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.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis.

BACKGROUND AND PURPOSE: Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis. EXPERIMENTAL APPROACH: Apremilast was tested in vitro against endotoxin- and superantigen-stimulated PBMC, bacterial peptide and zymosan-stimulated polymorphonuclear cells, immunonoglobulin and cytokine-stimulated NK cells, and ultraviolet B light-activated keratinocytes. Apremilast was orally administered to beige-severe combined immunodeficient mice, xenotransplanted with normal human skin and triggered with human psoriatic NK cells. Epidermal skin thickness, proliferation index and inflammation markers were analysed. KEY RESULTS: Apremilast inhibited PBMC production of the chemokines CXCL9 and CXCL10, cytokines interferon-gamma and tumour necrosis factor (TNF)-alpha, and interleukins (IL)-2, IL-12 and IL-23. Production of TNF-alpha by NK cells and keratinocytes was also inhibited. In vivo, apremilast significantly reduced epidermal thickness and proliferation, decreased the general histopathological appearance of psoriasiform features and reduced expression of TNF-alpha, human leukocyte antigen-DR and intercellular adhesion molecule-1 in the lesioned skin. CONCLUSIONS AND IMPLICATIONS: Apremilast displayed a broad pattern of anti-inflammatory activity in a variety of cell types and decreased the incidence and severity of a psoriasiform response in vivo. Inhibition of TNF-alpha, IL-12 and IL-23 production, as well as NK and keratinocyte responses by this phosphodiesterase-4 inhibitor suggests a novel approach to the treatment of psoriasis.

Ageing of human epidermis: the role of apoptosis, Fas and telomerase.

BACKGROUND: Aged human epidermis is characterized by morphological changes including flattening of the dermal-epidermal junction and a decrease in thickness. OBJECTIVES: To determine the roles of proliferation, apoptosis, Fas (CD95), Fas ligand (FasL) and telomerase in changes of human epidermis during ageing. METHODS: Human epidermis from aged subjects (n = 14; mean age 70.7 years) and young subjects (n = 14; mean age 23.4 years) was studied by histology, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay for apoptotic cells and reverse transcription-polymerase chain reaction to determine epidermal thickness, proliferation (Ki-67), apoptosis, expression of Fas and FasL, and telomerase activity. RESULTS: Aged skin was associated with thinning of the epidermis, decreased proliferation, and increased apoptosis below the granular layer. This was associated with increased epidermal expression of Fas and FasL. Telomerase activity was similar in aged and young epidermis. CONCLUSIONS: Fas/FasL-mediated apoptosis, along with decreased proliferation, may have a role in changes of human epidermis during ageing. Telomerase activity did not appear to be limiting in young vs. old human epidermis.

Smoking effect on skin wrinkling in the aged population.

OBJECTIVES: Smoking causes premature wrinkling and is one of the more potent risk factors for atherosclerosis. The aims of the present study were to verify: (i) whether there is a difference between the wrinkling appearance in aged smokers and the nonsmoking population; and (ii) whether the systemic effects of smoking, such as atherothrombotic disease and cerebrovascular disease, are associated with a more striking appearance of wrinkling. PARTICIPANTS: Eighty volunteers (mean age, 76 years) were included in the study, 40 of whom were smokers and 20 of whom had suffered a stroke. RESULTS: The mean value of the wrinkling score measured for the smokers' group (stroke and nonstroke) was significantly higher than that for the nonsmokers' group (analysis of variance, ANOVA; P < 0.01). There was no significant difference between the smokers who had suffered from stroke and smokers who had not. CONCLUSION: The study indicated that prominent facial wrinkling was significantly more common among smokers than nonsmokers, not only in the relatively young but also among the aged population.

The role of frozen storage in preserving adipose tissue obtained by suction-assisted lipectomy for repeated fat injection procedures.

BACKGROUND: The injection of autologous free fat obtained by suction-assisted lipectomy for the correction of soft tissue defects is a common procedure in plastic surgery. However, unpredictable partial absorption of the injected fat often necessitates repeated procedures. OBJECTIVE: To examine the role of frozen storage as a means of preserving the fat obtained by suction-assisted lipectomy for repeated procedures. METHODS: Human adipose tissue obtained by suction-assisted lipectomy was stored in a domestic refrigerator at -18 degrees C for 2 weeks. After thawing, the fat was injected into nude mice. In the control group, the fat was injected immediately after the harvesting procedure. Grafts were dissected out and compared 15 weeks postinjection. RESULTS: Injected fat survived in both study and control groups. No significant differences were found between fat graft weight and volume, or in any of the histologic parameters examined. CONCLUSION: Fat obtained by suction-assisted lipectomy may be preserved for future use by freezing.

Incidence of Candida in psoriasis--a study on the fungal flora of psoriatic patients.

The presence of Candida albicans and other Candida species in saliva and faeces of 50 psoriatic patients compared with a control group of 50 healthy donors was examined quantitatively. The quantity of Candida in saliva and faeces of the psoriatics proved to be significantly higher than in the controls. Candida was detected in 78% of the saliva samples of the psoriatics but in only 50% of the controls, and in the faeces samples in 72% of the psoriatics, but in only 46% of the controls. Qualitative analysis revealed a predominance of Candida albicans (saliva, 77%; faeces, 64%) and Candida rugosa (saliva, 28%; faeces, 28%). We did not find a correlation between the severity of the psoriasis according to the Psoriasis Area and Severity Index and the amount of Candida in the saliva or in the faeces. Our results reinforce the hypothesis that C. albicans is one of the triggers to both exacerbation and persistence of psoriasis. We propose that in psoriatics with a significant quantity of Candida in faeces, an antifungal treatment should be considered as an adjuvant treatment of psoriasis.

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.

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.

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.

Alopecia areata is a T-lymphocyte mediated autoimmune disease: lesional human T-lymphocytes transfer alopecia areata to human skin grafts on SCID mice.

Much evidence suggests that alopecia areata is a tissue restricted autoimmune disease. Alopecia areata responds to immunosuppressive agents, and is associated with other tissue restricted autoimmune diseases, including autoimmune thyroiditis and vitiligo. Furthermore, hair regrows when involved scalp is transplanted to nude mice. This study was undertaken to determine whether alopecia areata is mediated by T lymphocytes. Involved scalp from alopecia areata patients was grafted onto SCID mice. Additional biopsies from lesional scalp of the same patients were used to isolate T lymphocytes. These T lymphocytes were cultured with hair follicle homogenate, as well as autologous antigen presenting cells. The T lymphocytes were then injected into autologous scalp grafts on the SCID mice, which had regrown hair. Injection of scalp T lymphocytes resulted in hair loss. Hair loss was associated with the histologic and immunochemical changes of alopecia areata, including perifollicular infiltrates of T cells, along with HLA-DR and ICAM-1 expression by the follicular epithelium. Scalp T lymphocytes that had not been cultured with hair follicle homogenate did not have this effect. Preliminary data suggests hair loss requires a collaboration between CD8+ and CD4+T cells. These studies have demonstrated that alopecia areata can be induced by the transfer of T cells that recognize a hair follicle autoantigen.

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

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.

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.

Sebum and water content in the skin of aged immobilized patients.

In the present study we investigated the sebum content and hydration of the skin in aged immobilized patients. Healthy aged as well as young and aged immobilized patients were evaluated by photometry, using a sebum tester and capacitance meter to detect sebum and hydration, respectively, in various skin areas. Sebum content was significantly higher in the young groups as compared to the aged ones, including those of the immobilized patients. Similar values of water content were observed in the healthy young and aged volunteers. However, surprisingly, a significantly marked decrease was detected in each tested area of the immobilized patients. We may assume that a cascade of events caused by the immobilization status of the patients leads to a significant decrease in water content within the dermis.

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.