reveals a role of type I IFN in the clearance of recombinant VACV. This data also suggests that persistent VACV infection and prolonged antigen exposure in IFNaR deficient hosts is likely driving enhanced activation of melanocyte specific CD8 T cells and the subsequent development of severe vitiligo. Since melanocytes and melanoma cells express shared antigens that can be recognized by CD8 T cells, and because the development of vitiligo after melanoma immunotherapy is a positive prognostic factor for patients, we asked whether VACV vaccine therapy in IFNaR deficient mice would enhance the anti-tumor response to melanoma. B16-F10 inoculated wild type and IFNaR-deficient mice received adoptive transfer of melanocyte-specific CD8 T cells in combination with vaccinia virus expressing their cognate antigen to activate the cells in vivo. Treatment of adoptive T cell transfer and infection with VACV in IFNaR-deficient mice revealed significantly reduced tumor burden compared to wild type mice. Improved tumor regression in IFNaR-deficient hosts was characterized by increased infiltrating cytotoxic T lymphocytes and reduced PD-1 expression. These results further demonstrate that in the absence of type I IFN, hosts mount a robust cytotoxic CD8 T cell response against melanocyte/melanoma antigens and this is likely a result of persistent VACV that leads to prolonged CD8 T cell priming. As a result, IFNaR deficient hosts kill tumor cells more efficiently. To determine whether type I IFN regulates disease pathogenesis in the absence of virus infection, we generated a model of vitiligo in which bone marrow derived dendritic cells (BMDCs) pulsed with the cognate antigen were used to prime melanocyte-specific T cells in place of the viral vector. Induction of vitiligo in IFNaR-deficient hosts using BMDCs revealed no significant differences in disease score compared to wild type hosts. This data clearly demonstrates that type I IFN, in contrast to IFNg, is not required during the effector stage of vitiligo pathogenesis in mice. However, since we intentionally activate transferred melanocyte-specific CD8 T cells with VACV or BMDCs expressing their cognate antigen, our mouse models may circumvent the role of type I IFNs in initiating activation of autoreactive cells and driving autoimmunity. Type I IFN is critical for providing innate immune signals that drive the priming of autoreactive T cells through maturation of DCs by inducing antigen presentation, co-stimulatory molecule expression, and migration to the lymph nodes to encounter naïve T cells. Our mouse models of vitiligo may not capture this process. We have addressed this question by using a TLR ligand to activate BMDCs before transfer into hosts. In fact, activation of BMDCs before transfer leads to significantly enhanced vitiligo in mice and this is partially a result of type I IFN signaling on host cells. Thus, we provide evidence that type I IFNs can enhance the activation of melanocyte-specific CD8 T cells and drive autoimmunity. Collectively, our results show that type I IFN signaling has disparate effects on autoreactive T cell priming in a context dependent manner. We reveal that although type I IFN is not required for the effector phase of vitiligo in mice, maturation of DCs and subsequent type I IFN production can enhance the priming of autoreactive T cells and enhance vitiligo severity. Our studies also reveal that type I IFN is required to clear recombinant attenuated VACV infection and vaccine administration in IFNaR deficient hosts led to a robust autoreactive and anti-tumor response. These insights describing the role of type I IFN in autoimmunity and tumor immunology could have important implications for T cell dependent tumor immunotherapy.

Vitiligo is an idiopathic, non-contagious and often familial depigmentation disorder affecting both sexes equally. The mostly progressive and patchy loss of the inherited skin colour is not only a cosmetical problem, it has a profound impact on the patient¿s well-being. Stigmatisation and rejection often causes depression, self-consciousness, sexual problems and an impaired quality of life. To further substantiate earlier investigations and to introduce new aspects, we utilised the Dermatology Life Quality Index (DLQI), the Beck Depression Inventory (BDI) and the Adjustment to Chronic Skin Disorders Questionnaire (ACS) with its sub-scales on Social anxiety / avoidance, Helplessness and Anxious-depressive Mood in 422 patients and 55 healthy controls. We also included 103 children, their parents and 18 controls by using the Children¿s Dermatology Life Quality Index (CDLQI) and an adapted version of the ACS. We found that patients with vitiligo experience high levels of stigmatisation. They have an impaired quality of life and are more socially anxious / avoidant, helpless and (anxious-) depressive compared to healthy controls. The results correlate with disease severity, avoidant behaviour / hiding of vitiligo and the belief that psychological stress influences the disease. Female patients are generally more affected. Treatment with pseudocatalase PC-KUS improves quality of life and reduces anxious-depressive mood. Children also suffer from stigmatisation and an impaired quality of life. Parents are more socially anxious and helpless compared to the control group. / German Vitiligo Association (Deutscher Vitiligo Verein)