Effects of topical application of aqueous solutions of hexoses on epidermal permeability barrier recovery rate after barrier disruption

Abstract: Previous studies have suggested that hexose molecules influence the stability of phospholipid bilayers. Therefore, the effects of topical application of all 12 stereoisomers of dextro‐hexose on the epidermal barrier recovery rate after barrier disruption were evaluated. Immediately after tape stripping, 0.1 m aqueous solution of each hexose was applied on hairless mouse skin. Among the eight dextro‐aldohexoses, topical application of altose, idose, mannose and talose accelerated the barrier recovery, while allose, galactose, glucose and gulose had no effect. Among the four dextro‐ketohexoses, psicose, fructose, sorbose and tagatose all accelerated the barrier recovery. As the effects of hexoses on the barrier recovery rate appeared within 1 h, the mechanism is unlikely to be genomic. Instead, these hexoses may influence phase transition of the lipid bilayers of lamellar bodies and cell membrane, a crucial step in epidermal permeability barrier homeostasis.     

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.     

Lipopolysaccharide and cytokines induce nitric oxide synthase and produce nitric oxide in cultured normal human melanocytes

Abstract The role of nitric oxide in normal and pathological conditions of human skin is still poorly understood. In this study we have demonstrated by immunobloting the expression of an inducible nitric oxide synthase isoform (iNOS) in cultured normal human melanocytes treated with bacterial lipopolysaccharide, tumor necrosis factor-· and interferon-á. Nitric oxide was also detected in the culture medium and its formation was abolished upon treatment with N^G-monomethyl-l-arginine(l-NMMA), a competitive inhibitor of nitric oxide synthase. These results suggest that nitric oxide could led to autodestruction of melanocytes causing skin depigmentation. The therapeutic relevance of nitric oxide synthase inhibitors in treatment of vitiligo was suggested. Received: 15 September 2000 / Revised: 7 October 2000 / Accepted: 20 January 2001     

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

Kang-Rotondo CH, Major S, Chiang TM, Myers LK, Kang ES. Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. Photodermatol Photoimmunol Photomed 1996: 12: 57–65. © Munksgaard, 1996. Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [³H]l -citrulline using labeled l -arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2–48.3 mJ/cm² but was inhibitory after 64.4 and 80.5 mJ/cm². Thirty min after 10^?6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M_r 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.     

Skin wound healing in the SKH-1 female mouse following inducible nitric oxide synthase inhibition

BACKGROUND: The inducible isoform of the nitric oxide (NO) synthase (NOS) enzyme (iNOS) is upregulated by inflammatory mediators and/or other pathological stresses, generating high, sustained levels of NO. Cumulative data suggest a role for NO in the regulation of skin wound healing, although it is not clear to what extent NO generated by iNOS, and possibly endothelial NOS (eNOS), contribute to that healing process. Because of the current lack of understanding regarding the contribution of iNOS in wound healing, as well as the lack of wound healing data available for SC-842, an iNOS inhibitor, this in vivo study was conducted to investigate the possible role of SC-842 in interfering with wound healing. OBJECTIVES: This study evaluated whether inhibition of iNOS affects incisional skin wound healing. METHODS: Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, the role of iNOS in the wound healing process was evaluated by comparing in vivo effects of the iNOS inhibitor, SC-842, at various doses that result in selective inhibition of iNOS as well as nonselective NOS inhibition (as evidenced by elevated blood pressure resulting in inhibition of eNOS and/or neuronal NOS). Dexamethasone was used as a positive control. RESULTS: There were no differences in wound healing at day 28 postwounding, as evaluated by tensile strength and histology, between SC-842- and vehicle-treated animals. A decrease in tensile strength was noted at day 14 postwounding in wounds from the mid- and high-dose-treated animals as compared with vehicle-treated animals, but this difference was slight and was not associated with histological differences from vehicle-treated controls. CONCLUSIONS: These data indicate that iNOS inhibition does not adversely affect the healing of incisional wounds in SKH-1 mice as assessed over 28 days by wound tensile strength and histology.     

Extremely low frequency electromagnetic fields modulate expression of inducible nitric oxide synthase,endothelial nitric oxide synthase and cyclooxygenase‐2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing

Background Extremely low frequency (ELF) electromagnetic fields (EMF) are known to produce a variety of biological effects. Clinical studies are ongoing using EMF in healing of bone fractures and skin wounds. However, little is known about the mechanisms of action of ELF‐EMF. Several studies have demonstrated that expression and regulation of nitric oxide synthase (NOS) and cyclooxygenase‐2 (COX‐2) are vital for wound healing; however, no reports have demonstrated a direct action of ELF‐EMF in the modulation of these inflammatory molecules in human keratinocytes. Objectives The present study analysed the effect of ELF‐EMF on the human keratinocyte cell line HaCaT in order to assess the mechanisms of action of ELF‐EMF and to provide further support for their therapeutic use in wound healing. Methods Exposed HaCaT cells were compared with unexposed control cells. At different exposure times, expression of inducible NOS (iNOS), endothelial NOS (eNOS) and COX‐2 was evaluated by Western blot analysis. Modulation of iNOS and eNOS was monitored by evaluation of NOS activities, production of nitric oxide (NO) and O₂^? and expression of activator protein 1 (AP‐1). In addition, catalase activity and prostaglandin (PG) E₂ production were determined. Effects of ELF‐EMF on cell growth and viability were monitored. Results The exposure of HaCaT cells to ELF‐EMF increased iNOS and eNOS expression levels. These ELF‐EMF‐dependent increased expression levels were paralled by increased NOS activities, and increased NO production. In addition, higher levels of AP‐1 expression as well as a higher cell proliferation rate were associated with ELF‐EMF exposure. In contrast, ELF‐EMF decreased COX‐2 expression, PGE₂ production, catalase activity and O₂^? production. Conclusions Mediators of inflammation, such as reactive nitrogen and PGE₂, and keratinocyte proliferation are critical for the tissue regenerative processes. The ability of ELF‐EMF to upmodulate NOS activities, thus nitrogen intermediates, as well as cell proliferation, and to downregulate COX‐2 expression and the downstream intermediate PGE₂, highlights the potential therapeutic role of ELF‐EMF in wound healing processes.     

一氧化氮合酶在外阴慢性单纯性苔藓和外阴硬化性苔藓中的表达

目的：检测诱导型和内皮型一氧化氮合酶（iNOS 和eNOS）在外阴慢性单纯性苔藓和外阴硬化性苔藓中的表达。方法：采用免疫组织化学SABC法检测iNOS和eNOS在30例外阴慢性单纯性苔藓蜡块（LSC）、30例外阴硬化性苔藓蜡块（LS）和10例外阴正常皮肤中的表达,并以人原始造血细胞抗原（CD34）标记微血管内皮细胞,测量各组织的微血管密度（MVD）。结果：iNOS和eNOS在外阴正常皮肤中无表达;在LSC中iNOS和eNOS每视野平均阳性细胞数分别为14.83±3.79和17.86±4.82,高于LS的8.00±3.35和6.43±3.87,差异均有统计学意义（P＜0.05）;在LSC和正常皮肤中MVD分别为21.58±2.48和20.44±3.66,高于LS（10.34±2.83）。iNOS和eNOS的表达具有明显的正相关性（Kappa=0.811,P＜0.05）。结论：iNOS和eNOS可能与LSC炎症过程中的血管扩张有关;在LS皮损真皮中微血管减少,iNOS和eNOS可代偿性地改善LS的血液循环。     

Topical application of neuronal nitric oxide synthase inhibitor accelerates cutaneous barrier recovery and prevents epidermal hyperplasia induced by barrier disruption

The effect of nitric oxide (NO) on skin barrier recovery rate was evaluated in hairless mouse. Topical application of an NO synthase (NOS) inhibitor and a neuronal nitric oxide synthase (nNOS) inhibitor accelerated the barrier recovery after tape stripping, whereas application of an inducible NOS (iNOS) inhibitor had no effect. After tape stripping, the barrier recovery in nNOS-/- mice was significantly faster than in wild type. Topical application of the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) delayed the barrier recovery in hairless mice. Immediately after barrier disruption on skin organ culture, NO release from the skin was significantly increased. The increase was blocked by nNOS inhibitor, but not by iNOS inhibitor. Topical application of the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) accelerated the barrier recovery, whereas SIN-1 chloride, a guanylyl cyclase activator, delayed the barrier recovery. In cultured human keratinocytes, SNAP increased the intracellular calcium concentration. The increase was blocked by ODQ, but not by the calcium channel-blocker nifedipine. In calcium-free medium, SNAP increased the intracellular calcium concentration. Topical application of both nNOS inhibitor and ODQ also reduced the epidermal hyperplasia induced by barrier disruption under low environmental humidity. These results suggest that NO plays an important signaling role in cutaneous barrier homeostasis and in epidermal hyperplasia induced by barrier disruption.     

Expression of nitric oxide synthases in keratinocytes after UVB irradiation

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.     

诱生型一氧化氮合酶和血管内皮生长因子在皮肤血管瘤中的表达及其与血管增生的关系

目的：研究诱生型一氧化氮合酶（iNOS）及血管内皮生长因子（VEGF）在增生期皮肤血管瘤组织中的表达及相关性。探讨它们与增生期血管瘤血管生成的关系,研究iNOS产生的一氧化氮（NO）和VEGF的相互作用及NO在介导VEGF促血管瘤间质内血管生成中的作用机制。方法：应用免疫组化法检测51例增生期皮肤血管瘤标本中iNOS、VEGF和第Ⅷ因子相关抗原（FⅧRAg）,血管内皮细胞特异性染色计数肿瘤微血管密度（MVD）。结果：①42例血管瘤组织表达VEGF,32例表达iNOS,血管畸形表达较弱或不表达iNOS和VEGF;②VEGF与iNOS的表达呈正相关性：③VEGF、iNOS的表达与血管瘤组织MVD呈正相关性,血管瘤MVD明显高于血管畸形。结论：①VEGF表达与iNOS表达具有明显的相关性,提示iNOS对VEGF的表达和调节血管生成过程中可能具有重要作用;②MVD随着VEGF和iNOS表达的增强而增加,说明两者对血管瘤血管生成具有促进作用。     

Potassium channel openers accelerate epidermal barrier recovery

BACKGROUND: Maintenance of a competent permeability barrier in the face of external and internal stressors requires signals between the stratum corneum interface and the metabolic machinery in the underlying nucleated layers. For example, reductions in the ion gradients for Ca2+ after acute barrier disruption stimulate lamellar body (LB) secretion, a response required to restore barrier homeostasis. Although alterations in external K+ levels also regulate barrier recovery after acute insults, the mechanisms whereby K+ regulates barrier function remain unknown. OBJECTIVES: To evaluate effects of regulators of K+ channels on barrier homeostasis in hairless mice. METHODS: We tested a number of chemically different drugs that alter intracellular K+ levels. Results Single applications of either K+ channel openers (i.e. 1-EBIO, minoxidil, diazoxide) or the K+ ionophore, valinomycin, accelerated barrier recovery after acute insults to murine skin, paralleled by a reduction in intracellular K+ levels in cultured human keratinocytes. In contrast, applications of K+ channel blockers (i.e. gilbenclamide, dequalinium) delayed barrier recovery. Alterations in intracellular K+ regulated barrier homeostasis by either stimulating (reduced K+) or inhibiting (elevated K+) LB secretion. Finally, development of epidermal hyperplasia, a downstream consequence of barrier disruption, was also inhibited by agents that reduce intracellular K+ levels. CONCLUSIONS: These results demonstrate that changes in K+ levels that can be presumed to occur after barrier disruption signal metabolic responses, i.e. LB secretion, which accelerates normalization of barrier function.     

Inducible nitric oxide synthase is required for epidermal permeability barrier homeostasis in mice

Nitric oxide (NO) regulates a variety of epidermal functions, including epidermal proliferation, differentiation and cutaneous wound healing. However, whether nitric oxide (NO) and its synthetic enzymes regulate epidermal permeability barrier homeostasis is not clear. In the present study, we employed inducible nitric oxide synthase (iNOS) KO mice to explore the role of iNOS in epidermal permeability barrier homeostasis. Our results showed that iNOS mice displayed a comparable levels of basal transepidermal water loss rates, stratum corneum hydration and skin surface pH to their wild-type mice, but epidermal permeability barrier recovery was significantly delayed both 2 and 4 hours after acute barrier disruption by tape stripping. In parallel, expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes were lower in iNOS KO mice versus wild-type controls. Topical applications of two structurally unrelated NO donors to iNOS KO mice improved permeability barrier recovery kinetics and upregulated expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes. Together, these results indicate that iNOS and its product regulate epidermal permeability barrier homeostasis in mice.     

长波紫外线照射对HaCaT细胞iNOS/NO表达的影响

目的 探讨不同剂量长波紫外线 （UVA） 照射HaCaT细胞后不同时间点诱导型一氧化氮合成酶（iNOS）的表达情况。方法 1 J/cm2、5 J/cm2和10 J/cm2 UVA照射HaCaT细胞后继续培养24 h、48 h和72 h,倒置相差显微镜下观察细胞形态的变化;分别用RT-PCR、Western印迹和Griess法检测HaCaT细胞iNOS mRNA、蛋白及NO的表达。结果 所有UVA剂量组HaCaT细胞iNOSmRNA在光照后24 h有表达,48 h达高峰,72 h后下降,各时间点间表达量差异有统计学意义（P < 0.05）;1 J/cm2 UVA照射后3个时间点均未见iNOS蛋白表达,而5 J/cm2和10 J/cm2 UVA照射后iNOS蛋白在24 h增加,48 h达高峰且显著高于24 h（P < 0.05）,照射后72 h无iNOS蛋白表达。所有UVA剂量组HaCaT细胞NO表达量在24 h升高,48 h显著升高,72 h平稳升高,3个时间点NO表达量均比正常对照组明显增加（P < 0.05）。对照组HaCaT细胞无iNOS mRNA和蛋白表达,NO表达量低。结论 HaCaT细胞iNOS和NO的表达变化与UVA照射存在时间和剂量关系。     

Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin

Nitric oxide (NO) is a small molecule produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma.     

结节性红斑患者皮损中一氧化氮合酶、血管内皮生长因子的表达

目的 :　探讨一氧化氮合酶 (NOS)和血管内皮生长因子 (VEGF)在结节性红斑中的表达。方法 :用ABC免疫组化方法检测了 4 2例结节性红斑患者皮损和 15例正常人皮肤组织中NOS和VEGF。结果 :结节性红斑皮损中NOS和VEGF的检出率分别为 80 .95 %及 6 6 .6 7% ,阳性信号的表达与分布基本一致(χ2 =2 .2 2 <3.84 ,P >0 .5 ,u =0 .36 6 <0 .6 78,P >0 .5 ) ,主要位于表皮角质层和部分棘细胞、真皮及皮下脂肪层小血管内皮细胞和腺上皮细胞内 ,正常人皮肤组织中NOS和VEGF的表达微弱 ,分布于表皮角质层和部分棘细胞 ,真皮及皮下脂肪层未见。结论 :　结节性红斑皮损中有NOS和VEGF的强阳性表达 ,可能与病毒等微生物引起的免疫反应有关。     

Expression of neuronal nitric oxide synthase in dermal infiltrated eosinophils in eosinophilic pustular folliculitis

Nitric oxide (NO) is produced by a wide variety of human cells and affects physiological and pathophysiological processes. In this study, we demonstrated for the first time that neuronal NO synthase (NOS) is expressed in eosinophils infiltrating into the dermis and follicular epidermis in eosinophilic pustular folliculitis (EPF). EPF is a cutaneous inflammatory follicular disorder first described in Japan. The clinical and histological features of EPF are characterized by erythematous papules, infiltration of numerous eosinophils, and a spongiotic appearance of the follicular epidermis, but the pathophysiology of EPF remains unclear. Our results suggest that NO produced from eosinophils plays an important part in the pathogenesis of EPF. Furthermore, we speculate that NOS inhibitors may be useful in the management of EPF.     

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

BACKGROUND: Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. OBJECTIVES: We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. METHODS: This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). RESULTS: Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. CONCLUSIONS: The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.     

Barium sulphate with a negative zeta potential accelerates skin permeability barrier recovery and prevents epidermal hyperplasia induced by barrier disruption

BACKGROUND: Barium sulphate, a stable inorganic material, has been used in contrast media and cosmetic products because of its stability. As a negative external electric potential accelerates the skin barrier repair after barrier disruption, we hypothesized that topical application of barium sulphate may affect the skin barrier recovery rate depending on its zeta potential. OBJECTIVES: To investigate whether barium sulphate particles in aqueous solution have different zeta potentials depending on their surface structure, and to investigate the possible relation between zeta potential and skin barrier recovery rate. METHODS: Mice were subjected to tape stripping to disrupt barrier function, or were treated with acetone and kept in a dry environment to induce epidermal hyperplasia. They were then treated with different forms of barium sulphate, and barrier recovery was monitored by measurements of transepidermal water loss. RESULTS: There was a significant correlation between the barrier recovery rate and zeta potential of barium sulphate applied topically. Barium sulphate with a negative zeta potential significantly accelerated barrier recovery, but barium sulphate with a positive zeta potential did not accelerate or even delayed barrier repair. Barium sulphate with a negative zeta potential had an X-ray diffraction pattern different from that with a positive potential. The distribution of calcium in the epidermis was also influenced by the polarity of zeta potential. CONCLUSIONS: These findings suggest a new pharmacological approach towards altering barrier function or epidermal hyperplasia with inorganic particles in healthy and diseased skin.