Guidelines for clinical trials of frontal fibrosing alopecia: consensus recommendations from the International FFA Cooperative Group (IFFACG).

BACKGROUND: Frontal fibrosing alopecia (FFA) has become one of the most common causes of cicatricial alopecia worldwide. However, there is a lack of clear aetiology and robust clinical trial evidence for the efficacy and safety of agents currently used for treatment. OBJECTIVES: To enable data to be collected worldwide on FFA using common criteria and assessment methods. METHODS: A multicentre, international group of experts in hair loss was convened by email to create consensus recommendations for clinical trials. Consensus was defined at > 90% agreement on each recommended part of these guidelines. RESULTS: Standardized diagnostic criteria, severity rating, staging, and investigator and patient assessment of scalp hair loss and other clinical features of FFA were created. CONCLUSIONS: These guidelines should allow the collection of reliable aggregate data on FFA and advance efforts in both clinical and basic research to close knowledge gaps in this condition.

Update on cicatricial alopecia.

Cicatricial alopecia is an enigmatic group of hair disorders linked by the potential permanent loss of scalp hair follicles in involved areas. Progress in our understanding and treatment of these disorders has been stymied by the lack of clear diagnostic criteria for the current terms used to describe the various hair loss entities. Since all of these conditions evolve as the hair is destroyed or replaced, diagnosis is further made difficult by a lack of clinical and pathologic "snapshots" over the evolution of each disorder. Without some acceptance of general clinical and histological presentations in the early, mid and late stage of these disorders, one cannot begin to explore ways to make the diagnosis at a very early stage before significant follicular destraction has occurred (making the clinical diagnosis obvious) and when the damage is potentially repairable or progression preventable.

[The hair follicle as a target for gene therapy]. / Les follicules pilaires comme cibles de la thérapie génique.

The hair follicle possesses progenitor cells required for continuous hair follicle cycling and for epidermal keratinocytes, melanocytes and Langerhans cells. These different cell types can be the target of topical gene delivery in the skin of the mouse. Using a combination of liposomes and DNA, we demonstrate the feasibility of targeting hair follicle cells in human scalp xenografts. We consider liposome composition and stage of the hair cycle as important parameters influencing transfection of human hair follicles. Transfection is possible only during the early anagen phase. Factors and obstacles for the use of gene therapy in treating alopecia and skin diseases are discussed. A theoretical framework for future treatment of cutaneous and systemic disorders using gene therapy is presented.

Atrichia caused by mutations in the vitamin D receptor gene is a phenocopy of generalized atrichia caused by mutations in the hairless gene.

Generalized atrichia with papules is a rare disorder characterized by loss of hair shortly after birth and development of cutaneous cysts. Mutations in the hairless gene (HR) cause this phenotype in both mouse and human. Here we present a case of atrichia with papules in a patient with a normal HAIRLESS gene but with mutations in both alleles of the VITAMIN D RECEPTOR. The patient exhibited vitamin D resistant rickets, which was confirmed by an absent response of her fibroblasts to 1,25-dihydroxyvitamin D3 in vitro. Similar to individuals with HAIRLESS mutations, her skin showed an absence of normal hair follicles and the presence of follicular remnants and cysts. The cyst epithelium contained keratin-15- and keratin-17-positive cells suggesting derivation from the hair follicle bulge and the presence of epithelial stem cells. Although hair loss has been reported in association with hereditary vitamin D resistant rickets, we now characterize this alopecia as clinically and pathologically indistinguishable from generalized atrichia with papules, which was previously thought to be caused only by mutations in HAIRLESS. These findings suggest that VDR and HR, which are both zinc finger proteins, may be in the same genetic pathway that controls postnatal cycling of the hair follicle.

Towards a molecular understanding of hair loss and its treatment.

Most common forms of hair loss (alopecia) are caused by aberrant hair follicle cycling and changes in hair follicle morphology. However, current treatments for alopecia do not specifically target these processes. We are now beginning to identify the molecules and molecular pathways that control normal hair follicle formation, cycling and growth. In parallel, new techniques are being developed for delivering molecules to hair follicles. Here, we outline the characteristics of common hair loss diseases, and discuss ways in which recent advances in hair follicle biology could be translated into effective therapies for these conditions.

The hair follicle as a target for gene therapy.

The hair follicle possesses progenitor cells for continued hair follicle cycling and for epidermal keratinocytes, melanocytes and Langerhans cells. These different cell types can be targeted by topical gene delivery to mouse skin. Using a combination of liposomes and DNA, we demonstrated the feasibility of targeting hair follicle cells in human scalp xenografts as well. We defined liposome composition and stage of the hair cycle as important parameters influencing transfection of human hair follicles. Transfection occurred only during anagen onset. Considerations and obstacles for using gene therapy to treat alopecias and skin disease are discussed. A theoretical framework for future gene therapy treatments for cutaneous and systemic disorders is presented.

beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.

The human hair follicle immune system: cellular composition and immune privilege.

The immunology of the hair follicle, its relationship with the 'skin immune system' and its role in hair diseases remain biologically intriguing and clinically important. In this study, we analysed the immunoreactivity patterns of 15 immunodermatological markers to determine the cellular composition and immune privilege of the human hair follicle immune system in anagen VI (growth phase). The most prominent cells located in or around the hair follicle were Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells, whereas B cells, natural killer cells and gammadelta T cells were found very rarely. Langerhans cells (CD1a+, major histocompatibility complex, MHC class II+), and T cells (CD4+ or CD8+) were predominantly distributed in the distal hair follicle epithelium, whereas macrophages (CD68+, MHC class II+) and mast cells (Giemsa+) were located in the perifollicular connective tissue sheath. Transmission electron microscopy confirmed low numbers of immune cells in the proximal hair follicle epithelium, and very few macrophages and Langerhans cells were seen in the dermal papilla. Melanophages were observed in the connective tissue sheath and dermal papilla. MHC class I (HLA-A, -B, -C) and beta2-microglobulin immunoreactivity was found on most skin cells, but was substantially reduced on isthmus keratinocytes and virtually absent in the proximal hair follicle epithelium. Apart from the absence of Fas ligand immunoreactivity, the sharply reduced numbers of T cells and Langerhans cells, and the virtual absence of MHC class I expression all suggest that the anagen proximal hair follicle constitutes an area of immune privilege within the hair follicle immune system, whose collapse may be crucial for the pathogenesis of alopecia areata.

Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex.

The topical delivery of transgenes to hair follicles has potential for treating disorders of the skin and hair. Here we show that the topical administration of liposome-DNA mixtures (lipoplex) to mouse skin and to human skin xenografts resulted in efficient in vivo transfection of hair follicle cells. Transfection depended on liposome composition, and occurred only at the onset of a new growing stage of the hair cycle. Manipulating the hair follicle cycle with depilation and retinoic acid treatment resulted in nearly 50% transfection efficiency-defined as the proportion of transfected, newly growing follicles within the xenograft. Transgenes administered in this fashion are selectively expressed in hair progenitor cells and therefore have the potential to affect the characteristics of the follicle. These findings form a foundation for the future use of topical lipoplex applications to alter hair follicle phenotype and treat diseases of the hair and skin.

Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide.

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.

Cytokeratin 15 expression in trichoepitheliomas and a subset of basal cell carcinomas suggests they originate from hair follicle stem cells.

Trichoepitheliomas and many basal cell carcinomas appear to arise from the hair follicle, and in particular from the hair follicle bulge. This histogenesis is suggested from both morphological and immunohistochemical studies on tumor cells and stroma. Epithelial stem cells are thought to be important in tumorigenesis, and we previously localized a population of stem cells to the bulge area of the outer root sheath. We recently identified an anti-CD8 monoclonal antibody (DAKO clone C8/144B) that cross-reacts with cytokeratin 15 (K15), and serves as a specific marker for the bulge. In this study, we screened a series of trichoepitheliomas (n=13), basal cell carcinomas (n=37) and a variety of other skin tumors with this antibody. All trichoepitheliomas (100%) showed keratin 15 expression, while only a subset of basal cell carcinomas (27%) was K15-positive. Epidermal tumors, including squamous cell carcinomas, were K15-negative. Tumors of follicular derivation such as proliferating trichilemmal cysts were also K15-positive, while others such as pilomatricoma were K15-negative. Expression of K15 in trichoepitheliomas, some basal cell carcinomas and other follicular tumors suggests that these tumors are related to hair follicle stem cells in the bulge.

Epithelial stem cells in the skin: definition, markers, localization and functions.

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?

Human hair follicle bulge cells are biochemically distinct and possess an epithelial stem cell phenotype.

Stem cells are vital for the homeostasis of self-renewing tissues and their manipulation may have wide ranging applications, including gene therapy, wound repair, and tissue transplantation. Although rodent hair follicle stem cells have been localized to the follicle bulge, the location of human hair follicle stem cells is less clear, and their characterization has been hampered by a lack of cellular markers for the bulge area. We demonstrate that the C8/144B monoclonal antibody, originally raised against a CD8 peptide sequence, immunostains the human hair follicle bulge. We show that this antibody recognizes cytokeratin 15 (K15) in keratinocytes, and that K15-positive bulge cells possess a stem cell phenotype characterized by their slowly cycling nature, proliferation at the onset of new hair follicle growth, and high level of beta1 integrin expression. These results localize human hair follicle stem cells to the bulge and suggest that K15 is preferentially expressed in epithelial stem cells.

The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.

Stem cells are vital for the homeostasis of self-renewing tissues such as the hair follicle. Epithelial stem cells have been implicated in tumorigenesis and wound healing, and their manipulation may have wide ranging applications including gene therapy and tissue transplantation. Rodent hair follicle stem cells have been localized to an area of the follicle called the bulge, however, the identification and characterization of human hair follicle stem cells has been hampered by a lack of cellular markers for this area. We have determined that the C8/144B monoclonal antibody, originally generated against a short intracytoplasmic peptide of CD8, preferentially immunostains hair follicle bulge keratinocytes without staining the remaining hair follicle. Using expression cloning, we identified cytokeratin 15 as the keratinocyte protein recognized by the C8/144B monoclonal antibody. By delineating the bulge using this antibody, we demonstrated that bulge cells possess a stem cell phenotype characterized by their slowly-cycling nature, preferential proliferation at the onset of new hair follicle growth, high level of beta1 integrin expression, and expression of cytokeratin 19.

Desmoglein 3 anchors telogen hair in the follicle.

Little is known about the function of desmosomes in the normal structure and function of hair. Therefore, it was surprising that mice without desmoglein 3 (the autoantigen in pemphigus vulgaris) not only developed mucous membrane and skin lesions like pemphigus patients, but also developed hair loss. Analysis of this phenotype indicated that hair was normal through the first growth phase ('follicular neogenesis'). Around day 20, however, when the hair follicles entered the resting phase of the hair growth cycle (telogen), mice with a targeted disruption of the desmoglein 3 gene (DSG3-/-) lost hair in a wave-like pattern from the head to the tail. Hair then regrew and was lost again in the same pattern with the next synchronous hair cycle. In adults, hair was lost in patches. Gentle hair pulls with adhesive tape showed that anagen (growing) hairs were firmly anchored in DSG3-/- mice, but telogen hairs came out in clumps compared to that of DSG3+/- and +/+ littermates in which telogen hairs were firmly anchored. Histology of bald skin areas in DSG3-/- mice showed cystic telogen hair follicles without hair shafts. Histology of hair follicles in early telogen, just before clinical hair loss occurred, showed loss of cell adhesion (acantholysis) between the cells surrounding the telogen club and the basal layer of the outer root sheath epithelium. Electron microscopy revealed 'half-desmosomes' at the plasma membranes of acantholytic cells. Similar acantholytic histology and ultrastructural findings have been previously reported in skin and mucous membrane lesions of DSG3-/- mice and pemphigus vulgaris patients. Immunoperoxidase staining with an antibody raised against mouse desmoglein 3 showed intense staining on the cell surface of keratinocytes surrounding the telogen hair club in normal mice. Similar staining was seen in human telogen hair with an anti-human desmoglein 3 antibody. Finally, a scalp biopsy from a pemphigus vulgaris patient showed empty telogen hair follicles. These data demonstrate that desmoglein 3 is not only critical for cell adhesion in the deep stratified squamous epithelium, but also for anchoring the telogen hair to the outer root sheath of the follicle and underscore the importance of desmosomes in maintaining the normal structure and function of hair.

Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis.

PURPOSE: To determine the cell kinetic properties of epithelial cells from various zones of the conjunctiva. METHODS: The morphology and cell kinetics of bulbar, fornical, and palpebral conjunctival epithelium were studied in neonatal and adult SENCAR mice. To examine the proliferative rate of the conjunctival epithelium, a single administration of tritiated thymidine (3H-TdR) was used to detect cells in "S" phase. Proliferative rates were also assessed by determining mitotic activity after an intraperitoneal injection of colchicine to arrest cells in mitosis. To detect slow-cycling cells, mice received 3H-TdR continuously for 1 week. After a 4-week chase, animals were sacrificed and eyes were surgically removed. All tissues were immediately fixed in formalin and processed for histology and autoradiography. RESULTS: Slow-cycling cells, detected as label-retaining cells (LRCs), were identified in bulbar, fornical, and palpebral epithelia, as well as in limbal epithelium. The greatest number of LRCs was found in fornical epithelium. In addition, we found a number of label-retaining goblet cells. This cell population was shown to incorporate 3H-TdR after a single pulse administration, and mitotic figures were seen in goblet cells after colchicine treatment, indicating that conjunctival goblet cells have proliferative capabilities. CONCLUSIONS: These findings are consistent with earlier in vitro data that the fornical epithelium may be a zone enriched in conjunctival epithelial stem cells. This has important implications in conjunctival epithelial development and is relevant in wound repair. Furthermore, the concept that goblet cells are slow-cycling cells with proliferative capabilities provides new insights into the area of conjunctival homeostasis.

Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: heterogeneity and functional differences of various hair cycles.

Based on cell kinetic, morphological and several biological considerations, we have recently proposed that hair follicle stem cells reside in the bulge area of the upper follicle. We predicted that during early anagen the normally slow-cycling bulge stem cells may be activated by the abutting dermal papilla cells to undergo transient proliferation giving rise to keratinocytes of the lower follicle. In the present work, we performed tritiated thymidine-labeling of DNA-synthesizing cells and colcemid-arrest of mitotic figures on the skins of 20-23 and 75-80 day old SENCAR mice, when the follicles entered the anagen phase of the 2nd and 3rd hair cycles. The results clearly indicate that the normally slow-cycling bulge cells indeed undergo transient proliferation during early anagen. Similar results were obtained when the telogen follicles are experimentally induced to enter the 3rd hair cycle by plucking and by topical applications of phorbol ester or tretinoin. These results support the notion that bulge cells are follicular stem cells, and that transient proliferation of these cells is a critical feature of early anagen. However, the long duration of the 2nd telogen (> 30 days in mouse) suggests that a new anagen phase does not automatically result from the physical proximity of dermal papilla to the bulge cells, and that another 'factor' is required for the initiation of the 3rd anagen. The tremendous difference in the durations of the first and second telogen (lasting for 2-3 days and > 50 days, respectively) suggests that follicles can exist in a non-cycling state that may be conceptually equivalent to the G0 state of the cell cycle. Our results also underscore the fact that the first hair cycle is distinct from all the subsequent hair cycles in their cellular origin and morphological sequence, and thus should be regarded as a neogenic event.