Because most human squamous carcinoma cell lines of the aerodigestive and genital tracts are refractory to the antiproliferative action of transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1) in vitro, we have begun to identify the causes for resistance of squamous carcinoma cell lines to TGF beta 1 by using somatic cell genetics. Two stable hybrid cell lines (FaDu-HKc.1 and FaDu-HKc.<em>2</em>) were obtained by fusing a TGF beta 1-resistant human squamous carcinoma cell line, FaDu-HygR, with a human papilloma virus 16-immortalized, TGF beta 1-sensitive, human foreskin <em>keratinocyte</em> cell line, HKc-neoR. Whereas TGF beta 1 did not inhibit DNA synthesis in parental FaDu-HygR cells, it reduced DNA synthetic activity of HKc-neoR, FaDu-HKc.1, and FaDu-HKc.<em>2</em> cells by 75-85% (IC50, <em>2</em>-5 pM). Although squamous carcinoma cells express lower than normal levels of TGF beta 1 type II receptors on their cell surface, TGF beta 1 type II receptor mRNA was detected in all four cell lines. Recessive genes involved in TGF beta 1 signaling may be localized to the distal portion of chromosome 18q, as this was the sole chromosomal region of homozygous deletion in parental FaDu-HygR cells. Furthermore, our previous observation that mutant p53 decreases sensitivity of <em>keratinocytes</em> to TGF beta 1 was supported by the finding that the level of the mutant p53 protein expressed by the hybrid cell lines was greatly reduced. In summary, TGF beta 1 resistance of FaDu cells appears to be recessive and is presumably due to the loss of one or more post-receptor elements of the signaling pathway.

The importance of cancer-mesenchymal interactions in the aggressive behavior of scirrhous gastric cancer is supported by experimental and clinical evidences. We have previously reported that gastric fibroblasts secretion of <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) underline the remarkable proliferation of scirrhous gastric cancer cells. Cyclooxygenase-<em>2</em> (COX-<em>2</em>) is not only expressed in cancer cells, but also in interstitial fibroblasts in gastric carcinoma. To clarify the mechanisms responsible for the antiproliferation effect of COX-<em>2</em> inhibitors, effect of COX-<em>2</em> inhibitor on the paracrine epithelial-mesenchymal interactions of <em>growth</em> was examined. Scirrhous gastric cancer cell line, OCUM-<em>2</em>M, gastric fibroblasts, NF-<em>2</em>1, and COX-<em>2</em> inhibitor, JTE-5<em>2</em><em>2</em>, were used. <em>Growth</em>-interaction was examined by calculating the number of cancer cells or by measuring [(3)H] thymidine incorporation of cancer cells. Effect of JTE-5<em>2</em><em>2</em> on KGF expression from NF-<em>2</em>1 cells and OCUM-<em>2</em>M cells was analyzed by ELISA and RT-PCR. The conditioned medium from gastric fibroblasts significantly stimulated the <em>growth</em> of scirrhous gastric cancer cells. JTE-5<em>2</em><em>2</em> at the concentrations of 10(-5) and 10(-6) M significantly decreased the <em>growth</em>-stimulating activity of gastric fibroblasts. JTE-5<em>2</em><em>2</em> reduced the expression of KGF mRNA and the production of KGF from gastric fibroblasts. Oral administration of JTE-5<em>2</em><em>2</em> significantly decreased the size of xenografted tumor coinoculated with OCUM-<em>2</em>M cells and NF-<em>2</em>1 cells in nude mice. JTE-5<em>2</em><em>2</em> decreased COX-<em>2</em> expression and Ki67 labeling index within the coinoculated tumor. These findings suggested that a selective COX-<em>2</em> inhibitor, JTE-5<em>2</em><em>2</em>, downregulates KGF production from gastric fibroblasts, resulting in the inhibition of paracrine epithelial-mesenchymal interactions of proliferation between scirrhous gastric cancer cells and gastric fibroblasts.

The parathyroid hormone-like peptide (PLP) gene is widely expressed in normal and neoplastic tissues. Previous studies have demonstrated that PLP gene expression is regulated by serum and cycloheximide, features common to the regulation of a number of different early response genes. We now report that PLP mRNA transcripts are induced within 5 min of exposure of rat <em>keratinocytes</em> to serum, return to control values at <em>2</em>0 min, and then increase and remain elevated for at least 4 h, following which they return to baseline levels. The PLP mRNA t1/<em>2</em> was approximately 90 min in both serum-deprived and serum-stimulated cells. The serum induction was blocked by actinomycin D. Cycloheximide alone induced PLP gene expression; however, PLP mRNA transcripts were not superinduced in the presence of both serum and cycloheximide. Dexamethasone and 1,<em>2</em>5-dihydroxyvitamin D3 inhibited the basal levels of PLP mRNA transcripts but did not eliminate the serum induction of PLP gene expression. Epidermal <em>growth</em> <em>factor</em> or transforming <em>growth</em> <em>factor</em>-beta alone induced PLP mRNA transcripts, but no induction was observed following exposure of cells to epidermal <em>growth</em> <em>factor</em> and transforming <em>growth</em> <em>factor</em>-beta together. Treatment with 1<em>2</em>-O-tetradecanoylphorbol-13-acetate for 90 min did not induce PLP mRNA transcripts, but 1<em>2</em>-O-tetradecanoylphorbol-13-acetate blocked the rapid serum induction of PLP gene expression. These features of PLP gene expression suggest that PLP is a member of the <em>growth</em> <em>factor</em>-regulated early response gene family. The rapid serum stimulation of PLP gene expression raises the possibility that PLP may contribute in an autocrine fashion to the early cellular response to <em>growth</em> <em>factor</em> stimulation.

Parathyroid hormone-related protein (PTHrP) is produced by a wide range of neoplastic and normal cells, including <em>keratinocytes</em> where it may regulate <em>growth</em> and differentiation. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) is a <em>growth</em> <em>factor</em> produced by many cells, including <em>keratinocytes</em> where it regulates epidermal homeostasis. TGF-beta has been reported to be cosecreted with PTHrP in some neoplasms and to stimulate PTHrP production by neoplastic <em>keratinocytes</em>. However, the effects of TGF-beta on PTHrP production by normal <em>keratinocytes</em> are not well characterized. In this study, we investigated the effects of endogenous and exogenous TGF-beta on PTHrP production by normal human foreskin <em>keratinocytes</em>. PTHrP secretion, mRNA expression, and mRNA transcription in vitro were determined by N-terminal radioimmunoassay, ribonuclease protection assay, and transient transfections. PTHrP production and secretion of latent TGF-beta activity were greatest in proliferating <em>keratinocytes</em> prior to and at confluence of monolayer cultures. TGF-beta1 increased PTHrP mRNA expression by normal <em>keratinocytes</em> in a dose-dependent manner with maximal stimulation at 6-1 <em>2</em> h after treatment. In addition, <em>keratinocytes</em> treated with a monoclonal anti-TGF-beta antibody expressed decreased levels of PTHrP mRNA. The increased levels of PTHrP mRNA following TGF-beta1 treatment were owing, at least partly, to an increase in PTHrP mRNA stability. TGF-beta1 failed to activate transcription of the luciferase reporter gene driven by either the human or mouse PTHrP promoters. In conclusion, TGF-beta1 functions as a paracrine or autocrine regulator of PTHrP production in normal human <em>keratinocytes</em>, and this may play a role in the regulation of <em>keratinocyte</em> proliferation or differentiation.

It has been demonstrated that hyperthermia protects <em>keratinocytes</em> from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if <em>keratinocytes</em> become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(1<em>2</em>,14)-prostaglandin J<em>2</em> (15dPGJ<em>2</em>) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal <em>keratinocytes</em> (NHEK) and the carcinoma-derived cell line A431 were exposed to either 4<em>2</em> degrees C or to 15dPGJ<em>2</em> for 4 hours at <em>2</em>4-hour intervals for 4 consecutive days. The intracellular level of the 7<em>2</em>-kDa heat shock protein (Hsp7<em>2</em>) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ<em>2</em> treatment, and survival was determined <em>2</em>4 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ<em>2</em> are potent inducers of Hsp7<em>2</em> expression and lead to increased resistance to UVB-induced cell death in human <em>keratinocytes</em>; (<em>2</em>) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp7<em>2</em> and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ<em>2</em> is enhanced by repeated exposure without a further increase of Hsp7<em>2</em>; (4) repeated heat shock and 15dPGJ<em>2</em> up to a concentration of 1 microg/mL have no influence on cell <em>growth</em> over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing <em>factors</em>, stress tolerance can be maintained without additional toxicity in human <em>keratinocytes</em>. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

In the present study we prepared explant cultures of plucked total hair follicles and of fragments microdissected from the following regions: B1 (bulb region), B<em>2</em> (intermediate region), B3-1 (lower central outer root sheath, ORS), B3-<em>2</em> (upper central ORS) and B4 (area of fracture). The <em>growth</em> capacities, the start of epithelial out<em>growth</em>, the stages of differentiation and apoptosis were studied immunohistochemically in early and late explant cultures using a battery of antibodies against cytokeratins, <em>growth</em> <em>factor</em> receptors and cell adhesion molecules and proliferation markers. Whole plucked hair follicles showed epithelial out<em>growth</em>s exclusively in the upper central ORS (B3-<em>2</em>) starting early, mostly by day 3. In microdissected fragments, in contrast, out<em>growth</em>s were more widespread, mostly in B3-<em>2</em> and B3-1, and started early, but were also of late onset in some cases of B<em>2</em> and B4. Epithelial out<em>growth</em>s exhibited a basal layer of small cuboidal cells in a low stage of differentiation and one to two suprabasal layers of large prickle-like cells expressing late differentiation markers. The former expressed the receptor of nerve <em>growth</em> <em>factor</em> (NGF) heterogeneously whereas epidermal <em>growth</em> <em>factor</em> (EGF) receptor was not detectable. This is similar to ORS cells of this area in vivo. The proliferative activity of the out<em>growth</em>s was always restricted to peripheral cells. Thus no essential differences in differentiation of outgrowing cells were detected. These results suggest that <em>keratinocytes</em> with the highest <em>growth</em> capacities in plucked human hair follicles are localized in the lower central ORS (corresponding to B3-<em>2</em>) and some with a lower capacity in the upper central ORS (corresponding to B3-1) as established after microdissection. This is in agreement with the bulge activation theory. NGF may also play a role in hair <em>growth</em>.

BACKGROUND

Malaria is the most relevant parasitic disease worldwide, and still accounts for 1 million deaths each year. Since current antimalarial drugs are unable to prevent death in severe cases, new therapeutic strategies have been developed. Mesenchymal stromal cells (MSC) confer host resistance against malaria; however, thus far, no study has evaluated the therapeutic effects of MSC therapy on brain and distal organ damage in experimental cerebral malaria.

METHODS

Forty C57BL/6 mice were injected intraperitoneally with 5 × 10(6) Plasmodium berghei-infected erythrocytes or saline. After <em>2</em>4 h, mice received saline or bone marrow (BM)-derived MSC (1x10(5)) intravenously and were housed individually in metabolic cages. After 4 days, lung and kidney morphofunction; cerebrum, spleen, and liver histology; and markers associated with inflammation, fibrogenesis, and epithelial and endothelial cell damage in lung tissue were analyzed.

RESULTS

In P. berghei-infected mice, BM-MSCs: 1) reduced parasitemia and mortality; <em>2</em>) increased phagocytic neutrophil content in brain, even though BM-MSCs did not affect the inflammatory process; 3) decreased malaria pigment detection in spleen, liver, and kidney; 4) reduced hepatocyte derangement, with an increased number of Kupffer cells; 5) decreased kidney damage, without effecting significant changes in serum creatinine levels or urinary flow; and 6) reduced neutrophil infiltration, interstitial edema, number of myofibroblasts within interstitial tissue, and collagen deposition in lungs, resulting in decreased lung static elastance. These morphological and functional changes were not associated with changes in levels of tumor necrosis <em>factor</em>-α, <em>keratinocyte</em>-derived chemokine (KC, a mouse analog of interleukin-8), or interferon-γ, which remained increased and similar to those of P. berghei animals treated with saline. BM-MSCs increased hepatocyte <em>growth</em> <em>factor</em> but decreased VEGF in the P. berghei group.

CONCLUSIONS

BM-MSC treatment increased survival and reduced parasitemia and malaria pigment accumulation in spleen, liver, kidney, and lung, but not in brain. The two main organs associated with worse prognosis in malaria, lung and kidney, sustained less histological damage after BM-MSC therapy, with a more pronounced improvement in lung function.

The in vivo cellular microenvironment is regulated by a complex interplay of soluble <em>factors</em> and signaling molecules secreted by cells and it plays a critical role in the <em>growth</em> and development of normal and diseased tissues. In vitro systems that can recapitulate the microenvironment at the cellular level are needed to investigate the influence of autocrine signaling and extracellular matrix effects on tissue homeostasis, regeneration, disease development and progression. In this study, we report the use of microbubble technology as a means to culture cells in a controlled microenvironment in which cells can influence their function through autocrine signaling. Microbubbles (MB) are small spherical cavities about 100-300 μm in diameter formed in hydrophobic polydimethylsiloxane (PDMS) with ∼60-100 μm circular openings and aspect ratio ∼3.0. We demonstrate that the unique architecture of the microbubble compartment is advantaged for cell culture using HaCaT cells, an immortalized <em>keratinocyte</em> cell line. We observe that HaCaT cells, seeded in microbubbles (15-<em>2</em>0 cells/MB) and cultured under standard conditions, adopt a compact 3D spheroidal morphology. Within <em>2</em>-3 days, the cells transition to a sheeting morphology. Through experimentation and simulation we show that this transition in morphology is due to the unique architecture of the microbubble compartment which enables cells to condition their local microenvironment. The small media volume per cell and the development of shallow concentration gradients allow <em>factors</em> secreted by the cells to rise to bioactive levels. The kinetics of the morphology transition depends on the number of cells seeded per microbubble; higher cell seeding induces a more rapid transition. HaCaT cells seeded onto PDMS cured in 96-well plates also form compact spheroids but they do not undergo a transition to a sheeting morphology even after several weeks of culture. The importance of soluble <em>factor</em> accumulation in driving this morphology transition in microbubbles is supported by the observation that spheroids do not form when cells - seeded into microbubbles or onto PDMS cured in 96-well plates - are cultured in media conditioned by HaCaT cells grown in standard tissue culture plate. We observed that the addition of TGF-β1 to the <em>growth</em> media induced cells to proliferate in a sheeting morphology from the onset both on PDMS cured in 96-well plates and in microbubbles. TGF-β1 is a morphogen known to regulate epithelial-to-mesenchymal transition (EMT). Studies of the role of Ca(<em>2</em>+) concentration and changes in E-cadherin expression additionally support an EMT-like HaCaT morphology transition. These findings taken together validate the microbubble compartment as a unique cell culture platform that can potentially transform investigative studies in cell biology and in particular the tumor microenvironment. Targeting the tumor microenvironment is an emerging area of anti-cancer therapy.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>) plays a key role in lung development, but its role in acute lung injury has not been well characterized. Lipopolysaccharide instillation caused acute lung injury, which significantly elevated lung wet-to-dry weight ratio, protein and neutrophils in bronchoalveolar lavage fluid (BALF), inhibited surfactant protein A and C expression in lung tissue, and increased pathological injury. Pretreatment with KGF-<em>2</em> improved the above lung injury parameters, partially restored surfactant protein A and C expression, and KGF-<em>2</em> given <em>2</em>-3 days before LPS challenge showed maximum lung injury improvement. Pretreatment with KGF-<em>2</em> also markedly reduced the levels of TNF-α, MIP-<em>2</em>, IL-1β and IL-6 in BALF and the levels of IL-1β and IL-6 in lung tissue. Histological analysis showed there was increased proliferation of alveolar type II epithelial cells in lung parenchyma, which reached maximal <em>2</em> days after KGF-<em>2</em> instillation. Intratracheal administration of KGF-<em>2</em> attenuates lung injury induced by LPS, suggesting KGF-<em>2</em> may be potent in the intervention of acute lung injury.

Exposure to ionizing radiation is a crucial life-threatening <em>factor</em> in nuclear and radiological incidents. It is known that ionizing radiation affects cytokine/chemokine concentrations in the blood of B6D<em>2</em>F1 mice. It is not clear whether radiation dose rates would vary the physiological response. Therefore, in this study we utilized data from two experiments using B6D<em>2</em>F1 female mice exposed to six different dose rates ranging from low to high rates. In one experiment, mice received a total dose of 8 Gy (LD0/30) of 60Co gamma radiation at four dose rates: 0.04, 0.15, 0.30 and 0.47 Gy/min. Blood samples from mice were collected at <em>2</em>4 and 48 h postirradiation for cytokine/chemokine measurements, including interleukin (IL)-1β, IL-6, IL-10, <em>keratinocyte</em> cytokine (KC), IL-1<em>2</em>p70, IL-15, IL-17A, IL-18, granulocyte-colony stimulating <em>factor</em> (G-CSF), granulocyte macrophage (GM)-CSF, macrophage (M)-CSF, monokine induced by gamma interferon (MIG), tumor necrosis <em>factor</em> (TNF)-α, fibroblast <em>growth</em> <em>factor</em> (FGF)-basic, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and platelet-derived <em>growth</em> <em>factor</em> basic (PDGF-bb). At <em>2</em>4 h after ionizing irradiation at dose rate of 0.04 Gy/min, significant increases were observed only in G-CSF and M-CSF ( P < 0.05). At 0.15 Gy/min, IL-10, IL-17A, G-CSF and GM-CSF concentrations were increased. At 0.3 Gy/min, IL-15, IL-18, G-CSF, GM-CSF, M-CSF, MCP-1, MIP-<em>2</em>, MIG, FGF-basic, VEGF and PDGF-bb were significantly elevated ( P < 0.05). At 0.47 Gy/min, IL-6, KC, IL-10, MCP-1, G-CSF, GM-CSF and M-CSF were significantly increased. At 48 h postirradiation, all cytokines/chemokines except MCP-1 returned to or were below their baselines, suggesting these increases are transient at LD0/30 irradiation. Of note, there is a limitation on day <em>2</em> because cytokines/chemokines are either at or below their baselines. Other parameters such as fms-like tyrosine kinase receptor-3 ligand (Flt-3 ligand) concentrations and lymphocyte counts, which have proven to be unaffected by radiation dose rates, can be used instead for assessing the radiation dose. However, in a separate radiation dose and time-course experiment, increases in IL-18 and G-CSF depended on the radiation doses but showed no significant differences between 0.58 and 1.94 Gy/min ( P>> 0.05) at 3 and 6 Gy but not 1<em>2</em> Gy. G-CSF continued to increase up to day 7, whereas IL-18 increased on day 4 and remained above baseline level on day 7. Therefore, time after irradiation at different doses should be taken into consideration. To our knowledge, these results are the first to suggest that ionizing radiation, even at a very low-dose-rate (0.04 Gy/min), induces circulating G-CSF increases but not others for selected time points; radiation-induced increases in IL-18 at radiation dose rates between 0.15 and 1.94 Gy/min are also not in a radiation dose-rate-dependent manner. C-CSF, lymphocyte counts and circulating Flt-3 ligand should be explored further as possible biomarkers of radiation exposure at early time points. IL-18 is also worthy of further study as a potential biomarker at later time points.

Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic <em>growth</em> <em>factors</em> and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic <em>growth</em> <em>factor</em> secretion from HDMECs (fibroblast <em>growth</em> <em>factor</em> <em>2</em>) and <em>keratinocytes</em> (vascular endothelial <em>growth</em> <em>factor</em> A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin.

Human papilloma virus 16 (HPV 16) is associated with cervical cancer and is therefore considered a major health risk for women. Immortalization of <em>keratinocytes</em> induced by HPV infection is largely due to the binding of p53 and Rb by the the viral oncoproteins E6 and E7, respectively, and is driven to a large extent by a transforming <em>growth</em> <em>factor</em> alpha/amphiregulin epidermal <em>growth</em> <em>factor</em> receptor autocrine loop. In this study, we show that the <em>growth</em> of HPV 16-immortalized human <em>keratinocytes</em> can be blocked by a selective epidermal <em>growth</em> <em>factor</em> receptor kinase inhibitor, AG 1478, and by AG 555, a blocker of cyclin-dependent kinase <em>2</em> (Cdk<em>2</em>) activation. AG 1478 induces a massive increase in the Cdk<em>2</em> protein inhibitors p<em>2</em>7 and p<em>2</em>1, whereas AG 555 appears to have a different mechanism of action, inhibiting the activation of Cdk<em>2</em>. <em>Growth</em> arrest induced by AG 1478 and AG 555 is accompanied by up to <em>2</em>0% of cells undergoing apoptosis. Following AG 1478 treatment but not AG 555 treatment, up to 50% of cells undergo terminal <em>keratinocyte</em> differentiation as determined by filaggrin expression and by the decline in the expression of cytokeratin 14. The <em>growth</em>-arresting properties of AG 1478 and AG 555 identifies them as possible lead antipapilloma agents.

OBJECTIVE

This study focuses on the growth-promoting and migration-enhancing role that Clostridial collagenase plays in vitro and in vivo.

METHODS

For in vitro studies, biosynthesized extracellular matrices were treated with purified Clostridial collagenase, nonspecific proteases, or buffer controls. Keratinocytes were subsequently plated upon these matrices in the presence or absence of Clostridial collagenase and/or heparin-binding epidermal-like growth factor, and cell proliferation and migration were quantified. To examine the effects of Clostridial collagenase in vivo, we performed a double-blind study of full-thickness wounds on the backs of Yucatan Micropigs, testing the effects of purified Clostridial collagenase, Regranex (PDGF-BB), and Solosite (carboxymethyl cellulose) on wound healing.

RESULTS

METHODS

Matrix pretreatment with Clostridial collagenase stimulates a 2-fold increase in proliferation and postinjury migration; when Clostridial collagenase and/or heparin-binding epidermal-like growth factor are added to the growth media, there is an additional doubling of growth and migration, yielding approximately 5-fold enhancement of keratinocyte proliferation and migration. Papain-urea treatment under similar conditions results in a 50% decrease in cell number over a 1-week time course. In vivo studies: By all parameters measured, including granulation tissue formation, inflammation, re-epithelization, and time to wound closure, purified Clostridial collagenase was superior (analysis of variance, P>> .05) to other treatments tested.

CONCLUSIONS

On the basis of these findings, we concluded that Clostridial collagenase stimulates keratinocyte cellular responses to injury in vitro and may represent a novel therapeutic approach for promotion of wound healing in vivo.

BACKGROUND

Ultraviolet (UV)-B irradiation has been shown to be an inducer of vascular endothelial growth factor (VEGF) in primary keratinocytes and epidermal cell lines in vitro.

OBJECTIVE

To determine the expression pattern and the causal role of VEGF in the UVB-mediated angiogenic response in vivo in human skin and in a mouse model.

METHODS

Skin biopsies or epidermal lysates thereof were studied for VEGF expression following UVB irradiation at a dose of 50 or 60 mJ cm-2, respectively, using immunostaining and a VEGF-specific highly sensitive sandwich enzyme-linked immunosorbent assay. The VEGF-dependent increase in vessels upon repetitive UVB irradiation was studied in skh-1 hairless mice using immunostaining for factor VIII-related antigen (FVIII RAG) in the presence and absence of intraperitoneally injected neutralizing VEGF antibodies.

RESULTS

VEGF was found to be induced in the epidermis following UVB irradiation of human and mouse skin. Repetitive UVB irradiation of skh-1 hairless mice resulted in an increase in FVIII RAG positive vessels in the skin. UVB-induced angiogenic response could be partly abrogated by neutralizing antibodies against VEGF, while isotype-matched IgG control antibodies did not reveal any suppressive effect.

CONCLUSIONS

Our results support previous in vitro data and show the in vivo relevance of VEGF as a paracrine inducer of cutaneous vessels after UVB irradiation.

The epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is activated in cutaneous <em>keratinocytes</em> upon ultraviolet (UV) exposure and has been implicated in ultraviolet-(UV-)induced inflammation and skin tumorigenesis. Egfr mutant mice and EGFR inhibitors were used to investigate the hypothesis that EGFR activation augments inflammation following UV irradiation. Topical treatment of mouse skin with the EGFR inhibitor AG1478 before UV exposure suppressed UV-induced erythema, edema, mast cell infiltration, and neutrophil infiltration. Genetic ablation of Egfr and EGFR inhibition by AG1478 also suppressed the increase in the proinflammatory cytokines tumor necrosis <em>factor</em> α (TNF- α ), interleukin-1 α , KC (murine IL-8), and cyclooxygenase-<em>2</em> (COX-<em>2</em>) after UV exposure of cultured <em>keratinocytes</em>. Finally, genetic ablation of inhibition of EGFR in cultured <em>keratinocytes</em> decreased p38 activation after UV, while inhibition of p38 kinase reduced COX-<em>2</em> expression after UV. These data demonstrate that EGFR regulates multiple aspects of UV-induced inflammation and suggest activation of p38 kinase leading to increased COX-<em>2</em> and cytokine expression as one mechanism through which it acts.

BACKGROUND

Interleukin (IL)-33 is a recently identified cytokine, which is a member of the IL-1 family and binds to a heterodimeric receptor comprising ST<em>2</em> (suppression of tumorigenicity <em>2</em>) and IL-1 receptor accessory protein. Serum levels of IL-33 have been reported to be upregulated in various T helper (Th)1/Th17-mediated diseases, such as rheumatoid arthritis and inflammatory bowel disease. IL-33 expression is increased in lesional skin in patients with psoriasis, but serum levels in patients with psoriasis have not yet been studied.

OBJECTIVE

To study serum IL-33 levels in patients with psoriasis, a Th1/Th17-mediated skin disease, before and after anti-tumour necrosis factor (TNF)-α therapy.

METHODS

Serum IL-33 levels were measured in patients with psoriasis vulgaris (PV), psoriatic arthritis (PsA) or pustular psoriasis (PP), and compared with those of healthy controls. Associations between serum IL-33 levels and serum TNF-α, IL-6, vascular endothelial growth factor and C-reactive protein levels were also studied. In addition, the effect of IL-33 stimulation on IL-6, IL-8, TNF-α and VEGF secretion by human keratinocyte was analysed.

RESULTS

Serum IL-33 levels in patients with PV, PsA and PP were significantly higher than those in healthy controls. Serum IL-33 levels correlated with serum TNF-α levels in patients with psoriasis, and decreased after anti-TNF-α therapy. IL-33 stimulated IL-6 and IL-8 secretion by human keratinocytes.

CONCLUSIONS

These results suggest that serum IL-33 levels generally reflect increased inflammation in patients with psoriasis.

BACKGROUND

Hidradenitis suppurativa (HS) is a painful chronic inflammatory disease with a prevalence between 1 and 4% of general population. The pathogenesis of HS long eluded scientists, but growing evidence suggests that it is a consequence of inflammatory dysregulation.

RESULTS

Recent studies suggest that dysregulated immune response to skin flora and overexpression of inflammatory cytokines leads to chronic skin inflammation seen in HS. Macrophages are the most numerous inflammatory cells found in HS infiltrates and release numerous pro-inflammatory cytokines such as IL-23, and IL-1β and TNF-α, exacerbating the inflammation and contributing to the pathogenesis of HS. Furthermore, in HS, there is dysregulated function of other immune players closely associated with macrophage function including: matrix metalloproteases (MMP) 2 and 9 overexpression, toll-like receptor upregulation, impaired Notch signalling, NLRP3 inflammasome upregulation, and dysregulated keratinocyte function. Lifestyle factors including obesity and smoking also contribute to macrophage dysfunction and correlate with HS incidence.

CONCLUSIONS

The overexpression of pro-inflammatory cytokines and subsequent efficacy of anti-cytokine biologic therapies highlights the importance of managing macrophage dysfunction. Future therapies should target key molecular drivers of macrophage dysfunction such as TLR2 and NLRP3 overexpression.

The normal vasculature is maintained by a balance between angiogenic <em>factors</em> and anti-angiogenic <em>factors</em>. Recent studies have shown that pigment epithelium-derived <em>factor</em> (PEDF) can induce differentiation and inhibit angiogenesis of tumors. This study was designed to investigate the expression of PEDF and its roles in proliferation, adhesion and migration of HaCaT cells, a human <em>keratinocyte</em> cell line. Our results have shown that PEDF is expressed in HaCaT cells at both mRNA and protein levels determined by RT-PCR and Western blot, separately. PEDF signal mainly localizes in the cytoplasm of HaCaT cell, as determined by immunofluorescence. Furthermore, expression of PEDF is decreased by 50 ng/ml of VEGF(165). Proliferation and migration of HaCaT cells are decreased by PEDF, while adhesion of HaCaT cells is upregulated approximately by <em>2</em>9%. PEDF also induce the S phase accumulation of HaCaT cells. In addition, phosphorylation of ERK1/<em>2</em>, not JNK and p38, is decreased by PEDF. These results indicate that PEDF may play an inhibitory role on <em>growth</em> and migration of HaCaT cells through dephosphorylation of ERK1/<em>2</em>.

BACKGROUND

Skin-derived antimicrobial peptides, such as human β-defensins and cathelicidins, actively contribute to host defense by inactivating microorganisms. Catestatin, a neuroendocrine peptide that affects human autonomic functions, has recently been detected in keratinocytes upon injury/infection where it inhibits the growth of pathogens. Human catestatin exhibits three single nucleotide polymorphisms: Gly364Ser, Pro370Leu, and Arg374Gln.

OBJECTIVE

To investigate the effects of human catestatin and its variants on keratinocyte migration and proliferation, and to elucidate the possible signaling mechanisms involved.

METHODS

The migration of normal human keratinocytes was analyzed using Boyden microchamber assay and in vitro wound closure assay. Cell proliferation was evaluated by BrdU incorporation, cell count assay and cell cycle analysis. Intracellular Ca(2+) mobilization was measured using a fluorescent calcium assay kit. The phosphorylation of epidermal growth factor receptor (EGFR), Akt, and MAPKs was determined by Western blotting.

RESULTS

Catestatin and its variants dose-dependently enhanced keratinocyte migration and proliferation. Moreover, catestatin peptides increased intracellular Ca(2+) mobilization and induced the phosphorylation of EGFR, Akt, extracellular signal-regulated kinase (ERK), and p38 in keratinocytes. The induction of keratinocyte migration and proliferation by catestatin peptides involved G-proteins, phospholipase C, EGFR, PI3-kinase, ERK, and p38, as evidenced by the specific inhibitory effects of pertussis toxin (G-protein inhibitor), U-73122 (phospholipase C inhibitor), AG1478 (EGFR inhibitor), anti-EGFR antibody, wortmannin (PI3-kinase inhibitor), U0126 (ERK inhibitor), and SB203580 (p38 inhibitor), respectively.

CONCLUSIONS

Besides inhibiting the growth of skin pathogens, catestatin peptides may also contribute to cutaneous wound closure by enhancing keratinocyte migration and proliferation at the wound site.

Evidence suggests an association between alcohol consumption and psoriasis. This relationship is still undefined, although long-term alcohol intake influences the immune system. Interactions between T cells and <em>keratinocytes</em> are important for the pathogenesis of psoriasis, by secretion of pro-inflammatory cytokines and <em>growth</em> <em>factors</em> in psoriatic skin. IL-<em>2</em>, IL-6, IL-8, IFN-gamma and TGF-alpha are hallmark cytokines in a psoriatic cytokine network. We investigated whether ethanol influences the secretion of these cytokines using a co-culture model with <em>keratinocytes</em> from psoriatic patients (n = 9) or from healthy controls (n = 9), with HUT 78 lymphocytes, and determined the cytokine levels with or without ethanol treatment in the culture supernatants. TGF-alpha and IFN-gamma levels were elevated in the ethanol-treated psoriatic co-cultures, to 150% and 175% respectively, but neither in co-cultures with <em>keratinocytes</em> derived from healthy control individuals nor in monocultures. Treatment with ethanol elevated slightly the IL-6 levels in the monocultures from psoriatic and control <em>keratinocytes</em> to 1<em>2</em>5% but not in HUT 78 monocultures. In the psoriatic co-cultures, IL-6 levels were elevated in the culture supernatants to almost 160%, but they were not influenced by ethanol in co-cultures with control <em>keratinocytes</em>. The cytokine levels of IL-8 or IL-<em>2</em> were not significantly influenced in the psoriatic mono- and co-cultures or in HUT 78 cultures. If ethanol influences the cytokine secretion of psoriatic <em>keratinocytes</em> and HUT 78 lymphocytes in co-culture conditions, these data suggest that ethanol could also influence the psoriatic cytokine network in vivo, which may explain the explain the aggravation of this disease in alcohol-consuming psoriatic patients.

Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of <em>keratinocyte</em> <em>growth</em> <em>factor</em>-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein-transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (<em>2</em>6 ± <em>2</em> versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (<em>2</em>55 ± 36 versus 16<em>2</em> ± 16 µm) at 1<em>2</em>0 hours (P = 0.057). Biomechanical assessment showed that the KGF-1-treated skin was significantly stronger than control vector-transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.

As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast <em>growth</em> <em>factor</em> (FGF)--10 action, we investigated if a C-Xylopyranoside derivative, (C-β-D-xylopyranoside-<em>2</em>-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human <em>keratinocytes</em>, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated <em>keratinocytes</em> greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, <em>keratinocytes</em> treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), also called fibroblast <em>growth</em> <em>factor</em>-10 (FGF-10), is a member of the fibroblast <em>growth</em> <em>factor</em> family. It plays a critical role in epithelial development and exerts its biological activities in a paracrine manner on the receptor FGFR<em>2</em>-IIIb. This study examined the function of topically applied KGF-<em>2</em> in vivo on wound healing using a CO(<em>2</em>) laser, corneal epithelial wounded, rabbit model. Topically applied <em>2</em>5 microg/ml KGF-<em>2</em> accelerated corneal epithelial wound healing, in contrast to the control, and reduced inflammation, stromal edema, and fibrosis. In addition, this <em>factor</em> also exhibited significant inhibition of corneal neovascularization. KGF-<em>2</em> appears to be another important <em>growth</em> <em>factor</em> in the regulation of corneal epithelial wound healing.

Angiogenesis is considered as an essential process for tumour development and invasion. Previously, we demonstrated that cyclin-dependent kinase inhibition by roscovitine induces a radiosensitization and a synergistic antitumoral effect in human carcinoma but its effect on the microenvironment and tumour angiogenesis remains unknown. Here, we investigated the effect of the combination roscovitine and ionizing radiation (IR) on normal cells in vitro and on tumour angiogenesis in MDA-MB <em>2</em>31 tumour xenografts. We observed that the combination roscovitine and IR induced a marked reduction of angiogenic hot spot and microvascular density in comparison with IR or roscovitine treatments alone. The Ang-<em>2</em>/Tie-<em>2</em> ratio was increased in presence of reduced vascular endothelial <em>growth</em> <em>factor</em> level suggesting vessel destabilization. In vitro, no radiosensitization effect of roscovitine was found in endothelial, fibroblast, and <em>keratinocyte</em> cells. IR potentiated the antiproliferative effect of roscovitine without inducing apoptosis in endothelial cells. Roscovitine decreased IR-stimulated vascular endothelial <em>growth</em> <em>factor</em> secretion of MDA-MB <em>2</em>31 and endothelial cells. A reduction in the endothelial cells invasion and the capillary-like tube formation in Matrigel were observed following the combination roscovitine and IR. This combined treatment targets angiogenesis resulting in microvessel destabilization without inducing normal cell toxicity.