A tibia fracture cast immobilized for 4 weeks can induce exaggerated substance P and calcitonin gene-related peptide signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome (CRPS). Four weeks of hind limb cast immobilization can also induce nociceptive and vascular changes resembling CRPS. To test our hypothesis that immobilization alone could cause exaggerated neuropeptide signaling and inflammatory changes, we tested 5 cohorts of rats: 1) controls; <em>2</em>) tibia fracture and hind limb casted; 3) hind limb casted, no fracture; 4) tibia fracture with intramedullary pinning, no cast; and 5) tibia fracture with intramedullary pinning and hind limb casting. After 4 weeks, the casts were removed and hind limb allodynia, unweighting, warmth, edema, sciatic nerve neuropeptide content, cutaneous and spinal cord inflammatory mediator levels, and spinal c-Fos activation were measured. After fracture with casting, there was allodynia, unweighting, warmth, edema, increased sciatic nerve substance P and calcitonin gene-related peptide, increased skin neurokinin 1 receptors and <em>keratinocyte</em> proliferation, increased inflammatory mediator expression in the hind paw skin (tumor necrosis <em>factor</em>-α, interleukin [IL]-1β, IL-6, nerve <em>growth</em> <em>factor</em>) and cord (IL-1β, nerve <em>growth</em> <em>factor</em>), and increased spinal c-Fos activation. These same changes were observed after cast immobilization alone, except that spinal IL-1β levels were not increased. Treating cast-only rats with a neurokinin 1 receptor antagonist inhibited development of nociceptive and inflammatory changes. Four weeks after fracture with pinning, all nociceptive and vascular changes had resolved and there were no increases in neuropeptide signaling or inflammatory mediator expression.

CONCLUSIONS

Collectively, these data indicate that immobilization alone increased neuropeptide signaling and caused nociceptive and inflammatory changes similar to those observed after tibia fracture and casting, and that early mobilization after fracture with pinning inhibited these changes. Early limb mobilization after fracture may prevent the development of CRPS.

The effects exerted by the <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) on intestinal epithelial cells cultured in vitro are influenced by cell confluence and differentiation through the modulation of <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR) expression. In order to better define the contribution of KGF on the intestinal epithelial cell differentiation and proliferation, here we developed a coculture model, able to mimick in vitro the epithelial-mesenchymal interactions of the bowel. In consequence of its ability to produce KGF, demonstrated by real-time PCR and Western blot analysis, the human colon fibroblast cell line CCD-18 has been selected as coculture partner for the intestinal epithelial Caco-<em>2</em> cell line. Analysis of the expression of the differentiation and proliferation markers CEA and Ki67, through double immunofluorescence assays, showed that either the coculture with CCD-18 cells or the incubation with primary colon fibroblast-derived conditioned media (CM-F and CM-F<em>2</em>) induced an increase in differentiation and proliferation of confluent intestinal epithelial Caco-<em>2</em> or HT<em>2</em>9 cells, parallel to that obtained by KGF treatment. Use of anti-KGF blocking antibodies and of a tyrosine kinase KGFR inhibitor demonstrated the contribution of KGF and the direct role of its receptor in the regulation of epithelial <em>growth</em> and differentiation, indicating that KGF is a crucial paracrine <em>factor</em> involved in promoting these effects.

Human <em>keratinocytes</em> are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro <em>growth</em>-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-<em>2</em> against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether <em>keratinocytes</em> are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary <em>keratinocytes</em> to living conidia of T. rubrum. This led to conidia germination and mycelial <em>growth</em> which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-<em>2</em> were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the <em>keratinocytes</em> with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary <em>keratinocytes</em> incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the <em>keratinocytes</em> to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The <em>growth</em> of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that <em>keratinocytes</em> are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.

The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis <em>factor</em>-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in <em>keratinocytes</em>. TRIP mRNA expression was strongly downregulated in primary human <em>keratinocytes</em> undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-1<em>2</em>-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative <em>keratinocytes</em> suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. <em>Keratinocytes</em> undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused <em>growth</em> arrest in the G1/S phase. <em>Keratinocytes</em> with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD <em>keratinocytes</em>, indicating that NF-κB activity in <em>keratinocytes</em> is not regulated by TRIP. TRIP expression was increased by ∼<em>2</em>-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to <em>keratinocyte</em> proliferation.

Alcohol has been reported to be a risk <em>factor</em> in psoriasis mainly based on the observation that there is a higher prevalence of alcohol abuse in individuals with psoriasis. The mechanism by which alcohol affects this disease is still elusive. So far there are no reports describing the effects of metabolites relevant to alcohol metabolism on the <em>growth</em> of human <em>keratinocytes</em>. In the present study we examined the effects of ethanol and acetone, which exceeds its normal endogenous level in the blood of heavy drinkers, on the proliferation of HaCaT <em>keratinocytes</em>. HaCaT cells were incubated for 30 min in the presence of various concentrations of ethanol (<em>2</em>.14 m M-1.71 M) and acetone (1.7 mM-1.36 M). The numbers of viable and proliferating cells were determined at different times after ethanol and acetone treatment. The effects of ethanol and acetone on the mRNA levels of genes characteristic for proliferating <em>keratinocytes</em> such as alpha5 integrin, <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor and cyclin D1 were studied by reverse-transcriptase polymerase chain reaction. Both ethanol and acetone induced proliferation of HaCaT cells. The maximum increase in the number of viable cells and the maximum proliferative response was observed with 4.<em>2</em>8 m M ethanol and 13.6 m M acetone. The alpha5 integrin, <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor and cyclin D1 mRNA levels were higher compared to the controls as early as <em>2</em> h after ethanol and 30 min after acetone treatment of the cells. The stimulatory effect of ethanol and acetone on human <em>keratinocytes</em> may be one of the reasons why psoriasis can be precipitated by alcohol misuse.

The expression of the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR) has been analyzed on intestinal epithelial Caco-<em>2</em> cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal <em>growth</em> <em>factor</em> receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

Platelet-activating <em>factor</em> (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-<em>2</em> acetyl-GPC in a human <em>keratinocyte</em>-derived cell line (HaCaT <em>keratinocytes</em>). Approximately <em>2</em>8% of HaCaT <em>keratinocyte</em> GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-<em>2</em>-acyl-GPC were as follows: hexadecyl>> octadecenyl>> octadecyl. Ionophore (A<em>2</em>3187)-stimulated HaCaT <em>keratinocytes</em> synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-<em>2</em>acetyl-GPC at <em>2</em>.5 min were 1-hexadecyl-<em>2</em>-acetyl-GPC (<em>2</em>.<em>2</em> ng/10(6) cells) and 1-palmitoyl-<em>2</em>-acetyl-GPC (<em>2</em>.4 ng/10(6) cells). HaCaT <em>keratinocytes</em> also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide <em>growth</em> <em>factor</em> endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-<em>2</em>- acetyl-GPC and 1-palmitoyl-<em>2</em>-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-<em>2</em> acetyl-GPC act in autocrine fashion. These studies revealed that the human <em>keratinocyte</em>-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A<em>2</em>3187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in <em>keratinocyte</em> pathophysiology.

Mesenchymal stem cell therapies promote wound healing by manipulating the local environment to enhance the function of host cells. Aggregation of mesenchymal stem cells (MSCs) into three-dimensional spheroids increases cell survival and augments their anti-inflammatory and proangiogenic potential, yet there is no consensus on the preferred conditions for maximizing spheroid function in this application. The objective of this study was to optimize conditions for forming MSC spheroids that simultaneously enhance their anti-inflammatory and proangiogenic nature. We applied a design of experiments (DOE) approach to determine the interaction between three input variables (number of cells per spheroid, oxygen tension, and inflammatory stimulus) on MSC spheroids by quantifying secretion of prostaglandin E<em>2</em> (PGE<em>2</em> ) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), two potent molecules in the MSC secretome. DOE results revealed that MSC spheroids formed with 40,000 cells per spheroid in 1% oxygen with an inflammatory stimulus (Spheroid 1) would exhibit enhanced PGE<em>2</em> and VEGF production versus those formed with 10,000 cells per spheroid in <em>2</em>1% oxygen with no inflammatory stimulus (Spheroid <em>2</em>). Compared to Spheroid <em>2</em>, Spheroid 1 produced fivefold more PGE<em>2</em> and fourfold more VEGF, providing the opportunity to simultaneously upregulate the secretion of these <em>factors</em> from the same spheroid. The spheroids induced macrophage polarization, sprout formation with endothelial cells, and <em>keratinocyte</em> migration in a human skin equivalent model-demonstrating efficacy on three key cell types that are dysfunctional in chronic non-healing wounds. We conclude that DOE-based analysis effectively identifies optimal culture conditions to enhance the anti-inflammatory and proangiogenic potential of MSC spheroids. Stem Cells <em>2</em>017;35:1493-1504.

Dendritic epidermal T cells (DETC) are skin-specific members of the epithelial gamma delta T-cell family in mice. We have reported previously that the <em>growth</em> of DETC is promoted by interleukin (IL)-<em>2</em> in an autocrine fashion, or by IL-7, which is secreted by neighboring <em>keratinocytes</em>. Here we report that DETC <em>growth</em> is promoted by IL-15, a newly discovered T-cell <em>growth</em> <em>factor</em> that is produced in lymphoid as well as nonlymphoid tissues. Recombinant IL-15 promoted the <em>growth</em> of the 7-17 DETC line in a time- and dose-dependent fashion. Using monoclonal antibodies against alpha-, beta-, or gamma c-chains of the IL-<em>2</em> receptor complex, we observed that the combination of anti-beta chain and anti-gamma c chain antibodies blocked IL-15 responsiveness completely, whereas anti-alpha chain had no effect. These results indicate that this gamma delta T-cell line uses the beta/gamma c heterodimer for proliferative responses to IL-15. Antibodies against IL-<em>2</em> or IL-7 did not block IL-15-driven proliferation of 7-17 DETC, indicating that IL-15 promotes their <em>growth</em> in an IL-<em>2</em>- and IL-7-independent manner. Both the surface expression of beta/gamma c heterodimers and the IL-15 responsiveness of 7-17 DETC were highest 1 to 8 days after concanavalin A stimulation, and both declined substantially <em>2</em>1 days after stimulation, illustrating regulation by the state of cell activation. Working with epidermal cells that were freshly procured from CBA mice, we noted that IL-15 promoted conavalin-A-triggered <em>growth</em> of Thy-1+ cells (i.e., DETC), but not of the Thy-1- cells. The gamma c-chain was not expressed by freshly procured DETC, becoming detectable within 48 h after concanavalin A stimulation. We propose that IL-15 facilitates the <em>growth</em> of epithelial gamma delta T cells by a beta/gamma c receptor-dependent mechanism.

Dendritic epidermal T cells (DETC) are skin-specific members of the epithelial gamma delta T-cell family that reside normally in murine epidermis. Recent studies indicate that certain cytokines (e.g. IL-<em>2</em>, IL-7 and IL-15) secreted by neighboring cells promote their residence and regulate their immune function. Conversely, DETC regulate the function of neighboring <em>keratinocytes</em> and Langerhans cells by elaborating other cytokines (e.g. interferon-gamma, <em>keratinocyte</em> <em>growth</em> <em>factor</em> and colony-stimulating <em>factors</em>). This reciprocal interaction represents a unique model of cytokine-mediated intercellular communication by tissue-specific gamma delta T cells with nearby epithelial and antigen presenting cells.

Intestinal failure (IF), due to short bowel syndrome (SBS), results from surgical resection of a major portion of the intestine, leading to reduced nutrient absorption and need for parenteral nutrition (PN). The incidence is highest in infants and relates to preterm birth, necrotizing enterocolitis, atresia, gastroschisis, volvulus, and aganglionosis. Patient outcomes have improved, but there is a need to develop new therapies for SBS and to understand intestinal adaptation after different diseases, resection types, and nutritional and pharmacological interventions. Animal studies are needed to carefully evaluate the cellular mechanisms, safety, and translational relevance of new procedures. Distal intestinal resection, without a functioning colon, results in the most severe complications and adaptation may depend on the age at resection (preterm, term, young, adult). Clinically relevant therapies have recently been suggested from studies in preterm and term PN-dependent SBS piglets, with or without a functional colon. Studies in rats and mice have specifically addressed the fundamental physiological processes underlying adaptation at the cellular level, such as regulation of mucosal proliferation, apoptosis, transport, and digestive enzyme expression, and easily allow exogenous or genetic manipulation of <em>growth</em> <em>factors</em> and their receptors (e.g., glucagon-like peptide <em>2</em>, <em>growth</em> hormone, insulin-like <em>growth</em> <em>factor</em> 1, epidermal <em>growth</em> <em>factor</em>, <em>keratinocyte</em> <em>growth</em> <em>factor</em>). The greater size of rats, and especially young pigs, is an advantage for testing surgical procedures and nutritional interventions (e.g., PN, milk diets, long-/short-chain lipids, pre- and probiotics). Conversely, newborn pigs (preterm or term) and weanling rats provide better insights into the developmental aspects of treatment for SBS in infants owing to their immature intestines. The review shows that a balance among practical, economical, experimental, and ethical constraints will determine the choice of SBS model for each clinical or basic research question.

OBJECTIVE

Cellular and molecular mechanisms of esophageal ulcer healing remain unexplored. We studied the sequential cellular events and the expression of keratinocyte growth factor (KGF) and its receptor (KGF-R) during the healing of experimental esophageal ulcers.

METHODS

Esophageal ulcers were produced in rats by local application of acetic acid. Studies included (1) ulcer size, (2) quantitative histology, and (3) KGF and KGF-R messenger RNA and protein expression by reverse-transcription polymerase chain reaction, Western blotting, and immunostaining. In separate groups, ulcer size and esophageal epithelial proliferation were evaluated after a single injection of recombinant human KGF (1 mg/kg) around the ulcer.

RESULTS

Ulcers were fully developed 3 days after induction, and 58% of ulcers were re-epithelialized by 9 days. At 3 days, in esophageal tissue bordering the ulcers, KGF messenger RNA and protein were increased by 191% and 151%, respectively, and KGF-R messenger RNA and protein were increased by 357% and 237%, respectively. KGF was expressed in stromal cells, whereas KGF-R was expressed in epithelial cells. At 6 days, epithelial proliferation at the ulcer margin was increased by 216%, and treatment with KGF further enhanced cell proliferation and accelerated ulcer healing.

CONCLUSIONS

KGF is a likely mediator of esophageal epithelial proliferation and ulcer healing.

Protein phosphorylation and dephosphorylation are involved in regulation of cell <em>growth</em>. We tested the hypothesis that the <em>growth</em> inhibitory effect of transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta 1) involves activation of protein phosphatases. Exposure of human <em>keratinocytes</em> in culture to 400 pM TGF-beta 1 for 48 h led to 80% inhibition of DNA synthesis as measured by nuclear labeling. Incubation of cultured <em>keratinocytes</em> with 400 pM TGF-beta 1 rapidly activated (within 30 min) protein serine/threonine phosphatase, measured using phosphorylase as a substrate. Based on several criteria, including neutralization of activity with specific antibodies and inhibitor-<em>2</em>, TGF-beta 1-activated phosphorylase phosphatase was identified as protein phosphatase 1. TGF-beta 1 did not have rapid effects on protein serine/threonine phosphatase activity (type <em>2</em>A) measured with histone phosphorylated by protein kinase C or on protein tyrosine phosphatase activity. However, protein tyrosine phosphatase was activated at 48 h, coincident with <em>growth</em> arrest. Differentiation, induced by the combination of TGF-beta 1 plus calcium or by serum, was not accompanied by further serine/threonine or tyrosine phosphatase activation. We conclude that induction of <em>growth</em> arrest in <em>keratinocytes</em> by TGF-beta 1 involves acute activation of protein phosphatase 1, while activation of protein tyrosine phosphatase may represent an additional mechanism for maintaining cells in a <em>growth</em>-arrested state.

Desmosomes in <em>keratinocytes</em> are the most important intercellular adhering junctions that provide structural strength for the epidermis. These junctions are connected directly with desmosomal cadherin proteins. Desmosomal cadherins are divided into four desmogleins (Dsgs), Dsg1-4, and three desmocollins (Dscs), Dsc1-3, all of which are involved in desmosomal adhesion by homo- and/or heterophilic binding between Dsgs and Dscs in a Ca(<em>2</em>+)-dependent manner. Cadherins are present on the cell surface and anchor keratin intermediate filaments (KIFs) to their inner cytoplasmic surface to generate an intracellular KIF-skeletal scaffold through several associate proteins, including plakoglobin, plakophillin, and desmoplakins. As such, the desmosomal contacts between adjacent cells generate an intercellular KIF scaffold throughout the whole epidermal sheet. However, despite these critical roles in maintaining epidermal adhesion and integrity, desmosomes are not static structures. Rather, they are dynamic units that undergo regular remodeling, i.e., assembly and disassembly, to allow for cell migration within the epidermis in response to outside-in signaling during epidermal differentiation. Recently, two cell-cell adhesion states controlled by desmosomes have been recognized, including "stable hyperadhesion (Ca(<em>2</em>+)-independent)" and "dynamic weak-adhesion (Ca(<em>2</em>+)-dependent)" conditions. These conditions are mutually reversible through cell signaling events involving protein kinase C (PKC) and epidermal <em>growth</em> <em>factor</em> receptor. Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-Dsg3 antibodies. Binding of these antibodies to Dsg3 causes endocytosis of Dsg3 from the cell surface and results in the specific depletion of Dsg3 from desmosomes, an event linked to acantholysis in the epidermis. This binding of anti-Dsg3 antibody to Dsg3 in epidermal <em>keratinocytes</em> activates PKC, to generate the "weak-adhesion (Ca(<em>2</em>+)-dependent)" state of desmosomes. The weak-adhesion desmosomes appear to be the susceptible desmosomal state and a prerequisite for Dsg3 depletion from desmosomes, pivotal and specific events leading to PV blistering. These observations allow us to propose a concept for pemphigus blistering disorders as a "desmosome-remodeling impairment disease" involving a mechanism of Dsg3 nonassembly and depletion from desmosomes through PV immunoglobulin G-activated intracellular signaling events.

OBJECTIVE

Epidermal growth factor receptor inhibitor (EGFRI) induced skin toxicity has a prognostic value suggesting skin toxicity can be a useful surrogate marker for successful epidermal growth factor receptor (EGFR) inhibition, improved response and survival. But the pathophysiology of EGFRI induced skin toxicity remains elusive. However the involvement of immunological mechanisms has been speculated. This study investigates the possible underlying mechanism of EGFR inhibition associated cytokine production in keratinocytes as well as in patients after treatment with epidermal growth factor receptor inhibitors (EGFRIs).

METHODS

Normal human epidermal keratinocytes (NHEK) were incubated for 2 and 24h with different concentrations of EGFRI (erlotinib) for Western blot analysis and cytokine expression analysis, respectively. Inhibition of EGFR, extracellular-signal-regulated kinase 1/2 (Erk 1/2) and c-Jun was examined by Western blot analysis. Cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the NHEK cell supernatant and also in the serum of 186 cancer patients who are followed up for EGFRI induced skin rash.

RESULTS

A significant inhibitory effect of EGFRI was seen on EGFR (Y845), Erk 1/2 and c-Jun in a dose dependent manner. Further downstream, increased CC-chemokine ligand 2 (CCL2), CC-chemokine ligand 5 (CCL5) and decreased interleukin-8 (IL-8) or CXCL8 expression was observed in keratinocytes. In EGFRI treated patients, low levels of serum CXCL8 corresponding to stronger EGFR inhibition were associated with a higher grade of skin toxicity (p=0.0016) and a prolonged overall survival (p=0.018).

CONCLUSIONS

The results obtained in this study indicate that EGFRI can regulate cytokines by modulating EGFR signalling pathway in keratinocytes. Moreover, serum levels of CXCL8 in EGFRI treated patients may be important for individual EGFRI induced skin toxicity and patient's survival.

BACKGROUND

Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization.

OBJECTIVE

To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence.

METHODS

Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6).

RESULTS

Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine.

CONCLUSIONS

Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

BACKGROUND

Total parenteral nutrition (TPN) induces epithelial cell (EC) apoptosis. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) increases EC <em>growth</em>; however, little is known of its effect on apoptosis. This study aimed to determine the effect of recombinant human KGF (rHuKGF) on small bowel EC apoptosis. We further determined mRNA expression of Bcl-<em>2</em> family members (major mediators of epithelial cell apoptosis) with TPN and whether KGF administration influences Bcl-<em>2</em> family expression in EC.

METHODS

C57BL/6J mice (n = 6/group) received oral feeding (Control); TPN; or TPN plus daily intravenous rHuKGF (TPN+KGF). After 7 days, intestines were harvested and EC isolated. Villus height was determined by microscopy and EC proliferation by immunohistochemistry using incorporation of 5-bromodeoxyuridine (BrdU). Apoptosis was identified by Annexin V as well as by TUNEL staining. EC mRNA expression of Bcl-<em>2</em> family members was measured by reverse-transcriptase polymerase chain reaction and Bcl-<em>2</em> protein level by immunoblot analysis.

RESULTS

Villus height in Controls was 310 +/- 4<em>2</em> microm. This decreased with TPN to <em>2</em>10 +/- 45 microm; however, villus height was preserved in TPN + KGF mice (<em>2</em>73 +/- 39 microm). EC proliferation rates decreased significantly in TPN mice, and this decline was prevented by administration of rHuKGF. EC apoptotic rate in Controls was 14.4 +/- 5.1%; TPN administration resulted in doubling of largely prevented TPN-induced EC apoptosis (<em>2</em>9.4 +/- 11.3%) rHuKGF administration largely prevented TPN-induced EC apoptosis (17.<em>2</em> +/- 5.6%). Proapoptotic Bcl-<em>2</em> members changed minimally with TPN or rHuKGF; however, the anti-apoptotic Bcl-<em>2</em> changed significantly: Control 0.78 +/- 0.<em>2</em>4; TPN 0.10 +/- 0.13; rHuKGF administration prevented the decline in Bcl-<em>2</em> expression observed with TPN (0.76 +/- 0.36). EC Bcl-<em>2</em> protein levels were: Control 0.16 +/- 0.13; TPN 0.18 +/- 0.16; and TPN+KGF 0.47 +/- 0.19.

CONCLUSIONS

TPN-induced apoptosis was associated with decreased Bcl-<em>2</em> mRNA expression. rHuKGF decreased TPN-induced EC apoptosis and increased Bcl-<em>2</em> expression. rHuKGF administration may have benefit in patients on TPN.

Transforming <em>growth</em> <em>factor</em>-betas (TGF-betas) have been shown to enhance the expression of extracellular matrix genes, including several collagens. In this study, the effects of TGF-beta 1 and TGF-beta <em>2</em> on the expression of the gene for type VII collagen, the major component of anchoring fibrils, in human epidermal cell cultures were examined. Incubation of human epidermal <em>keratinocytes</em> or oral epidermoid carcinoma KB cells with TGF-beta 1 or TGF-beta <em>2</em> markedly (up to 6.3-fold) elevated the alpha 1(VII) collagen mRNA levels. This elevation was accompanied by enhanced synthesis of type VII collagen, as demonstrated by indirect immunofluorescence with a monoclonal antibody. The results indicate that TGF-beta 1 and TGF-beta <em>2</em> have similar biological activities with respect to enhanced type VII collagen gene expression.

Recently, the <em>keratinocyte</em> IL-8/IL-8 receptor (IL-8R) pathway has been implicated in the pathogenesis of psoriasis, and there is evidence that the potent macrolide immune suppressant tacrolimus (formerly FK506) can inhibit this pathway in vitro. In this study, determination of the expression of cytokine mRNAs in lesional skin of patients with active disease by reverse transcriptase polymerase chain reaction revealed transcripts for IL-1 beta, tumour necrosis <em>factor</em>-alpha (TNF-alpha), IL-6, IL-8, IL-8R, IL-10, interferon-gamma (IFN-gamma), IL-<em>2</em>R and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), but not IL-<em>2</em> or IL-4. IL-8 was the only cytokine expressed in affected skin of all patients but not in clinically normal skin of healthy subjects. In seven CD4+ T cell clones propagated from the lesional skin of an untreated psoriasis patient, IL-8 was expressed by the skin-derived T lymphocytes and not by feeder cells (irradiated autologous blood lymphocytes); IL-1 beta, IL-<em>2</em>, IL-6 and IL-10 were also expressed by some or all of the T cell clones. IL-8 mRNA was not detected in the skin of any patient after the start of systemic tacrolimus therapy; IL-1 beta, IL-6 and IFN-gamma transcripts were also reduced. By 1<em>2</em> weeks, the mean psoriasis area and severity index (PASI) had decreased from 18.8 to 3.8, a reduction of 80%. In the same post-treatment biopsies, however, message for IL-8R persisted. Estimation of circulating IL-8 levels by enzyme immunoassay showed that all patients with detectable IL-8 before treatment had decreased levels in response to treatment with tacrolimus; reductions in PASI scores were accompanied by decreases in IL-8 levels, that varied both in rate and extent. Partial relapse, which in a minority of patients followed the initial period of remission, and was precipitated by drug dose reduction, was accompanied by an increase in circulating IL-8. These findings add credence to the view that the IL-8/IL-8R autocrine/paracrine pathway may be important in the pathogenesis of psoriasis. They further suggest that interference with IL-8 production and/or that of other key chemokines may be an important mechanism underlying the therapeutic efficacy of tacrolimus, and other agents such as cyclosporin A, with similar molecular actions.

The topical application of recombinant <em>growth</em> <em>factors</em> such as epidermal <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>-BB homodimer (rPDGF-BB), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (rKGF), and neu differentiation <em>factor</em> has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these <em>growth</em> <em>factors</em> to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.<em>2</em>% control; P = 0.016) 1<em>2</em> days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.0<em>2</em>) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for KGFR in porcine burns revealed strong expression of KGFR on hair follicles and basal epidermis, confirming direct rKGF action on follicular as well as epidermal <em>keratinocytes</em>. Although the epithelial proliferation induced by rKGF resulted in marked neoepidermal psoriasiform hyperplasia with exaggerated rete ridges and neoepidermal and follicular maturation as assessed by expression of cytokeratin 10, a marker of <em>keratinocyte</em> terminal differentiation was not delayed and appeared to be accelerated in some rKGF-treated burns. Recombinant epidermal <em>growth</em> <em>factor</em> induced a trend toward increased new epithelial area in deep partial thickness burns, but had no effect on reepithelialization. The recombinant neu differentiation <em>factor</em>-alpha <em>2</em> isoform had no significant biological effects in either full or deep partial thickness burns.(ABSTRACT TRUNCATED AT <em>2</em>50 WORDS)

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal <em>keratinocytes</em> to breach the epidermal barrier through mechanisms not fully understood. By use of primary <em>keratinocytes</em> from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with <em>keratinocyte</em> <em>growth</em> <em>factor</em> increased their uptake <em>2</em>.5-fold. ILK-deficient mouse <em>keratinocytes</em> internalized bacteria <em>2</em>- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was <em>2</em>.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of <em>keratinocytes</em>, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection.

BACKGROUND

New strategies for treating Pseudomonas aeruginosa pulmonary infection are urgently needed. Adipose tissue-derived mesenchymal stem cells (ASCs) may have a potential therapeutic role in P. aeruginosa-induced pulmonary infection.

METHODS

The therapeutic and mechanistic effects of ASCs on P. aeruginosa pulmonary infection were evaluated in a murine model of P. aeruginosa pneumonia.

RESULTS

ASCs exhibited protective effects against P. aeruginosa pulmonary infection, evidenced by reduced bacterial burdens, inhibition of alveolar neutrophil accumulation, decreased levels of myeloperoxidase, macrophage inflammatory protein-<em>2</em> and total proteins in broncho-alveolar lavage fluid (BALF), and attenuated severity of lung injury. ASCs had no effects on BALF and serum levels of <em>keratinocyte</em> <em>growth</em> <em>factor</em> or Ang-1. ASCs had no effects on the levels of insulin <em>growth</em> <em>factor</em> 1 (IGF-1) in BALF, but increased IGF-1 levels in serum. ASCs inhibited the overproduction of prostaglandin E<em>2</em> (PGE<em>2</em> ) by decreasing the expression of cyclooxygenase-<em>2</em> (COX<em>2</em>) and enhancing the expression of 15-PGDH. In addition, the addition of exogenous PGE<em>2</em> with ASCs abolished many of the protective effects of ASCs, and administrating PGE<em>2</em> alone exacerbated lung infection. By inhibiting production of PGE<em>2</em> , ASCs improved phagocytosis and the bactericidal properties of macrophages. Furthermore suppressing PGE<em>2</em> signaling by COX<em>2</em> inhibition or EP<em>2</em> inhibition exhibited protective effects against pulmonary infection as well.

CONCLUSIONS

In a murine model of P. aeruginosa pneumonia, ASCs exhibited protective effects by inhibiting production of PGE<em>2</em> , which subsequently improved phagocytosis and the bactericidal properties of macrophages. ASCs may provide a new strategy for managing pulmonary infection caused by P. aeruginosa.

Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein <em>2</em> (Dsg<em>2</em>), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg<em>2</em> increased EV release and mitogenic content including epidermal <em>growth</em> <em>factor</em> receptor and c-Src. Inhibiting ectodomain shedding of Dsg<em>2</em> with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg<em>2</em> in EVs and reduced EV release. When cocultured with Dsg<em>2</em>/green fluorescence protein-expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/<em>2</em> and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg<em>2</em> was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg<em>2</em> C-terminal fragment and epidermal <em>growth</em> <em>factor</em> receptor. This study defines a mechanism by which Dsg<em>2</em> expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.-Overmiller, A. M., Pierluissi, J. A., Wermuth, P. J., Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, L. A., Wahl, J. K. III, South, A. P., Mahoney, M. G. Desmoglein <em>2</em> modulates extracellular vesicle release from squamous cell carcinoma <em>keratinocytes</em>.

Exosomes are nano-scale and closed membrane vesicles which are promising for therapeutic applications due to exosome-enclosed therapeutic molecules such as DNA, small RNAs, proteins and lipids. Recently, it has been demonstrated that mesenchymal stem cell (MSC)-derived exosomes have capacity to regulate many biological events associated with wound healing process, such as cell proliferation, cell migration and blood vessel formation. This study investigated the regenerative potentials for cutaneous tissue, in regard to <em>growth</em> <em>factors</em> associated with wound healing and skin cell proliferation and migration, by exosomes released from primary MSCs originated from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) under serum- and xeno-free condition. We found crucial wound healing-mediated <em>growth</em> <em>factors</em>, such as vascular endothelial <em>growth</em> <em>factor</em> A (VEGF-A), fibroblast <em>growth</em> <em>factor</em> <em>2</em> (FGF-<em>2</em>), hepatocyte <em>growth</em> <em>factor</em> (HGF), and platelet-derived <em>growth</em> <em>factor</em> BB (PDGF-BB) in exosomes derived from all three MSC sources. However, expression levels of these <em>growth</em> <em>factors</em> in exosomes were influenced by MSC origins, especially transforming <em>growth</em> <em>factor</em> beta (TGF-β) was only detected in UCMSC-derived exosomes. All exosomes released by three MSCs sources induced <em>keratinocyte</em> and fibroblast proliferation and migration; and, the induction of cell migration is a dependent manner with the higher dose of exosomes was used (<em>2</em>0 μg), the faster migration rate was observed. Additionally, the influences of exosomes on cell proliferation and migration was associated with exosome origins and also target cells of exosomes that the greatest induction of primary dermal fibroblasts belongs to BMMSC-derived exosomes and <em>keratinocytes</em> belongs to UCMSC-derived exosomes. Data from this study indicated that BMMSCs and UCMSCs under clinical condition secreted exosomes are promising to develop into therapeutic products for wound healing treatment.

Keywords: ADMSC-derived exosomes; BMMSC-derived exosomes; UCMSC-derived exosomes; exosomes; growth factors; mesenchymal stem cells; wound healing.