Stem cell <em>factor</em> (SCF), characterized as mast cell <em>growth</em> <em>factor</em>, is known to be produced by fibroblasts, <em>keratinocytes</em> and endothelial cells. Two different splice variants encode for either a soluble (SCF-1) or a membrane-bound (SCF-<em>2</em>) form. In order to explore whether mast cells themselves can produce SCF, we examined cultured cord blood (CBMC) and peripheral-blood-derived mast cells (PBMC), mast/basophil cell lines (HMC-1 and KU-81<em>2</em>), and skin mast cells for SCF expression. On immunocytochemistry, cytoplasmatic SCF-reactivity was observed in HMC-1 cells, with additional cell membrane staining in KU-81<em>2</em>, skin and cultured mast cells. Low amounts of SCF could be detected by ELISA in lysates of isolated and unstimulated mast cells and in supernatants of skin cells stimulated with anti-IgE or Ca-ionophore A<em>2</em>3187. SCF mRNA was detected in all cells, although marked quantitative differences were observed among the various cell types. SCF-<em>2</em> mRNA expression was low in HMC-1 cells while it was marked in skin mast cells, KU-81<em>2</em> cells, CBMC and PBMC. A time-dependent, increasing induction of both SCF forms was seen in CBMC and PBMC during culture. After stimulation with A<em>2</em>3187 and phorbol myristate acetate, an up-regulation of SCF mRNA was noted in HMC-1 and KU-81<em>2</em> cells, without changes in the relationship of the two splice variants. The differential expression of SCF-specific mRNA splice variants in immature and mature human mast cells and the secretion of this molecule by these cells may play a role in autocrine stimulation, maintenance of survival and the differentiation of tissue mast cells.

The novel protein PTPIP51 (protein tyrosine phosphatase-interacting protein 51), which has been found to interact with protein tyrosine phosphatases of the PTP1B/TcPTP subfamily, is expressed in all suprabasal layers of human epidermis. Hence, a human <em>keratinocyte</em> cell line (HaCaT) grown on culture slides was used as a simplified model system to study the influence of hormonal agents on the regulation of PTPIP51 expression. Results were obtained by immunocytochemistry and subsequent statistical analysis. Additionally, immunoblotting was performed to detect the possible occurrence of distinct molecular weight forms as described previously. Subcellular localization of PTPIP51 protein was analyzed by specific staining of cellular organelles. HaCaT cells were subjected to treatment with <em>factors</em> that are crucial for the regulation of proliferation and differentiation of <em>keratinocytes</em> in human epidermis: epidermal <em>growth</em> <em>factor</em> (EGF), transforming <em>growth</em> <em>factor</em>-beta(TGF-beta), retinoic acid (RA) and 1,<em>2</em>5-dihydroxyvitamin D(3) [1,<em>2</em>5(OH)(<em>2</em>)D(3)]. Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) expressed in HaCaT cells was inhibited by PD153035. Only about 35% of untreated HaCaT cells were immunoreactive for the PTPIP51 protein. Whereas cells treated with increasing concentrations of 1,<em>2</em>5 (OH)(<em>2</em>)D(3) showed a stepwise numerical increase of PTPIP51-positive cells, treatment with RA did not influence the number of PTPIP51-positive cells except when supraphysiological concentrations were applied. Concentration-dependent increase of cells stained positive for PTPIP51 was also observed when HaCaT cells were subjected to EGF treatment. Additional treatment of these cells with PD153035 led to a slight decrease in the fraction of PTPIP51-positive cells, which was not statistically significant. Immunoblotting results suggest a specific pattern of different molecular weight forms of PTPIP51 being expressed in HaCaT cells. Subcellular analysis revealed an association of the protein with mitochondria in nonconfluent cells, whereas confluent cells lack such correlation. The intracellular distribution of PTPIP51 resembled the localization of its interacting partner TcPTP. Furthermore, PTPIP51 was found to be present in both the nucleus and cytoplasm of HaCaT cells. In summary, the results indicate a possible association of PTPIP51 expression with differentiation as well as with apoptosis of <em>keratinocytes</em>.

Binding of the coagulation protease <em>factor</em> VIIa to its receptor Tissue <em>Factor</em> (TF) induces intracellular signals in several cell types including HaCaT <em>keratinocytes</em>. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR<em>2</em> is involved in <em>factor</em> VIIa and <em>factor</em> Xa signal transduction. Our results indicate that the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and the proline rich tyrosine kinase <em>2</em> (PYK<em>2</em>) participate in TF/FVIIa signalling as formation of the TF/FVIIa complex increased the phosphorylation of these proteins. Both FVIIa protease activity and available TF were necessary for generation of the signal. Increased tyrosine phosphorylation of the EGFR was observed following TF/FVIIa complex formation on the cell surface. The EGFR kinase inhibitor tyrphostin AG1478 abrogated the TF/FVIIa-complex induced MAP kinase activation and mRNA increase of egr-1, heparin-binding EGF, and interleukin-8 following FVIIa addition. Using specific antibodies, increased phosphorylation of PYK<em>2</em> tyrosine residues 40<em>2</em> and 580 was observed. The first site is the major autophosphorylation site and the docking site for Src family kinases. The second site is important for the kinase activity. The Src family kinase Yes and the tyrosine phosphatase SHP-<em>2</em> were detected in immunoprecipitates using either anti-PYK<em>2</em> or anti-EGFR antibodies. Their coprecipitation with EGFR increased in the presence of FVIIa. Moreover, the coprecipitation of EGFR and PYK<em>2</em> increased with FVIIa stimulation. Together, these data suggest that EGFR, PYK<em>2</em>, Yes, and SHP-<em>2</em> are involved in transduction of the TF/FVIIa signal possibly via transactivation of the EGF receptor.

The role of matrix metalloproteases and their regulation in the pathology of middle ear cholesteatoma is still unclear. Recently we have demonstrated that incubation of <em>keratinocytes</em> with cholesteatoma debris and granulation tissue extracts causes induction of gelatinase B (matrix metalloproteinase-9, MMP-9) secretion in vitro. Antibodies against a variety of <em>growth</em> <em>factors</em> revealed some inhibitory effect on MMP-9 induction, caused by debris or granulation tissue extracts. In order to investigate the coherence of <em>growth</em> <em>factor</em> expression and matrix metalloproteinase activity in vivo in middle ear cholesteatoma, we performed quantitative gelatin zymographic analysis with tissue homogenates of 37 cholesteatoma and nine external ear canal skin (EACS) samples. Furthermore we quantified levels of the cytokines IL-1alpha, IL-1beta, TNF-alpha, TGF-beta and EGF present in tissue extracts, using enzyme-linked immunosorbent assays (ELISA), and correlated cytokine concentrations with gelatinolytic activities. Zymographic analysis revealed a highly heterogeneous expression of gelatinase A and B in cholesteatoma specimens. As shown previously, MMP-9, but not MMP-<em>2</em>, was increased in cholesteatoma when compared to EACS samples. ELISA studies revealed a significantly elevated IL-1alpha level in cholesteatoma. Regression analysis involving gelatinolytic activity and cytokine concentrations in tissue homogenates showed no statistically significant correlation between expression of gelatinases and the cytokines IL1-alpha, IL1-beta, TNF-alpha, TGF-beta or EGF. The discrepancy between in vitro observations and the situation in vivo is discussed critically.

OBJECTIVE

To determine the effect of treatment with keratinocyte growth factor (KGF) on the survival of cells in cultured preantral follicles and on the growth and differentiation of preantral follicles.

METHODS

Preantral follicles (140-150 microm) were dissected mechanically from the ovaries of 14-day-old rats and cultured for 24 hours with and without KGF. Genomic DNA was extracted, labeled with [32P]-dideoxyadenosine triphosphate, and fractionated through agarose gels. For growth studies, the follicles were cultured individually in 96-well dishes. After 72 hours, the follicles were collected and their protein or DNA content was evaluated and their inhibin-alpha content was determined.

RESULTS

Keratinocyte growth factor suppressed apoptosis in cultured preantral follicles by 60%. Treatment with KGF or FSH increased follicle diameter by 8% and 16%, respectively, and combined treatment with KGF and FSH increased follicle diameter by 26%. Western blot analysis demonstrated increased expression of inhibin-alpha content after treatment with KGF (2-fold), treatment with FSH (4-fold), and combined treatment with FSH and KGF (12-fold), demonstrating the effect of KGF on preantral follicle differentiation.

CONCLUSIONS

Treatment with KGF promotes the survival, growth, and differentiation of cultured preantral follicles. Keratinocyte growth factor produced by theca cells may play a role in the progression of early follicle development.

Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human <em>keratinocyte</em> <em>growth</em> <em>factor</em> would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of <em>keratinocyte</em> <em>growth</em> <em>factor</em>, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When <em>keratinocyte</em> <em>growth</em> <em>factor</em> treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with <em>2</em>% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of <em>keratinocyte</em> <em>growth</em> <em>factor</em>, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.

To investigate the role of transforming <em>growth</em> <em>factor</em> alpha (TGF alpha) in tumor development, we introduced the human TGF alpha (hTGF alpha) cDNA into cultured primary mouse epidermal cells or papilloma cells using a replication-defective retroviral vector and analyzed skin grafts constructed with such cells. Expression of the exogenous gene was confirmed by detection of hTGF alpha mRNA by northern RNA blot analysis, and the secreted hTGF alpha was measured by ELISA of culture supernatants. Tumor cells expressing hTGF alpha produced benign tumors (papillomas), which were 1.5- to <em>2</em>-fold larger than tumors of parental cells when tested as skin grafts on nude mice. Grafts of normal cells that expressed hTGF alpha produced normal skin. When mixtures of parental tumor cells and normal mouse <em>keratinocytes</em> were grafted to nude mice, papilloma formation was suppressed and tumors that did form were small. Grafts of hTGF alpha-producing papilloma cells combined with either normal epidermal cells or hTGF alpha-producing epidermal cells yielded large tumors. Mixed grafts containing <em>keratinocytes</em> expressing hTGF alpha and parental papilloma cells also produced large tumors. While the tumor size was substantially increased by hTGF alpha expression, the tumors that developed in all groups were histologically benign and reached a stable size 4-5 wk after grafting. These results indicate that expression of hTGF alpha by either tumor cells (autocrine) or adjoining normal cells (paracrine) can stimulate tumor <em>growth</em>, particularly when tumor <em>growth</em> is suppressed by normal tissue. However, expression of this <em>growth</em> <em>factor</em> did not appear to influence tumor progression directly.

BACKGROUND

Cellular therapy is a novel treatment option for intestinal ischemia. Bone marrow-derived mesenchymal stromal cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion (I/R) injury. We therefore hypothesized that (1) human BMSCs (hBMSCs) would produce more beneficial <em>growth</em> <em>factors</em> and lower levels of proinflammatory mediators compared to differentiated cells, (<em>2</em>) direct application of hBMSCs to ischemic intestine would decrease mortality after injury, and (3) decreased mortality would be associated with an altered intestinal and hepatic inflammatory response.

METHODS

Adult hBMSCs and keratinocytes were cultured on polystyrene flasks. For in vitro experiments, cells were exposed to tumor necrosis factor, lipopolysaccharides, or <em>2</em>% oxygen for <em>2</em>4 h. Supernatants were then analyzed for <em>growth</em> <em>factors</em> and chemokines by multiplex assay. For in vivo experiments, 8- to 1<em>2</em>-wk-old male C57Bl6J mice were anesthetized and underwent a midline laparotomy. Experimental groups were exposed to temporary superior mesenteric artery occlusion for 60 min. Immediately after ischemia, <em>2</em> × 10(6) hBMSCs or keratinocytes in phosphate-buffered saline were placed into the peritoneal cavity. Animals were then closed and allowed to recover for 6 h (molecular/histologic analysis) or 7 d (survival analysis). After 6-h reperfusion, animals were euthanized. Intestines and livers were harvested and analyzed for inflammatory chemokines, <em>growth</em> <em>factors</em>, and histologic changes.

RESULTS

hBMSCs expressed higher levels of human interleukin (IL) 6, IL-8, vascular endothelial growth factor (VEGF), and epidermal growth factor and lower levels of IL-1, IL-3, IL-7, and granulocyte-monocyte colony-stimulating factor after stimulation. In vivo, I/R resulted in significant mortality (70% mortality), whereas application of hBMSCs after ischemia decreased mortality to 10% in a dose-dependent fashion (P = 0.004). Keratinocyte therapy offered no improvements in mortality above I/R. Histologic profiles were equivalent between ischemic groups, regardless of the application of hBMSCs or keratinocytes. Cellular therapy yielded significantly decreased murine intestinal levels of soluble activin receptor-like kinase 1, betacellulin, and endothelin, whereas increasing levels of eotaxin, monokine induced by gamma interferon (MIG), monocyte chemoattractant protein 1, IL-6, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein 10 (IP-10) from ischemia were appreciated. hBMSC therapy yielded significantly higher expression of murine intestinal VEGF and lower levels of intestinal MIG compared to keratinocyte therapy. Application of hBMSCs after ischemia yielded significantly lower murine levels of hepatic MIG, IP-10, and G-CSF compared to keratinocyte therapy.

CONCLUSIONS

Human BMSCs produce multiple beneficial growth factors. Direct application of hBMSCs to the peritoneal cavity after intestinal I/R decreased mortality by 60%. Improved outcomes with hBMSC therapy were not associated with improved histologic profiles in this model. hBMSC therapy was associated with higher VEGF in intestines and lower levels of proinflammtory MIG, IP-10, and G-CSF in liver tissue after ischemia, suggesting that reperfusion with hBMSC therapy may alter survival by modulating the systemic inflammatory response to ischemia.

Hyperproliferative diseases of the epidermal <em>keratinocytes</em>, such as psoriasis vulgaris, are characterized by overexpression and altered distribution of the epidermal <em>growth</em> <em>factor</em>/transforming <em>growth</em> <em>factor</em> (EGF/TGF)-alpha receptor, and of TGF-alpha itself. It is believed that overexpression of this lignad/receptor system contributes to the hyperproliferative state of <em>keratinocytes</em> in an autocrine fashion. However, little is known about the <em>factors</em> that regulate expression of the EGF/TGF-alpha receptor, as well as expression of TGF-alpha in stratified epithelium. We examined modulation of the immunoreactive EGF/TGF-alpha receptor and TGF-alpha expression in normal neonatal foreskin explants by a variety of cytokines present in psoriatic lesions. Human (hu) recombinant (r) tumor necrosis <em>factor</em> (TNF)-alpha and interferon (IFN)-gamma induced EGF/TGF-alpha receptor and TGF-alpha expression by <em>keratinocytes</em> as determined by immunohistochemistry. Neutralizing antibodies to TNF-alpha and IFN-gamma inhibited upregulation of EGF/TGF-alpha receptors and TGF-alpha by the respective cytokines. Interleukin (IL)-8 induced expression of TGF-alpha, but not of its receptor. Other cytokines (TNF-beta, IFN-beta, IL-1 alpha, IL-<em>2</em>, IL-3, IL-5, IL-6, granulocyte/macrophage colony-stimulating <em>factor</em>, and macrophage colony-stimulating <em>factor</em>) did not alter the expression patterns of EGF/TGF-alpha receptors or TGF-alpha in normal neonatal skin explants. These experiments demonstrate that specific cytokines known to be present in psoriatic lesions can induce normal epidermis to express TGF-alpha and its receptor in a pattern similar to that observed in psoriatic skin.

The skin-associated lymphoid tissue is composed of <em>keratinocytes</em>, Langerhans cells, skin trophic T cells, and lymphatic endothelial cells of the skin. The epidermis, which is involved in many viral infections, contains all of the components needed for an effective immune response: antigen-presenting Langerhans cells, T cells, and cytokines from leukocytes and <em>keratinocytes</em>. There have been some recent advances in the study of the cutaneous immunology involved in infections with the human immunodeficiency virus (HIV), human papillomavirus (HPV), and herpes simplex virus (HSV). In general, viral diseases with cutaneous manifestations lead to a decline in epidermal Langerhans cell numbers, which probably reflects Langerhans cell emigration out of the epidermis and entry into regional lymph nodes, leading to Langerhans cell activation and antigen presentation to T cells. In HSV, there is a subsequent T-cell infiltration of the epidermis, composed of CD4+ cells that have both immune modulatory action and direct cytotoxic action. In HIV, where there is a systemic depletion of CD4+ cells, the epidermis is left with reduced numbers of T cells. Intradermal injection of interleukin-<em>2</em>, however, leads to an epidermal cellular infiltration in HIV+ individuals. In HPV-induced condyloma, intralesional interferon increases Langerhans cells and CD4+ and CD8+ cells in the skin, as well as transforming <em>growth</em> <em>factor</em> beta 1, tumor necrosis <em>factor</em>-alpha, pRB, and p53. Therefore, viral infections involving the epidermal immune system have certain similar characteristics, whereas other <em>factors</em> are unique to the infecting virus.

OBJECTIVE

Regulation of epithelial cell behavior associated with periodontitis is not well elucidated but many responses will ultimately be regulated by growth factor receptors. Using a rat experimental periodontitis model, protein and gene expression of select growth factor receptors in junctional and pocket epithelium were examined.

METHODS

Periodontal disease was induced by daily topical application of lipopolysaccharide using an established protocol. Animals were killed at time 0 (control), and at 2 and 8 wk. Frozen tissue samples were collected from the right palatal gingival soft tissue, and the left periodontal tissues were decalcified and embedded in paraffin. Laser microdissection and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify keratinocyte growth factor receptor (KGFR), hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) gene expression, and in situ RT-PCR localized these increases to specific epithelial cells. Receptor protein expression was examined immunohistochemically. In cell culture, induction of HGFR and KGFR protein expression by serum, lipopolysaccharide and pro-inflammatory cytokines were examined using flow cytometry.

RESULTS

Eight-week tissue samples exhibited histological changes consistent with periodontitis. KGFR and HGFR gene and protein expression were significantly induced at the 8 wk time point. KGFR expression was significantly up-regulated in basal and parabasal pocket epithelial cells, but HGFR was up-regulated throughout the pocket epithelium. In cell culture serum, lipopolysaccharide and pro-inflammatory cytokines, interleukin-1beta and tumour necrosis factor-alpha significantly induced KGFR protein receptor expression, but HGFR expression was only induced by serum.

CONCLUSIONS

KGFR and HGFR are highly up-regulated in this model of periodontal disease and may play a significant role in regulating the proliferation and migration of pocket epithelium.

<em>Keratinocytes</em> are the cells in vertebrates that form the frontline barrier to the environment, and are also the most common origin of human cancer. They normally retain tight cell-cell adhesion and low motility, allowing them to terminally differentiate as they stratify. However, they must be able to respond to tissue damage by migrating into and across wounds. This requires reduced mutual adhesion, suppressed terminal differentiation and increased motility, processes driven by the Snail family of transcriptional repressors. The quantity, location and activity of Snail proteins are regulated by <em>growth</em> <em>factors</em> and cytokines to mediate these responses and invoke an inflammatory response. Subversion of these same pathways can promote carcinoma invasion and metastasis. Signaling network facts: • Snail1 and Snail<em>2</em> in <em>keratinocytes</em> are important in promoting migration, inflammation and carcinogenesis, and suppressing terminal differentiation. • Extracellular stimuli, including TGFR and EGFR ligands, regulate Snails transcriptionally, via SMAD and MAPK pathways, and post-translationally, by modulating GSK3 and PAK1 activity, which determine Snail stability and intracellular location. • Snails directly repress transcription of genes important for cell-cell adhesion and cornified envelope formation. • Down-regulation of epithelial cadherins by Snails allows LIMDPs to relocate from adherens junctions to the cytoplasm, where they stimulate MAPK pathways, and to the nucleus, where they bind directly to Snails and act as corepressors. • Snail<em>2</em> is essential for re-epithelialization of healing wounds and can be up-regulated in the <em>keratinocytes</em> at wound margins by p38, ERK1/<em>2</em> and ERK5 MAPKs, and the arylhydrocarbon receptor. • Further information on signaling related to Snail proteins can be found online at KEGG: http://www.genome.jp/kegg-bin/show pathway?hsa045<em>2</em>0 http://www.genome.jp/kegg-bin/show_pathway?hsa04350 http://www.genome.jp/kegg-bin/show pathway?hsa0401<em>2</em>.

Routine treatment of burns with cultured skin substitutes (CSS) has been limited by poor engraftment and by scarring. Hypothetically, topical application of essential nutrients and/or <em>growth</em> <em>factors</em> may support epithelial survival temporarily during graft vascularization. CSS, composed of human epidermal <em>keratinocytes</em> and dermal fibroblasts attached to collagen-glycosaminoglycan substrates, were incubated for 19 d in media optimized for <em>keratinocytes</em>. CSS, human xenografts, murine autografts, or no grafts were applied orthotopically to full-thickness skin wounds (<em>2</em> x <em>2</em> cm) in athymic mice. Wounds were irrigated for 14 d with 1 ml/d modified cell culture medium or with saline containing epidermal <em>growth</em> <em>factor</em>, or were treated with dry dressings. After 6 weeks, treated sites were scored for percentage original wound area (mean +/- SEM) and percentage HLA-ABC-positive healed wounds [(number positive/n) x 100], and tested for significance (analysis of variance, p < 0.0001; Tukey test, p < 0.05). The data showed that CSS irrigated with nutrient medium were not statistically different in wound area (67.8 +/- 5.1%) from murine autografts (63.3 +/- <em>2</em>.9%) but were statistically larger than human xenograft, no graft, or CSS treated with saline irrigation or dry dressings. HLA-ABC expression was 100% in CSS with nutrient irrigation, 86% in CSS with saline irrigation, 83% in CSS without irrigation, and 75% in xenografts with nutrient irrigation. These findings suggest that availability of essential nutrients supports <em>keratinocyte</em> viability during graft vascularization of CSS.

Meshed split-thickness skin grafts, especially when required to be widely spread, do not obtain immediate biologic wound closure. In cases of patients with burns that cover a large percentage of the body surface area, this leaves the patient at risk for metabolic problems and life-threatening infection. Several cytokines and <em>growth</em> <em>factors</em> could theoretically affect the rate of epithelialization and, therefore, the rate of meshed graft interstitial closure. With the use of human meshed skin grafts explanted onto athymic "nude" rats, the epithelialization kinetics of interleukin-4 (IL-4), macrophage colony-stimulating <em>factor</em> (MCSF), <em>keratinocyte</em> <em>growth</em> <em>factor</em>-1 (KGF-1), <em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), basic fibroblast <em>growth</em> <em>factor</em> (bFGF), and transforming <em>growth</em> <em>factor</em> beta-<em>2</em> (TGF(B<em>2</em>)) were investigated; the results were compared with the rates of epithelialization of grafts treated with a vehicle control. On postoperative day 3, wounds treated with IL-4, KGF-<em>2</em>, bFGF, and TGF(B<em>2</em>) showed a significantly increased rate of interstitial closure (P < .05). On postoperative days 5 and 7, wounds treated with KGF-<em>2</em>, bFGF, and TGF(B<em>2</em>) all exhibited a significantly higher rate of interstitial closure than the grafts in the control group (P < .05). These data suggest that epithelialization kinetics can be accelerated with the use of several topical <em>growth</em> <em>factors</em>, and they provide support for a future clinical trial.

Epidermal <em>growth</em> <em>factor</em> receptor tyrosine kinase (EGFR-TK) is a transducer of mitogenic signals, and is involved in the pathogenesis and progression of a number of cancers, including non-small cell lung cancer (NSCLC). Gefitinib is an EGFR-TK inhibitor that is clinically used to treat NSCLC; however, this drug frequently causes adverse effects, including skin eruptions. The mechanism underlying these skin reactions is elusive, although it is assumed that they are caused by the inhibition of EGFR-TK signalling in epidermal and adnexal cells. In this article, we demonstrate by immunocytochemistry that the skin lesions of patients treated with oral gefitinib had higher expression of CCL<em>2</em> and CCL5 compared to normal human epidermis. Further, PD153035, a gefitinib prototype, induced CCL<em>2</em> and CCL5 mRNA and protein expression in HaCaT and HSC-1 <em>keratinocyte</em> cell lines with or without interleukin-1 (IL-1) treatment in vitro. PD153035 also reduced the levels of interleukin-1 receptor <em>2</em> (IL-1R<em>2</em>), an IL-1 decoy receptor. Moreover, we demonstrate that reduction in IL-1R<em>2</em> by RNA interference increased IL-1-mediated CCL<em>2</em> and CCL5 mRNA and protein expression. Taken together, our data strongly suggest that IL-1-mediated signalling is activated to induce the high expression of CCL<em>2</em> and CCL5 via reduction in IL-1R<em>2</em> in the skin lesions caused by gefitinib.

The importance of epidermal <em>growth</em> <em>factor</em> (EGF) receptor expression level and autophosphorylation sites in src homology and collagen protein (SHC) tyrosine phosphorylation has been studied. In contrast to EGF-induced tyrosine phosphorylation of the GTPase-activating protein for ras (rasGAP) and phospholipase C-gamma 1 (PLC-gamma 1), SHC tyrosine phosphorylation occurs at a very low receptor density in parental NIH3T3 mouse fibroblasts expressing less than 1 x 10(4) EGF receptors per cell. In transfected NIH3T3 cells expressing human EGF receptors (approximately 4 x 10(5) receptors per cell), maximal levels of SHC and PLC-gamma 1 tyrosine phosphorylation occur when approximately 4 x 10(4) receptors or more are occupied by ligand. At lower levels of receptor occupancy only SHC phosphorylation was significant. Also, EGF treatment of mouse <em>keratinocytes</em>, which represent a physiological target of EGF, express a low number of EGF receptors (approximately <em>2</em> x 10(4) receptors per cell), and stringently require EGF to grow, results in intense SHC tyrosine phosphorylation, compared to rasGAP or PLC-gamma 1. SHC is also efficiently tyrosine phosphorylated by an EGF receptor deletion mutant (Dc<em>2</em>14) that is devoid of autophosphorylation sites, but which remains mitogenically responsive to EGF. The EGF receptor mutant Dc<em>2</em>14 is able to activate the ras guanine nucleotide exchanger and phosphorylate mitogen-activated protein kinase (MAPK), presumable as a result of complex formation between tyrosine phosphorylated SHC and GRB<em>2</em>. These results indicate that potent EGF-induced SHC tyrosine phosphorylation can be triggered in cells having relatively few receptors. Also, our data show that EGF receptors are able to phosphorylate SHC, activate the exchange of guanine nucleotide on ras and phosphorylate MAPK by a mechanism that does not require receptor autophosphorylation sites and, therefore, the src homology <em>2</em> (SH<em>2</em>):phosphotyrosine-dependent interaction of SHC or GRB<em>2</em> with the EGF receptor.

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human <em>keratinocyte</em> <em>growth</em> <em>factor</em> (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured <em>2</em> days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.0<em>2</em>8 transduction events/mm<em>2</em>, respectively). Amphotropic vectors use Pit-<em>2</em> for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-<em>2</em> for cell entry. By in situ hybridization the retroviral receptor Pit-<em>2</em> (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.

The interaction of several of the fibroblast <em>growth</em> <em>factors</em> (FGFs) with polyanions is thought to be of physiological significance and has been exploited to create more stable pharmaceutical formulations of FGF-1 and -<em>2</em>. The extent of such phenomena throughout the <em>2</em>3-member FGF family is, however, unknown. In these studies, we examine the effect of several polyanions on the structure and stability of <em>keratinocyte</em> <em>growth</em> <em>factor</em> <em>2</em> (KGF-<em>2</em>, FGF-10), a candidate for use as a wound-healing agent. Employing a variety of methods sensitive to the protein's structure including circular dichroism (CD), intrinsic fluorescence, derivative near-UV absorption spectroscopy, bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid) fluorescence, differential scanning calorimetry (DSC), and dynamic light scattering (DLS), we find that a variety of polyanions (e.g., heparin, sucrose octasulfate (SOS), and inositol hexaphosphate (IHP)) stabilize KGF-<em>2</em> by increasing the thermal-unfolding temperature by approximately 9-15 degrees C. Negatively charged liposomes produce a similar effect, arguing for relatively nonspecific interactions of polyanions with KGF-<em>2</em>. Unlike some other FGFs, no evidence for the presence of a molten globule state is found during thermal perturbation of this <em>growth</em> <em>factor</em>. The generality of this polyanion/protein interaction is discussed as well as its potential role in various cellular events such as protein folding and transport.

In vitro, normal human <em>keratinocytes</em> reconstitute a differentiated stratified epidermis, maintaining the same gene expression pattern as its in vivo counterpart and are suitable for permanent grafting onto patients. <em>Keratinocyte</em> adhesion to basal lamina and lateral interactions among basal epidermal cells are also mediated by integrin receptors that are sorted to defined plasma membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized at the basal surface of basal cells and codistributes with laminin and nicein/kalinin; the alpha <em>2</em> beta 1 and alpha 3 beta 1 integrins are enriched laterally and play crucial roles in cell-cell interaction and proper colony morphology. During wound healing, proliferating and migrating <em>keratinocytes</em> express on their plasma membrane alpha v beta 5 and alpha 5 beta 1, which allow <em>keratinocyte</em> attachment and migration over the provisional matrix present in the wound. TGF beta, which is an autocrine and paracrine mediator in wound healing, specifically increases the synthesis and expression of alpha v beta 5 and alpha 5 beta 1, induces the de novo expression of alpha v beta 6, and determines the loss of integrin polarization. In hyperproliferative skin diseases, such as skin cancer or psoriasis vulgaris, and in normal <em>keratinocytes</em> forced into more frequent cell cycles, the polarized expression of integrins is lost, and alpha 5 beta 1 becomes costitutively expressed on the plasma membrane. In addition, the alpha 6 beta 4 integrin becomes associated with focal contacts. Nerve <em>growth</em> <em>factor</em> (NGF) is a potent autocrine stimulator of <em>keratinocyte</em> <em>growth</em> and induces melanocyte migration toward the leading edge of a healing wound.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a <em>keratinocyte</em> stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose <em>growth</em> was stimulated by epidermal <em>growth</em> <em>factor</em> (EGF) and basic fibroblast <em>growth</em> <em>factor</em> (FGF-<em>2</em>). FGF-<em>2</em> stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the <em>growth</em>-stimulating effect of FGF-<em>2</em> was mediated through FGFR1. EGF and FGF-<em>2</em> also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the <em>factors</em> that regulate the <em>growth</em> and differentiation of lingual and gustatory epithelial cells in vitro.

Recent studies have shown a correlation between an increased number of mast cells in patients with atopic dermatitis (AD) resulting in raised plasma levels of nerve <em>growth</em> <em>factor</em> (NGF), pointing to a possible key role of their interaction in the pathogenesis of AD. It is well known that mast cells synthesize, store and release NGF. Mast cells and NGF both appear to be involved in tissue inflammation and neuroimmune interactions, with NGF acting as a general "alert" molecule capable of recruiting and priming both local tissue and systemic defense processes following stressful events. Also, NGF has been demonstrated to increase mast cell histamine content and intracellular tryptase activity in a dose- and time-dependent fashion. Endogenous aliamides are capable of down-regulating mastocyte reactivity by their action through the vanilloid (VR1) receptors, and <em>keratinocytes</em>, and through the CB1 and CB<em>2</em> cannabinoid receptors linked to G-protein, also expressed by sensitive nerve endings, macrophages, and epithelial cells. Therefore, aliamide action should be regarded as a multifaceted mechanism interfering with the inflammatory process occurring in AD further beyond the known and controversial anti-histamine pharmacologic effect. In this regard, the reduction of mast cell degranulation by adelmidrol, as demonstrated by in vitro and in vivo investigations in animals, would interfere with the release of other inflammatory mediators, including NGF. Based on these considerations, a pilot study aimed to assess the efficacy and safety of twice daily application of a topical emulsion containing adelmidrol <em>2</em>%, a novel aliamide, in a series of <em>2</em>0 patients (11 male and 9 female, mean age 8 (range 3-16) years) affected by mild AD was performed. Complete resolution with no side effects was observed in 16 (80%) patients after 4 weeks of treatment, with no relapses at 8-week follow up. Six patches in six subjects with multiple lesions that had not been treated and served as controls showed no improvement. Controlled clinical studies in larger series are warranted to confirm the efficacy of aliamide in the management of AD.

Gram-negative bacteria naturally produce and secrete nanosized outer membrane vesicles (OMVs). In the human gastrointestinal tract, OMVs produced by commensal Gram-negative bacteria can mediate interactions amongst host cells (including between epithelial cells and immune cells) and maintain microbial homeostasis. This OMV-mediated pathway for host-microbe interactions could be exploited to deliver biologically active proteins to the body. To test this we engineered the Gram-negative bacterium <i>Bacteroides thetaiotaomicron</i> (Bt), a prominent member of the intestinal microbiota of all animals, to incorporate bacteria-, virus- and human-derived proteins into its OMVs. We then used the engineered Bt OMVs to deliver these proteins to the respiratory and gastrointestinal (GI)-tract to protect against infection, tissue inflammation and injury. Our findings demonstrate the ability to express and package both <i>Salmonella enterica</i> ser. Typhimurium-derived vaccine antigens and influenza A virus (IAV)-derived vaccine antigens within or on the outer membrane of Bt OMVs. These antigens were in a form capable of eliciting antigen-specific immune and antibody responses in both mucosal tissues and systemically. Furthermore, immunisation with OMVs containing the core stalk region of the IAV H5 hemagglutinin from an H5N1 strain induced heterotypic protection in mice to a 10-fold lethal dose of an unrelated subtype (H1N1) of IAV. We also showed that OMVs could express the human therapeutic protein, <em>keratinocyte</em> <em>growth</em> <em>factor</em>-<em>2</em> (KGF-<em>2</em>), in a stable form that, when delivered orally, reduced disease severity and promoted intestinal epithelial repair and recovery in animals administered colitis-inducing dextran sodium sulfate. Collectively, our data demonstrates the utility and effectiveness of using Bt OMVs as a mucosal biologics and drug delivery platform technology.

Our recent epidemiological study (Ahonen et al., Cancer Causes Control 11(<em>2</em>000) (847-85<em>2</em>)) suggests that vitamin D deficiency may increase the risk of initiation and progression of prostate cancer. The nested case-control study was based on a 13-year follow-up of about 19000 middle-aged men free of clinically verified prostate cancer. More than one-half of the serum samples had <em>2</em>5OH-vitamin D (<em>2</em>5-VD) levels below 50 nmol/l, suggesting VD deficiency. Prostate cancer risk was highest among the group of younger men (40-51 years) with low serum <em>2</em>5-VD, whereas low serum <em>2</em>5-VD appeared not to increase the risk of prostate cancer in older men (>51 years). This suggests that VD has a protective role against prostate cancer only before the andropause, when serum androgen concentrations are higher. The lowest <em>2</em>5-VD concentrations in the younger men were associated with more aggressive prostate cancer. Furthermore, the high <em>2</em>5-VD levels delayed the appearance of clinically verified prostate cancer by 1.8 years. Since these results suggest that vitamin D has a protective role against prostate cancer, we tried to determine whether full spectrum lighting (FSL) during working hours could increase serum <em>2</em>5-VD concentrations. After 1-month exposure, there was no significant increase in the serum <em>2</em>5-VD level, although there was a bias towards slightly increasing values in the test group as opposed to decreasing values in controls. There was no significant change in the skin urocanic acid production. The possibility to use FSL in cancer prevention is discussed. In order to clarify the mechanism of VD action on cell proliferation and differentiation, we performed studies with the rat and human prostates as well prostate cancer cell lines. It is possible that <em>2</em>5-VD may have a direct role in the host anticancer defence activity, but the metabolism of vitamin D in the prostate may also play an important role in its action. We raised antibodies against human 1alpha-hydroxylase and <em>2</em>4-hydroxylase. Our preliminary results suggest that vitamin D is actively metabolised in the prostate. Vitamin D appears to upregulate androgen receptor expression, whereas androgens seem to upregulate vitamin D receptor (VDR). This may at least partially explain the androgen dependence of VD action. VD alone or administered with androgen causes a suppression of epithelial cell proliferation. VD can activate mitogen-activated kinases, erk-1 and erk-<em>2</em>, within minutes and p38 within hours. Also, auto/paracrine regulation might be involved, since <em>keratinocyte</em> <em>growth</em> <em>factor</em> (mRNA and protein) was clearly induced by VD. Based on these studies, a putative model for VD action on cell proliferation and differentiation is presented.

In order to isolate genes that might be involved in regulating human <em>keratinocyte</em> (HKc) <em>growth</em> and/or differentiation, we constructed a cDNA library by subtractive hybridization between primary HKc and FaDu head-and-neck squamous cell carcinoma cells. Among the first set of independent cDNAs that we have isolated, ten correspond to known genes, and two represent novel sequences. Nine of the ten known genes are expressed at significantly lower levels in the majority of the SqCC cell lines in comparison with primary HKc. These include cDNAs that encode keratins K5 and K14 which are cytoskeletal proteins normally expressed in lining epithelia, the 14-3-3 protein stratifin/HME-1, lipocortin-II and CaN19 which are calcium-binding proteins that may play a role in HKc differentiation by regulating protein kinase C, plasminogen-activator inhibitor-<em>2</em> which is a serine-proteinase inhibitor, HBp17 which is a HKc-specific secreted inhibitor of fibroblast <em>growth</em> <em>factors</em>, integrin alpha 3 which plays a role in the anchoring of <em>keratinocytes</em> to basement membrane, and YL-8, a ras-like protein that probably mediates intracellular protein trafficking. In addition, we isolated two cDNAs, LIS-1 which encodes the 45-kDa intracellular subunit of the platelet-activating <em>factor</em> acetyl-hydrolase, and the unknown sequence HFBCB84 which showed reduced expression in only a small number of tumor lines as compared to HKc. Inactivation or loss of any of these proteins may confer a selective advantage onto squamous epithelial cells and contribute to their malignant transformation.