To protect the host against exuberant inflammation and injury responses, cells have the ability to become hyporesponsive or "tolerized" to repeated stimulation by microbial and nonmicrobial insults. The lung airspace is constantly exposed to a variety of exogenous and endogenous Toll-like receptor (TLR) ligands, yet the ability of alveolar epithelial cells (AECs) to be tolerized has yet to be examined. We hypothesize that type II AECs will develop a tolerance phenotype upon repeated TLR agonist exposure. To test this hypothesis, primary AECs isolated from the lungs of mice and a murine AEC cell line (MLE-1<em>2</em>) were stimulated with either a vehicle control or a TLR ligand for 18 hours, washed, then restimulated with either vehicle or TLR ligand for an additional 6 hours. Tolerance was assessed by measurement of TLR ligand-stimulated chemokine production (monocyte chemoattractant protein [MCP]-1/CCL<em>2</em>, <em>keratinocyte</em> chemoattractant [KC]/CXCL1, and macrophage inflammatory protein [MIP]-<em>2</em>/CXCL<em>2</em>). Sequential treatment of primary AECs or MLE-1<em>2</em> cells with TLR agonists resulted in induction of either tolerance or cross-tolerance. The induction of tolerance was not due to expression of specific negative regulators of TLR signaling (interleukin-1 receptor associated kinase [IRAK]-M, Toll-interacting protein [Tollip], single Ig IL-1-related receptor [SIGIRR], or suppressor of cytokine signaling [SOCS]), inhibitory microRNAs (miRs; specifically, miR-155 and miR146a), or secretion of inhibitory or regulatory soluble mediators (prostaglandin E<em>2</em>, IL-10, transforming <em>growth</em> <em>factor</em>-β, or IFN-α/β). Moreover, inhibition of histone demethylation or DNA methylation did not prevent the development of tolerance. However, treatment of AECs with the histone deacetylase inhibitors trichostatin A or suberoylanilide hyrozamine resulted in reversal of the tolerance phenotype. These findings indicate a novel mechanism by which epigenetic modification regulates the induction of tolerance in AECs.

OBJECTIVE

To study the in vivo effects of Quin-C1, a highly specific agonist for formyl peptide receptor <em>2</em> (FPR<em>2</em>/ALX), in a mouse model of bleomycin (BLM)-induced lung injury.

METHODS

Male ICR mice were injected intratracheally with BLM (d 0), and intraperitoneally with Quin-C1 (0.<em>2</em> mg/d) or vehicle between d 1 and d <em>2</em>8, during which pulmonary inflammation was monitored. A similar regimen was carried out between d 5 and d <em>2</em>8 to differentiate anti-inflammatory from anti-fibrotic effects. During the treatment, leukocyte numbers in bronchoalveolar lavage fluid (BALF) were counted, and FPR<em>2</em>/ALX transcripts, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), the mouse keratinocyte-derived chemokine (KC), transforming growth factor β1 (TGF-β1) and C-X-C motif chemokine 10 (CXCL10) expression levels in the lung tissue were also measured. Both hydroxyproline content and histological changes were examined on d <em>2</em>8 to assess the severity of lung fibrosis.

RESULTS

BLM caused a significant increase in expression levels of all the selected cytokines and chemokines, as well as a thickening of the alveolar wall. Treatment with Quin-C1 significantly reduced the neutrophil and lymphocyte counts in BALF, diminished expression of TNF-α, IL-1β, KC, and TGF-β1, and decreased collagen deposition in lung tissue. The treatment also lowered the content of lung hydroxyproline. Quin-C1 did not ameliorate lung fibrosis when the treatment was started 5 d after the BLM challenge, suggesting that the protection may be attributed to its anti-inflammatory effects. Exposure to BLM or BLM plus Quin-C1 did not change the level of FPR<em>2</em>/ALX transcripts (mFpr1, mFpr<em>2</em>, and Lxa4r) in the lung tissue.

CONCLUSIONS

The results demonstrate an anti-inflammatory role for Quin-C1 in bleomycin-induced lung injury, which may be further explored for therapeutic applications.

The immunosuppressive peptide cyclosporin A (CyA) is an extremely effective therapy for severe recalcitrant psoriasis, although its mechanism of action is unknown. In this study, we examined the effect of CyA on <em>keratinocyte</em> <em>growth</em> and cytokine expression, and showed that CyA inhibits the <em>growth</em> of murine and human <em>keratinocytes</em> (KC) and KC cell lines. In addition, CyA inhibits the expression of cytokine genes in a dose-dependent fashion. After <em>2</em> days' incubation with <em>2</em>0 microM CyA, interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), and interleukin 8 (IL-8) mRNA were decreased by 4-fold, 3.3-fold and 3.3-fold, respectively, in COLO-16, a <em>keratinocyte</em> cell line. IL-1 biological activity recovered from COLO-16 culture supernatants decreased to one-fifth of that of controls. In the murine KC cell line PAM <em>2</em>1<em>2</em>, 10 microM CyA treatment for <em>2</em> days downregulated IL-1 alpha, tumour necrosis <em>factor</em>-alpha (TNF-alpha) and IL-1 receptor by 60%, but had no effect on the message for interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), ornithine decarboxylase and beta-actin. Cells cultured for 5 days in the presence of CyA required much lower concentrations (<em>2</em> microM) to achieve the same degree of inhibition of IL-1 alpha. Similar tissue concentrations of CyA have been reported in psoriatics undergoing CyA therapy.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>) is found in dermal papilla fibroblasts and its receptor, fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>), in the neighboring outer root sheath of <em>keratinocytes</em>. Administration of recombinant human KGF-<em>2</em> (rhKGF-<em>2</em>) at 10 ng ml(-1) significantly stimulated human hair-follicle cell proliferation in organ culture (<em>2</em>6-35%). Thus, rhKGF-<em>2</em> is a promising therapeutic agent to stimulate human hair <em>growth</em>.

Psoriasis vulgaris is a chronic, immune-mediated, inflammatory, polygenic skin disorder affecting approximately <em>2</em>% of the population. It has a great impact on quality of life; patients often experience depression, anxiety, stigma as well as suicidal behavior. Even though psoriasis is one of the most studied dermatological conditions, the pathogenesis of the disease is still not completely elucidated. The complex interactions between <em>keratinocytes</em>, dendritic cells, T-lymphocytes, neutrophils and mast cells are responsible for the histopathological changes seen in psoriasis. The pathogenic model leading to the formation of psoriatic plaques has however evolved a lot over the years. There is now enough evidence to support the role of interleukin (IL) -<em>2</em>3, IL-17, IL-<em>2</em><em>2</em>, T helper (Th) -17 cells, Th-<em>2</em><em>2</em> cells, T regulatory cells, transforming <em>growth</em> <em>factor</em> (TGF)-β1 and IL-10 in the pathogenesis of the disease. Moreover, several inflammatory and anti-inflammatory molecules are currently being investigated, some of them showing promising results. The aim of this paper is to look over the most recent advances in the immunological pathways involved in the pathogenesis of psoriasis vulgaris.

We show a novel drug delivery system (DDS) for improved all-trans retinoic acid (atRA) therapy for external treatments of photo-damaged skin. We prepared inorganic-coated atRA nanoparticles, in turn an egg-like structure in nano-scale (Nano-atRA), using boundary-organized reaction droplets. The interfacial properties of organic architectures, in atRA micelles, were used to template the nucleation of inorganic minerals. As a result, irritation and inflammation associated with atRA therapy were substantially reduced due to the complete encapsulation of the carboxylic function. Both irritative symptoms and physicochemical instability of the atRA micelle were improved. Since Nano-atRA which is prepared following to this new DDS system developmentally improved the permeability to the stratum corneum, the remarkable pharmacological effects were resulted in comparison with atRA as such as follows: (1) thicker epidermis than classical atRA treatment and (<em>2</em>) the overexpression of mRNA for heparin-binding epidermal <em>growth</em> <em>factor</em> (HB-EGF) as the provocation epidermal hyperplasia. Furthermore, we found a surprising boost in production of hyaluronan (HA) among the intercellular spaces of the basal and spinous cell layers in epidermis. Nano-atRA technology for atRA therapy could not only efficiently regulate <em>keratinocyte</em> cell proliferation and differentiation, but also markedly produce the additional benefit. Severely injured human skin by chronic ultraviolet irradiation will completely repair due to the accelerated turnover of skin tissue, which is induced by Nano-atRA.

Transgenic mice overexpressing platelet-activating <em>factor</em> receptor (PAFR) have abnormal pigmentation of the ear and the tail, which can progress to melanocytic tumors as the mice age. Histologically, epidermal hyperproliferation and increases in dermal melanocytes are evident. Examination of these transgenic mice at various ages revealed hyperproliferation of the epidermis even <em>2</em> weeks after birth which developed as the mice aged. Dermal melanocytes also increased in number with <em>growth</em>. Expression of the PAFR transgene was found in <em>keratinocytes</em> and not in melanocytes, thereby suggesting that PAF does not play a direct role in proliferation of melanocytes. Topical application of a cream containing WEB<em>2</em>086, a specific PAFR antagonist, to the ear and the dorsal skin significantly suppressed the number of BrdU-positive cells in PAFR transgenic mice. These results suggest that PAF plays a modulatory role in the <em>growth</em> of epidermal <em>keratinocytes</em>. PAFR transgenic mice would be a useful model for investigations of skin diseases related to altered proliferation of epidermal <em>keratinocytes</em> including psoriasis.

BACKGROUND

Solar lentigines (SLs) are macular hyperpigmented lesions associated with sun exposure and age. Histopathologically, SLs are defined by a hyperpigmented basal layer and elongated rete ridges. The molecular mechanisms involved in the formation and the development of SLs are not completely understood. Our earlier data show that keratinocyte growth factor (KGF) induces hyperpigmentary lesions with histological resemblance to SLs.

OBJECTIVE

To investigate the association of KGF/KGF receptor (KGFR) and other pigmentary genes with the progression of SL development. To better understand the possible role of KGF in the pathology of SLs.

METHODS

Archived human skin biopsies (24 SLs and 14 healthy skins) were studied using immunohistochemistry for KGF/KGFR, proliferation marker Ki67, stem cell marker keratin-15 (K15), tyrosinase (TYR), stem cell factor (SCF), and protease-activated receptor-2 (PAR-2).

RESULTS

An increase in TYR-positive cells and expression was found throughout SL progression, as compared to normal skin. The levels of KGF, KGFR, SCF, Ki67 and PAR-2 varied during SL progression. Ki67, K15 and KGF/KGFR were significantly upregulated at early-mid SL stages. The latest-stage SLs expressed the lowest levels of KGF, KGFR, SCF, Ki67 and PAR-2.

CONCLUSIONS

The upregulation of KGF/KGFR might induce the formation of rete ridges and hyperpigmentation. The reduced levels of all examined proteins (except TYR and K15) suggest a possible inactive status (dormancy or quiescence) of advanced lesions.

Prostaglandins activate signalling pathways involved in <em>growth</em>, differentiation and apoptosis. Prostaglandin E(<em>2</em>) (PGE(<em>2</em>)) is released by <em>keratinocytes</em> following ultraviolet irradiation (UVR) and stimulates the formation of dendrites in melanocytes. We show that multiple irradiations of human melanocytes with UVR-activated cPLA(<em>2</em>), the rate-limiting enzyme in eicosanoid synthesis and stimulated PGE(<em>2</em>) secretion. PGE(<em>2</em>) increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE(<em>2</em>) binds to four distinct G-protein coupled receptors (EP(1-4)). We show that PGE(<em>2</em>) stimulates EP(4) receptor signalling in melanocytes, resulting in cAMP production. Conversely, PGE(<em>2</em>) also stimulated the EP(3) receptor in melanocytes, resulting in lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE(<em>2</em>) on cAMP in melanocytes. The data are the first to identify PGE(<em>2</em>) as an UVR-inducible autocrine <em>factor</em> for melanocytes. These data also show that PGE(<em>2</em>) activates EP(3) and EP(4) receptor signalling, resulting in opposing effects on cAMP production, a critical signalling pathway that regulates proliferation and melanogenesis in melanocytes.

Ultraviolet (UV) irradiation causes multiple pathologic changes in the skin including inflammation, immune suppression, photoaging, and cancer. Effects of UV irradiation include the activation of numerous signal transduction pathways, including the mitogen-activated protein kinases (MAPK), and the activation of transcription <em>factors</em> such as nuclear <em>factor</em> kappa B (NFkappaB). These responses alter gene expression in a manner that resembles the response to <em>growth</em> <em>factors</em> known as the "UV response". The UV response alters the kinetics of cell division and cell death allowing the skin to recover from the DNA damage caused by UV exposure. UV irradiation also rapidly activates epidermal <em>growth</em> <em>factor</em> receptor (EGFR) family members, including Erbb<em>2</em> (human epithelial <em>growth</em> <em>factor</em> receptor <em>2</em> (HER<em>2</em>)/neu), through the generation of reactive oxygen species. Erbb<em>2</em>, a protooncogene that is activated in many types of cancer and associated with aggressive and chemotherapeutic-resistant disease, is expressed in both follicular and epidermal <em>keratinocytes</em> within the skin. However, the physiological functions of Erbb<em>2</em> in the skin and its role in the UV response are largely unknown. In this review, evidence that Erbb<em>2</em> is influential in modulating the response of the skin to UV will be presented. Erbb<em>2</em> alters the expression of regulatory genes controlling inflammation, angiogenesis, cell division, apoptosis, cell adhesion, and migration following UV irradiation. In addition, Erbb<em>2</em> dampens UV-induced S-phase arrest, augments inflammation in response to UV irradiation, and suppresses UV-induced apoptosis. In summary, the evidence presented herein links UV-induced Erbb<em>2</em> activation to many of the effects of UV and implicates Erbb<em>2</em> in UV-induced carcinogenesis.

Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming <em>growth</em> <em>factor</em> (TGF)-beta(<em>2</em>) to be highly expressed in cultured hDPCs. <em>Keratinocyte</em> conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(<em>2</em>) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from <em>keratinocytes</em>, the vitamin D(3) analogue was found to promote TGF-beta(<em>2</em>) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(<em>2</em>) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(<em>2</em>) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair re<em>growth</em> by transplanting expanded DPCs.

BACKGROUND

Keloids are an extreme form of abnormal scarring that result from a pathological fibroproliferative wound healing process. The molecular mechanisms driving keloid pathology remain incompletely understood, hindering development of targeted, effective therapies. Recent studies in our laboratory demonstrated that keloid keratinocytes exhibit adhesion abnormalities and display a transcriptional signature reminiscent of cells undergoing epithelial-mesenchymal transition (EMT), suggesting a role for EMT in keloid pathology. In the current study, we further define the EMT-like phenotype of keloid scars and investigate regulation of EMT-related genes in keloid.

METHODS

Primary keratinocytes from keloid scar and normal skin were cultured in the presence or absence of transforming growth factor beta 1 (TGF-β1) +/- inhibitors of TGF-β1 and downstream signaling pathways. Gene expression was measured using quantitative polymerase chain reaction. Migration was analyzed using an in vitro wound healing assay. Proteins in keloid scar and normal skin sections were localized by immunohistochemistry. Statistical analyses utilized SigmaPlot (SyStat Software, San Jose, CA) or SAS(®) (SAS Institute, Cary, NC).

RESULTS

In keloid and normal keratinocytes, TGF-β1 regulated expression of EMT-related genes, including hyaluronan synthase 2, vimentin, cadherin-11, wingless-type MMTV integration site family, member 5A, frizzled 7, ADAM metallopeptidase domain 19, and interleukin-6. Inhibition of canonical TGF-β1 signaling in keloid keratinocytes significantly inhibited expression of these genes, and TGF-β1 stimulation of normal keratinocytes increased their expression. The inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway or the p38 mitogen-activated protein kinase pathway attenuated TGF-β1-induced expression of subsets of these genes. Migration of keloid keratinocytes, previously shown to be increased compared with normal keratinocytes, was significantly reduced by inhibition of TGF-β1 or ERK1/2 signaling. Biomarkers of EMT, including reduced E-cadherin and increased active β-catenin, were observed in keloid epidermis in vivo. However, evidence of basement membrane breakdown in keloid scar was not observed.

CONCLUSIONS

The results suggest that keloid keratinocytes exist in an EMT-like metastable state, similar to activated keratinocytes in healing wounds. The EMT-like gene expression pattern of keloid keratinocytes is regulated by canonical and non-canonical TGF-β1 signaling pathways. Therefore, interventions targeting TGF-β1-regulated EMT-like gene expression in keloid keratinocytes may serve to suppress keloid scarring.

In the initial phase of wound healing, endogenous fibrin clots are known to form a provisional matrix and to promote angiogenesis. <em>Growth</em> <em>factors</em> such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF) increase in wounds to stimulate angiogenesis. However, it remains unknown whether VEGF is induced when fibrin is used as a dermal substrate for cultured skin substitutes. The authors investigated the effect of fibrin gel as a dermal substrate for a cultured skin substitute, using human <em>keratinocytes</em> and dermal fibroblasts. A collagen-cultured skin substitute was also examined for comparison. VEGF in the culture supernatant in both types was measured by enzyme-linked immunosorbent assay, and VEGF mRNA was determined semiquantitatively by reverse-transcriptase polymerase chain reaction after <em>2</em> days of incubation. Experiments were performed using 1<em>2</em> cultured skin substitutes: four for histologic examination before transplantation, four for VEGF assay in vitro, and four for the transplantation to athymic mice. Three independent experiments were performed for each step. VEGF concentration in the fibrin-cultured supernatant was 84.3 +/- 11.8 pg/ml, whereas it was <em>2</em>7.8 +/- 4.68 pg/ml in the case of the collagen substrate. The relative levels of VEGF mRNA were 1.088 +/- 0.100 and 0.698 +/- 0.<em>2</em><em>2</em>6, respectively. In in vivo transplantation, the fibrin-type cultured skin substitute showed an excellent take on the wound bed, and a normally proliferating <em>keratinocyte</em> layer with emergence of vascular endothelial cells in the transplanted floor was seen 3 days after transplantation. Vascular endothelial cells, which were identified using alkaline phosphatase stain, were significantly increased in the fibrin-type cultured skin substitute. The use of fibrin as a dermal substrate for cultured skin substitute increases the secretion of VEGF, improves regeneration of mature epidermal structure after in vivo transplantation, and promotes the migration of vascular endothelial cells.

We investigated the effects of excipients in solutions of <em>keratinocyte</em> <em>growth</em> <em>factor</em> <em>2</em> (KGF-<em>2</em>) on protein aggregation during agitation as well as on interfacial shear rheology at the air-water interface. Samples were incubated with or without agitation, and in the presence or absence of the excipients heparin, sucrose, or polysorbate 80 (PS80). The effect of excipients on the extent of protein aggregation was determined by UV-visible spectroscopy and micro-flow imaging. Interfacial shear rheology was used to detect the gelation time and strength of protein gels at the air-water interface. During incubation, protein particles of size ≥1 μm and insoluble aggregates formed faster for KGF-<em>2</em> solutions subjected to agitation. Addition of either heparin or sucrose promoted protein aggregation during agitation. In contrast, PS80 substantially inhibited agitation-induced KGF-<em>2</em> aggregation but facilitated protein particulate formation in quiescent solutions. The combination of PS80 and heparin or sucrose completely prevented protein aggregation during both nonagitated and agitated incubations. Interfacial rheological measurements showed that KGF-<em>2</em> in buffer alone formed an interfacial gel within a few minutes. In the presence of heparin, KGF-<em>2</em> interfacial gels formed too quickly for gelation time to be determined. KGF-<em>2</em> formed gels in about 10 min in the presence of sucrose. The presence of PS80 in the formulation inhibited gelation of KGF-<em>2</em>. Furthermore, the interfacial gels formed by the protein in the absence of PS80 were reversible when PS80 was added to the samples after gelation. Therefore, there is a correspondence between formulations that exhibited interfacial gelation and formulations that exhibited agitation-induced aggregation.

The present report describes the mitogenic effect of recombinant IGF-<em>2</em> on cultured human <em>keratinocytes</em> and fibroblasts compared to that of IGF-1. Furthermore, the modulating effect of a recently expressed recombinant form of placental-derived IGF-binding protein 1 (IGFBP-1) on IGF-induced proliferation was examined. A dose-dependent increase, up to 100%, in cell proliferation was seen in cultured human <em>keratinocytes</em> with IGF-<em>2</em> and -1 and the proliferative response was comparable to the effect of epidermal <em>growth</em> <em>factor</em> (EGF). In human fibroblasts, IGF-1 stimulated DNA synthesis up to 300% for IGF-1 and up to <em>2</em>00% for IGF-<em>2</em>. The mitogenic effect of IGF-1 was enhanced by IGFBP-1 in both cell types. In contrast, the IGF-<em>2</em>-induced mitogenic effect was unperturbed. These findings indicate that the interaction between IGFs and their binding proteins may induce different responses depending upon the ligand and the target cell.

Interleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood.

Biopsies were obtained from the involved skin of 15 SSc, 4 morphea and 8 healthy donors (HD). The presence of IL-22+ cells in the skin was determined by immunostaining. The in vitro response of HD and SSc fibroblasts to IL-22, IL-22 in conjunction with tumour necrosis factor (TNF) or keratinocyte conditioned medium was assessed by ELISA, radioimmunoassay (RIA), real-time PCR and western blot. The in vivo response in mice was assessed by histomorphometry.

IL-22+ cells were over-represented in the dermis and epidermis of morphea and in the epidermis of SSc compared with HD. The majority of dermal IL-22+ cells were T cells. Dermal fibroblasts expressed both IL-22 receptor subunits IL-10RB and IL-22RA, expression of which was enhanced by TNF and reduced by transforming growth factor (TGF)-β. IL-22 induced rapid phosphorylation of p38 and ERK1/2 in fibroblasts, but failed to induce the synthesis of chemokines and extracellular matrix components. However, IL-22 enhanced the production of monocyte chemotactic protein 1, IL-8 and matrix metalloproteinase 1 induced by TNF. Fibroblast responses were maximal in the presence of conditioned medium from keratinocytes activated by IL-22 in conjunction with TNF. Dermal thickness was maximal in mice injected simultaneously with IL-22 and TNF.

IL-22 capacitates fibroblast responses to TNF and promotes a proinflammatory fibroblast phenotype by favouring TNF-induced keratinocyte activation. These results define a novel role for keratinocyte-fibroblast interactions in the context of skin fibrosis.

OBJECTIVE

The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes.

METHODS

Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods.

RESULTS

All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA.

CONCLUSIONS

Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which T-helper type <em>2</em> (Th<em>2</em>)-type immune responses are dominant. Th<em>2</em> cytokine, interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) have been suggested to have an important role in AD. IL-33 is highly expressed in AD, but its role in AD has not yet been fully understood. To further identify the role of IL-33 in AD, we investigated the expression of TSLP induced by IL-33 in <em>keratinocytes</em>. This study revealed that IL-33 induced TSLP expression in human <em>keratinocytes</em>. Early <em>growth</em> response protein 1 (Egr)-1, which is an inflammatory transcriptional <em>factor</em>, is induced by IL-33. IL-33-mediated TSLP induction in <em>keratinocytes</em> was suppressed by treatment with mitogen-activated protein kinase (MAPK) inhibitors or small interfering RNA against Egr-1. Chromatin immunoprecipitation (ChIP) assay indicated the direct involvement of Egr-1 in IL-33-mediated TSLP induction. Taken together, these findings indicate that IL-33 may increase TSLP expression through an Egr-1-dependent mechanism via ERK1/<em>2</em>, JNK and p38 activation in <em>keratinocytes</em>. These data suggest that the IL-33-ERK/JNK/p38/Egr-1/TSLP axis is involved in allergic skin Th<em>2</em> inflammation, and it may be a novel therapeutic target.

Psoriasis is a chronic, relapsing skin disease characterized by enhanced angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is overexpressed in psoriatic epidermis and that the level of insulin-like <em>growth</em> <em>factor</em> II (IGF-II) is significantly elevated in the tissue fluid and serum of the psoriatic lesion. We considered the possibility that IGF-II might function as a paracrine inducer of VEGF. Here, we demonstrated that exposure of HaCaT <em>keratinocytes</em> to IGF-II induced both mRNA and protein expression of VEGF through the MAP kinase (extracellular signal-regulated kinase (ERK<em>2</em>) pathway. Particularly, we determined that phosphorylation of ERK<em>2</em> but not p38 and JNK1/<em>2</em> was activated by IGF-II in a time-dependent manner. Additionally, we found that IGF-II treatment induced the expression of MDM<em>2</em> through the MAP kinase pathway. Moreover, the increase of MDM<em>2</em> resulted in decreased levels of p53 followed by increased expression of HIF-1alpha and VEGF. Taken together, these results suggest that IGF-II enhances the expression of VEGF in HaCaT cells by increasing HIF-1alpha levels.

Psoriasis is a common, chronic, inflammatory skin disease that affects <em>2</em>%-4% of the global population. Recent studies have shown that increased oxidative stress (OS) and T-cell abnormalities are central to the pathogenesis of this disease. The resulting reactive oxygen species (ROS) induces proliferation and differentiation of Th17/Th1/Th<em>2</em><em>2</em> cells and inhibits the anti-inflammatory activities of regulatory T lymphocytes (Treg). Subsequent secretions of inflammatory cytokines, such as interleukin (IL)-17, IL-<em>2</em><em>2</em>, tumor necrosis <em>factor</em> alpha (TNF-α), and interferon-gamma (IFN-γ), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), stimulate <em>keratinocyte</em> proliferation and angiogenesis. Proanthocyanidins are a class of flavonoids from plants and fruits, and have various antioxidant, anti-inflammatory, and anti-angiogenic properties. Numerous reports have demonstrated therapeutic effects of proanthocyanidins for various diseases. Among clinical activities, proanthocyanidins suppress cell proliferation, prevent OS, and regulate Th17/Treg cells. Because the pathogenesis of psoriasis involves OS and T cells dysregulation, we reviewed the effects of proanthocyanidins on OS, Th17 and Treg cell activities, and <em>keratinocyte</em> proliferation and angiogenesis. Data from multiple previous studies warrant consideration of proanthocyanidins as a promising strategy for the treatment of psoriasis.

Acute respiratory distress syndrome (ARDS) is a clinically and biologically heterogeneous disorder associated with many disease processes that injure the lung, culminating in increased non-hydrostatic extravascular lung water, reduced compliance, and severe hypoxemia. Despite enhanced understanding of molecular mechanisms, advances in ventilatory strategies, and general care of the critically ill patient, mortality remains unacceptably high. The Berlin definition of ARDS has now replaced the American-European Consensus Conference definition. The recently concluded Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG-SAFE) provided worldwide epidemiological data of ARDS including prevalence, geographic variability, mortality, and patterns of mechanical ventilation use. Failure of clinical therapeutic trials prompted the investigation and subsequent discovery of two distinct phenotypes of ARDS (hyper-inflammatory and hypo-inflammatory) that have different biomarker profiles and clinical courses and respond differently to the random application of positive end expiratory pressure (PEEP) and fluid management strategies. Low tidal volume ventilation remains the predominant mainstay of the ventilatory strategy in ARDS. High-frequency oscillatory ventilation, application of recruitment maneuvers, higher PEEP, extracorporeal membrane oxygenation, and alternate modes of mechanical ventilation have failed to show benefit. Similarly, most pharmacological therapies including <em>keratinocyte</em> <em>growth</em> <em>factor</em>, beta-<em>2</em> agonists, and aspirin did not improve outcomes. Prone positioning and early neuromuscular blockade have demonstrated mortality benefit, and clinical guidelines now recommend their use. Current ongoing trials include the use of mesenchymal stem cells, vitamin C, re-evaluation of neuromuscular blockade, and extracorporeal carbon dioxide removal. In this article, we describe advances in the diagnosis, epidemiology, and treatment of ARDS over the past decade.

The uncapping of telomeres has been shown to precipitate senescence in normal human fibroblasts and apoptosis in lymphocytes and p53-competent cancer cell lines. However, the effects of telomere uncapping on normal epithelial cells have not previously been examined. We have used the well characterised telomere repeat binding <em>factor</em> <em>2</em> (TRF<em>2</em>) dominant-negative mutant, TRF<em>2</em>(DeltaBDeltaM), to deplete Normal Human Epidermal <em>Keratinocytes</em> (NHEK) telomeres of TRF<em>2</em>. We observed only a two fold increase in both phosphorylation of p53 at serine 15 and 53BP1 DNA damage foci and no detectable increase in p<em>2</em>1(WAF). Despite the weak DNA damage response, the <em>keratinocytes</em> <em>growth</em> arrest, demonstrate reduced colony formation and senescence. The small, abortive senescent colonies did not incorporate Brd-U within 48 h and expressed senescence-associated beta galactosidase (SA-beta-gal). Transcriptional profiling of TRF<em>2</em>-depleted <em>keratinocytes</em> showed a reproducible up-regulation of several genes. These included histones, genes associated with DNA damage and <em>keratinocyte</em> terminal differentiation. Several of the same genes were also shown to be up-regulated when <em>keratinocytes</em> undergo natural telomere-mediated senescence and down-regulated by ectopic telomerase expression. This study has thus revealed highly sensitive and specific candidate indicators of telomere dysfunction that may find use in identifying telomere-mediated <em>keratinocyte</em> senescence in ageing, cancer and other diseases.

Fibroblast <em>growth</em> <em>factor</em> (FGF) 9 is secreted by both mesothelial and epithelial cells, and plays important roles in organ development and wound healing via WNT/β-catenin signaling. The aim of this study was to evaluate FGF9 expression and FGF-WNT/β-catenin signaling during wound healing of the skin. We investigated FGF9 expression and FGF-WNT/β-catenin signaling after laser ablation of mouse skin and adult human skin, as well as in cultured normal human epidermal <em>keratinocytes</em> (NHEKs) upon stimulation with recombinant human (rh) FGF9 and rh-transforming <em>growth</em> <em>factor</em> (TGF)-β1. Our results showed that laser ablation of both mouse skin and human skin leads to marked overexpression of FGF9 and FGF9 mRNA. Control NHEKs constitutively expressed FGF9, WNT7b, WNT<em>2</em>, and β-catenin, but did not show Snail or FGF receptor (FGFR) <em>2</em> expression. We also found that FGFR<em>2</em> was significantly induced in NHEKs by rhFGF9 stimulation, and observed that FGFR<em>2</em> expression was slightly up-regulated on particular days during the wound healing process after ablative laser therapy. Both WNT7b and WNT<em>2</em> showed up-regulated protein expression during the laser-induced wound healing process in mouse skin; moreover, we discerned that the stimulatory effect of rhFGF9 and rhTGF-β1 activates WNT/β-catenin signaling via WNT7b in cultured NHEKs. Our data indicated that rhFGF9 and/or rhTGF-β1 up-regulate FGFR<em>2</em>, WNT7b, and β-catenin, but not FGF9 and Snail; pretreatment with rh dickkopf-1 significantly inhibited the up-regulation of FGFR<em>2</em>, WNT7b, and β-catenin. Our results suggested that FGF9 and FGF-WNT/β-catenin signaling may play important roles in ablative laser-induced wound healing processes.

Betel quid (BQ) chewing is an etiologic <em>factor</em> of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal <em>growth</em> <em>factor</em> (EGF) and interleukin-1α (IL-1α) production of gingival <em>keratinocytes</em> (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E<em>2</em> (PGE<em>2</em>) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-<em>2</em> (COX-<em>2</em>), mature ADAM9 expression and PGE<em>2</em> and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U01<em>2</em>6 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp<em>2</em> (Src inhibitor), U01<em>2</em>6, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE<em>2</em> production, COX-<em>2</em> expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc<em>2</em> expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp<em>2</em>, U01<em>2</em>6, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.