Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium.

Immune cells and epithelium form sophisticated barrier systems in symbiotic relationships with microbiota. Evidence suggests that immune cells can sense microbes through intact barriers, but regulation of microbial commensalism remain largely unexplored. Here, we uncovered spatial compartmentalization of skin-resident innate lymphoid cells (ILCs) and modulation of sebaceous glands by a subset of RORγt+ ILCs residing within hair follicles in close proximity to sebaceous glands. Their persistence in skin required IL-7 and thymic stromal lymphopoietin, and localization was dependent on the chemokine receptor CCR6. ILC subsets expressed TNF receptor ligands, which limited sebocyte growth by repressing Notch signaling pathway. Consequently, loss of ILCs resulted in sebaceous hyperplasia with increased production of antimicrobial lipids and restricted commensalism of Gram-positive bacterial communities. Thus, epithelia-derived signals maintain skin-resident ILCs that regulate microbial commensalism through sebaceous gland-mediated tuning of the barrier surface, highlighting an immune-epithelia circuitry that facilitates host-microbe symbiosis.

The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

Preventative effects of antioxidants on changes in sebocytes, outer root sheath cells, and Cutibacterium acnes-pretreated mice by particulate matter: No significant difference among antioxidants.

OBJECTIVES: Particulate matter (PM) is an air pollutant that can damage human skin; antioxidants have shown some efficacy in alleviating PM-induced skin inflammation. We investigated the antioxidant effects of punicalagin, epigallocatechin-3-gallate (EGCG), and resveratrol on PM-induced changes in cultured human sebocytes, outer root sheath (ORS) cells, and Cutibacterium acnes-pretreated mice. METHODS: Sebocytes and ORS cells were cultured with 100 µg/mL PM10 and 5 µM punicalagin, 1 µM EGCG, or 1 µM resveratrol for 24 h. In C. acnes-pretreated mice, inflammatory nodules were treated with 100 µg/mL PM10 and 5 µM punicalagin, 1 µM EGCG, or 1 µM resveratrol. Cell viability was measured using an MTT assay. Antioxidant effects were analyzed according to RNA expression, using real-time PCR, as well as reactive oxygen species (ROS) and sebum measurements. RESULTS: Antioxidants inhibited the upregulation of inflammatory cytokines, matrix metalloproteinase, aryl hydrocarbon receptor, and NF-kB as well as the production of ROS induced by PM10 in cultured sebocytes and ORS cells. The preventative effects of punicalagin and EGCG on biomarker expression in cultured sebocytes and ORS cells were slightly greater than those of resveratrol, though the difference was not significant. In C. acnes-pretreated mice, the antioxidants inhibited inflammatory cytokine and matrix metalloproteinase expression as well as sebum production. CONCLUSIONS: Antioxidants effectively reduced the expression of inflammatory biomarkers and sebum production in cultured sebocytes, ORS cells, and C. acnes-pretreated mice.

Augmentation of gene expression and production of promatrix metalloproteinase 2 by Propionibacterium acnes-derived factors in hamster sebocytes and dermal fibroblasts: a possible mechanism for acne scarring.

Aberrant extracellular matrix (ECM) remodeling in sebaceous glands and pilosebaceous units in the skin is associated with scar formation under acne conditions. To investigate the involvement of Propionibacterium acnes (P. acnes), a Gram-positive anaerobic microbial species, in ECM remodeling in sebaceous glands and pilosebaceous units, we examined the effects of P. acnes culture media, formalin-fixed P. acnes, and peptidoglycan (PGN) from Gram-positive bacteria walls on the production of promatrix metalloproteinase 2 (proMMP-2)/progelatinase A in hamster sebocytes and dermal fibroblasts. When hamster sebocytes (1.8×10(5) cells) and dermal fibroblasts (1×10(5) cells) were treated with P. acnes culture media and formalin-fixed P. acnes (corresponding to 1×10(6) and 1×10(7) bacterial cells), the production of proMMP-2 was augmented. In addition, PGN (5-50 µg/ml) dose-dependently augmented the production of proMMP-2 in both cells. Furthermore, the PGN (50 µg/ml)-augmented proMMP-2 production was resulted from an increase of its transcript. In contrast, there were no changes in cell proliferative activity in either the P. acnes or PGN-treated sebocytes and dermal fibroblasts, indicating that the augmented proMMP-2 production was not due to an increase in cell numbers. Therefore, these results provide novel evidence that PGN transcriptionally up-regulates the production of proMMP-2 in hamster sebocytes and dermal fibroblasts. Given an increase in the quantity of Gram-positive bacteria, including P. acnes in acne lesions, the aberrant ECM degradation may progress in sebaceous glands and pilosebaceous units, which is associated with acne scar formation.

Quantitative PCR of Propionibacterium acnes DNA in samples aspirated from sebaceous follicles on the normal skin of subjects with or without acne.

To elucidate whether people with hair follicles containing many Propionibacterium acnes cells are prone to acne, we developed a novel method to count the number of P. acnes in hair follicles. We sampled sebaceous material in hair follicles by aspiration at a constant negative pressure from the nose, forehead, and upper arm of 86 patients with acne vulgaris and 209 control subjects with healthy skin, including 84 subjects age-matched to the patients. Genome-equivalents of P. acnes in samples were estimated by real-time quantitative PCR (TaqMan). Numbers of P. acnes genome-equivalents were extremely low in control subjects less than 10 years of age and generally higher at greater ages, with much variation in subjects in the same decade of life. In men, the median count was highest in controls aged 15-19 years; in women, it peaked twice, in controls aged 15-19 years and again in those aged 40 years or older. P. acnes counts on the forehead and nose were higher in the acne patients aged 10-14 years than in the age-matched controls in both sexes. The counts at three sites were similar in acne patients and controls aged 15 to 29 years in both sexes. The results suggest that people with hair follicles containing many P. acnes cells are not particularly prone to acne, except for younger teenagers. Our aspiration method with estimation by real-time PCR can be used to examine the cutaneous microflora of P. acnes.

New developments in our understanding of acne pathogenesis and treatment.

Interest in sebaceous gland physiology and its diseases is rapidly increasing. We provide a summarized update of the current knowledge of the pathobiology of acne vulgaris and new treatment concepts that have emerged in the last 3 years (2005-2008). We have tried to answer questions arising from the exploration of sebaceous gland biology, hormonal factors, hyperkeratinization, role of bacteria, sebum, nutrition, cytokines and toll-like receptors (TLRs). Sebaceous glands play an important role as active participants in the innate immunity of the skin. They produce neuropeptides, excrete antimicrobial peptides and exhibit characteristics of stem cells. Androgens affect sebocytes and infundibular keratinocytes in a complex manner influencing cellular differentiation, proliferation, lipogenesis and comedogenesis. Retention hyperkeratosis in closed comedones and inflammatory papules is attributable to a disorder of terminal keratinocyte differentiation. Propionibacterium acnes, by acting on TLR-2, may stimulate the secretion of cytokines, such as interleukin (IL)-6 and IL-8 by follicular keratinocytes and IL-8 and -12 in macrophages, giving rise to inflammation. Certain P. acnes species may induce an immunological reaction by stimulating the production of sebocyte and keratinocyte antimicrobial peptides, which play an important role in the innate immunity of the follicle. Qualitative changes of sebum lipids induce alteration of keratinocyte differentiation and induce IL-1 secretion, contributing to the development of follicular hyperkeratosis. High glycemic load food and milk may induce increased tissue levels of 5alpha-dihydrotestosterone. These new aspects of acne pathogenesis lead to the considerations of possible customized therapeutic regimens. Current research is expected to lead to innovative treatments in the near future.

Acne vaccines targeting Propionibacterium acnes.

Acne vulgaris is one of the most common skin diseases and can affect a large number of individuals at some point in their lives. Though the disease is multi-factorial, the Gram-positive, anaerobic bacterium Propionibacterium acnes (P. acnes), a member of resident skin microflora, is implicated in acne inflammation and associated with acne lesions. Common treatments such as antibiotic or benzoyl peroxide nonspecifically reduce bacteria population on the skin, which may disrupt homeostasis and cause further complications such as promoting growth of antibiotic-resistant bacteria strains. A component vaccine and an inactivated whole bacteria vaccine are made to target specifically P. acnes. The component vaccine targeting P. acnes surface sialidase and heat-inactivated P. acnes vaccine have both been shown to reduce P. acnes- induced inflammation in vivo and neutralize P. acnes in vitro, suggesting their potentials as new treatment for acne vulgaris. To facilitate acne studies, a bioengineering approach was utilized to design a new human acne model using tissue chamber. The tissue chamber of human sebocytes is shown to produce in mice a microenvironment similar to human acne inflammation. This approach can also be utilized in future studies in developing therapeutic acne vaccines and designing possible combined treatment of acne vaccine with alternative acne treatments.

Acne, the Skin Microbiome, and Antibiotic Treatment.

Acne vulgaris is a chronic skin disorder involving hair follicles and sebaceous glands. Multiple factors contribute to the disease, including skin microbes. The skin microbiome in the follicle is composed of a diverse group of microorganisms. Among them, Propionibacterium acnes and Malassezia spp. have been linked to acne development through their influence on sebum secretion, comedone formation, and inflammatory response. Antibiotics targeting P. acnes have been the mainstay in acne treatment for the past four decades. Among them, macrolides, clindamycin, and tetracyclines are the most widely prescribed. As antibiotic resistance becomes an increasing concern in clinical practice, understanding the skin microbiome associated with acne and the effects of antibiotic use on the skin commensals is highly relevant and critical to clinicians. In this review, we summarize recent studies of the composition and dynamics of the skin microbiome in acne and the effects of antibiotic treatment on skin microbes.

Host-microbiome interactions and recent progress into understanding the biology of acne vulgaris.

Acne is one of the most common skin diseases worldwide and results in major health care costs and significant morbidity to severely affected individuals. However, the pathophysiology of this disorder is not well understood. Host-microbiome interactions that affect both innate and adaptive immune homeostasis appear to be a central factor in this disease, with recent observations suggesting that the composition and activities of the microbiota in acne is perturbed. Staphylococcus epidermidis and Cutibacterium acnes (C. acnes; formerly Propionibacterium acnes) are two major inhabitants of the skin that are thought to contribute to the disease but are also known to promote health by inhibiting the growth and invasion of pathogens. Because C. acnes is ubiquitous in sebaceous-rich skin, it is typically labeled as the etiological agent of acne yet it fails to fulfill all of Koch's postulates. The outdated model of acne progression proposes that increased sebum production promotes over-proliferation of C. acnes in a plugged hair follicle, thereby driving inflammation. In contrast, growing evidence indicates that C. acnes is equally abundant in both unaffected and acne-affected follicles. Moreover, recent advances in metagenomic sequencing of the acne microbiome have revealed a diverse population structure distinct from healthy individuals, uncovering new lineage-specific virulence determinants. In this article, we review recent developments in the interactions of skin microbes with host immunity, discussing the contribution of dysbiosis to the immunobiology of acne and newly emerging skin microbiome-based therapeutics to treat acne.

Corynebacterium heidelbergense sp. nov., isolated from the preen glands of Egyptian geese (Alopochen aegyptiacus).

Two strains (pedersoliT and girotti) of a new species of bacteria were isolated from the preen glands of wild Egyptian geese (Alopochen aegyptiacus) from the river Neckar in southern Germany in two subsequent years. The strains were lipophilic, fastidious, Gram-positive rods and belonged to the genus Corynebacterium. Phylogenetically, the isolates were most closely related to Corynebacterium falsenii DSM 44353T which has been found to be associated with birds before. 16S rRNA gene sequence similarity to all known Corynebacterium spp. was significantly <97%. Corresponding values of rpoB showed low levels of similarity <87% and ANIb was <73%. G+C content of the genomic DNA was 65.0mol% for the type strain of the goose isolates, as opposed to 63.2mol% in Corynebacterium falsenii. MALDI-TOF MS analysis of the whole-cell proteins revealed patterns clearly different from the related species, as did biochemical tests, and polar lipid profiles. We therefore conclude that the avian isolates constitute strains of a new species, for which the name Corynebacterium heidelbergense sp. nov. is proposed. The type strain is pedersoliT (=DSM 104638T=LMG 30044T).

Involvement of adenosine triphosphate-binding cassette subfamily B member 1 in the augmentation of triacylglycerol excretion by Propionibacterium acnes in differentiated hamster sebocytes.

An onset of acne, a common inflammatory skin disease, is associated with excess sebum production and secretion in sebaceous glands. Because Propionibacterium acnes has been reported to augment intracellular sebum accumulation in sebaceous glands in hamsters, it remains unclear whether P. acnes influences sebum secretion from differentiated sebocytes. Both P. acnes culture media (Acnes73-CM) and formalin-killed P. acnes (F-Acnes73) dose-dependently increased the extracellular levels of triacylglycerol (TG), a major sebum component, and Rhodamine 123, a substrate of adenosine triphosphate-binding cassette (ABC) transporter, from differentiated hamster sebocytes (DHS). In addition, the gene expression of the ABC subfamily B member 1 (ABCB1) was dose-dependently augmented by adding Acnes73-CM and F-Acnes73 into DHS. Furthermore, the F-Acnes73-induced increase of TG excretion was suppressed by PSC833, a selective ABCB1 inhibitor. On the other hand, peptidoglycan (PGN), which is a Toll-like receptor 2 (TLR2) ligand in P. acnes, increased extracellular TG levels, transporter activity and ABCB1 mRNA expression in DHS. The PGN-augmented TG excretion was suppressed by PSC833. Thus, these results provide novel evidence that P. acnes facilitates sebum secretion due to the activation of ABCB1 concomitantly with the increased ABCB1 expression, which may result from the activation of the TLR2 pathway in DHS. Therefore, the ABCB1 inhibitor is likely to become a candidate as a possible therapeutic for the treatment of acne.

Propionibacterium acnes and lipopolysaccharide induce the expression of antimicrobial peptides and proinflammatory cytokines/chemokines in human sebocytes.

Acne is a common skin disorder of the pilosebaceous unit. In addition to genetic, hormonal and environmental factors, abnormal colonization by Propionibacterium acnes has been implicated in the occurrence of acne via the induction of inflammatory mediators. To gain more insight into the role that sebocytes play in the innate immune response of the skin, particularly in acne, we compared the antimicrobial peptide and proinflammatory cytokine/chemokine expression at mRNA and protein levels, as well as the viability and differentiation of SZ95 sebocytes in response to co-culture with representative isolates of P. acnes type IA and type IB as well as Escherichia coli-derived lipopolysaccharide (LPS). We found that, in vitro, P. acnes type IA and IB isolates and LPS induced human beta-defensin-2 and proinflammatory cytokine/chemokine expression, and influenced sebocyte viability and differentiation. Our results provide evidence that sebocytes are capable of producing proinflammatory cytokines/chemokines and antimicrobial peptides, which may have a role in acne pathogenesis. Furthermore, since P. acnes types IA and IB differentially affect both the differentiation and viability of sebocytes, our data demonstrate that different strains of P. acnes vary in their capacity to stimulate an inflammatory response within the pilosebaceous follicle.

Nest Bacterial Environment Affects Microbiome of Hoopoe Eggshells, but Not That of the Uropygial Secretion.

The study of associations between symbiotic bacterial communities of hosts and those of surrounding environments would help to understand how bacterial assemblages are acquired, and how they are transmitted from one to another location (i.e. symbiotic bacteria acquisition by hosts). Hoopoes (Upupa epops) smear their eggshells with uropygial secretion (oily secretion produced in their uropygial gland) that harbors antibiotic producing bacteria. Trying to elucidate a possible role of nest material and cloaca microbiota in determining the bacterial community of the uropygial gland and the eggshells of hoopoes, we characterized bacterial communities of nest material, cloaca, uropygial gland and eggshells by the ARISA fingerprinting. Further, by adding material with scarce bacteria and antimicrobial properties, we manipulated the bacterial community of nest material and thus tested experimentally its effects on the microbiomes of the uropygial secretion and of the eggshells. The experiment did not influence the microbiome of the uropygial secretion of females, but affected the community established on eggshells. This is the first experimental evidence indicating that nest material influences the bacterial community of the eggshells and, therefore, probability of embryo infection. Some of the bacterial strains detected in the secretion were also in the bacterial communities of the nest material and of cloaca, but their occurrence within nests was not associated, which suggests that bacterial environments of nest material and cloaca are not sources of symbiotic bacteria for the gland. These results do not support a role of nest environments of hoopoes as reservoirs of symbiotic bacteria. We discuss possible scenarios explaining bacterial acquisition by hoopoes that should be further explored.

Cell-free extracts of Propionibacterium acnes stimulate cytokine production through activation of p38 MAPK and Toll-like receptor in SZ95 sebocytes.

AIMS: Propionibacterium acnes has been considered to influence the acne lesions. The present study intended to elucidate the underlying signaling pathways of P. acnes in human sebaceous gland cells relative to the generation of proinflammatory cytokines. MAIN METHODS: Cell-free extracts of P. acnes under stationary growth phase were co-incubated with human immortalized SZ95 sebocytes. Then, cell-free P. acnes extracts-induced cytokine expression was evaluated by measuring mRNA and protein levels using quantitative RT-PCR and ELISA. Changes of phosphorylated cell signaling proteins and transcription factors were measured by Western blots and Milliplex assay. The interactive molecular mechanisms of P. acnes and sebocytes were examined through use of shRNA and the specific inhibitors of signaling pathways. KEY FINDINGS: Cell-free extracts of P. acnes significantly stimulated secretion of interleukin (IL)-8 and IL-6 in SZ95 sebocytes. The degradation of IκB-α and increased phosphorylation of IκB-α, p38 mitogen activated protein kinase (MAPK), CREB, and STAT3 were demonstrated. Quantitative RT-PCR measurements revealed that gene expression of IL-8 and Toll-like receptor 2 (TLR2) was enhanced by cell-free extracts of P. acnes. In addition, the NF-κB inhibitor BMS345541, p38 MAPK inhibitor SB203580, or anti-TLR2 neutralizing antibody prevented cell-free P. acnes extracts-induced secretion of IL-8. Knockdown of TLR2 using shRNA exerted similar inhibitory effects on IL-8 expression. Moreover, inhibition of STAT3 activity by STA-21 enhanced P. acnes-mediated secretion of IL-8. SIGNIFICANCE: Cell-free extracts of P. acnes are capable to activate NF-κB and p38 MAPK pathways and up-regulate secretion of IL-8 through TLR2-dependent signaling in human SZ95 sebocytes.

Quantification of bacteria in isolated pilosebaceous follicles in normal skin.

A technique for quantitating bacteria in isolated pilosebaceous follicles is described. This involves microdissection of the follicles from biopsies of skin, using the method of chemical pretreatment of skin to facilitate the separation of the epidermis and epidermal appendages from the dermis. The aerobic cocci and anaerobic diphtheroids in pilosebaceous follicles in 66 biopsies of scalp and 48 biopsies of skin of the upper back were quantitated using this technique. On the back, aerobic staphylococci were very sparse in normal follicles, indicating that their primary habitat on the skin must be on the skin surface rather than within follicles. Of 138 isolated follicles from skin of the upper back, 94 contained no aerobic cocci. Anaerobic organisms were present in high numbers within normal follicles. The geometric mean density of anaerobes in 138 isolated follicles from skin of the upper back was 3.8 X 10(4) diphtheroids per follicle. Eighty-eight follicles contained more than 10(4) anaerobic diphtheroids. Using data from scalp biopsies we found that there was a correlation between the weight of sebaceous glands and the density of anaerobes within the follicles attached to these glands (coefficient of correlation = 0.6).

Esterification of (4--14C)cholesterol by cutaneous bacteria (Staphylococcus epidermidis, Propionibacterium acnes, and Propionibacterium granulosum).

Cholesterol and cholesteryl esters constitute 3 to 5% of the lipids derived from human sebaceous glands. The present study demonstrates that cutaneous bacteria, Staphylococcus epidermidis, and to a lesser extent Propionibacterium acnes, and Propionibacterium granulosum, esterify [4--14C]cholesterol to steryl esters in vitro. The degree of esterification is a characteristic of the strain of organism, ranging from 0 to 32.7% in the organisms tested. Cholesterol esterification in these experiments was dependent on the addition of homogenates of isolated, pooled sebaceous glands to the incubation medium. Indirect evidence suggests that the fatty acids from sebaceous triglycerides are utilized for cholesterol esterification by the bacteria. These results indicate that the proportion of steryl esters in skin surface lipids may be influenced by the density of the microbial population in the skin.

The relationship between bacteria and the abnormal follicular keratinization in acne vulgaris.

The structural organization and bacteriological profile of follicular casts and early comedones in prepuberal children were investigated. Follicular casts were present in all samples but were not as abundant as usually seen in older individuals with acne. When examined with the light microscope, all casts and comedones were composed of numerous layers of horny cells and sebum, but were devoid of bacteria. Neither follicular casts nor comedones yielded bacteria when cultured. Electron microscopy of the follicular casts revealed the presence of small round, discrete lipid droplets, and alternating dense and less dense lamellar configurations within the horny cells. Some horny cells closest to the sebum-filled lumen contained large lipid masses, resulting in "balloon-shaped" regions. Prepuberal follicular horny cells contained all of the abnormalities usually seen in follicular casts and biopsy material from acne patients, which suggests that these casts are potential comedones. These aberrations occurred in the complete absence of bacteria indicating that bacteria are not essential to the formation of casts or comedones. Furthermore, our findings indicate that bacteria play little if any role in the initial events of pathological keratinization.

The localization and distribution of gram-positive cocci in normal skin and in lesions of acne vulgaris.

The localization of gram-positive cocci in the normal skin and in the lesions of acne vulgaris was investigated using fluorescein-labeled antiserum raised to gram-positive, coagulase-negative cocci. The cocci were found in 10 of 19 specimens from normal facial skin and in 3 of 11 specimens from the normal skin of the rest of the body. The bacteria were found mostly in the openings of follicles, but in 6 of 10 facial skin specimens, they were also present deeply in the lumina of the dilated sebaceous follicles near the sebaceous glands. Cocci were found in 5 of 6 noninflammatory acne comedones. In inflammatory acne they were demonstrated not only in the follicular canals but also sparsely in the infiltrate surrounding the follicles.

Propionibacterium acnes and the pathogenesis of progressive macular hypomelanosis.

BACKGROUND: Progressive macular hypomelanosis is a common hypopigmentation mainly on the central parts of the trunk, predominantly in young adults, especially women. It is often mistaken for pityriasis versicolor and pityriasis alba. It occurs in all races and has been described in many parts of the world. We discovered follicular red fluorescence restricted to lesional skin. We suspected a relation with a porphyrin-producing bacteria residing in sebum of the pilosebaceous duct, and we therefore performed a study in 8 patients. Observation In all biopsy specimens taken from lesional skin of 8 women, we could demonstrate gram-positive bacteria in the pilosebaceous duct, and a mild perifollicular lymphocytic infiltrate was seen. In all but 1 patient, Propionibacterium acnes was yielded from cultured biopsy specimens taken from follicular lesional skin. Healthy follicular skin did not show bacteria in histological sections, and cultures did not yield anaerobic bacteria. CONCLUSIONS: There seems to be a relation between the presence of P acnes and the hypopigmented macules. We propose that a factor is produced by these strains of P acnes, which interfere with melanogenesis. Based on these observations, we are undertaking a clinical trial to find a treatment for this troubling, intractable disease.