I. Caroline Le Poole

Autoimmune etiology of generalized vitiligo

Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therapy.

Reduced skin homing by functional Treg in vitiligo

In human vitiligo, cutaneous depigmentation involves cytotoxic activity of autoreactive T cells. It was hypothesized that depigmentation can progress in the absence of regulatory T cells (Treg). The percentage of Treg among skin infiltrating T cells was evaluated by immunoenzymatic double staining for CD3 and FoxP3, revealing drastically reduced numbers of Treg in non‐lesional, perilesional and lesional vitiligo skin. Assessment of the circulating Treg pool by FACS analysis of CD4, CD25, CD127 and FoxP3 expression, and mixed lymphocyte reactions in presence and absence of sorted Treg revealed no systemic drop in the abundance or activity of Treg in vitiligo patients. Expression of skin homing receptors CCR4, CCR5, CCR8 and CLA was comparable among circulating vitiligo and control Treg. Treg from either source were equally capable of migrating towards CCR4 ligand and skin homing chemokine CCL22, yet significantly reduced expression of CCL22 in vitiligo skin observed by immunohistochemistry may explain failure of circulating, functional Treg to home to the skin in vitiligo. The paucity of Treg in vitiligo skin is likely crucial for perpetual anti‐melanocyte reactivity in progressive disease.

A symbiotic concept of autoimmunity and tumour immunity: lessons from vitiligo

Vitiligo is a skin disease in which melanocytes (MCs) are eradicated from lesional epidermis, resulting in disfiguring loss of pigment. MCs are destroyed by MC-reactive T cells, as well as other non-immune and immune components. Similarities exist between the autoimmunity observed in vitiligo and the tumour immunity observed in melanoma immuno-surveillance. An analysis of these mechanisms might lead to the development of new therapies for both vitiligo and melanoma.

Notch Signaling Regulates Mouse and Human Th17 Differentiation

Th17 cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. In this study, we report that Notch1 is activated in both mouse and human in vitro-polarized Th17 cells and that blockade of Notch signaling significantly downregulates the production of Th17-associated cytokines, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies, as well as chromatin immunoprecipitation, that IL-17 and retinoic acid-related orphan receptor γt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in vivo inhibition of Notch signaling reduced IL-17 production and Th17-mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.

Cervical cancer vaccines: recent advances in HPV research.

Carcinomas of the anogenital tract, particularly cancer of the cervix, account for almost 12% of all cancers in women, and so represent the second most frequent gynecological malignancy in the world (48). It is well established that chronic infection of cervical epithelium by human papillomaviruses (HPV) is necessary for the development of cervical cancer. In fact, HPV DNA has been demonstrated in more than 99.7% of cervical cancer biopsy specimens, with high-risk HPV16 and HPV18 sequences being most prevalent (45,73). Therefore, an effective vaccine that would mount an immune response against HPV-related proteins might contribute to the prevention or elimination of HPV expressing lesions. This review will concentrate on the most recent advances in vaccine-mediated prevention and immunotherapy of HPV-induced cervical cancer, including presentations from the 20(th) International HPV Conference held in October 2002 in Paris.

HSP70i Accelerates Depigmentation in a Mouse Model of Autoimmune Vitiligo

Vitiligo is a T-cell-mediated autoimmune disease of the skin. Progressive depigmentation accelerates in response to stress. Personal trauma, contact with bleaching phenols, overexposure to UV, and mechanical injury can lead to progressive loss of melanocytes. This study was focused on the role of stress protein heat shock protein (HSP)70 for translating stress into an autoimmune disease to melanocytes. Intracellular HSP70 can act as a cytoprotectant, preventing apoptosis in cells under stress. Isoform HSP70i can be secreted by live cells, and in prior in vitro studies, HSP70 has been shown to activate dendritic cells and elicit an immune response to chaperoned proteins and peptides. Here, the role of HSP70 in precipitating and perpetuating vitiligo was assessed in vivo in a mouse model of autoimmune vitiligo. In this model, depigmentation was introduced by gene gun vaccination with eukaryotic expression plasmids encoding melanocyte differentiation antigens. Inclusion of human and mouse-derived inducible HSP70 in the vaccination protocol significantly increased and accelerated depigmentation in this model, accompanied by the induction of prolonged humoral responses to HSP70. Cytotoxicity toward targets loaded with a K(b)-restricted tyrosinase-related protein 2-derived peptide correlated with depigmentation. The data presented strongly support a role for HSP70i in progressive depigmentation in vivo.

Monobenzyl Ether of Hydroquinone and 4-Tertiary Butyl Phenol Activate Markedly Different Physiological Responses in Melanocytes: Relevance to Skin Depigmentation

Monobenzyl ether of hydroquinone (MBEH) is a Food and Drug Administration approved drug used for depigmentation therapy of advanced vitiligo. Here, the working mechanism of MBEH is explored in comparison to 4-tertiary butyl phenol (4-TBP), a known causative agent for occupational vitiligo mediating apoptotic melanocytic death. Cytotoxic experiments reveal that similar to 4-TBP, MBEH induces specific melanocyte death. To compare death pathways initiated by 4-TBP and MBEH, classical apoptotic hallmarks were evaluated in treated melanocytes. MBEH induced cell death without activating the caspase cascade or DNA fragmentation, showing that the death pathway is non-apoptotic. Release of High Mobility Group Box-1 protein by MBEH-treated melanocytes and ultrastructural features further confirmed a necrotic death pathway mediated by MBEH. A negative correlation between MBEH-induced cell death and cellular melanin content supports a cytoprotective role for melanin. Moreover, MBEH exposure upregulated the levels of melanogenic enzymes in cultured melanocytes and skin explants, whereas 4-TBP reduced the expression of the same. In summary, exposure to MBEH or 4-TBP has profoundly different consequences for melanocyte physiology and activates different death pathways. As the mode of cell death defines the nature of the immune response that follows, these findings help to explain the relative efficacy of these agents in mediating depigmentation.

Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

Vitiligo can be reversed through immune targeting with mutant heat shock protein 70. New Treatment Makes Vitiligo Beat It Whether your grant application is due, you have a paper that needs to be submitted, or your patient load is too high, medical science is not a relaxing profession. High stress is known to negatively affect your health at both the whole body and cellular level. One way the body responds to cellular stressors is through the induction of heat shock proteins (HSPs). Now, Mosenson et al. suggest that mutant HSP70 could be a potential treatment for autoimmune vitiligo. The authors noticed that mutant inducible HSP70 (HSP70i) could prevent T cell–mediated depigmentation in a mouse model of vitiligo, perhaps by shifting dendritic cells from an inflammatory to a regulatory phenotype. Moreover, a DNA vaccine of the mutant HSP70i could be used therapeutically to partially restore pigmentation in a second model of depigmentation. The authors then took these studies into ex vivo human skin, showing that their mutant HSP70i could prevent the disease-related shift from quiescent to effector T cell phenotype. Although these observations still need to be translated into the clinic, they form the basis for a new potential treatment for autoimmune vitiligo. Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

Generation of a human melanocyte cell line by introduction of HPV16 E6 and E7 genes

SummaryAvailability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.

Efficient debridement of necrotic wounds using proteolytic enzymes derived from Antarctic krill: A double-blind, placebo-controlled study in a standardized animal wound model

Wound healing can be accelerated by removing necrotic tissue. Various methods of wound debridement have been developed, including enzymatic debridement. Recently potent proteolytic enzymes were isolated from the intestine of Euphausia superba (Antarctic krill) that might be useful for degrading necrotic tissue. The purpose of this study was to evaluate the debriding properties of krill enzymes, using a specially designed animal model and a computerized analysis system. In 10 female domestic pigs, each weighing 20 kg, 6 artificial ulcers were made on each animals back using electrokeratome, followed by application of trichloracetic acid. Ulcers were treated twice daily for 7 days with either krill enzymes at different concentrations or with saline. Reduction of necrotic tissue was measured daily using computerized wound analysis. Histological examination included the determination of bromodeoxyuridine incorporation in order to detect cell proliferation as well as routine stains. The debriding effect of krill enzymes at a concentration of ≥ 3.0 casein units per ml was significantly better than saline control treatment (p < 0.05). The effect was dose dependent, and granulation tissue formation was enhanced. In conclusion, krill enzymes are effective in wound debridement, as measured in this animal model.