An osteopontin-derived peptide inhibits human hair growth at least in part by decreasing fibroblast growth factor-7 production in outer root sheath keratinocytes.

BACKGROUND: Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. OBJECTIVES: To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. METHODS: Our hypothesis was tested ex vivo and in vivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. RESULTS: In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L-1 to 3 µmol L-1 ) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. CONCLUSIONS: With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth ex vivo and in vivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic? The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add? We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) ex vivo and in vivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both ex vivo and in vivo. What is the translational message? High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth.

Oncogenic role of microRNA-155 in mycosis fungoides: an in vitro and xenograft mouse model study.

BACKGROUND: MicroRNA (miR)-155 contributes to the proliferation of mycosis fungoides (MF) in vitro and is upregulated in tumours of MF compared with early MF lesions. OBJECTIVES: To investigate the contribution of miR-155 to the cancerous phenotype and drug resistance of MF/Sézary cell lines. METHODS: miR-155 was inhibited in MF cell lines (MyLa and MJ) by transduction of miRZip anti-miR-155, and overexpressed in Hut78 cells by transduction of miRVec-miR-155; empty plasmids served as controls. Cells were analysed for response to inducers of apoptosis and cell-cycle arrest, using fluorescence-activated cell sorting. Transduced MyLa cells were subcutaneously injected into severe combined immunodeficient mice, and tumours were analysed immunohistochemically and for final size. RESULT: MyLa and MJ cells expressed a high level of miR-155; Hut78 cells expressed a low level. MF cell lines stably expressing miR-155 inhibitor showed increased G2/M arrest in response to N-p-tolyl-2-(3,4,5-trimethoxyphenyl quinazolin-4-amine) (SL111), an inducer of cell-cycle arrest, followed by increased apoptosis. Additionally, they showed increased apoptosis in response to suberoylanilide hydroxamic acid (SAHA). Tumours formed in mice from injected anti-miR-155-expressing MyLa cells had a significantly lower volume and higher occurrence of apoptosis than controls. Stable overexpression of miR-155 in Hut78 cells had no effect. CONCLUSIONS: Oncogenic miR-155 appears to contribute to the cancerous phenotype of MyLa and MJ cells, but not of Hut78 cells, by interrupting activation of the G2/M checkpoint in response to SL111, and decreasing apoptosis in response to SL111 and SAHA, thereby facilitating tumour growth. These findings have implications for the potential development of novel therapeutic modalities for MF incorporating miR-155 inhibitors.

What causes alopecia areata?

The pathobiology of alopecia areata (AA), one of the most frequent autoimmune diseases and a major unsolved clinical problem, has intrigued dermatologists, hair biologists and immunologists for decades. Simultaneously, both affected patients and the physicians who take care of them are increasingly frustrated that there is still no fully satisfactory treatment. Much of this frustration results from the fact that the pathobiology of AA remains unclear, and no single AA pathogenesis concept can claim to be universally accepted. In fact, some investigators still harbour doubts whether this even is an autoimmune disease, and the relative importance of CD8(+) T cells, CD4(+) T cells and NKGD2(+) NK or NKT cells and the exact role of genetic factors in AA pathogenesis remain bones of contention. Also, is AA one disease, a spectrum of distinct disease entities or only a response pattern of normal hair follicles to immunologically mediated damage? During the past decade, substantial progress has been made in basic AA-related research, in the development of new models for translationally relevant AA research and in the identification of new therapeutic agents and targets for future AA management. This calls for a re-evaluation and public debate of currently prevalent AA pathobiology concepts. The present Controversies feature takes on this challenge, hoping to attract more skin biologists, immunologists and professional autoimmunity experts to this biologically fascinating and clinically important model disease.

Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice.

Alopecia areata is a tissue-restricted autoimmune disease of the hair follicle, which results in hair loss and baldness. It is often psychologically devastating. The role of T lymphocytes in this disorder was investigated with cell transfer experiments. Scalp explants from patients were transplanted to severe combined immunodeficiency (SCID) mice and injected with autologous T lymphocytes isolated from involved scalp. T lymphocytes which had been cultured with hair follicle homogenate along with antigen-presenting cells were capable of inducing the changes of alopecia areata, including hair loss and perifollicular infiltrates of T cells, along with HLA-DR and ICAM-1 expression of the follicular epithelium. Similar changes were not noted in grafts injected with scalp-derived T cells that had not been cultured with follicular homogenate. These data indicate that alopecia areata is mediated by T cells which recognize a follicular autoantigen.

Melanocytes and Langerhans cells in aged versus young skin before and after transplantation onto nude mice.

Previous studies have demonstrated decreased numbers of melanocytes and Langerhans cells (LC) in aged skin. In the present study, we employed dopa and indirect immunoperoxidase techniques in epidermal sheets to determine the fate of melanocytes and LC of aged versus young donors after skin transplantations onto nude mice. The detection of positive homologous leucocytic antibody reaction of degeneration (HLA-DR) of LC indicates an age-associated reduction in sun-protected thigh skin in aged versus young subjects (263 +/- 63 versus 589.25 +/- 142.643, p less than 0.001). The mean number of LC four weeks after transplantation remained almost constant. Prior to skin engraftment, a decreased number of melanocytes was found in aged versus young epidermis (160.77 +/- 51.7 versus 255.83 +/- 81.2, respectively, p less than 0.05). A significantly increased number of melanocytes was noted four weeks following engraftment in epidermis from aged (307.44 +/- 174, p less than 0.05) and young human donors (402.16 +/- 139, p less than 0.02). The marked increase in density of dopa-positive melanocytes following engraftment onto nude mice may indicate the existence of circulating factors in nude mice that perhaps both stimulates and enhances proliferation and activity of these cells.

In vivo destruction of melanocytes by the IgG fraction of serum from patients with vitiligo.

There are conflicting results regarding the role of autoantibodies in the pathogenesis of vitiligo. To examine their in vivo effect, human skin was transplanted onto nude mice injected with purified IgG obtained from patients with vitiligo and from controls. The effect was evaluated by several techniques. Dihydroxyphenylalanine staining revealed a marked decrease in the number of melanocytes in skin grafted onto mice injected with patients' IgG. Direct immunofluorescence staining demonstrated the presence of human IgG throughout the epidermis in specimens injected with purified IgG from vitiligo patients. No staining was observed when control IgG was injected. Electron microscopy studies demonstrated a marked decrease in melanin pigmentation with only rare melanosomes and melanocytes detected in grafts injected with patients' IgG. Thus all three techniques showed the destructive effect of vitiligo patients' serum on melanocytes. Our study highlights the important role of autoantibodies in the pathogenesis of vitiligo.

T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.

Considerable indirect evidence suggests that T lymphocytes have a role in the pathogenesis of psoriasis. The goal of this study was to directly test the ability of T cells to maintain psoriasis pathology. Psoriatic skin was transplanted onto SCID mice, which were then injected with autologous T cells. T cells were cultured from either psoriatic skin lesions or peripheral blood and injected intradermally or intravenously. Control SCID mice transplanted with psoriasis grafts were not injected with T cells. After 10 wk, control psoriatic skin grafts not injected with T cells lost many of the features of psoriasis. Injection of peripheral blood T cells was not able to maintain these psoriatic features. In contrast, the injection of T cells derived from psoriatic skin was able to maintain the psoriatic phenotype. Psoriatic features that were maintained included epidermal thickness and labeling index and expression of HLA-DR, involucrin, and ICAM-1, as well as loss of expression of filaggrin. Injection of skin infiltrating T cells into skin of normal donors on SCID mice did not induce changes of psoriasis. The ability of T cells from lesional skin, but not peripheral blood, to maintain psoriasis suggests that psoriasis is mediated by an autoantigen reactive T cell, which is present at a higher frequency in the psoriatic lesion.

Melanocyte-associated T cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc(scid) mice.

Alopecia areata is a tissue restricted autoimmune condition affecting the hair follicle, resulting in hair loss. The goal of this study was to test the hypothesis that the autoantigen of alopecia areata is melanocyte associated. Potential autoantigens were tested in the human scalp explant/Prkd(scid) CB-17 mouse transfer system. Scalp T cells from lesional (bald) alopecia areata scalp were cultured with antigen-presenting cells, and antigen, along with interleukin-2. The T cells were then injected into autologous lesional scalp grafts on SCID mice, and hair regrowth was measured. Hair follicle homogenate was used as an autoantigen control. T cells cultured with melanoma homogenate induced statistically significant reduction in hair growth (p <0.01 by ANOVA). HLA-A2-restricted melanocyte peptide epitopes were then tested with lesional scalp T cells from HLA-A2-positive alopecia areata patients. Melanocyte-peptide-activated T cells significantly reduced the number of hairs regrowing in two experiments with six patients (p <0.001 by ANOVA). Injected scalp grafts showed histologic and immunochemical changes of alopecia areata. The most consistent peptide autoantigens were the Gp100-derived G9-209 and G9-280 peptides, as well as MART-1 (27-35). Melanocyte peptide epitopes can function as autoantigens for alopecia areata. Multiple peptides were recognized, suggesting epitope spreading.

Effect of antiinsulin-like growth factor 1 on epidermal proliferation of human skin transplanted onto nude mice treated with growth hormone.

Recently it has been demonstrated that long-term administration of GH leads to increase of skin thickness. The aim of the present study was to determine whether this effect of GH is mediated by insulin growth factor 1 (IGF-1), which enhances epidermal proliferation. In order to address this question, human split-thickness grafts obtained from aged skin were grafted onto nude mice. One group of mice was treated systemically with GH, whereas a second group was treated with intradermal graft injections of anti-IGF-1 in addition to GH. A third group received distilled water and served as a control group. Histological and autoradiographic analyses were performed before and after engraftment. The GH-treated mice showed a significant increase in epidermal proliferation measured by epidermal thickness (analysis of variance with repeated measurements, P < 0.01) and labeled index (analysis of variance, P < 0.01) as compared to the control group. The intradermal injections of anti-IGF-1 reduced significantly the proliferative stimulatory effect of GH (P < 0.01). The present study emphasizes the role of IGF-1 in the increased skin thickness observed after GH administration and provides a useful model for determining the effect of various compounds, including GH, on human skin.

Epidermal Langerhans' cells in Behçet's disease.

Langerhans' cells were studied in the epidermis of two patients with active Behçet's disease and compared with those in two normal controls. Ultrastructural morphology and the percentage of Langerhans' cells found were similar in patients' (1.88%) and the control epidermis (1.79%). The density of Langerhans' cells in adjacent sites of the same epidermis was not homogeneous, being in the range of 0.8-2.8% in Behçet's disease and 0.6-4% in the controls. In the controls, Langerhans' cells were distributed unevenly. Some were located near the basal layer of the epidermis while the rest were in the mid and upper layers. In Behçet's disease most Langerhans' cells were in the mid-epidermis, but some were immediately beneath the stratum granulosum. In the Behçet's disease epidermis the area occupied by Langerhans' cells was increased by about 25% and the number of granules found increased by about 44%. It is suggested that in Behçet's disease the Langerhans' cells are in a more active state.

Hair growth in scalp grafts from patients with alopecia areata and alopecia universalis grafted onto nude mice.

A basic question in both mild and severe forms of alopecia areata (AA) relates to whether the disease is inherent to the affected tissue or secondary to circulating factors. This question has been addressed by grafting 2-mm grafts of scalp from affected areas of seven patients with AA or alopecia universalis (AU) onto congenitally athymic (nude) mice. Hair growth in these grafts has been compared with that of 2-mm grafts from hair-bearing skin remnants from two individuals undergoing elective plastic surgical procedures. Because cyclosporine seems to directly affect hair growth, a group of grafted mice was treated with this agent. By day 48, hair growth was present in many surviving grafts. Cyclosporine affected hair growth; this was most prominent by day 78 when the number of hairs per graft and the mean length of hair had increased significantly over untreated groups. Grafts from patients with AU had more hairs per graft and had greater hair length than did similar grafts from patients with AA. These experiments show that hair growth ability in situ is likely normal in AA and AU, and that the factors causative to this disease in situ are mediated humorally. Furthermore, cyclosporine seems to directly influence hair growth in this model system.

Possible role of cytokines in cellular proliferation of the skin transplanted onto nude mice.

BACKGROUND AND DESIGN: In recent studies on the behavior of aged skin transplanted onto nude mice, the epidermis of aged and young skin showed an increase in proliferation and thickness following engraftment, and became almost identical. The aim of this study was to ascertain a possible role for the release of local cytokines in this phenomenon. Grafted human skin was injected intradermally with anti-interleukin-6 (IL) and anti-IL-1 alpha, and comparisons of epidermal thymidine incorporation and thickness were made. Grafts injected with irrelevant antibodies served as control. RESULTS: Interleukin-6 and IL-1 alpha expression were studied in grafts by immunoperoxidase staining. Only IL-6 expression was found in the 1-month grafts. Intradermal injections of anti-IL-1 alpha and anti-IL-6 showed an inhibitory effect on cellular proliferation in the epidermis. A significant difference in the response of epidermal proliferation and, consequently, in thickness was found in samples injected with anti-IL-1 alpha and anti-IL-6 compared with those injected with irrelevant antibodies. CONCLUSIONS: These data may indicate that local cytokines released by the keratinocytes are involved in the cellular proliferative activity in skin engrafted onto the mice.

Vitiligo and idiopathic guttate hypomelanosis. Repigmentation of skin following engraftment onto nude mice.

Several diseases are included in the category of hypomelanosis. Their clinical course as well as the pathogenesis are diverse and in many cases poorly understood. The aim of the present study is to use the nude mice model to determine whether the primary defect in various pigmentary skin disorders is inherent to the tissue itself or is secondary to systemic factors. Split-thickness skin grafts obtained from patients with vitiligo, acquired hypomelanosis guttata, and tyrosinase-negative albinism were grafted onto nude mice. Histologic examination and dopa staining were performed prior to and following the engraftment. The dopa staining was performed on the epidermal sheet following separation from the dermis. The depigmented area of the vitiligo became completely pigmented 6 to 10 weeks after skin transplantation. The dopa reaction that was negative prior to skin engraftment became completely positive after the transplantation. The number of melanocytes (expressed per square millimeter of skin surface) 8 weeks after transplantation was 197 +/- 73 mm2. Dopa reaction in acquired hypomelanosis guttata showed reduction of the number of melanocytes in the depigmented macula as compared with the surrounding area (55.25 +/- 18.00 mm2 vs 220 +/- 28.28 mm2. Twenty days after skin transplantation, repigmentation of the area was observed. The number of melanocytes increased significantly (388.75 +/- 213 mm2). The grafted skin obtained from patients with tyrosinase-negative albinism showed persistence of the depigmentation after skin transplantation. Dopa reaction was negative prior to and 8 weeks after transplantation. The results of the present study suggest that systemic factors may play a role in the pathogenesis of vitiligo and acquired hypomelanosis guttata.

Vitiliginous vs pigmented skin response to intradermal administration of interferon gamma.

BACKGROUND AND DESIGN: Decreased sensitization and elicitation of contact allergens in vitiliginous skin has been described. This may be related to altered epidermal Langerhans cell migration with bound hapten to dermis and draining lymph nodes. The aim of the present study was to detect the potential ability of vitiliginous skin to respond to an in vivo immunologic stimulus such as intradermal injections of interferon gamma (IFN-gamma). Vitiliginous and normal pigmented skin of each patient was injected intradermally with 10 micrograms of recombinant IFN-gamma diluted in 0.1 mL of sterile water for 3 consecutive days. On day 5, punch biopsy specimens were obtained from the injected sites. Histologic and immunohistochemical staining was performed on all sections. The cryostat sections were stained with adenosine triphosphatase as well as with the indirect immunoperoxidase technique employing murine monoclonal antibodies to HLA-DR, ICAM-1, CD1, CD11a, and CD18. RESULTS: HLA-DR and ICAM-1 expression by epidermal cells, combined with perivascular accumulation of mononuclear cells with CD11a and CD18 expression, was observed in all sites injected with IFN-gamma. However, absence of an effect on the epidermal Langerhans cell population was noted only on the vitiliginous skin. CONCLUSION: The reactivity of depigmented and pigmented skin was found to be different after IFN-gamma administration, with fewer CD1-positive cells in the depigmented skin. As adenosine triphosphatase staining also showed fewer positive cells, it may be concluded that no effect on the migration of epidermal Langerhans cells was noted in the involved skin. This may shed light on the immunologic aberration seen in vitiliginous skin.

Calcitriol-resistant rickets with alopecia.

Four children of two kindred had alopecia associated with severe rickets, resistant to treatment, and caused by defective cytoplasmic and nuclear receptors for the active vitamin D metabolite calcitriol (1,25-dihydroxyvitamin D3). Scalp biopsy specimen revealed a normal number and light microscopic features of the hair and hair follicles. Calcitriol-resistant rickets should be added to the list of inherited disorders of hair growth, and the association of rickets with alopecia needs to be considered in the differential diagnosis of hair loss.

Favourable melanoma prognosis associated with the expression of the tumour necrosis factor receptor and the alpha1beta1 integrin: a preliminary report.

Recently we encountered a patient (p1) with a Clark's level IV melanoma associated with recurrent cutaneous metastasis who subsequently experienced a complete remission after a period of therapy with dichloronitrobenzene (DCNB) and interferon-alpha (IFNalpha). Another patient (p2) with a similar Clark's level of disease but with a fatal prognosis and melanoma cells from the Sk-Mel 28 and MeWo cell lines served as control groups. Since cytokines and members of the alpha1 integrin family have been implicated in the growth and metastatic behaviour of melanomas, we evaluated the cytokine effects and integrins expressed by melanoma cells in the patients' tumours. P1, but not p2 nor MeWo melanoma cells, expressed HLA-DR, alpha1beta1 and the tumour necrosis factor receptor (TNF-R). Culturing p1 melanoma cells with TNFalpha, but not MeWo or p2 melanoma cells, increased their expression of alpha1beta1 integrin and was cytotoxic for the cells. Significant in vivo growth of metastatic p1 and p2 melanoma explants as well as MeWo cells grafted subcutaneously onto nude mice was noted over 36 days. Intralesional injection of TNFalpha induced complete regression of p1 explants, but not of p2 or MeWo explants. Intralesional injection of IFNalpha or anti-alpha1beta1 integrin arrested p1 graft growth. These results suggest that the slow growth of the melanoma cells in nude mice, as well as the susceptibility to tumour killing by TNFalpha and the dependence of the tumour on signals mediated by the alpha1beta1 integrin are features that may have contributed to the beneficial therapeutic response in patient 1.

New topical antiandrogenic formulations can stimulate hair growth in human bald scalp grafted onto mice.

The purpose of this study was to test the ability of topical formulations of finasteride and flutamide to re-enlarge hair follicles in male-pattern baldness. This was evaluated by an experimental model of human scalp skin graft transplanted onto SCID mice. A comparison was made between formulations containing finasteride and flutamide, and a vehicle formulation in terms of the mean hairs per graft, length, diameter of the shafts, and structures of the growth stages of the hair. Flutamide and finasteride had a significantly higher effect (P<0.05) than the placebo in all the tested parameters, but flutamide demonstrated more hair per graft and longer hair shafts than finasteride (P<0.05). The number of hairs per graft for flutamide and finasteride groups were 1.22+/-0. 47 and 0.88+/-0.95 hairs/0.5 mm2 graft, respectively, versus 0. 35+/-0.6 hairs/graft for vehicle-treated graft. Similarly, hair lengths for flutamide and finasteride were 5.82+/-0.50 and 4.50+/-0. 32 mm, respectively, versus 2.83+/-0.18 mm for the vehicle-treated grafts. An in vitro diffusion study of flutamide gel using hairless mouse skin demonstrated the beneficial effect of the vehicle composition in comparison with a hydroalcoholic solution or a gel containing no penetration enhancer. It is therefore suggested that this topical composition containing flutamide or finasteride may effectively result in regression of male-pattern baldness.

The effect of hyperbaric oxygenation on the viability of human fat injected into nude mice.

Autologous free-fat injection for the correction of soft-tissue defects has become a common procedure in plastic surgery. The main shortcoming of this method for achieving permanent soft-tissue augmentation is the partial absorption of the injected fat, an occurrence that leads to the need for both overcorrection and repeated fat reinjection. Improving the oxygenation of the injected fat has been suggested as a means of helping to overcome the initial critical phase that occurs postinjection (when the fat cells are nourished by osmosis), increasing phagocyte activity, accelerating fibroblast activity and collagen formation, and enhancing angiogenesis. In addition, the hyperbaric oxygen-mediated decrement in endothelial leukocyte adhesion will decrease cytokine release, thereby reducing edema and inflammatory responses. The purpose of the present study was to examine the effect of hyperbaric oxygenation on improving the viability of injected fat. Adipose tissue obtained from human breasts by suction-assisted lipectomy was injected into the subcuticular nuchal region in nude mice. The mice were then exposed to daily hyperbaric oxygen treatments, breathing 100% oxygen at 2 atmospheres absolute (ATA) for 90 minutes. The duration of the administered hyperbaric oxygen therapy was 5, 10, or 15 days, according to the study group. Mice exposed to normobaric air alone served as the control group, and each group included 10 animals. The rats were killed 15 weeks after fat injection. The grafts were dissected out, weight and volume were measured, and histologic evaluation was performed. In all of the study groups, at least part of the injected fat survived, giving the desired clinical outcome. No significant differences could be found between the groups regarding fat weight and volume. Histopathologic examination of the dissected grafts demonstrated a significantly better integrity of the fat tissue in the group that received hyperbaric oxygen for 5 days (p = 0.047). This finding was manifested by the presence of well-organized, intact fat cells, along with a normal appearance of the fibrous septa and blood vessels. The worst results were found in animals treated by hyperbaric oxygenation for 15 consecutive days. An inverse correlation was found between an increased dose of the high-pressure oxygen and fat tissue integrity (r = -0.87, p = 0.076). The toxic effects of highly reactive oxygen species on fat cells might explain the failure of an excessively high dose of hyperbaric oxygen to provide any beneficial outcome. The clinical relevance of these results should be further investigated.