Trans-species hair growth induction by human hair follicle dermal papillae.

A series of experimental bioassays has shown that the dermal papilla of the adult rodent vibrissa hair follicle retains unique inductive properties. In view of the many phenotypic and functional differences between specific hair follicle types, and the growing interest in hair follicle biology and disease, it remains important to establish that the human hair follicle dermal papilla has equivalent capabilities. In this study we tested the ability of human hair follicle papillae to induce hair growth when implanted into transected, athymic mouse vibrissa follicles. The implanted papillae that interacted with mouse follicle epithelium created new fibre-producing follicle end bulbs. The origin of the papillae in the recombinant structures was confirmed using laser capture microdissection and human specific gender determination by PCR. The demonstration that intact adult human dermal papillae can induce hair growth has implications for molecular analysis of basic hair growth mechanisms, particularly since the study involved common epithelial-mesenchymal signalling and recognition properties across species. It also improves the prospects for a cell-based clinical approach to hair follicle disorders.

Partial restoration of hair growth in the DEBR model for Alopecia areata after in vivo depletion of CD4+ T cells.

Alopecia areata (AA) is widely believed to be an autoimmune disease. Hair loss is associated with a peri- and intrafollicular inflammatory infiltrate of anagen hair follicles primarily composed of CD4 + and CD8 + cells. A previous investigation involved in vivo depletion of CD8 + cells in the DEBR rat model to examine the cells' potential pathogenic activity in AA. The rat model is used here in a comparable study of CD4 + cell pathogenic activity. Eight AA affected DEBR rats were given intraperitoneal injections of a CD4 + cell depleting OX-35/OX-38 monoclonal antibody (MoAb) cocktail over a 15-day therapy course. A further eight AA-affected rats comprised a control group and were injected with equivalent volumes of an irrelevant MoAb, OX-21. Changes in both CD4 + and CD8 + peripheral blood cell populations were analysed by flow cytometry, and macrophotography was used to record any changes in hair growth. Of the eight CD4 + cell-depleted rats six responded with hair growth. The rats revealed significant hair growth within 23 days of treatment initiation. With rapid replacement of the CD4 + cell population the newly generated pelage hair was eventually lost. Two control rats also showed limited hair growth within the 112-day study period. In vivo depletion of CD4 + cells partially restores hair growth in AA affected rats. The response suggests that CD4 + cells may be actively involved in the pathogenesis of AA. Further research may elucidate whether CD4 + cells have a direct effect on hair follicles or exert their influence through their classic T helper cell supporting role for CD8 + cells.

Alopecia areata-like hair loss in C3H/HeJ mice and DEBR rats can be reversed using topical diphencyprone.

This study demonstrates the ability to treat successfully alopecia areata-like hair loss in both mouse and rat models using topical immunotherapy with diphencyprone.

Comparison of alopecia areata in human and nonhuman mammalian species.

Alopecia areata (AA) is a nonscarring form of inflammatory hair loss in humans. AA-like hair loss has also been observed in other species. In recent years the Dundee experimental bald rat and the C3H/HeJ mouse have been put forward as models for human AA. AA in all species presents with a wide range of clinical features from focal, locally extensive, diffuse hair loss, to near universal alopecia. Histologically, all species have dystrophic anagen stage hair follicles associated with a peri- and intrafollicular inflammatory cell infiltrate. Autoantibodies directed against anagen stage hair follicle structures are a consistent finding. Observations on AA pathogenesis suggest nonhuman species can provide excellent models for the human disease. Ultimately, animal models will be used to determine the genetic basis of AA, potential endogenous and/or environmental trigger(s), mechanism(s) of disease initiation and progression, and allow rapid evaluation of new and improved disease treatments.

Topical FK506: a potent immunotherapy for alopecia areata? Studies using the Dundee experimental bald rat model.

We elected to examine the efficacy of the topically applied immunosuppressive agent FK506 (Prograf) in the treatment of alopecia areata (AA) using the Dundee experimental bald rat (DEBR) model. Thirty lesional DEBR rats were allocated to five groups of six. Group 1 rats received 0.1 mL of a 0.25% solution of FK506 within a 2 x 2 cm marked area on one bald flank twice a week (125 micrograms FK506/cm2 per week) for 8 weeks, while the contralateral flank was left untreated. In group II, 0.05 mL of a 0.1% solution of FK 506 was applied 5 days per week on one flank (62.5 micrograms FK506/ cm2 per week) and control vehicle to the opposite flank for 8 weeks. Group III rats were treated as in group II except that drug and vehicle were applied twice a week (25 micrograms FK506/cm2 per week) for 4 weeks. A positive control group received orally administered cyclosporin A (CsA) (10 mg/kg daily) for 8 weeks and a further group was left untreated. Rats were regularly examined and photographed with skin biopsies taken from groups II and III. All FK 506-treated rats regrew hair at the site of drug application within 14-21 days. Growth continued for 3 weeks beyond termination of treatment after which gradual hair loss was observed. No hair growth was seen as a result of vehicle application and hair loss continued on untreated areas and in the untreated control group. Immunohistology revealed a drastic reduction in the follicular inflammatory infiltrate at the site of the FK506 application. The oral CsA group responded by simultaneous regrowth of hair over the whole body. Our findings suggest that FK506 may have considerable potential as a topical treatment for AA.

Human hair follicle regeneration following amputation and grafting into the nude mouse.

In this study we investigated the capacity of the human hair follicle to regenerate a fiber-forming bulb after its amputation. We removed the bases from terminal follicles from a variety of sites and transplanted the follicles onto athymic mice, either still attached to a skin graft or as subcutaneous implants of individual follicles. External hair growth was observed on the skin grafts, and histology of the follicles revealed restoration of dermal papillae and follicle bulb structures. This result suggests that the capacity of hair follicles to regenerate their lower structures after removal, which was first demonstrated on whisker follicles, may be a general phenomenon. It emphasizes the importance of specific cellular subpopulations within the follicle and the role of dermal-epidermal interactions in adult follicle activities.

In vivo depletion of CD8+ T cells restores hair growth in the DEBR model for alopecia areata.

Alopecia areata (AA) is a putative autoimmune disease in which anagen hair follicles are the target of immune cell attack. While both CD4+ and CD8+ T lymphocytes are prominent in the infiltrate, their respective roles in the pathogenesis of AA remain unknown. Here we directly investigated the activity of CD8+ cells in the inhibition of hair growth using the Dundee experimental bald rat (DEBR) model for AA. Eight lesional DEBRs were fully depleted of their CD8+ cells by intraperitoneal injection of OX-8 monoclonal antibody (MoAb) specific for these cells over a 15-day therapy course. A control group of eight lesional rats was injected with the irrelevant MoAb OX-21. Sequential blood samples were analysed by flow cytometry to observe changes in the CD8+ cell population and macrophotography used to record changes in hair growth activity. All eight CD8+ depleted rats started to regrow hair within 29 days from the start of treatment, the final response ranging from sparse regrowth to a near normal coat. While two rats maintained their new pelage, the remainder lost hair as the CD8+ population in peripheral blood increased. Two of the control rats also showed hair regrowth over the experimental period of 156 days. These results suggest that CD8+ cells play an active part in the pathogenesis of AA. As hair production did not fully recover in all animals, immune mechanisms other than CD8+ cells may be involved in effecting hair loss. However, analysis of CD8+ cell levels in the skin of CD8+ depleted rats may help resolve their full importance in AA.

The DEBR rat, alopecia areata and autoantibodies to the hair follicle.

Many attempts have been made to implicate hair follicle-specific autoantibodies in the pathogenesis of alopecia areata (AA), a suspected autoimmune disease. Using the DEBR rat model for AA, we developed a refined indirect immunofluorescent technique to examine the sera from individual rats for the presence of autoantibodies to the hair follicle and to other tissues. Sera were tested on cryostat sections from normal PVG/Ola rats and DEBR rats. We found that DEBR sera contained IgG autoantibodies specific for hair follicle epidermal differentia. While individual sera revealed detailed differences, the target tissues identified were hair cortex and cuticle and the inner root sheath, especially the Henle's layer. Some sera also contained autoantibodies specific for skeletal muscle and nuclear components. Of 10 young prelesional rats with apparently normal coats of hair, three had hair follicle autoantibodies and seven had skeletal muscle autoantibodies. Nine of 10 active lesional rats with progressing hair loss had follicle autoantibodies and four had skeletal muscle autoantibodies. All 10 established lesional rats had follicle autoantibodies and one had muscle autoantibodies. Control sera from eight PVG/Ola rats showed no specific positive staining for hair follicle components or other tissues. Autoantibodies to intracellular hair follicle differentiation products were readily detected in DEBR rat sera. As these antibodies appeared to be generated after the appearance of the mononuclear follicular infiltrate, such autoantibodies may be a secondary effect. We conclude that, while the presence of autoantibodies in the DEBR rat model is associated with autoimmune activity, their role in the pathogenic progression of AA has yet to be ascertained.

Oral cyclosporin A restores hair growth in the DEBR rat model for alopecia areata.

The Dundee experimental bald rat (DEBR) undergoes hair loss associated with the development of peri- and intrafollicular mononuclear cell infiltrates, as occurs in human alopecia areata. We studied the effect of orally administered cyclosporin A (10 mg/kg; 5 days/week for 7 weeks) on established lesional DEBR rats displaying extensive areas of hair loss. New hairs appeared after 10 days and there was simultaneous regrowth of hair over the whole body with restoration of a full pelt by 5 weeks. Semiquantitative histological examination of flank skin biopsies revealed early reduction of the cellular infiltrate associated with conversion of dystrophic anagen follicles to normal, hair-producing follicles. These results confirm the value of the DEBR model of alopecia areata in evaluating existing and new therapies for this disease in humans.

Immunohistological study of the development of the cellular infiltrate in the pelage follicles of the DEBR model for alopecia areata.

The Dundee experimental bald rat (DEBR) undergoes hair loss associated with perifollicular infiltrates of mononuclear cells (MNC), a pathological characteristic of human alopecia areata (AA). To investigate further the pathogenesis of the disease in this animal model, we have studied the development, composition and extent of the perifollicular MNC infiltration in young (6-week-old), prelesional (3-month-old), active lesional, and established lesional DEBR rats, using 6-week- and 6-month-old Wistar rats as normal controls. The proportions of hair follicles showing infiltration by MNC and their main subsets were determined using immunohistochemical staining of serial cryostat sections of flank skin biopsies. There was a good correlation between the degree of leucocyte (OX-1+) infiltration of anagen hair follicles and the development of hair loss. In 6-week-old DEBR skin, there were few perifollicular cells expressing MHC class II, with positively stained dendritic cells in the dermis above the sebaceous gland. There was a sparse perifollicular distribution of CD4+ cells (W3/25) and macrophages (ED-1+). No CD8+ cells (OX-8+) were seen associated with DEBR hair follicles, and only small numbers were present in Wistar rats. In prelesional DEBR rats there was an increased perifollicular presence of MHC class II+ cells, macrophages, and particularly of CD8+ cells, with little change in CD4+ cells. Active and established lesional rats, i.e. animals with overt loss of hair, showed a significant increase in the degree of MNC infiltration and the proportion of infiltrated follicles, the majority of which were in dystrophic anagen. In the perifollicular infiltrate the CD4+:CD8+ ratio was approximately 2:1. An intrafollicular infiltrate was prominent, and was composed of CD8+ cells and macrophages, with bulbar and suprabulbar keratinocytes expressing MHC class II antigens. CD4+ cells were not detected in follicular epithelium. ICAM-1 expression correlated with MNC infiltration. These results show marked similarities to lesional human AA. They also focus on a possible active role for CD8+ cells in the pathogenesis of hair loss in the DEBR rat.

Induction of hair growth in ear wounds by cultured dermal papilla cells.

In the adult hair follicle the dermal papilla plays a crucial role in the dermal-epidermal interactions that control hair production and events of the growth cycle. It has previously been shown that cultured cells from rat vibrissa follicle dermal papillae can stimulate hair growth when implanted into amputated follicles. This study investigated the effects of implanting low-passage cultured papilla cells into small incisional wounds in the rat ear pinna. The groups of fibers that emerged from wound sites were much larger than local hairs, and often had vibrissa-type characteristics. Later-passage papilla cells or cultured skin fibroblasts failed to elicit the same response. Histology revealed that big follicles were formed when papilla cells were trapped between the cut edges of the epidermis. Abnormally large follicles were seen at wound sites many months post-operatively. Independent of epidermal influence, cultured papilla cells in the wound dermis formed rounded papilla-like aggregates that also persisted until biopsy. A previously described method of wrapping papilla cells in glabrous epidermis was less successful in percentage terms but resulted in the production of one very large vibrissa-type follicle and fiber. These results further illustrate that the inductive powers and developmental information retained by cultured dermal papilla cells parallel the properties of their embryonic precursors; the findings may have implications for human hair growth.

The DEBR rat: an animal model of human alopecia areata.

The Dundee experimental bald rat (DEBR) is reported as a model for human alopecia areata. Parallels with human alopecia areata were observed in relation to the gradual and patchy loss of hair and the penetration of both pelage and vibrissa follicles by mononuclear cells. In particular, the apparent disruption of the follicles within the precortical region of the epidermal component and consequent alterations of normal geometrical relationships between dermal and epidermal components relate directly to similar studies on human alopecia areata. In comparison with other previously described hypotrichotic rodent mutants, the DEBR rat exhibits a unique mechanism of hair loss which may provide important information regarding the pathomechanism of human alopecia areata.

Dermal cell populations show variable competence in epidermal cell support: stimulatory effects of hair papilla cells.

A study was made of the comparative abilities of adult rat vibrissa dermal papilla cells, skin fibroblasts and 3T3 cells to support the initial attachment and subsequent growth and division of directly associated newborn rat skin basal epidermal cells. These associations were made under sub-optimal conditions; that is, in the absence of specific epidermal growth-promoting supplements, in order to assess more accurately the epidermal sustaining capacities of each dermal support. Analysis of epidermal cell counts and close photographic scrutiny revealed that low-passage dermal papilla cells, closely followed by transformed dermal papilla cells, were conducive to the successful attachment and subsequent proliferation of epidermal cell populations under three different experimental protocols. In contrast, skin fibroblasts did not support epidermal cell growth under any circumstances. These findings are particularly interesting in that they constitute a rare in vitro example of epidermal cells that are not only supported, but also encouraged to proliferate, by an actively dividing adult skin-derived dermal cell population.

Immunobiological studies on the alopecic (DEBR) rat.

The Dundee experimental bald rat (DEBR) has been proposed as an animal model of human alopecia areata, which is suspected of being an autoimmune disease. This study was carried out to establish whether the immunological changes observed in the lesional DEBR rat correlated with studies of human alopecia areata. The immune infiltrate was characterized using immunoperoxidase techniques on cryostat sections of vibrissa follicles. Indirect immunofluorescence was used to quantify the peripheral blood leucocytes. Some parallels were observed in the infiltration of human and DEBR rat follicles by T lymphocytes. In contrast, pre-lesional DEBR rat follicles, which are not available for investigation in human alopecia areata, were not penetrated by leucocytes and MHC class II antigens were expressed in the precortical region of the epidermal component of these follicles. Quantification of peripheral blood leucocytes showed significant increases in both T-lymphocyte subsets during lesional expression. We consider that the pre-lesional form of the rat may provide important information as a model for the pre-lesional and uninvestigated form of alopecia areata in man.