Calcitriol May Down-Regulate mRNA Over-Expression of Toll-Like Receptor-2 and -4, LL-37 and Proinflammatory Cytokines in Cultured Human Keratinocytes

BACKGROUND: Although vitamin D analogs have been used in the topical treatment of psoriasis, their mechanisms of action are not well understand. Calcitriol, the hormonally active vitamin D3 metabolite, has been demonstrated to exert immunomodulatory effects in the skin by down-regulating the expression of Toll-like receptors (TLRs) and proinflammatory cytokines. OBJECTIVE: We investigated the effects of calcitriol on the expression of TLR2, TLR4, antimicrobial peptide LL-37, and proinflammatory cytokines in cultured human keratinocytes. METHODS: The mRNA expression levels of TLR2, TLR4, tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta and LL-37 in cultured human keratinocytes were measured by real-time polymerase chain reaction (PCR) and reverse transcription (RT). Furthermore, we measured supernatant TNF-alpha levels by an enzyme-linked immunosorbent assay (ELISA) to confirm the effects of calcitriol on TLR2 and TLR4. RESULTS: As measured by RT-PCR and real-time PCR, calcitriol was found to suppress the lipopolysaccharide- and ultraviolet B radiation-mediated induction of expression of TLRs, LL-37 and proinflammatory cytokines such as TNF-alpha and IL-1beta in normal human keratinocytes. The supernatant TNF-alpha levels measured by ELISA were also suppressed after treatment with calcitriol. CONCLUSION: Calcitriol may down-regulate inflammatory stated over-expression of LL-37 and proinflammatory cytokines.

Genetic Polymorphism of FLG in Korean Ichthyosis Vulgaris Patients

BACKGROUND: Filaggrin is a key protein that facilitates the formation of skin barrier by forming a stratum corneum. Mutations in the gene encoding filaggrin (FLG) have recently been reported in patients with ichthyosis vulgaris (IV). Interestingly, there are ethnic differences between FLG mutations identified in Asians and Europeans, and few FLG mutations are overlapping between Chinese and Japanese IV patients. OBJECTIVE: The aim of this study was to investigative the genetic polymorphism of FLG in Korean IV patients. METHODS: Genomic DNA was extracted from whole venous blood specimen of Korean patients with IV and a control group, and the full sequence of FLG was determined via overlapping long-range polymerase chain reaction method. RESULTS: Analysis of base sequence previously unreported reveal new nonsense mutation p.Y1767X in a Korean IV patient, and additional new single nucleotide polymorphisms. CONCLUSION: On the basis of this study, it is anticipated that analysis of FLG gene sequence be extended to other dermatoses associated with FLG, such as atopic dermatitis.

Changes of Antimicrobial Peptides and Transepidermal Water Loss After Topical Application of Tacrolimus and Ceramide-dominant Emollient in Patients with Atopic Dermatitis

Increased transepidermal water loss (TEWL) and downregulated antimicrobial peptides (AMPs) are observed in patients with atopic dermatitis (AD). Tacrolimus and ceramide-dominant emollients are effective in the treatment of AD by preventing the production of inflammatory cytokines and by correcting skin barrier dysfunctions, respectively. Present study was designed to investigate the relationship between antimicrobial and barrier factors by measuring the changes of AMPs and TEWL after topical application of tacrolimus and ceramide-dominant emollient in the patients with AD. A total of three patients with AD were treated with tacrolimus in one lesion and ceramide-dominant emollient in another lesion for 4 weeks. RT-PCR and western blotting revealed that the mRNA and protein expression levels of hBD-2 and LL-37 were increased on the both study sites. Immunohistochemical analysis showed significant increase of AMPs and IL-1alpha, while, IL-4 was decreased on the both study sites. The mean changes of TEWL and AMPs showed no statistical difference between both sites. Tacrolimus and ceramide-dominant emollient influence on both TEWL and AMPs expression in patients with AD, namely they have similar effects on both of the two. This study shows that restoration of permeability barrier function is accompanied by the concomitant improvement of antimicrobial defense in patients with AD.

Time-dependent deformation of polymer-based provisional crown and fixed partial denture materials / 대한치과보철학회지

STATEMENT OF PROBLEM: One of the common problems of provisional crown and fixed partial denture materials is that when they are subjected to constant loads for a long period of time, they exhibit a dimensional change (creep). PURPOSE: The aim of this study was to investigate the viscoelastic behaviour of polymer-based provisional crown and fixed partial denture materials with time at constant compressive load. MATERIAL AND METHODS: Three dimethacrylate-based materials (Protemp 3 Garant, Temphase, Luxatemp) and one monomethacrylate-based material (Trim) were selected. Dimensional changes of the specimens were recorded by a LVDT to evaluate their viscoelastic behavior and creep strain. For all specimens, two loading procedures were used. At first, static compressive stress of 4 MPa was applied for 30 minutes and followed by 1 hour of strain recovery. Then, after 24 hours of water storage, the specimens were loaded again. The creep values between materials were statistically analyzed using one-way ANOVA and multiple comparison Scheffe'test. Independent samples t-test was also used to identify the difference of creep strain between first and secondary loading conditions at the significance level of 0.05. RESULTS: Following application of the first loading, Trim showed the highest maximum creep strain (32.7%) followed by Luxatemp, Protemp 3 Garant and Temphase, with values of 3.78%, 2.86% and 1.77%, respectively. Trim was significantly different from other materials (P0.05). The highest recovery and permanent set of Trim, were significantly different from those of others (P<0.05). At the secondary loading of the dimethacrylate-based materials, creep deformation, recovery and permanent set decreased and the percentage of recovery increased, while in Trim, all values of the measurements increased. This result showed that the secondary loading at 24 hours produced a significant creep magnitude. CONCLUSION: The dimethacrylate-based provisional crown and fixed partial denture materials showed significantly higher creep resistance and lower deformation than the monomethacrylate-based material. Thus, monomethacrylate-based materials should not be used in long-term stress-bearing situations.

Expression and Modulation of LL-37 in Normal Human Keratinocytes, HaCaT cells, and Inflammatory Skin Diseases

Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are negligible, but their expression can be markedly increased in inflammatory skin disease such as psoriasis. We designed this study to identify the expressions of LL-37 in normal human keratinocyte (NHK) and HaCaT cells after exposure to stimulants and to investigate difference of LL-37 expression accompanied with cell differentiation status, and come to understand difference of susceptibility to infection in atopic dermatitis and psoriasis. Expressions of LL-37 in NHKs and HaCaT cells were evaluated by using RT-PCR, Western blotting, and immunohistochemical (IHC) staining at 6, 12, and 24 hr post stimulation after exposure to Ultraviolet B irradiation and lipopolysaccharide. And expression of LL-37 in skin biopsy specimens from patients with atopic dermatitis and psoriasis was determined by immunohistochemical analysis. In time-sequential analyses of LL-37 expression revealed that LL-37 was expressed in NHKs, but not in HaCaT cells. IHC analysis confirmed the presence of abundant LL-37 in the epidermis of psoriasis. Therefore we deduced that expression of LL-37 is affected by UV irradiation, bacterial infection, and status of cell differentiation.

Expression of Human beta Defensin-2 in HaCaT Cell by Lipoteichoic Acid and Peptidoglycan / 대한피부과학회지

BACKGROUND: Defensin, a major family of antimicrobial peptides, is small cationic, cysteine rich peptides with wide range of antimicrobial activity against Gram negative and Gram positive bacteria, fungi, yeast, and virus. Expression of human defensin-2 is upregulated by bacteria, virus, fungus and pro-inflammatory cytokines. However, this peptide was found to be only bacteriostatic, but not bactericidal, against the Gram positive bacteria. OBJECTIVE: To evaluate human defensin-2 (hBD-2) expression after exposure of human skin keratinocytes to the cell wall component of Gram positive bacteria such as lipoteichoic acid (LTA) and peptidoglycan(PEN), and to compare quantitatively the amount of expression with that after their exposure to the cell wall component of Gram negative bacteria. METHODS: Expression of hBD-2 was measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry(IHC). RESULTS: 1. In RT-PCR results, the amount of hBD-2 expression after exposure to LPS was larger than those of PEN and LTA at 6 and 12 hours (p=0.02). At 24 hours, hBD-2 expression showed a peak in PEN stimulated group (p=0.09). 2. In Western blot analysis, hBD-2 expressions, when treated with PEN and LTA, were stronger than that treated with LPS at 6 and 12 hours. 3. In IHC, hBD-2 was stained much stronger in LPS stimulated group than PEN or LTA stimulated groups at 12 hours. CONCLUSION: Our study demonstrated that exposure of human skin keratinocytes to the cell wall components of Gram positive bacteria such as LTA and PEN triggered production of hBD-2 in addition to the cell wall component of Gram negative bacteria such as LPS, however, the amounts of expression were relatively stronger in LPS treated group.