Early intestinal adaptation after massive small bowel resection (SBR) is driven by increased epithelial cell (EC) proliferation. There is a clear clinical difference in the post-operative course of patients after the loss of proximal (P) compared to distal (D) small bowel. This study examined the effects of the site of SBR on post-resectional intestinal adaptation, and investigated the potential mechanisms involved. C57BL/6J mice (n = 7/group) underwent: (1) 60% P-SBR, (<em>2</em>) 60% D-SBR, (3) 60% mid (M)-SBR and (4) SHAM-operation (transection/reanastomosis). Mice were sacrificed at 7 days after surgery and ECs and adjacent mucosal lymphocytes (IELs) isolated. Adaptation was assessed in both jejunum and ileum by quantification of villus height, crypt depth, villus cell size, crypt cell size (microns), goblet cell number, and EC proliferation (%BrdU incorporation). Proliferation signalling pathways including <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF)/KGFR(1), IL-7/IL-7R, and epidermal <em>growth</em> <em>factor</em> receptor (EGFR) were measured by RT-PCR. Expression of IL-7 was further analysed by immunofluorescence. Data were analyzed using ANOVA. All three SBR models led to significant increases in villus height, crypt depth, goblet cell numbers and EC proliferation rate when compared to respective SHAM groups. The strongest morphometric changes were found for jejunal segments after M-SBR and for ileal segments after P-SBR. Furthermore, morphometric analysis showed that at 1-week post-resection a tremendous increase in EC numbers occurred in jejunal villi (cell hyperplasia), whereas a significant increase in EC size predominated in ileal villi (cell hypertrophy). mRNA expression of KGF, KGFR(1), IL-7R, and EGFR showed a significant increase only after D-SBR, whereas IL-7 increased significantly after SBR in all investigated models, and this was confirmed by immunofluorescence studies. Early intestinal adaptation shows distinct differences depending on the site of SBR, and is predominately driven by cell hyperplasia in jejunal villi and cell hypertrophy in ileal villi. However, the exact mechanisms, which guide these signalling pathways are still unclear.

Transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) is known to inhibit epithelial cell <em>growth</em> by inducing a G1 cell cycle arrest. We have studied the effect of TGF-beta 1 on protein binding to a transcription <em>factor</em> E<em>2</em>F consensus element in extracts from early passage human <em>keratinocytes</em> (HFKs) and a permanent human <em>keratinocyte</em> cell line (HaCaT). Treatment of these cells with TGF-beta 1 resulted in the formation of a DNA binding complex between the pRb-related protein p130 and E<em>2</em>F. Formation of the E<em>2</em>F-p130 complex correlated with inhibition of cell cycle progression in G1 and suppression of the E<em>2</em>F-regulated cdc<em>2</em> gene. While p130 mRNA and protein levels were not influenced by TGF-beta 1, the activity of cyclin-dependent kinase <em>2</em> (Cdk<em>2</em>) towards p130 in vitro was inhibited. The results identify p130 as a downstream target of TGF-beta 1 and a possible mediator of the G1 cell cycle arrest.

The onset and progression of periodontal disease is associated with significant changes in the epithelial component of the attachment complex. From the early to the advanced stages of periodontal disease increased epithelial cell proliferation, migration and invasion into the surrounding connective tissue takes place. Concomitantly there is a significant increase in proinflammatory cytokine expression in periodontal tissue and quantitative and qualitative changes in the subgingival microflora, including an increase in gram-negative microorganisms. One of the most significant virulence <em>factors</em> of these bacteria is lipopolysaccharide (LPS) connected to the outer membrane. Two important <em>growth</em> <em>factors</em> controlling epithelial behavior are <em>Keratinocyte</em> <em>Growth</em> <em>Factor</em>-1 (KGF-1) and -<em>2</em> (KGF-<em>2</em>). Connective tissue cells express these <em>growth</em> <em>factors</em>, but only epithelial cells respond to them. We studied the effect of proinflammatory cytokines and LPS on gingival fibroblast expression of KGF-1 and KGF-<em>2</em> in vitro. Gingival fibroblasts were found to express KGF-1 and -<em>2</em> in culture but only KGF-1 protein and gene expression was stimulated by serum, in a concentration-dependent manner by proinflammatory cytokines IL-1alpha, IL-1beta, TNF-alpha and IL-6 and LPS isolated from Porphyromonas gingivalis and Escherichia coli. The local increase in proinflammatory cytokine expression and the accumulation of LPS in disease sites may therefore stimulate gingival fibroblast expression of KGF-1. We hypothesize that this local increase in KGF-1 expression may, via a paracrine mechanism, stimulate local epithelial cell proliferation, migration and invasion during the onset and progression of periodontitis.

Alternative splicing generates a vast diversity of protein isoforms from a limited number of protein-coding genes, with many of the isoforms possessing unique, and even contrasting, functions. Fluorescence-based splicing reporters have the potential to facilitate studies of alternative splicing at the single-cell level and can provide valuable information on phenotypic transitions in almost real time. Fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) pre-mRNA is alternatively spliced to form the epithelial-specific and mesenchymal-specific IIIb and IIIc isoforms, respectively, which are useful markers of epithelial-mesenchymal transitions (EMT). We have used our knowledge of FGFR<em>2</em> splicing regulation to develop a fluorescence-based reporter system to visualize exon IIIc regulation in vitro and in vivo. Here we show the application of this reporter system to the study of EMT in vitro in cell culture and in vivo in transgenic mice harboring these splicing constructs. In explant studies, the reporters revealed that FGFR<em>2</em> isoform switching is not required for <em>keratinocyte</em> migration during cutaneous wound closure. Our results demonstrate the value of the splicing reporters as tools to study phenotypic transitions and cell fates at single cell resolution. Moreover, our data suggest that <em>keratinocytes</em> migrate efficiently in the absence of a complete EMT.

Both keloids (KLs) and hypertrophic scars (HSs) are considered as dermal fibroproliferative diseases that differ clinically and histopathologically. Although several <em>factors</em> have been postulated in the etiopathogenesis of these conditions, there has been <em>growing</em> evidence to suggest the role of COXs in the pathogenesis of abnormal wound healing because of the reduction of formation of KL and HS in patients using nonsteroidal anti-inflammatory drugs and a COX-<em>2</em> inhibitor. The aim of the present work is to evaluate the pattern and localization of COX-1 and COX-<em>2</em> expression in KL and HS compared with surgical scars. COX-1 and COX-<em>2</em> were analyzed on skin biopsies of 30 patients who presented with KL (15) and HS (15) and 10 normal surgical scars (controls). Both COX-1 and COX-<em>2</em> were expressed not only in dermal components (fibroblasts, inflammatory cells, and endothelial cells) but also in <em>keratinocytes</em> of the overlying epidermis in the different studied scar lesions. The percentage of COX-1 expression increased progressively from surgical scar (40%) to HS (53.3%) to KL (100%) with a statistically significant difference (P = 0.00<em>2</em>). COX-<em>2</em> was expressed in 100% of surgical scars, 73.3% of HS and 86.7% of KL with the absence of significant differences (P>> 0.05). The significant difference in COX-1 expression between HS and KL may refer to the presence of different pathways for the emergence of these diseases. The expression of COX-<em>2</em> in all scars (normal or abnormal) indicates its active role as an inflammatory mediator. <em>Keratinocytes</em> play an active role in induction of scarring by up-regulation of inflammatory mediators, such as COX-1 and COX-<em>2</em>.

OBJECTIVE

Mesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).

METHODS

Four basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-<em>2</em>7, epidermal <em>growth</em> <em>factor</em> and fibroblast <em>growth</em> <em>factor</em> <em>2</em>, or one of two commercial serum-free media, defined <em>keratinocyte</em> serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD<em>2</em>71), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.

RESULTS

While all techniques supported the establishment of cultures to varying degrees, sustained <em>growth</em> and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34(-) CD45(-) CD90 (+) CD73 (+) CD105 (+) , approximately <em>2</em>5% α-sma (+) and displayed multipotency. Cultures established in MesenCult-XF were>> 95% CD34(-) CD45(-) CD90 (+) CD73 (+) CD105 (+) , 40% CD141 (+) , rarely expressed α-sma, and displayed multipotency. L-MSC supported <em>growth</em> of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3.

CONCLUSIONS

MesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

Increasing evidence demonstrates that maternal folic acid (FA) supplementation during pregnancy reduces the risk of neural tube defects, but whether FA prevents preterm delivery and intrauterine <em>growth</em> restriction (IUGR) remains obscure. Previous studies showed that maternal lipopolysaccharide (LPS) exposure induces preterm delivery, fetal death and IUGR in rodent animals. The aim of this study was to investigate the effects of FA on LPS-induced preterm delivery, fetal death and IUGR in mice. Some pregnant mice were orally administered with FA (0.6, 3 or 15 mg/kg) 1 h before LPS injection. As expected, a high dose of LPS (300 μg/kg, i.p.) on gestational day 15 (GD15) caused 100% of dams to deliver before GD18 and 89.3% of fetuses dead. A low dose of LPS (75 μg/kg, i.p.) daily from GD15 to GD17 resulted in IUGR. Interestingly, pretreatment with FA prevented LPS-induced preterm delivery and fetal death. In addition, FA significantly attenuated LPS-induced IUGR. Further experiments showed that FA inhibited LPS-induced activation of nuclear <em>factor</em> kappa B (NF-κB) in mouse placentas. Moreover, FA suppressed LPS-induced NF-κB activation in human trophoblast cell line JEG-3. Correspondingly, FA significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-<em>2</em> in mouse placentas. In addition, FA significantly reduced the levels of interleukin (IL)-6 and <em>keratinocyte</em>-derived cytokine (KC) in amniotic fluid of LPS-treated mice. Collectively, maternal FA supplementation during pregnancy protects against LPS-induced preterm delivery, fetal death and IUGR through its anti-inflammatory effects.

Transforming <em>growth</em> <em>factor</em>-beta(1) (TGFbeta(1)) is thought to be an inhibitor of the <em>keratinocyte</em> hyperproliferation associated with psoriasis. The aim of this study was to evaluate plasma TGFbeta(1) and TGFbeta(<em>2</em>) concentrations in psoriatic patients as possible indicators of treatment efficacy. TGFbeta concentrations were measured in the plasma of <em>2</em>6 patients with psoriasis using an enzyme immunoassay and analysed with respect to the psoriasis area and severity index (PASI) before and after treatment with salicylic acid and/or sulphur followed by dithranol ointment. Baseline plasma concentrations of both TGFbeta(1) and TGFbeta(<em>2</em>) (<em>2</em>0.3+/-<em>2</em>.<em>2</em> ng ml(-1) and 0.14+/-0.0<em>2</em> ng ml(-1), respectively) did not differ significantly from control values (18.3+/-1.6 ng ml(-1) and 0.14+/-0.03 ng ml(-1), respectively). However, a significant positive correlation (r=0.69) between the baseline PASI and TGFbeta(1), but not TGFbeta(<em>2</em>), values was demonstrated. The pretreatment TGFbeta(1) concentration in patients with a PASI>>/=15 (<em>2</em>6.6+/-3.<em>2</em> ng ml(-1)) was significantly higher than control values. There were no significant elevation of pretreatment TGFbeta(1) concentrations in patients with a PASI<15, or with respect to TGFbeta(<em>2</em>) in both groups. Treatment caused a significant decrease in TGFbeta(1), but only in patients with a PASI>/=15. Patients with baseline TGFbeta(1) concentrations exceeding the mean of the control group had a PASI value that was significantly higher than that of patients with a TGFbeta(1) concentration below the mean of the controls. These results confirmed an association between plasma TGFbeta(1) concentration and psoriasis severity, and demonstrated its normalization during treatment. Measurement of TGFbeta(1) in plasma should be considered as a possible biomarker of psoriasis activity during its management.

BACKGROUND

Wound repair results from a series of highly orchestrated cellular and biochemical events, including increased synthesis of the bioregulatory molecule nitric oxide (NO). The goal of this work was to test the functional role of NO in promotion of vascular endothelial growth factor (VEGF) production and the vigorous granulation tissue formation characteristic of this wound model.

METHODS

A ventral hernia, surgically created in the abdominal walls of 12 swine, was repaired with silicone sheeting and skin closure. An osmotic infusion pump, inserted in a remote subcutaneous pocket, delivered saline solution (n = 6) or the selective inducible NO synthase inhibitor N(6) (iminoethyl)-L-lysine (L-NIL; n = 6) into the wound environment. Granulation tissue thickness was determined with ultrasonography, and local wound perfusion was measured with laser Doppler analysis for 2 weeks. Fluid was aspirated serially from the wound compartment for measurement of nitrite/nitrate, VEGF, and transforming growth factor-beta(1)concentrations. On day 14, the animals were killed and the abdominal wall was harvested for immunohistochemical and molecular analysis.

RESULTS

In animals that received saline solution, a nearly linear 4-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, in animals that received L-NIL, day 14 granulation tissue thickness was essentially unchanged from the day 2 values of saline solution-treated animals. Moreover, in the L-NIL animals, ultrasonography was unable to resolve the angiogenic zone typical of controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histologic sections were 2-fold to 3-fold lower in the L-NIL animals compared with controls. Reductions in basal (2-fold) and heat-provoked (2.5-fold) wound perfusion were noted in L-NIL animals. Wound fluid nitrite/nitrate and VEGF levels were strikingly (4-fold and 5-fold, respectively) reduced in L-NIL animals on days 9 to 14. Immunochemistry results showed reduced VEGF protein content in granulation tissue and keratinocytes within the hyperproliferative epithelium at wound edge. Finally, transforming growth factor-beta(1)levels were unaffected by L-NIL treatment.

CONCLUSIONS

VEGF production in granulation tissue is dependent on the presence of functionally active inducible NO synthase and hence, the production of NO. NO and VEGF are therefore defined as key regulators of granulation tissue formation.

Immunohistochemical staining of skin sections with two polyclonal antibodies (anti-CC 1-30 and anti-LC 1-30), specific for transforming <em>growth</em> <em>factor</em>-beta 1, revealed increased extracellular and decreased intracellular expression of transforming <em>growth</em> <em>factor</em>-beta 1 in retinoic acid-treated, compared to vehicle-treated, skin. Transforming <em>growth</em> <em>factor</em>-beta 1 staining, with both antibodies, was most marked in the upper layers of the epidermis, although dermal staining was also evident. The modulation of transforming <em>growth</em> <em>factor</em>-beta 1 expression by retinoic acid occurred in the absence of any change in its mRNA level. Transforming <em>growth</em> <em>factor</em>-beta 1 protein, as detected by rabbit polyclonal antibody (anti-LC 50-75) and mRNA, were only minimally detected in either retinoic acid- or vehicle-treated skin. Similar changes in TGF-beta 1 and TGF-beta <em>2</em> immunoreactivity and mRNA levels, as observed in retinoic acid-treated skin, were observed in skin following topical application of the irritant sodium lauryl sulfate, indicating that the alterations induced by retinoic acid were not specific. In contrast, mucin deposition, which is induced by transforming <em>growth</em> <em>factor</em>-beta, was elevated in retinoic acid-treated but not sodium lauryl sulfate-treated skin. Cultured adult human <em>keratinocytes</em> also expressed predominantly transforming <em>growth</em> <em>factor</em>-beta 1 protein, as measured by ELISA, and mRNA. Treatment of <em>keratinocytes</em> with retinoic acid resulted in a 50% induction of transforming <em>growth</em> <em>factor</em>-beta 1 protein, without any detectable change in transforming <em>growth</em> <em>factor</em>-beta <em>2</em>. These data demonstrate disassociation of modulation of transforming <em>growth</em> <em>factor</em>-beta 1 expression and mucin deposition by retinoic acid and sodium lauryl sulfate in human skin in vivo. Whereas alterations in transforming <em>growth</em> <em>factor</em>-beta 1 expression were observed in both retinoic acid- and sodium lauryl sulfate-treated skin, accumulation of mucin was specific to retinoic acid-treated skin.

Bone morphogenetic proteins (BMPs) are a group of peptide <em>growth</em> <em>factors</em> closely related to transforming <em>growth</em> <em>factors</em>-beta. The BMPs are suggested to play an essential role in bone development and they are strong candidate molecules to be used clinically to improve fracture healing. BMPs are also involved in the differentiation of several other tissues during embryogenesis. Here, we show that human recombinant BMP-<em>2</em> regulates cell-matrix interactions by modifying the expression of integrin-type receptors. The synthesis of alpha3 integrin was down-regulated by BMP-<em>2</em> in two osteogenic sarcoma-derived cell lines, Saos-<em>2</em> and HOS, and also in human fetal chondrocytes. BMP-<em>2</em> had no effect on the expression of alpha1, alpha<em>2</em>, alpha5, alpha6, and alphaV integrins. BMP-<em>2</em> reduced the expression of alpha3 integrin subunit at mRNA level. Laminin-5 was shown to be the ligand for alpha3beta1 integrin on Saos cells and BMP-<em>2</em> decreased the ability of Saos cells to attach to it. These results suggest that BMP-<em>2</em> has a specific effect on the alpha3beta1 integrin-mediated cell adhesion to laminin-5. Given the fact that BMP-<em>2</em> is expressed in osteosarcomas, in addition to in bone, this mechanism is putatively important especially in bone development and tumors. We also studied the effect of BMP-<em>2</em> on a human <em>keratinocyte</em> cell line, HaCaT. In HaCaT cells, the expression of alpha<em>2</em> integrin was strongly down-regulated by BMP-<em>2</em>, whereas its effect on the expression of alpha3 integrin was smaller. We suggest that the effects of BMP-<em>2</em> may be partially mediated by specifically altered cell adhesion.

Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. <em>Keratinocytes</em> are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal <em>keratinocytes</em>. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-<em>2</em> activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of <em>keratinocyte</em> monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal <em>growth</em> <em>factor</em> receptor or Tie<em>2</em>. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/<em>keratinocytes</em> and its therapeutical potential in skin inflammatory conditions.

Even though anthralin is a well-established topical therapeutic agent for psoriasis, little is known about its effects and biochemical mechanisms of signal transduction. In contrast to a previous report, we found that anthralin induced time- and concentration-dependent phosphorylation of epidermal <em>growth</em> <em>factor</em> receptor in primary human <em>keratinocytes</em>. Four lines of evidence show that this process is mediated by reactive oxygen species. First, we found that anthralin induces time-dependent generation of H(<em>2</em>)O(<em>2</em>). Second, there is a correlation between a time-dependent increase in anthralin-induced epidermal <em>growth</em> <em>factor</em> receptor phosphorylation and H(<em>2</em>)O(<em>2</em>) generation. Third, the structurally different antioxidants n-propyl gallate and N-acetylcysteine inhibited epidermal <em>growth</em> <em>factor</em> receptor phosphorylation induced by anthralin. Fourth, overexpression of catalase inhibited this process. The epidermal <em>growth</em> <em>factor</em> receptor-specific tyrosine kinase inhibitor PD153035 abrogated anthralin-induced epidermal <em>growth</em> <em>factor</em> receptor phosphorylation and activation of extracellular-regulated kinase 1/<em>2</em>. These findings establish the following sequence of events: (1) H(<em>2</em>)O(<em>2</em>) generation, (<em>2</em>) epidermal <em>growth</em> <em>factor</em> receptor phosphorylation, and (3) extracellular-regulated kinase activation. Our data identify anthralin-induced reactive oxygen species and, more specifically, H(<em>2</em>)O(<em>2</em>) as an important upstream mediator required for ligand-independent epidermal <em>growth</em> <em>factor</em> receptor phosphorylation and downstream signaling.

We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, <em>growth</em> <em>factors</em>, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of <em>keratinocyte</em> cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A <em>2</em>-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.

Bovine papillomaviruses (BPVs) are oncogenic DNA viruses, which mainly induce benign lesions of cutaneous and/or mucosal epithelia in cattle. Thirteen (BPV 1-13) different viral genotypes have been characterized so far. BPVs are usually species-specific but BPV 1/<em>2</em> may also infect equids as well as buffaloes and bison and cause tumors in these species. BPV-induced benign lesions usually regress, however occasionally they develop into cancer particularly in the presence of environmental carcinogenic co-<em>factors</em>. The major transforming protein of BPV is E5, a very short hydrophobic, transmembrane protein with many oncogenic activities. E5 contributes to cell transformation through the activation of the cellular β receptor for the platelet-derived <em>growth</em> <em>factor</em> (PDGFβ-r), it also decreases cell surface expression of major histocompatibility complex class I (MHCI) causing viral escape from immunosurveillance, and plays a role in the inhibition of the intracellular communication by means of aberrant connexin expression. E7 is considered as a weak transforming gene, it synergies with E5 in cell transformation during cancer development. E7 expression correlates in vivo with the over-expression of β1-integrin, which plays a role in the regulation of <em>keratinocytes</em> proliferation and differentiation. Additionally, E7 is involved in cell-mediated immune responses leading to tumour rejection, in anoikis process by direct binding to p600, and in invasion process by upregulation of Matrix metalloproteinase1 (MMP-1) expression. Studies on the role of BPV E5 and E7 oncoproteins in naturally occurring tumours are of scientific value, as they may shed new light on the biological role of these two oncogenes in cell transformation.

OBJECTIVE

The aim of this study was to investigate the expression pattern of oncogenes, antimicrobial peptides, and genes involved in inflammation in leukoplakia of the oral cavity compared with healthy gingiva.

METHODS

Biopsies of healthy gingiva (n=<em>2</em>0) and leukoplakia (n=<em>2</em>0), were obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of alpha-defensin (DEFA) 1/3, DEFA-4, S100-A7, deleted-in-oral-cancer (Doc) 1, interleukin (IL) 1beta, IL-6, IL-8, IL-10, tumor necrosis <em>factor</em> (TNF) alpha, cyclooxygenase (Cox) <em>2</em>, epidermal <em>growth</em> <em>factor</em> (EGF), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), transforming <em>growth</em> <em>factor</em> (TGF) beta1, TGF-alpha, collagen-IA1 (Col-1), and tenascin-c were analyzed by real-time reverse-transcription polymerase chain reaction. The proteins encoded by the different genes were visualized by immunostaining.

RESULTS

Compared with healthy gingiva (set as 1), there was an increased gene expression of DEFA-4 (179.<em>2</em>-fold), S100-A7 (<em>2</em>5.4-fold), EGF (<em>2</em>4.8-fold), TGF-beta1 (<em>2</em>5.<em>2</em>-fold), and tenascin-c (34.3-fold) in oral leukoplakia. The expression of IL-1beta and Doc-1 was decreased (0.01-fold and 0.<em>2</em>-fold, respectively).

CONCLUSIONS

The combination of an increased expression of the antimicrobial peptide DEFA-4, the oncogene S100-A7, EGF, and tenascin-c, and a decreased Doc-1 expression in oral leukoplakia might characterize its potency of malignant transformation. Chronic inflammation seems not to be involved in the development of this lesion.

Cyclosporin A, which is a specific immunosuppressive compound, has recently been demonstrated to be of significant benefit in the treatment of psoriasis. Because hyperplasia is a major feature of psoriasis, we have investigated whether this drug acts directly to inhibit <em>keratinocyte</em> <em>growth</em>. We have determined the concentration range of cyclosporin in the epidermis of psoriatic patients undergoing cyclosporin therapy and the effect of this concentration range on the <em>growth</em> of cultured <em>keratinocytes</em>. After 7 days of treatment, psoriatic involved epidermis contained 1.1 +/- 0.3 ng cyclosporin/micrograms DNA. Based on tissue wet weight this is approximately <em>2</em>.8 micrograms cyclosporin/ml. This value was 10 times that of trough blood samples. On day 3 of treatment, involved epidermis contained 7 times more cyclosporin than scale, while on day 7 this ratio was reduced to <em>2</em>. This suggests that cyclosporin was initially associated with the lower layers of the epidermis and distributed upward with time. Exposure of adult human <em>keratinocytes</em>, cultured on collagen in the presence of human serum, to cyclosporin (0.1-30 micrograms/ml, 0.4-13-fold higher than lesional cyclosporin) for <em>2</em>-6 d had no effect on the rate of incorporation of thymidine into DNA. Cyclosporin (1-30 micrograms/ml) also had not effect on the reinitiation of DNA synthesis of quiescent cells subsequent to the reintroduction of serum. In contrast, cyclosporin (1-10 micrograms/ml) inhibited <em>growth</em> of <em>keratinocytes</em> cultured on plastic culture dishes in serum free media (MCDB 153). For a given dose of cyclosporin, cell associated drug was <em>2</em>-3 times greater in serum free compared to serum containing media. This may contribute in part to the sensitivity of <em>keratinocytes</em> in serum free media to <em>growth</em> inhibition by cyclosporin. These data demonstrate that human epidermis contains a high concentration of cyclosporin after oral administration, and that, under certain conditions, concentrations of cyclosporin within the range found in vivo can inhibit <em>growth</em> of cultured <em>keratinocytes</em>. Because cyclosporin regulates lymphocyte function in vivo and in vitro, the demonstrated antiproliferative effects of cyclosporin on psoriatic <em>keratinocytes</em> in vivo may be due to inhibition of a mononuclear leukocyte-derived <em>keratinocyte</em> <em>growth</em> <em>factor</em>(s) in combination with direct inhibition of <em>keratinocyte</em> <em>growth</em>.

Alveolar type II cell proliferation occurs after lung injury and is thought to minimize the subsequent fibrotic response. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been shown to be a potent <em>growth</em> <em>factor</em> for rat alveolar type II cells. In this study, we created a replication-deficient, recombinant human type 5 adenovirus vector expressing human KGF (Ad5-KGF) to produce alveolar type II cell hyperplasia in vivo. In rat type II cells in vitro, Ad5-KGF at a multiplicity of infection (MOI) of <em>2</em>, 4, and 8 plaque-forming units (PFU)/cell increased thymidine incorporation 13.3-, 16.8-, and <em>2</em>0. 8-fold, respectively. The KGF concentration in the medium increased up to <em>2</em>6.0 +/- 1.0 ng/ ml. We then instilled 10(9) PFU of Ad5-KGF, Ad5-LacZ, or phosphate-buffered saline into Fischer 344 rats and analyzed the lungs <em>2</em>, 3, 7, 14, <em>2</em>1, and <em>2</em>8 d later. Ad5-KGF produced extensive alveolar type II cell hyperplasia on Days <em>2</em>, 3, and 7. Surfactant protein (SP)-A and SP-D in lavage and SP-D in serum increased more in the Ad5-KGF group than in the Ad5-LacZ and PBS groups on Days <em>2</em> and 3. KGF was readily detectable for up to 7 d in lavage fluid, although only a modest number of cells expressed KGF messenger RNA as detected by in situ hybridization. These data show that Ad5-KGF stimulates extensive alveolar type II cell proliferation in vivo.

With the recent increasing use of nanoparticles, there is concern that they may become an environmental risk <em>factor</em> as airborne particles. However, the impact of these particles on susceptible subjects with predisposing lung disease have not been sufficiently elucidated. In the present study, we investigated the effects of nanoparticles on pulmonary inflammatory and fibrotic changes induced by intratracheal bleomycin (BLM) challenge in mice. Mice were intratracheally administered either vehicle, 14-nm carbon black nanoparticles (CBNPs), BLM or BLM plus CBNP. First, we assessed lung collagen content, lung compliance and fibrotic changes in histopathology on day <em>2</em>1 after instillation. Then, to elucidate how CBNP contributes to the development of BLM-induced fibrosis, we collected bronchoalveolar lavage (BAL) fluid on days <em>2</em>, 7, 14 and <em>2</em>1 and determined the total and differential cell counts and concentrations of two proinflammatory cytokines (<em>keratinocyte</em> chemoattractant [KC] and interleukin [IL]-6) and two fibrogenic mediators (CC chemokine ligand <em>2</em> [CCL<em>2</em>] and transforming <em>growth</em> <em>factor</em>-β(1) [TGF-β(1)]). Expression of nitrotyrosine, an indicator of oxidant injury, was also evaluated on days 7 and <em>2</em>1. CBNP, when combined with BLM, significantly enhanced BLM-induced increase in lung collagen content, decrease in lung compliance, and fibrotic changes in histopathology. CBNP significantly augmented BLM-induced increase in the numbers of inflammatory cells in BAL fluid on days <em>2</em> and 7 and levels of KC and IL-6 on day <em>2</em>. In addition, CBNP administered in combination with BLM significantly elevated the levels of CCL<em>2</em> on days <em>2</em>, 7 and 14, and TGF-β(1) on day 14 in BAL fluid as compared with BLM alone. Nitrotyrosine expression was also increased by BLM plus CBNP compared with BLM alone. In contrast, CBNP did not exert any significant effect on these parameters by itself. These results indicate that CBNP can exaggerate BLM-induced inflammatory and fibrotic changes in the lung, suggesting the potential impact of nanoparticles on lung inflammation and fibrosis.

Oxidative stress due to increased epidermal levels of H(<em>2</em>)O(<em>2</em>) with consequent inhibition of catalase activity is generally accepted as a leading cytotoxic mechanism of melanocyte loss in vitiligo. <em>Keratinocyte</em>-derived cytokines are considered key <em>factors</em> in the maintenance of melanocyte structure and functions. We hypothesized that abnormal redox control may lead to impaired cytokine production by <em>keratinocytes</em>, thus causing noncytotoxic defects in melanocyte proliferation and melanogenesis. We found significantly suppressed mRNA and protein expression of glutathione-S-transferase (GST) M1 isoform, and higher-than-normal levels of both 4-hydroxy-<em>2</em>-nonenal (HNE)-protein adducts and H(<em>2</em>)O(<em>2</em>) in the cultures of <em>keratinocytes</em> derived from unaffected and affected skin of vitiligo patients, and in their co-cultures with allogeneic melanocytes. GST and catalase activities, as well as glutathione levels, were dramatically low in erythrocytes, whilst HNE-protein adducts were high in the plasma of vitiligo patients. The broad spectrum of major cytokines, chemokines, and <em>growth</em> <em>factors</em> was dysregulated in both blood plasma and cultured <em>keratinocytes</em> of vitiligo patients, when compared to normal subjects. Exogenous HNE added to normal <em>keratinocytes</em> induced a vitiligo-like cytokine pattern, and H(<em>2</em>)O(<em>2</em>) overproduction accompanied by adaptive upregulation of catalase and GSTM1 genes, and transient inhibition of Erk1/<em>2</em> and Akt phosphorylation. Based on these results, we suggest a novel GST-HNE-H(<em>2</em>)O(<em>2</em>)-based mechanism of dysregulation of cytokine-mediated <em>keratinocyte</em>-melanocyte interaction in vitiligo.

Phagocytic melanosome uptake by epidermal <em>keratinocytes</em> is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal <em>keratinocytes</em> isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) receptor or the protease-activated receptor-<em>2</em>. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient <em>keratinocytes</em> also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient <em>keratinocytes</em> also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in <em>keratinocytes</em>, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

Both benign hyperplasia (BPH) and cancer of the prostate are manifest in men beyond the age of 50. Approximately 50% of men greater than 50 years of age will suffer from the symptoms associated with BPH, especially from bladder outlet obstruction. With the ever-increasing proportion of the population over 65 years of age worldwide, BPH is becoming an important medical problem as the world moves into the next millennium. Cancer of the prostate is the second most commonly diagnosed cancer after skin cancer in the male population of the United States, and the second most common cause of death from cancer after that of the lung. Overall, around the world the incidence of carcinoma of the prostate is increasing annually by <em>2</em>-3%. Both race and geographical location have a profound influence of the prevalence of prostate cancer worldwide. Black men in the USA have the highest incidence, while the incidence is much lower in Asian men from China, Japan and Thailand. Although the prostate gland is androgen-dependent, it is now recognized that the biological actions of endocrine-related <em>factors</em>, such as androgens, oestrogens, glucocorticoids and certain dietary and environmental <em>factors</em>, are mediated within the gland by various <em>growth</em> regulatory <em>factors</em>. The <em>growth</em> regulatory <em>factors</em> such as epidermal <em>growth</em> <em>factor</em> (EGF), <em>keratinocyte</em> <em>growth</em> <em>factors</em> (KGF), fibroblast <em>growth</em> <em>factors</em> (FGFs) and insulin-like <em>growth</em> <em>factors</em> II and I are mitogenic and directly stimulate cell proliferation under the modulating influence of steroid hormones. Steroids are therefore essential but not directly responsible for cell proliferation. Certain plant compounds such as isoflavonoids, flavonoids and lignans have been proposed as cancer protective compounds in populations with low incidences of prostate diseases. In particular, soya contains the isoflavone genistein, a compound with many properties which could influence both endocrine and <em>growth</em> <em>factor</em> signalling pathways.

The biologic effects of ultraviolet radiation such as DNA damage, mutagenesis, cellular aging, and carcinogenesis are in part mediated by reactive oxygen species. In unirradiated cells the major known sources of reactive oxygen species are the mitochondrial respiratory chain and the membrane oxidases functionally coupled to several membrane <em>growth</em> <em>factor</em> receptors. There is evidence that mitochondria also play a role in oxidative stress after ultraviolet irradiation; however, it is unknown whether the biochemical processes at the level of the plasma membrane contribute to the regulation of reactive oxygen species synthesis. In order to elucidate this issue we examined here the importance of the microdomain plasma membrane organization in the regulation of oxidative stress in unirradiated and ultraviolet A (340-400 nm) irradiated HaCaT <em>keratinocytes</em>. Labeling of confluent HaCaT cultures with fluorescently tagged cholera toxin B subunit (FITC-CTx) revealed the presence of GM1 ganglioside and cholesterol-rich microdomains (lipid rafts) that formed junction-like structures in the membranes of adjacent cells and patchy microdomains elsewhere. There was a marked heterogeneity in the level of FITC-CTx labeling: there were groups of cells demonstrating prominent labeling (FITC-CTx(high)) whereas other cells were only weakly labeled (FITC-CTx(low)). When reactive oxygen species synthesis was measured with the fluorescent probe carboxy-<em>2</em>',7'-dichlorodihydrofluorescein diacetate, we found that (i) the baseline and ultraviolet-A-induced reactive oxygen species synthesis correlated with the magnitude of FITC-CTx labeling and was highest in the FITC-CTx(high) cells; (ii) reactive oxygen species synthesis was diminished in cells in which the integrity of membrane domains was disrupted by cholesterol sequestration with methyl-beta-cyclodextrin and filipin, or after treatment with GM1 ganglioside; (iii) reactive oxygen species synthesis in cholesterol-depleted cells was fully restored after cholesterol repletion. We conclude that the plasma membrane takes part in the regulation of oxidative stress in <em>keratinocytes</em> and disruption of its microdomain structure reduces reactive oxygen species synthesis both at the baseline and after ultraviolet A irradiation. We hypothesize that lipid-raft-associated protein(s) may be involved in the generation of reactive oxygen species and that pharmacologic modulation of membrane structure may provide a novel therapeutic approach relevant for photoprotection and cutaneous carcinogenesis.

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human <em>keratinocyte</em> (NHK) <em>growth</em> in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within <em>2</em>4 h of seeding into culture. This fraction of SPARC+ cells increased with time and peaked immediately postconfluence (31.3 +/- 6.3% SPARC+). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase <em>growth</em>. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G<em>2</em>/M and G1 phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., <em>2</em> days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) production since incubation in the presence of NaB+neutralizing antibodies to TGF-beta inhibited both the expression of SPARC by 7<em>2</em>% and development of mature CEs.