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Background: Cancer stem cells are responsible for tumour progression and chemoresistance. Fibroblasts surrounding a tumour also promote progression and fibroblast "activation" is an independent prognostic marker in oral cancer. Cancer stem cells may therefore promote tumourigenesis through communication with stromal fibroblasts., Methods: Cancer stem cells were isolated from oral cancer cell lines by adherence to fibronectin or cisplatin resistance. Fibroblasts were exposed to conditioned medium from these cells, and the activation markers, alpha smooth muscle actin and interleukin-6, were assessed using qPCR and immunofluorescence. Stem cell markers and smooth muscle actin were examined in oral cancer tissue using immunohistochemistry., Results: Adherent and chemoresistant cells expressed increased levels of stem cell markers CD24, CD44 and CD29 compared with unsorted cells. Adherent cells exhibited lower growth rate, higher colony forming efficiency and increased cisplatin resistance than unsorted cells. Smooth muscle actin and Interleukin-6 expression were increased in fibroblasts exposed to conditioned medium. In oral cancer tissue, there was a positive correlation between expression of αSMA and stem cell markers., Conclusions: Adherence to fibronectin and chemoresistance isolates stem-like cells that can activate fibroblasts, which together with a correlation between markers of both in vivo, provides a mechanism by which such cells drive tumourigenesis., (© 2020 The Authors. Oral Diseases published by John Wiley & Sons Ltd.)

Background: The expression of XCR1 receptor and its metamorphic ligand lymphotactin (hLtn) has been shown in cancers but their precise role in tumorigenesis is poorly understood including the significance of the physiologically existing hLtn monomeric (CC3) and dimeric (W55D) confirmations where the latter thought to function as the receptor antagonist. The aim of this study was to explore the functional role of bioengineered hLtn variants and the role of fibroblasts in XCR1/hLtn expression regulation in oral cancer cells (OCCL)., Material and Methods: qRT-PCR and flow cytometry were performed to evaluate mRNA and protein expression of XCR1 and hLtn. Recombinant hLtn variants (wild-type, CC3 and W55D mutant) were designed, expressed, purified and evaluated using proliferation, adhesion and chemotaxis assays. XCR1 and hLtn expression regulation by fibroblasts was determined using indirect co-culture. XCR1 and hLtn expression in primary and metastatic OSCC tissue was assessed using immunohistochemistry., Results: hLtn caused a significant decrease in OCCL XCR1 surface protein expression. hLtn CC3 mutant was highly functional facilitating proliferation and migration. Conditioned media from primary cancer-associated and senescent fibroblasts significantly upregulated XCR1 and hLtn mRNA expression in OCCL. Immunohistochemistry revealed higher XCR1 and hLtn expression in metastatic tumour deposits and surrounding stroma compared to primary OSCC tissue., Conclusions: The development of hLtn biological mutants, regulation of XCR1 expression by its ligand hLtn and crosstalk with fibroblasts are novel findings suggesting an important role for the XCR1/hLtn axis within the OSCC tumour microenvironment. These discoveries build upon previous studies and suggest that the hLtn/XCR1 axis has a significant role in stromal crosstalk and OSCC progression., (© 2020 The Authors. Oral Diseases published by John Wiley & Sons Ltd.)

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host-cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host-cell identity (cell line vs. primary), host-cell lysis method, and host-cell passage number had no significant effect on bacterial invasion. However, incubation time, host-cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5β1 integrin. Interestingly, this may represent a population of cells with stem cell-like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported., (© 2018 Eur J Oral Sci.)

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor β1 (TGF-β1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-β1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFβ signalling and endocytosis. TGF-β1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-β1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-β receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-β1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Porphyromonas gingivalis, a key periodontal pathogen, is capable of invading a variety of cells, including oral keratinocytes, by exploiting host cell receptors, including alpha-5 beta-1 (α5β1) integrin. Previous studies have shown that P. gingivalis accelerates the cell cycle and prevents apoptosis of host cells, but it is not known whether the cell cycle phases influence bacterium-cell interactions. The cell cycle distribution of oral keratinocytes was characterized by flow cytometry and BrdU (5-bromo-2-deoxyuridine) staining following synchronization of cultures by serum starvation. The effect of cell cycle phases on P. gingivalis invasion was measured by using antibiotic protection assays and flow cytometry, and these results were correlated with gene and surface expression levels of α5 integrin and urokinase plasminogen activator receptor (uPAR). There was a positive correlation (R = 0.98) between the number of cells in S phase and P. gingivalis invasion, the organism was more highly associated with cells in S phase than with cells in G2 and G1 phases, and S-phase cells contained 10 times more bacteria than did cells that were not in S phase. Our findings also show that α5 integrin, but not uPAR, was positively correlated with cells in S phase, which is consistent with previous reports indicating that P. gingivalis invasion of cells is mediated by α5 integrin. This study shows for the first time that P. gingivalis preferentially associates with and invades cells in the S phase of the cell cycle. The mechanism of targeting stable dividing cells may have implications for the treatment of periodontal diseases and may partly explain the persistence of this organism at subgingival sites., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2)., Methods: mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays., Results: mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL., Conclusions: Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The neurotrophin Nerve Growth factor (NGF) is known to influence the phenotype of mature nociceptors, for example by altering synthesis of neuropeptides, and changes in NGF levels have been implicated in the pathophysiology of chronic pain conditions such as neuropathic pain. We have tested the hypothesis that after partial nerve injury, NGF accumulates within the skin and causes 'pro-nociceptive' phenotypic changes in the remaining population of sensory nerve fibres, which could underpin the development of neuropathic pain., Results: Eleven days after chronic constriction injury of the rat mental nerve the intra-epidermal nerve fibre density of the chin skin from had reduced from 11.6 ± 4.9 fibres/mm to 1.0 ± 0.4 fibres/mm; this slowly recovered to 2.4 ± 2.0 fibres/mm on day 14 and 4.0 ± 0.8 fibres/mm on day 21. Cold hyperalgesia in the ipsilateral lower lip was detectable 11 days after chronic constriction injury, although at this time skin [NGF] did not differ between sides. At 14 days post-injury, there was a significantly greater [NGF] ipsilaterally compared to contralaterally (ipsilateral = 111 ± 23 pg/mg, contralateral = 69 ± 13 pg/mg), but there was no behavioural evidence of neuropathic pain at this time-point. By 21 days post-injury, skin [NGF] was elevated bilaterally and there was a significant increase in the proportion of TrkA-positive (the high-affinity NGF receptor) intra-epidermal nerve fibres that were immunolabelled for the neuropeptide Calcitonin Gene-related peptide., Conclusions: The temporal mismatch in behaviour, skin [NGF] and phenotypic changes in sensory nerve fibres indicate that increased [NGF] does not cause hyperalgesia after partial mental nerve injury, although it may contribute to the altered neurochemistry of cutaneous nerve fibres.

A disintegrin and metalloprotease (ADAM) proteins are upregulated in cancer and can interact with integrin receptors. We investigated whether such interactions may have functional significance in oral squamous cell carcinoma (OSCC). ADAM 10 expression was increased in OSCC tissue and cell lines compared to normal oral mucosa. Silencing of ADAM 10 reduced migration and invasion specifically in OSCC cells over-expressing αvβ6 integrin. This may result from ADAM 10-induced up-regulation of MMPs. We conclude ADAM 10 may influence OSCC invasion by functionally interacting with αvβ6 integrin which we have previously shown is over expressed in OSCC., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Staphylococcus aureus can invade epithelial cells, and the host-cell receptor α(5)β(1) integrin is thought to mediate this process. The aim of this study was to investigate S. aureus invasion of epithelial cell lines derived from oral (H357), skin (UP) and nasopharyngeal (Detroit 562) sites and to determine whether any differences were due to the levels of α(5)β(1) integrin expressed. While the adhesion and invasion of two S. aureus strains were similar in both oral and skin-derived keratinocytes, this was markedly reduced in the nasopharyngeal cell line, despite it expressing similar levels of α(5)β(1). While this might be explainable on the basis of availability of cell receptor, adhesion to and invasion of H357 and UP cells by S. aureus were enhanced when the epithelial cells were in suspension rather than on a surface, and levels of α(5) integrin subunit mRNA were also increased. Detroit 562 cells exhibited a similar α(5) gene upregulation, but this did not result in enhanced adhesion and invasion of S. aureus. The Detroit 562 cells also showed reduced adhesion to fibronectin compared with the other cell types. This, and the low S. aureus invasion, may result from reduced α(5)β(1) integrin activity or from variation in an as-yet-unidentified additional receptor or accessory molecule. These studies shed further light on the mechanisms of S. aureus invasion of human cells.

Aims: The aims of this study were to examine the role of endothelin-1 (ET-1), a pleiotropic peptide found at elevated levels in a number of malignancies and which has been shown to influence oral cancer cell behaviour via paracrine signalling pathways, on the phenotype of oral fibroblasts., Main Methods: The effect of ET-1 on proliferation and migration of human primary oral fibroblasts was assessed using MTS and scratch assays, respectively. The ability of ET-1 to affect fibroblast contractility was analysed using type-I collagen gels. Changes in gene expression in oral fibroblasts exposed to ET-1 were examined using quantitative PCR. The invasiveness of oral cancer cells in the presence of conditioned media collected from ET-1 treated fibroblasts was determined using 2D Matrigel assays., Key Findings: Here we provide evidence that ET-1 increases the migration of oral fibroblasts and induces a more contractile phenotype which is not associated with changes in gene expression indicative of myofibroblast transdifferentiation. In addition we provide evidence that conditioned medium of ET-1-stimulated oral fibroblasts promotes invasion of OSCC cells in vitro., Significance: In oral squamous cell carcinoma, a frequently fatal and increasingly common epithelial malignancy of the oral cavity, ET-1 is known to contribute to pro-migratory paracrine signalling between stromal fibroblasts and cancer cells. The ability of ET-1 to modulate the phenotype of human oral stromal fibroblasts, however, has not previously been reported. The findings presented here suggest that targeting the stromal endothelin system may be a viable and novel therapeutic strategy for invasive oral cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The invasion and migration of cancer cells is increasingly recognised to be influenced by factors derived from adjacent tumour-associated stroma. The contextual signals regulating stromal-tumour interactions, however, remain poorly understood. Here, we identify a role for endothelin-1 (ET-1), a mitogenic peptide elevated in a number of malignancies, in promoting pro-metastatic cross-talk between head and neck cancer cells and adjacent fibroblasts. We demonstrate that treatment of oral fibroblasts with ET-1 activates ADAM17-mediated release of epidermal growth factor receptor (EGFR) ligands, triggering EGFR signalling and increased motility in neighbouring head and neck cancer cells. ET-1-mediated paracrine transactivation of EGFR also increased cyclo-oxygenase-2 levels in the cancer cells, providing a molecular insight into the mechanisms by which the elevated levels of ET-1 observed in head and neck cancers may contribute to disease progression., (Copyright © 2011 UICC.)

Background: Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin., Methods: H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry., Results: Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions., Conclusions: The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases., (© 2011 John Wiley & Sons A/S.)

The integrin αE(CD103)β7 (αEβ7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEβ7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, 'open' and an inactive, 'closed' conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEβ7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn(2+) , which is essential for integrin function in vitro. The locked 'open' conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEβ7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEβ7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn(2+) dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked 'open'αE I domain, independently of the β7 subunit.

Alterations in the levels of molecules which interact with the extracellular matrix, such as integrins, are associated with invasion of oral squamous cell carcinomas (OSCC). The molecular mechanisms underlying dysregulation of integrin expression in OSCC, however, remain unclear. Here, we show that microRNA-124, a small non-coding RNA down-regulated in OSCC, is able to downregulate expression of integrin beta-1 (ITGB1) by interacting with its 3' untranslated region. Over-expression of miR-124 attenuates endogenous ITGB1 expression and reduces the adherence and motility of OSCC cells, suggesting disruption of miR-124-mediated repression of ITGB1 may be a key factor in OSCC progression., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Bistable populations of bacteria give rise to two or more subtypes that exhibit different phenotypes. We have explored whether the periodontal pathogen Porphyromonas gingivalis exhibits bistable invasive phenotypes. Using a modified cell invasion assay, we show for the first time that there are two distinct subtypes within a population of P. gingivalis strains NCTC 11834 and W50 that display differences in their ability to invade oral epithelial cells. The highly invasive subtype invades cells at 10-30-fold higher levels than the poorly invasive subtype and remains highly invasive for approximately 12-16 generations. Analysis of the gingipain activity of these subtypes revealed that the highly invasive type had reduced cell-associated arginine-specific protease activity. The role of Arg-gingipain activity in invasion was verified by enhancement of invasion by rgpAB mutations and by inclusion of an Arg-gingipain inhibitor in invasion assays using wild-type bacteria. In addition, a population of ΔrgpAB bacteria did not contain a hyperinvasive subtype. Screening of the protease activity of wild-type populations of both strains identified high and low protease subtypes which also showed a corresponding reduction or enhancement, respectively, of invasive capabilities. Microarray analysis of these bistable populations revealed a putative signature set of genes that includes oxidative stress resistance and iron transport genes, and which might be critical to invasion of or survival within epithelial cells.

Chemokines are chemoattractant cytokines which act on specific receptors and play an important role in leukocyte migration as well as physiological and pathological processes. We investigated the role of the chemokine receptor XCR1 and its ligand lymphotactin (Lptn/XCL1) in the regulation of oral epithelial cell behaviour. In vitro XCR1 mRNA and cell surface protein expression was detected in normal oral keratinocytes and oral squamous cell carcinoma cell lines. Lymphotactin mediated intracellular activation of the ERK1/2 signalling pathway and stimulated migration, invasion, and proliferation of all cells through XCR1. Oral cancer cells showed a greater response to lymphotactin than normal keratinocytes and a direct relationship between receptor expression and migration, invasion, and proliferation was observed. Exposure of normal keratinocytes to lymphotactin resulted in increased adhesion to fibronectin but not collagen and stimulated MMP-2 and MMP-9 but not MMP-7 release, whereas exposure of cancer cells resulted in increased adhesion to both collagen and fibronectin and stimulated production of MMP-2, MMP-9, and MMP-7. We observed XCR1 but not lymphotactin to be expressed by epithelial cells in normal oral mucosa in vivo, whilst both were expressed and up-regulated in inflammatory oral disease and oral cancer including primary and metastatic disease. Lymphotactin mRNA and constitutive intracellular protein were detected in normal keratinocytes and oral cancer cell lines in vitro. These findings show that XCR1 and its ligand, lymphotactin, are expressed by oral epithelial cells and suggest that they play a role in regulating the behaviour of these cells.

Fibroblast-epithelium interactions are crucial for successful tissue engineering of skin and oral mucosal equivalents. In this study, we assessed early force generation in organotypic fibroblast-epithelium co-cultures, using normal human keratinocytes (NHK) and HPV16-transformed (UP) cells. During the initial 2 h period, organotypic co-cultures containing both epithelial cell types produced significantly more force than fibroblasts alone (p < 0.05). After 2 h, the epithelial contribution became diminished and did not significantly contribute to intrinsic force generation by fibroblasts, and no differences were observed when using UP vs. NHK. We then measured protease gene expression at the end of the experimental period. Distinct differences were evident in protease expression both between NHK-human skin fibroblast (HSF) vs. UP-HSF co-cultures and compared to fibroblasts alone. We conclude that whilst the very early contractile response of fibroblasts is enhanced by the overlying epithelium, this becomes diminished as the fibroblast response becomes predominant and it does contribute to tissue remodelling via regulation of protease expression.

Changes in the extracellular matrix, integrin expression, and protease secretion occur in wound healing and cancer and these systems are thought to play a crucial role in such processes. In this study, experiments were performed to examine the interaction of epithelial cells with the 120 kDa cell-binding fibronectin fragment. Cell migration was significantly increased in response to the 120 kDa fragment when compared with the full-length molecule, but only in cells overexpressing the beta-6 integrin (VB6). This involved the up-regulation of matrix metalloproteinases (MMPs) 2 and 9, the levels of which are increased in the supernatant from VB6 cells plated on the 120 kDa fragment. Inhibition of MMP activity with both tissue inhibitor of metalloproteinase-1 (TIMP-1) and a chemical inhibitor (Ro32-3580) resulted in a significant reduction in migration on the fibronectin fragment. In vitro this fragment can be generated by incubating full-length fibronectin with purified MMP-9. Expression of the beta6 integrin subunit, which is only seen in wound healing and cancer, enhances the motility of and MMP secretion by epithelial cells in response to the 120 kDa fibronectin fragment. These enzymes can then further degrade fibronectin to generate additional fragments, leading to the creation of a positive feedback loop, which may be of significance in disease processes.

Aims: CD40 expression is restricted to Keratinocytes of normal epidermis or stratified squamous epithelium of oral mucosa. Ligation of CD40 inhibits keratinocyte proliferation and apoptosis. The aim of this study was to investigate the functional significance of CD40 in the proliferation, apoptosis, adhesion and migration of human oral keratinocytes in vitro., Methods: The CD40-negative oral keratinocyte line OSC19, its CD40-positive transfected derivative (OSC19T-CD40) and null transfectants (OSC19T-control), with and without stimulation by soluble protein CD40 ligand (sCD40L) or anti-CD40 antibodies were used., Results: OSC19T-CD40 showed significantly (P < 0.001) slower growth than the null transfectants and parent cells. OSC19T-CD40 proliferation was inhibited by ligation with sCD40L and blocking by two anti-CD40 antibodies, but stimulated by a third. Binding of CD40 with ligand or antibody had no effect on keratinocyte apoptosis in any cell line. The capacity of OSC19T-CD40 cells to adhere to CD40L-coated wells was significantly greater (P < 0.001) than that of parent OSC19 and OSC19T-control cells, and the migration rate of OSC19T-CD40 cells was significantly higher than parent OSC19 (P = 0.038 on fibronectin, P = 0.004 on Matrigel) or OSC19T-control (P =0.017 on fibronectin, P = 0.013 on Matrigel) cells., Conclusions: CD40 is an important molecule in keratinocyte homeostasis, and has more than one ligand. The ligand that is bound may be critical in oral epithelial homeostasis, the development of malignancy and the behaviour of the subsequent tumour.

Background: There is upregulation of class II molecules of the major histocompatibility complex (MHC) by keratinocytes in oral squamous cell carcinoma (OSCC) and inflammatory diseases such as lichen planus. The significance of this expression, or whether it is accompanied by upregulation of membrane-bound costimulatory molecules, is unknown., Objectives: To compare the expression of CD40, CD80, CD86, MHC class II and intercellular adhesion molecule-1 (ICAM-1), and the ability to induce allogeneic T-lymphocyte proliferation in vitro, of a CD40- OSCC cell line, its CD40+ transfected derivative and null transfectants., Methods: OSCC cell lines and purified T lymphocytes were cocultured and T cell proliferation recorded. Phenotypes were analysed by flow cytometry., Results: After T lymphocyte proliferation, which all OSCC cell lines were able to induce, there was upregulation of MHC class II and ICAM-1. However, the CD40+ transfectants were the most immunologically potent and were the only cells to show increased expression of CD86 (as well as further upregulation of CD40 and a statistically insignificant rise in CD80). The effects of blocking antibodies on T-cell proliferation were only statistically significant with the CD40+ transfectants., Conclusions: While not essential, expression of CD40 by OSCC cells is necessary for optimal induction of allogeneic T lymphocytes, possibly because of concurrent upregulation of other membrane-bound costimulatory molecules.

Staphylococcus aureus is a common wound-infecting organism which can interact with cells via the extracellular matrix protein fibronectin (FN). The aim of this study was to determine the effect of the FN-binding protein (FnBP) of S. aureus on the behaviour of a human skin keratinocytes cell line (UP). FN-coated plates and Transwell membranes were exposed to a recombinant protein encompassing the D1-D4 repeat region of S. aureus FnBPB (rFnBPBD1-D4) before cell adhesion and migration assays. The influence of this protein on migration into a wounded area, cell cycle progression and endogenous cellular FN assembly was also assessed. The rFnBPBD1-D4 protein potently inhibited S. aureus entry into UP keratinocytes but had no effect on cell adhesion to FN substrate. It inhibited UP keratinocyte Transwell migration by 17% (P= 0.04) and 31% (P=0.02) at 10 and 100 microg/ml, respectively. In the wound assay, 100 microg/ml of rFnBPBD1-D4 protein reduced the migration area by approximately 30%. No differences in cell cycle progression were observed. In the presence of rFnBPBD1-D4, most of the cellular FN matrix on the cell surface and along the cell filopodia seen in untreated cells was absent. Interaction of S. aureus FnBPs with FN may influence cell behaviour and thus play a role in delayed epithelial closure in infected healing wounds.

Staphylococcus aureus is among the most important human pathogens and causes various superficial and systemic infections. The ability of S. aureus to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of S. aureus by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of S. aureus FnBPs, and an anti-alpha5beta1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of S. aureus by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin alpha5beta1. In contrast to internalization by immortalized keratinocytes, internalization of S. aureus by primary keratinocytes could occur through FnBP-dependent and -independent pathways. S. aureus clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of S. aureus FnBPs and host cell alpha5beta1, has important implications for the design of therapies targeted to bacterial uptake by host cells.

Over-expression of components of the urokinase system is well documented in cancer and is thought to enable tumour cells to migrate and invade. Changes in integrin expression are also a common feature of tumours and have been linked to changes in protease activity. It has been shown that the alphavbeta6 integrin is neo-expressed in a number of epithelial carcinomas and in wound healing situations. We therefore investigated whether alphavbeta6 is able to modulate a key regulator of proteolysis, the urokinase receptor. We report that epithelial cells expressing full-length alphavbeta6 exhibit decreased urokinase receptor expression and function. Furthermore, this novel modulation requires the C-terminal 11 amino acids of the cytoplasmic tail of the beta6 integrin subunit. Cells expressing alphavbeta3, however, did not affect urokinase receptor expression. De novo expression of beta6 by melanoma cells and beta3 by epithelial cells did not influence urokinase receptor expression or function, suggesting that modulation of urokinase system is both integrin subunit and cell-specific.

We have demonstrated that tissue plasminogen activator (tPA) binds specifically to human vascular smooth muscle cells (VSMC) in a functionally relevant manner, both increasing plasminogen activation and decreasing tPA inhibition (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322; Werner, F., Razzaq, T. M., and Ellis, V. (1999) J. Biol. Chem. 274, 21555-21561). To further understand this system we have now identified and characterized the protein responsible for this binding. Rat VSMC were surface-labeled with 125I, and cell lysates were subjected to an affinity chromatography scheme based on the previously identified tPA binding characteristics. A single radiolabeled protein of 63 kDa bound specifically and was eluted at low pH. This protein was isolated from large scale preparations of VSMC and unambiguously identified as the rat homologue of the human type-II transmembrane protein p63 (CKAP4) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. In confirmation of this, a monoclonal antibody raised against authentic human p63 recognized the isolated protein in Western blotting. Immunofluorescence microscopy demonstrated that p63 was located principally in the endoplasmic reticulum but was also detected in significant quantities on the surface of human VSMC. In support of the hypothesis that p63 is the functional tPA binding site on VSMC, an anti-p63 monoclonal antibody was found to block tPA binding. Furthermore, heterologous expression of an N-terminally truncated mutant of p63, which targets exclusively to the plasma membrane, led to an increase in tPA-catalyzed plasminogen activation. Therefore, p63 on the surface of VSMC may contribute to the functional regulation of the plasminogen activation system in the vessel wall.

Integrin receptors and the growth factor, scatter factor (SF; also known as hepatocyte growth factor) have been shown to modulate similar cellular processes including embryogenesis, wound healing and tumour invasion. The purpose of this study was to examine the role of SF in the regulation of integrin expression, migration and adhesion in normal human oral keratinocytes (NHK). Integrin expression was examined using flow cytometry, SF did not alter levels of expression but had a dramatic effect on cell morphology, inducing migratory filopodia and lamellipodia. SF selectively induced migration towards fibronectin, but not towards collagen I. Integrin function was further investigated by measuring the ability of NHK to adhere and migrate on various integrin ligands. SF reduced adhesion of NHK to collagen types I and IV, laminins 1 and 5 and fibronectin. The inclusion of function-blocking antibodies revealed that SF mediated upregulation of migration through alpha(5)beta(1) integrin and downregulation of adhesion through alpha(v) integrins. SF increased tyrosine phosphorylation of FAK protein in NHK after a 30-min treatment. These results show that SF can affect keratinocyte behaviour by modifying integrin function but not expression.

The integrin alphavbeta6 is a fibronectin receptor whose expression is not detectable on normal oral epithelium but is increased significantly in healing and in oral epithelial dysplasia and oral squamous cell carcinoma, suggesting it may promote changes associated with tumor development. To study whether alphavbeta6 may drive invasive behavior we have used transfection and retroviral infection to create a panel of epithelial cell lines expressing various levels of alphavbeta6. We report that increased expression of alphavbeta6 in malignant keratinocytes promotes invasion and leads to an increased capacity for migration towards fibronectin. alphavbeta6 expression may have a significant role in contributing to the malignant behavior of epithelial cells.

Matrix metalloproteinases (MMPs) are Zn2+ dependent proteases produced by a variety of cell types. They have a fundamental role in tissue remodelling, tumour invasion and metastasis. Scatter factor (SF), secreted by fibroblasts, has a paracrine action on epithelial cells and binds the trans-membrane c-met receptor inducing loss of adhesion, cell motility and invasiveness in vitro. The purpose of this study was to test if SF can regulate the production of MMPs by epithelial cells. Supernatants from oral squamous cell carcinoma-derived cells (H375 and H376), a human keratinocyte line (UP), and primary cultures of oral mucosal keratinocytes, grown in the presence or absence of SF, were analysed using 0.1% gelatin zymography. MMPs were characterised by comparison with human recombinant enzymes and by the use of specific inhibitors. Oral mucosal keratinocytes, UP, and H357 cells expressed MMP-2 and MMP-9, whilst H376 cells only expressed MMP-2. SF increased the expression of MMP-9 in UP and MMP-2 in H376 supernatants. Both MMP-2 and MMP-9 activity were increased in H357 and normal keratinocyte supernatants. This could be blocked using a human recombinant anti-SF antibody. In all epithelial lines tested, c-Met, the cell surface receptor for SF, could be detected. The results indicate that SF stimulates MMP expression in UP, H376, H357, and normal oral mucosal cells and points to a role for SF in the regulation of oral keratinocyte behaviour in wound healing and neoplasia.

We describe the human ACT genomic and cDNA sequence which like its murine counterpart contains the defining secondary structure of the FHL (Four-and-a-Half LIM-domain) LIM-protein family. The coding region of the human ACT gene spans five exons. This distribution is very similar to the FHL1 gene and includes the arrangement of split codons across exon boundaries suggesting that these genes share a common ancestor. The human ACT gene was not detected by Northern analysis in the adult testis although this is the only known site of expression found with its murine counterpart. However, the human ACT gene was found to be expressed in a panel of human tumor cell lines derived from squamous cell carcinomas, melanomas, and leukemias. Interestingly, FHL1, FHL2, and FHL3 were also found to be expressed in some of these cell lines and the results suggest an important role for FHLs in tumor biology., (Copyright 2000 Academic Press.)

The binding of the zymogenic form of urokinase-type plasminogen activator (pro-uPA) to its specific cellular receptor, uPAR, leads to a large potentiation of plasmin generation. This is dependent on the concurrent cellular binding of plasminogen, and is completely abrogated by the plasminogen lysine-binding site ligand, 6-aminohexanoic acid. Previous data have provided circumstantial evidence for the formation of specific complexes to mediate the kinetically favorable reciprocal interactions between the protease and zymogen components [Ellis, V., and Dano, K. (1993) J. Biol. Chem. 268, 4806-4813]. To further investigate the formation of these putative complexes, we have studied the effect of various lysine-binding site ligands on the binding and activation of plasminogen on U937 cells. Lysine-binding site ligands resembling internal lysine residues, such as Nalpha-acetyl-L-lysine methyl ester, were found to specifically inhibit uPAR-mediated cell-surface plasminogen activation at concentrations up to 40-fold lower than those inhibiting the cellular binding of 125I-labeled plasminogen (IC50s 300 microM vs 8.5 mM). By contrast, 6-aminohexanoic acid, resembling a C-terminal lysine residue, did not display this disparity (IC50s 25 vs 30 microM). These lysine analogues were also found to compete a non-active-site interaction between uPA and plasminogen, detected by surface plasmon resonance (Kd 50 nM), at concentrations correlating with their effect on cell-surface plasminogen activation, suggesting that this interaction is part of the kinetic mechanism. Consistent with this, synthetic peptides corresponding to the sequence uPA149-158 (GQKTLRPRFK) and uPA149-157 (GQKTLRPRF) specifically abolished the amplification of cell-surface plasminogen activation. These data demonstrate that a novel non-active-site interaction between uPA and plasminogen is necessary for the assembly and efficiency of cell-surface plasminogen activation complexes.

Primary closure of the pericardium affords some protection against adhesion formation and the consequent hazards of resternotomy. However, its completion may be impractical and hazardous, and therefore the pursuit of an ideal pericardial substitute has prompted much research. Twenty calves were divided into 3 groups for the study. All animals underwent right posterolateral thoracotomy. The test group (group X), consisting of 6 animals, received a poly-beta-hydroxybutyrate patch (PHB) to close the pericardium following cardiopulmonary bypass (CPB). In group Y (9 animals) the pericardium was left open following CPB. Group Z (5 animals) also had their pericardium left open but did not undergo CPB (non-CPB). The plasminogen activating activity (PAA) of homogenates of pericardial tissue samples were measured in 5 animals in group X, and 5 in group Z. Samples were taken at three time points from the time of pericardiotomy, and at reoperation 4 weeks later. In group X (CPB) there was a significant reduction in the PAA during the operation with some recovery at reoperation. The reduction in the pericardial PAA of group Z (non-CPB) animals did not reach significance. For both group X and group Z the progress of mesothelial damage, compared with that at zero time, showed a significant increase. In addition, their pericardial inflammatory features became more apparent in the later samples but more significantly in group Z. This study demonstrated no significant short-term differences in adhesion formation or postoperative coronary anatomy visibility between any of the groups. At reoperation the patch material contained pronounced macrophage activity but no regenerative mesothelium. There were no infective episodes in any of the animals studied. Furthermore, this study suggests that CPB in comparison to non-CPB has a significant affect on pericardial PAA.

Plasminogen activators play a role in the response of the vessel wall to injury, presumably by mediating the degradation of extracellular matrix (ECM) by vascular smooth muscle cells (VSMCs) that is necessary for their migration and proliferation. We have therefore investigated the ability of VSMCs to assemble specific cell surface plasminogen-activating systems. Urokinase-type plasminogen activator (uPA) bound to a single class of site on VSMCs (kd, 2 nmol/L), binding of pro-uPA resulted in a large potentiation of plasmin generation and both were competed by antibodies to the uPA receptor (uPAR). Tissue-type plasminogen activator (tPA) also bound to VSMCs as determined by functional assay, with the binding isotherms showing two classes of binding site with apparent kds of 25 and 300 nmol/L. tPA binding to the higher affinity site caused a greater than 90-fold enhancement of the activation of cell bound plasminogen, whereas the lower affinity binding, mediated primarily by the ECM, had little effect on tPA activity. The high-affinity binding of tPA to VSMCs resulted in an eightfold greater potential for plasmin generation than the binding of uPA, with this difference increasing to 15-fold after thrombin stimulation of the cells due to a 1.8-fold increase in tPA binding. These data show a novel specific tPA receptor on VSMCs that may be important for the regulation of plasminogen activation in various vascular pathologies.

Aims: To investigate the fibrinolytic activity of normal and calculous human bile., Methods: Fibrinolytic properties of the biliary tract were studied in patients with gall bladder stones (n = 7) compared with acalculous gall bladders (n = 8)., Results: Bile plasminogen activating activity was detected in a wide range in both groups (calculous bile median 0.35 IU/ml; range: 0.06-6.59, versus normal bile 0.70 IU/ml; 0.19-3.56). There was no difference in the bile concentration of tissue plasminogen activator between the two groups (calculous bile median 21.5 ng/ml versus normal bile 9.5 ng/ml), which was present in much greater concentrations than urokinase (calculous bile median 0.10 ng/ml versus normal bile 0.36 ng/ml). Both plasminogen activators were detected in low concentrations in gall bladder mucosa. Plasminogen activator inhibitors-1 and 2 were detected in bile in significantly greater concentrations in patients with gall bladder stones (plasminogen activator inhibitor-1: calculous bile median 15 ng/ml versus normal bile < 2 ng/ml, plasminogen activator inhibitor-2: 157 ng/ml versus < 6 ng/ml, p < 0.05)., Conclusions: Human bile possesses fibrinolytic activity and the principal plasminogen activator in bile seems to be tissue plasminogen activator. Plasminogen activator inhibitors were present in greater concentrations in stone bile and may be a factor in the pathogenesis of gall stone formation.

In an attempt to appreciate the changes that favour adhesion formation we compared the morphological and fibrinolytic changes that occur in human primary and reoperative pericardium. Ten patients undergoing primary elective open heart surgery and ten undergoing first time reoperative open heart surgery were studied. Pericardial samples were taken at four time points. At 0 (time A) and 30 (time B) minutes from the time of pericardiotomy (before the commencement of CPB), 30-50 minutes (time C) after the commencement of CPB, and then finally 10 minutes (time D) after the patient had been rewarmed. The fibrinolytic activity, as measured by the plasminogen activating activity (PAA), in the pericardial samples of the ten primary cases was compared with that in 5 of the reoperative cases. For the primary group, the PAA after 30 minutes of exposure (median 6.65 IU/cm2, range 3.85-11.89 IU/cm2, p = 0.14, n = 10) was not significantly reduced when compared to the initial activity (median 8.74 IU/cm2, range 2.22-17.68 IU/cm2, n = 10). After 30-50 minutes CPB the PAA was significantly reduced (median 3.93 IU/cm2, range 1.5-13.24 IU/cm2, p = 0.028, n = 10) and still reduced after rewarming for 10 minutes (median 3.12 IU/cm2, range 0.88-19.93 IU/cm2, p = 0.047, n = 10). The simultaneous plasma tissue-type plasminogen activator activity showed a significant (p < 0.05) increase after 30-50 minutes bypass with a later decline. The changes in the reoperative pericardial PAA were similar. In addition, the degree of PAA in reoperative pericardium was consistently lower than that observed in primary tissue. The extent of primary pericardial mesothelial damage at times B, C, and D compared with that at time A showed a significant (p < 0.01 for times B, C, and D) increase. Similarly there was a significant worsening of the degree of inflammation. Compared with primary pericardium, the reoperative samples showed a significant (p < 0.01 for times A, B, and C) preponderance of damaged mesothelium at the earlier stages of the operation. It appears that, following the initial bypass surgery, the processes that cause pericardial and mesothelial healing with recovery of PAA compete with those leading to pericardial adhesions and fibrosis. The histological and biochemical outcome seen in reoperative pericardium is the result of these competitive actions.

Plasminogen activator inhibitors are thought to be responsible for the abolition of fibrinolytic activity in inflamed peritoneum. This reduction in the fibrin clearing capacity of the peritoneum promotes the formation of intra-abdominal adhesions. High concentrations of plasminogen activator inhibitor-2 (PAI-2) have been previously found in inflamed peritoneal tissue using immunoassays, but it is undetectable in normal peritoneum. The aim of this study was to localize plasminogen activator inhibitor-2 production in tissue by in situ mRNA hybridisation. Sections of normal and inflamed human appendix were hybridised with a digoxigenin labelled cDNA probe. In normal appendix staining was confined to macrophages in the mucosa. Macrophage staining was also seen in inflamed tissue but with a wider distribution throughout the appendix wall. PAI-2 was also localized to mesothelial cells of inflamed but not normal appendix. Cell identities were confirmed using immunohistochemistry directed against cell specific markers. Staining was absent from control slides incubated with plasmid DNA or PAI-2 probe following ribonuclease digestion. The identification of the cells expressing the PAI-2 gene in peritoneum increases our understanding of the pathophysiological process leading to fibrin deposition within the abdomen during peritonitis.

Objective: To measure changes in the fibrinolytic properties of human peritoneum during operation., Design: Open study., Setting: University hospital, UK., Subjects: 20 patients undergoing elective operations for non-inflammatory disease., Interventions: Peritoneum was biopsied at the beginning and end of operation., Main Outcome Measures: Peritoneal plasminogen activating activity (PAA) and the concentrations of tissue plasminogen activator (t-PA), urokinase, and plasminogen activator inhibitors 1 and 2 were measured at both time points., Results: Peritoneal PAA was reduced over the time of the operation (p < 0.05) as was the concentration of t-PA (p < 0.05). The urokinase concentration rose significantly (p < 0.05), but plasminogen activator inhibitors 1 and 2 were not detected., Conclusions: Elective abdominal operation caused an immediate reduction in peritoneal PAA which seemed to be secondary to a reduced concentration of t-PA. Such a reduction in peritoneal fibrinolytic activity allows the early deposition of fibrinous deposits within the peritoneal cavity.

Objective: To see what effect the inflammatory cytokines tumour necrosis factor (TNF), interleukin 1 (IL-1), and interleukin 6 (IL-6) had, both alone and in combination, on the production of plasminogen activator inhibitor 1 (PAI-1) by human mesothelial cells., Design: Laboratory study., Setting: University hospital, UK., Material: Six cell cultures of human mesothelial cells., Main Outcome Measures: Concentrations of PAI-1 in conditioned media and cell lysates after culture for 24 hours., Results: TNF, IL-1, and IL-6 all significantly increased the amount of PAI-1 in the media compared with controls (81%, p < 0.05; 77%, p < 0.05; and 60%, p < 0.05, respectively). TNF and IL-1 together produced significantly more release than each cytokine alone (190%, p < 0.05) and a combination of the three increased release even further (290%, p < 0.05). The presence of endotoxin did not significantly affect the release of PAI-1., Conclusion: Inflammatory cytokines mediate the release of PAI-1 by mesothelial cells, and so may affect the deposition of fibrin within the peritoneal cavity.

The deposition of fibrin in the peritoneal cavity leads to fibrous adhesion formation. Recombinant tissue plasminogen activator (rtPA), delivered locally, was investigated as a method of preventing adhesion formation. Six standardised areas of peritoneal ischaemia were formed in each of 36 male Wistar rats randomised to three intraperitoneal treatments: (A) no treatment control; (B) carboxymethylcellulose gel; (C) rtPA-carboxymethylcellulose gel combination. At 1 week all animals underwent relaparotomy and the number of ischaemic sites with an adhesion counted by an independent observer. rtPA-treated animals formed fewer adhesions compared with gel alone or controls (median number of adhesions 1.5 versus 2.5 versus 5, P < 0.001, ANOVA). Intraperitoneal rtPA in a slow-release formulation is able to reduce adhesion formation significantly in an animal model and may prove to have clinical benefit.

Objective: Measurement of the fibrinolytic response of the peritoneum to experimental peritonitis and ischaemia., Design: Controlled study, Setting: Academic surgical unit, UK MATERIAL: Male Wistar rats, Interventions: Peritoneal injuries were caused in four groups of male Wistar rats (n = 35 in each group): (1) control group ("open and close" laparotomy); (2) bacterial peritonitis (mixed faecal flora); (3) chemical peritonitis (10 mg/ml tetracycline) and; (4) ischaemic peritoneum (ligated peritoneal buttons). Peritoneal biopsy specimens were taken from five animals in each group at seven time intervals and plasminogen activating activity (PAA) measured by fibrin plate assay., Results: Compared with the control group the three peritoneal injuries produced a uniform reduction in PAA during the first 6 and 12 hours: at 6 hours the median PAA was 0.029 IU/cm2 for bacterial peritonitis, 0.021 IU/cm2 for chemical peritonitis, and 0.05 IU/cm2 for ischaemic peritoneum compared with 0.112 IU/cm2 for the control group; p < 0.001, ANOVA. At 12 hours the median PAA was 0.024 IU/cm2 for bacterial peritonitis, < or = 0.014 IU/cm2 for chemical peritonitis, and 0.05 IU/cm2 for ischaemic peritoneum compared with 0.112 IU/cm2 for the control group; p < 0.001, ANOVA. There then followed a rebound peak in all groups, maximal at 4-7 days, before a return to baseline values at two weeks., Conclusion: Peritoneal fibrinolysis was appreciably inhibited after three different standardised peritoneal injuries. The data support the hypothesis that there is a single pathophysiological mechanism of adhesion formation.

The presence of pericardial adhesions prolongs the operation time and increases the risk of serious damage to the heart and other major vascular structures during resternotomy. The reported incidence of such damage is 2% to 6%. Pericardial mesothelial cells exhibit fibrinolytic activity, and therefore have an actual or potential role in the breakdown of the fibrinous adhesions that serve as the initial scaffolding for the firm collagenous adhesions seen at reoperation. Ten patients undergoing primary cardiac procedures were studied to assess the morphologic changes that take place within the pericardium and to relate these to accompanying changes in the pericardial plasminogen activating activity. Samples were taken at 0, 75, and 135 minutes after pericardiotomy. Compared with samples obtained at the time of pericardiotomy, those taken at 75 and 135 minutes demonstrated a significant progression in the mesothelial cell damage (p < 0.01), together with increasing evidence of pericardial inflammation (p < 0.01). The findings from electron microscope studies confirmed and supplemented these findings. Furthermore, compared with its initial levels (median, 2.06 IU/cm2; range, 1.28 to 6.48 IU/cm2), the plasminogen activating activity of pericardial biopsy specimens was significantly reduced at 75 minutes (median, 0.64 IU/cm2; range, 0.12 to 2.44 IU/cm2; P < 0.05), with some recovery at 135 minutes (median, 1.45 IU/cm2; range, 0.12 to 4.39 IU/cm2; p = 0.059). This study has revealed that, during cardiac procedures, the pericardium undergoes inflammatory changes with concomitant damage to its mesothelium, together with a reduction in the pericardial mesothelial fibrinolytic potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Human mesothelial cells synthesize plasminogen activator inhibitor (PAI) 1 in inflamed peritoneal tissue. The role of tumour necrosis factor (TNF) in the mediation of this response was studied. Postoperative peritoneal drain fluid contained both TNF and PAI-1. Peak levels of TNF at 4 h (median 271 pg/ml) preceded a rise in PAI-1 concentration, which peaked at 18 h (median 943.1 ng/ml). Thus TNF may mediate increased PAI-1 release in inflamed peritoneum. TNF significantly increased the mean(s.e.m.) release of PAI-1 by human peritoneal mesothelial cells in vitro at 4 h (control 1.84(0.17) ng/micrograms versus TNF 2.37(0.17) ng/micrograms, P < 0.05), 6 h (2.53(0.09) versus 3.88(0.46) ng/micrograms, P < 0.05), 18 h (0.50(0.02) versus 1.04(0.11) ng/micrograms, P < 0.05) and 24 h (0.87(0.05) versus 1.35(0.11) ng/micrograms, P < 0.05). TNF may be an important mediator of PAI-1 production by human mesothelial cells during peritoneal inflammation.

The presence of pericardial adhesions at resternotomy not only increases the operation time but also increases the risk of serious damage to the heart, great vessels, and extracardiac grafts. The reported prevalence of damage is 2% to 6%. The fibrinolytic activity of pericardial tissue may be a crucial factor in determining the extent of adhesion formation following primary operation. Ten patients undergoing cardiac operations were studied to assess the plasminogen activating activity of homogenates of pericardial tissue samples. Samples were taken at three times during the operation and the plasminogen activating activity was measured by means of a standard fibrin plate technique. Tissue-type plasminogen activator, urokinase-type plasminogen activator, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2 were also measured by means of enzyme-linked immunosorbent assays. Compared with its initial levels (median 2.06 IU/cm2, range 1.28 to 6.48 IU/cm2), the plasminogen activating activity of pericardial biopsy tissue was significantly reduced at 75 minutes (median 0.64 IU/cm2, range 0.12 to 2.44 IU/cm2, p < 0.01) and at 135 minutes (median 1.45 IU/cm2, range 0.12 to 4.39 IU/cm2, p < 0.05). The major plasminogen activator present was tissue-type plasminogen activator. Compared with its initial levels (median 2.34 ng/ml, range 1.03 to 6.42 ng/ml), subsequent tissue-type plasminogen activator values were also significantly reduced at 75 minutes (median 0.83 ng/ml, range 0.75 to 5.13 ng/ml, p < 0.005) and at 135 minutes (median 1.24 ng/ml, range 0.75 to 6.67 ng/ml, p < 0.05). Low levels of urokinase-type plasminogen activator were found in 5 of 10 patients. However, neither plasminogen activator inhibitor-1 nor plasminogen activator inhibitor-2 was detected. Examination with a light microscope showed both increasing pericardial mesothelial damage and increasing features of acute inflammatory changes with time. This study shows that plasminogen activating activity is present in pericardial tissue and that tissue-type plasminogen activator is the major plasminogen activator. The observed inflammatory changes and concomitant damage to the pericardial mesothelium, and the significant reductions in pericardial tissue-type plasminogen activator and plasminogen activating activity seen during cardiac operations, may be important factors contributing to the early development of pericardial adhesions.

Coagulopathy is a well recognised complication of peritoneovenous shunting for ascites. The relative contributions of primary fibrinolysis and disseminated intravascular coagulation remain controversial. Plasminogen activating activity was significantly lower in malignant ascites (n = 10, median < 0.02 (range < 0.02-1.26) IU/ml) than in alcoholic ascites (n = 10, 1.07 (0.30-1.49) IU/ml) (p < 0.05). Fibrinolytic activity was determined by a balance between tissue plasminogen activator and plasminogen activator inhibitor-1. There was no significant difference between the two groups in the concentration of tissue plasminogen activator (34 (12-64) ng/ml in malignant ascites v 29 (12-43) ng/ml in alcoholic ascites), but the concentration of plasminogen activator inhibitor-1 was significantly higher in malignant ascites (736 (213-1651) ng/ml) than in alcohol ascites (29 (12-43) ng/ml) (p < 0.05). Malignant ascites contained significantly higher concentrations of urokinase (0.7 (< 0.1-1.3) ng/ml v 0.2 (< 0.1-0.6) ng/ml in alcoholic ascites) and plasminogen activator inhibitor-2 (33 (< 6-140) ng/ml v 9 (< 6-28) ng/ml alcoholic ascites). The plasminogen activating activity of alcohol ascites may lead to primary fibrinolysis after peritoneovenous shunting. The considerably lower activity found in malignant ascites may explain why coagulopathy after shunting is less pronounced in this group of patients.

Fibrinolysis in renal transplant rejection has not been systematically investigated but is known to be impaired. By an in situ hybridization technique, we have studied gene expression of plasminogen activator inhibitor 1 (PAI-1) in human renal tissue showing severe acute vascular rejection (n = 8), clinically irreversible (chronic) vascular rejection (n = 3), mild vascular rejection (n = 8), parenchymal rejection (n = 4), 'normal' kidneys (n = 6), and non-rejecting kidneys (n = 6). The results showed that in 7/8 cases showing severe acute vascular rejection and in all three chronic vascular rejection cases, PAI-1 mRNA was expressed by endothelial cells of arterioles and arteries, and interstitial inflammatory cells but was not detectable in any other groups. The expression of PAI-1 was frequently associated with areas of haemorrhage. Expression of PAI-1 might be expected to promote thrombosis and ischaemia, the catastrophic consequence of severe vascular rejection. In irreversible chronic rejection, this seems to be the principal mode of action. However, our observation that there is expression of PAI-1 in areas of haemorrhage in severe acute vascular rejection may suggest an additional potentially protective role, if it were produced as a response to haemorrhage.

Fibrinolysis in peritoneal tissue may play a role in the development of intra-abdominal adhesions. The plasminogen-activating capacity of human peritoneum results largely from the presence of tissue plasminogen activator (tPA). Inflammation reduces peritoneal plasminogen-activating activity and leads to the appearance of plasminogen activator inhibitor (PAI) type 1. The role of PAI-2 in the inhibition of peritoneal fibrinolysis during inflammation was investigated in this study. The plasminogen-activating activity of peritoneal biopsy homogenates (seven inflamed, seven normal), measured using a fibrin plate technique, was reduced in inflamed compared with normal tissue (median < 0.07 versus 13.9 units/cm2, P < 0.01); tPA antigen levels were not significantly different (median 1.02 versus 1.34 ng/ml). PAI-1 and PAI-2 antigens were not detected in normal human peritoneum but were present in inflamed peritoneum (median concentration 8.8 ng/ml for PAI-1, 26.7 ng/ml for PAI-2). These inhibitors may be important factors in adhesion formation by contributing to the abolition of peritoneal plasminogen-activating activity.

The peritoneum has been shown to possess fibrinolytic activity which is thought to play a role in the prevention of intra-abdominal adhesion formation. Recently inflamed peritoneal tissue has been shown to have reduced fibrinolytic activity secondary to increased levels of plasminogen activator inhibitor-1 (PAI-1). The aim of this study was to localize the production of PAI-1 in appendix tissue using in situ mRNA hybridization. Sections of normal and inflamed appendix were hybridized with a digoxigenin-labelled cDNA probe. PAI-1 production was localized to both mesothelium and serosal blood vessel endothelium in all inflamed appendix samples. Cell identities were confirmed using immunohistochemistry directed against mesothelial and endothelial cell markers. Staining was not seen on 1 probe following ribonuclease digestion). The identification of the cells expressing the PAI-1 gene in peritoneum increases our understanding of the pathophysiological changes in fibrinolytic activity which occur in inflammation and may lead to adhesion formation.

The mechanisms leading to reduction of peritoneal fibrinolytic activity in conditions that are associated with the formation of intra-abdominal adhesions were studied. Tissue plasminogen activator was found, by antibody inhibition techniques, to be the activator of fibrinolysis in homogenates of control peritoneum (n = 6). Homogenates of control (n = 10) and inflamed peritoneum (n = 10) were analysed. Plasminogen activating activity was much lower in inflamed peritoneum (median 0.07 IU/cm2) than in control tissue (median 12.0 IU/cm2) (p less than 0.001). Levels of tissue plasminogen activator and alpha 2-antiplasmin were similar in both control and inflamed tissue. Plasminogen activator inhibitor-1, not detectable in control peritoneum, was present in inflamed tissue and might be the reason for the reduction in functional fibrinolytic activity.