Autoimmune Vitiligo Research Articles

Seven cases of vitiligo complicated by atopic dermatitis: suggestive new spectrum of autoimmune vitiligo

ejd.2012.1656 Auteur(s) : Atsushi Tanemura1 tanemura@derma.med.osaka-u.ac.jp, Tomoko Yajima1, Mayuko Nakano1, Megumi Nishioka1, Saori Itoi1, Yorihisa Kotobuki1, Mari Higashiyama2, Ichiro Katayama1 1 Department of Dermatology, Osaka University School of Medicine, 2-2 Yamadaoka Suita, Osaka 565-0871, Japan 2 Department of Dermatology, Nissei Hospital, Osaka, Japan A recent report strongly suggests that genetic polymorphisms such as the NALP1 gene are closely associated with generalized vitiligo, especially [...]

Dysregulation of melanocyte function by Th17‐related cytokines: significance of Th17 cell infiltration in autoimmune vitiligo vulgaris

The aim of this study was to determine whether CD4(+) IL-17A(+) Th17 cells infiltrate vitiligo skin and to investigate whether the proinflammatory cytokines related to Th17 cell influence melanocyte enzymatic activity and cell fate. An immunohistochemical analysis showed Th17 cell infiltration in 21 of 23 vitiligo skin samples in addition to CD8(+) cells on the reticular dermis. An in vitro analysis showed that the expression of MITF and downstream genes was downregulated in melanocytes by treatment with interleukin (IL)-17A, IL-1β, IL-6, and tumor necrosis factor (TNF)-α. Treatment with these cytokines also induced morphological shrinking in melanocytes, resulting in decreased melanin production. In terms of local cytokine network in the skin, IL-17A dramatically induced IL-1β, IL-6, and TNF-α production in skin-resident cells such as keratinocytes and fibroblasts. Our results provide evidence of the influence of a complex Th17 cell-related cytokine environment in local depigmentation in addition to CD8(+) cell-mediated melanocyte destruction in autoimmune vitiligo.

A Current Viewpoint of Lymphangioleiomyomatosis Supporting Immunotherapeutic Treatment Options

Lymphangioleiomyomatosis (LAM) leads to hyperproliferation of abnormal smooth muscle cells in the lungs, associated with diffuse pulmonary parenchymal cyst formation and progressive dyspnea on exertion. The disease targets women of child-bearing age. Complications include pneumothoraces and chylous pleural effusions. Ten-year survival is estimated at 70%, and lung transplantation remains the only validated treatment. It has been observed that LAM cells express markers associated with melanocytic differentiation, including gp100 and MART-1. Other melanocytic markers have also been observed. The same proteins are targeted by T cells infiltrating melanoma tumors as well as by T cells infiltrating autoimmune vitiligo skin, and these antigens are regarded as relatively immunogenic. Consequently, vaccines have been developed for melanoma targeting these and other immunogenic melanocyte differentiation proteins. Preliminary data showing susceptibility of LAM cells to melanoma derived T cells suggest that vaccines targeting melanosomal antigens can be successful in treating LAM.

Necrobiosis lipoidica in diabetic patients: pathogenetic and clinical features

Necrobiosis lipoidica (NL) is a chronic skin disease (dermatosis) associated with various metabolic disorders including carbohydrate metabolism.In the last years, NL has been increasingly frequently diagnosed by practitioners probably due to the rise in type 2 diabetes mellitus morbidity in thegeneral population. Moreover, association of NL with autoimmune thyroid diseases, Crohns disease, ulcerative colitis, sarcoidosis, and vitiligo hasbeen documented. Such cases may be attributed to phenotypic and functional defects of cellular immunity.

Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

BackgroundWe have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma.Methodology and Principal FindingsTo induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision.Conclusions and SignificanceThis work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer.

Tongue Hyperpigmentation Resulting from Peginterferon Alfa-2b and Ribavirin Treatment in a Patient with Chronic Hepatitis C

Hepatitis C virus (HCV) infection has been associated with several cutaneous diseases such as lichen planus, porphyria cutanea tarda, chronic pruritus, and cutaneous necrotizing vasculitis (Doutre, Arch Dermatol 135:1401-1403, 1999). The antiviral treatment for chronic HCV with interferon alfa (INF) or peginterferon alfa (PEG-INF) combined with rivabirin also leads to many skin side effects including injection site reaction, generalized skin rashes, pruritus, dry skin, alopecia, and exacerbation of autoimmune processes, particularly psoriasis, lichen planus or vitiligo (Dalekos et al., Eur J Gastroenterol Hepatol 10:933-939, 1998; Sookoian et al., Arch Dermatol 135:1000-1000, 1999). There are case reports of tongue hyperpigmentation during combination therapy of PEG IFN and RBV in chronic hepatitis C both in dark-skined as well as Caucasian. We report the first case of tongue hyperpigmentation associated with PEG-INF-2b plus ribavirin administration in a non-Caucasian patient with genotype 4.

Engagement of NK receptor NKG2D, but not 2B4, results in self-reactive CD8+T cells and autoimmune vitiligo

In this study, we demonstrate that engagement of two different natural killer receptors (NKRs) can lead to contrasting effects in the development of self-reactive CD8+T cells and autoimmune vitiligo. Specifically, using a mouse model, we show that CD8+T-cell targeting of a melanocyte antigen, tyrosinase-related protein-1 (TRP-1) in combination with delivery of the NKG2D ligands (Rae-1ϵ or H60), results in strong CD8+T-cell responses against TRP-1 and in the development of autoimmune vitiligo. In contrast, targeting of TRP-1 in combination with delivery of CD48, the natural ligand for the NKR 2B4, leads to reduced formation of TRP-1-reactive CD8+T-cell responses and decreased development of vitiligo. These data indicate that autoimmune vitiligo is limited by insufficient signals, despite plentiful self-reactive T cells in the peripheral immune system. To our knowledge, this is the first experimental evidence supporting the role of NKRs in modulating CD8+T-cell autoimmune vitiligo. This study supports the utilization of NKR signaling as a therapeutic avenue toward prevention of vitiligo and other autoimmune diseases.

Systemic sclerosis in a patient with pityriasis rubra pilaris

Pityriasis rubra pilaris (PRP) is a rare, chronic erythematous squamous disorder of unknown etiology. It has been found in association with several autoimmune diseases, including thyroiditis, myositis, myasthenia gravis and vitiligo. Herein we report a case of systemic sclerosis in a patient with classic adult pityriasis rubra pilaris. A 38 year old woman with classic adult type 1 pityriasis rubra pilaris (PRP) developed progressive skin thickening of the trunk, face, upper and lower extremities after 2 years of PRP treatment with topical emollients and steroids. Clinical examination and immunological findings were consistent with SSc. Co-existence of these two rare conditions is documented for the first time.

Impact of the hypomethylating agent 5-azacytidine on dendritic cells function

Recent evidence suggested that 5-azacytidine (5-aza) can impact important immune functions via epigenetic modifications, making it an attractive candidate for pharmacologic manipulation of the immune system. The aim of this work was to study the effects of 5-aza on human dendritic cells (DC) generated from peripheral blood monocytes, and to test the type of immune response induced in patients treated with 5-aza. On the phenotypic level, CD40 and CD86 expression was significantly increased on mature DC exposed to 5-aza (5-aza-DC), compared with control untreated DC. Mature control DC and mature 5-aza-DC secreted comparable amounts of interleukin (IL)-6, IL-12p70, IL-23, and tumor necrosis factor-α. However, mature 5-aza-DC secreted significantly lower levels of IL-10 and IL-27 compared to mature control DC (p = 0.04 and p = 0.005, respectively). In the peripheral blood of 14 patients (7 males and 7 females; age range, 53-81 years) with advanced myeloid malignancies (8 acute myeloid leukemia and 6 myelodysplastic syndrome) treated with 5-aza, there was a significant decrease of IL-4-secreting CD4(+) T cells (p = 0.001), and a significant increase of IL-17A- and IL-21-secreting CD4(+) T cells (p = 0.003 and p = 0.01, respectively, compared to 5 healthy donors) suggesting a Th17 response pattern in the blood of patients receiving 5-aza. In all, these data suggest potentially novel mechanisms of action of epigenetic therapies, such as 5-aza, which may have broader implications for immunotherapeutic strategies.

Maintenance of immune hyporesponsiveness to melanosomal proteins by DHICA-mediated antioxidation: Possible implications for autoimmune vitiligo

Melanocyte destruction in the skin of vitiligo patients has been considered to be a consequence of an autoimmune response against melanosomal proteins. However, little is known about the molecular mechanisms by which the immune system recognizes these sequestered intracellular self-proteins, which are confined in specialized organelles termed melanosomes, and is provoked into an autoimmune response to melanocytes. Here, we utilize a sucrose density-gradient ultracentrifugation protocol to enrich melanosomal components from dopachrome tautomerase (Dct)-mutant or wild-type melanocytes exposed to a pulse of hydrogen peroxide at a noncytotoxic concentration to evaluate their immunogenicity in mice challenged with the corresponding melanosomal proteins. The results demonstrate that enhanced humoral and cellular immune responses to a challenge with late-stage melanosomal proteins, especially with those derived from Dct-mutant melanocytes, are found in the immunized mice. To elucidate whether a reduced 5,6-dihydroxyindole-2-carboxylic acid (DHICA) content in melanin might cause a loss in antioxidative protection to the proteins, we incubated these melanosomal proteins in vitro with synthetic 5,6-dihydroindole (DHI)–melanin or DHI/DHICA (1:1)–melanin and then used them to immunize mice. T cell proliferation and IgG antibody responsiveness to the challenges were significantly induced by melanosomal proteins treated with DHI–melanin, but not by those treated with DHI/DHICA (1:1)–melanin. Moreover, we observed that melanosomal proteins derived from Dct-mutant melanocytes are subject to oxidative modifications that alter their antigenic configurations to attain an enhanced immunogenicity compared with those derived from wild-type melanocytes. From these results, we conclude that DHICA-mediated antioxidation plays a critical role in the maintenance of immune hyporesponsiveness to melanosomal proteins.

Stimulation of the Glucocorticoid-Induced TNF Receptor Family-Related Receptor on CD8 T Cells Induces Protective and High-Avidity T Cell Responses to Tumor-Specific Antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.

Transplantation of mouse HSCs genetically modified to express a CD4-restricted TCR results in long-term immunity that destroys tumors and initiates spontaneous autoimmunity

The development of effective cancer immunotherapies has been consistently hampered by several factors, including an inability to instigate long-term effective functional antitumor immunity. This is particularly true for immunotherapies that focus on the adoptive transfer of activated or genetically modified mature CD8+ T cells. In this study, we sought to alter and enhance long-term host immunity by genetically modifying, then transplanting, mouse HSCs. We first cloned a previously identified tumor-reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen tyrosinase-related protein 1 (Tyrp1), then constructed both a high-expression lentivirus vector and a TCR-transgenic mouse expressing the genes encoding this TCR. Using these tools, we demonstrated that both mouse and human HSCs established durable, high-efficiency TCR gene transfer following long-term transplantation into lethally irradiated mice transgenic for HLA-DR4. Recipients of genetically modified mouse HSCs developed spontaneous autoimmune vitiligo that was associated with the presence of a Th1-polarized memory effector CD4+ T cell population that expressed the Tyrp1-specific TCR. Most importantly, large numbers of CD4+ T cells expressing the Tyrp1-specific TCR were detected in secondary HLA-DR4-transgenic transplant recipients, and these mice were able to destroy subcutaneously administered melanoma cells without the aid of vaccination, immune modulation, or cytokine administration. These results demonstrate the creation of what we believe to be a novel translational model of durable lentiviral gene transfer that results in long-term effective immunity.

Transgenic mice with a diverse human T cell antigen receptor repertoire

Because of tolerance mechanisms, it has been hard to identify the T cell receptors (TCRs) of high-avidity T cells against self (for example, tumor) antigens. TCRs that are specific for foreign human antigens from the nontolerant T cell repertoire can be identified in mice. Moreover, if mice are constructed to express the human TCR repertoire, they can be used to analyze the unskewed repertoire against human self antigens. Here we generated transgenic mice with the entire human TCRalphabeta gene loci (1.1 and 0.7 Mb), whose T cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. A human major histocompatibility class I transgene increases the generation of CD8+ T cells with human compared to mouse TCRs. Functional CD8+ T cells against several human tumor antigens were induced, and those against the Melan-A melanoma antigen used similar TCRs to those that have been detected in T cell clones from individuals with autoimmune vitiligo or melanoma. These mice will allow researchers to identify pathogenic and therapeutic human TCRs.

Potentiating Cancer Immunotherapy Using an Oncolytic Virus

Oncolytic viruses (OVs) are highly immunogenic and this limits their use in immune-competent hosts. Although immunosuppression may improve viral oncolysis, this gain is likely achieved at the cost of antitumoral immunity. We have developed a strategy wherein the immune response against the OV leads to enhanced therapeutic outcomes. We demonstrate that immunization with an adenoviral (Ad) vaccine before treatment with an oncolytic vesicular stomatitis virus (VSV) expressing the same tumor antigen (Ag) leads to significantly enhanced antitumoral immunity. Intratumoral replication of VSV was minimally attenuated in Ad-immunized hosts but extending the interval between treatments reduced the attenuating effect and further increased antitumoral immunity. More importantly, our combination approach shifted the immune response from viral Ags to tumor Ags and further reduced OV replication in normal tissues, leading to enhancements in both efficacy and safety. These studies also highlight the benefits of using a replicating, OV to boost a pre-existing antitumoral immune response as this approach generated larger responses versus tumor Ag in tumor-bearing hosts than could be achieved in tumor-free hosts. This strategy should be applicable to other vector combinations, tumor Ags, and tumor targets.

A phase II clinical trial of a novel combinatorial antitumor immunotherapy for patients with high-risk resected stage III and metastatic melanoma.

8586 Background: Patients with advanced melanoma have a uniformly poor outcome, despite all current treatment modalities. Cancer immunotherapy is designed to activate the host immune system and destroy viable tumor cells, often through novel immunologic combinatorial approaches. We hypothesize that such an approach can be an effective treatment option for patients with high-risk resected stage III, recurrent, refractory or stage IV melanoma. Methods: We designed a phase II clinical trial comprised of the HyperAcute Melanoma (HAM) vaccine (NewLink Genetics Corp.) combined with PEG-Intron (Schering-Plough Corp.). Trial design consists of a 12-week regimen, the first 4 weeks with HAM vaccine alone (intradermally) followed by weeks 5-12 with HAM vaccine + PEG-Intron (once weekly, subcutaneously at 6 μ g/kg). Trial endpoints include correlative studies for observed anti-tumor effect and secondarily to determine the overall safety and response rates. Planned sample size is 25 patients. Results: A total of 15 patients have enrolled, with 12 completing the trial and 3 in active treatment. RECIST criteria was utilized for 12 evaluable patients, with 5 developing progressive disease, 5 with no evidence of disease, one with a complete response and one with an ongoing partial response of > 12 months, with marked regression of metastatic disease. Relapse-free interval ranges from 15 months to 3 months. The development of autoimmune antibodies (anticardiolipin, antithyroglobulin) following the completion of the trial was seen in all 11 patients (first patient not evaluated). Vitiligo has developed in 4/12 patients, correlating with 2 patients who developed clinical tumor regression. Conclusions: The combined immunotherapy of advanced melanoma patients with PEG-Intron and HAM Vaccine shows evidence of synergistic immune activation that translates into clinical efficacy as seen with tumor regression combined with development of autoimmune antibodies and vitiligo. However, we will need to await final analysis of all patients to determine the ultimate significance of host immune activation as it relates to tumor regression in melanoma patients with advanced disease. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration NewLink Genetics NewLink Genetics NewLink Genetics, Schering-Plough

Global transcriptome analysis of feathers from Smyth line chickens revealed different gene-expression profiles at various stages of autoimmune vitiligo (83.17)

Abstract The Smyth line (SL) chicken is an animal model for autoimmune vitiligo (SLV). SL chickens develop spontaneous, post-hatch, autoimmune melanocyte loss in growing feathers (GF). With repeatable access to GF and the high incidence (85-95%) of SLV, SLV development can be examined in the same bird. A microarray study was conducted to investigate the global transcriptomic profile in GF from SL and Brown line (BL, parental control) chickens. RNA pools were made from GF of BL chickens, SL chickens that never developed SLV (S0), and SL chickens before (S1), during (S2) and at complete (S3) SLV. Complementary RNA probes were generated from BL and SL (S0~S3) RNA, labeled with Cy3 and Cy5, respectively and hybridized on a chicken 4x44 K Agilent microarray. The slide was scanned and normalized signal intensities were analyzed to identify differentially expressed (DE, >2x) genes between BL and SL samples. There were 504, 518, 1124, and 1151 DE genes containing 262, 226, 658, and 680 unique DE genes in S0, S1, S2 and S3, respectively. S0 and S1 had similar proportions of up-and down-regulated DE genes while up-regulated DE genes predominated in S2 and S3. The highest expressed DE genes were related to immune activity and cell death. Ingenuity Pathway Analysis software was used to determine biological functions and pathways of DE genes. The study provides comprehensive information from global gene expression to functionalities and biological networks of DE genes in the process of SLV.

Abnormal morphology of melanosomes in the autoimmune vitiligo-prone Smyth line chicken does not appear to be due to alteration in lipid composition (83.16)

Abstract The Smyth line (SL) chicken is an important animal model for autoimmune vitiligo. SL vitiligo is multifactorial in nature including genetic susceptibility, immune system components and environmental triggers. Genetic susceptibility to SL vitiligo appears to be manifested in part by an inherent melanocyte defect. SL melanosomes (pigment-carrying organelles) have irregular-shaped membranes with pigmented extensions that may be due to abnormal lipid composition. Lipids can affect protein topology and trafficking (and hence protein degradation and presentation), membrane stability and oxidative stress resistance. To compare melanosomal lipids from SL and control lines of chickens, melanosomes were isolated from growing feathers of 4-week-old SL, Brown line (parental control) and Light Brown Leghorn (vitiligo-resistant) chickens and lipids were extracted. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) combined with thin-layer chromatography (TLC) was used to determine expression differences in different classes of lipids by using TLC-MALDI imaging technique. Total fatty acid composition was examined by gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters. Statistical analysis of preliminary data shows no significant differences in total fatty acid and phospholipid composition in melanosomes from SL and control chickens. Hence, lipid composition may not explain the melanosomal abnormalities observed in SL melanocytes.

Mechanisms of Spatial and Temporal Development of Autoimmune Vitiligo in Tyrosinase-Specific TCR Transgenic Mice

Generalized vitiligo is thought to have an autoimmune etiology and has been correlated with the presence of CD8 T cells specific for melanocyte differentiation Ag. However, limited animal models for the disease have hampered its understanding. Thus, we generated TCR transgenic mice that recognize an epitope of the melanocyte protein, tyrosinase. These animals develop vitiligo with strikingly similar characteristics to the human disease. Vitiligo develops temporally and spatially, with juvenile lesions forming bilaterally in head and facial areas, and only arising later in the body of adult animals. Vitiligo is entirely dependent on CD8 T cells, whereas CD4 T cells exert a negative regulatory effect. Importantly, CD8 T cells can be pervasively present in the skin in the steady state without inducing vitiligo in most areas. This points to developmental differences in melanocyte susceptibility and/or immunological effector mechanisms over time, or in different body locations. Disease is strongly dependent on both IFN-gamma and CXCR3, whereas dependence on CCR5 is more limited, and both CCR4 and perforin are dispensable. Genetic ablation of CXCR3 or IFN-gamma also resulted in scarce CD8 T cell infiltration into the skin. Our results identify unexpected complexity in vitiligo development and point toward possible therapeutic interventions.

Serum ovotransferrin as a biomarker of inflammatory diseases in chickens

Infectious and metabolic disorders are common in poultry and cause stress, poor performance, and mortality that results in considerable economic loss. Identifying the nature of stress in chickens will assist the development of appropriate measures to improve health and welfare. Acute phase proteins are hepatic proteins, the blood concentrations of which change significantly in the event of many health problems including inflammation and physical injuries. Thus, acute phase proteins are used as nonspecific diagnostic markers for various health disorders. Our previous studies showed that serum ovotransferrin (OVT) is an acute phase protein in chickens. Therefore, in the present study, we investigated whether OVT concentration can be a marker of physiological stress using sera from chickens with different infectious and metabolic disorders. A competitive enzyme immunoassay was developed to measure serum OVT concentrations. The results show that with experimentally induced pulmonary hypertension syndrome and tibial dyschondroplasia, there were no significant changes in OVT levels compared with matched controls. In contrast, when chickens were infected with microbes such as the bacterium Escherichia coli, or protozoan parasites such as Eimeria maxima and Eimeria tenella, there was a significant increase in the levels of OVT in the serum. Chickens with spontaneous autoimmune vitiligo also showed a significant increase in blood OVT levels. These studies suggest that blood OVT concentration is modulated under inflammatory and microbial stress and can therefore be used as a diagnostic marker of infection and inflammation in chickens.

A Hard Diagnosis

A 56-year-old woman presented to the emergency room with a 4-week history of malaise. The patient reported no dyspnea, chest pain, weight loss, nausea, abdominal pain, hematuria, myalgias, or arthralgias. For many years, she periodically had an urticarial rash on her arms; the most recent episode was 4 weeks previously. Her medical history was notable only for autoimmune thyroiditis and vitiligo.