Abdelhabib Semlali

Thymic stromal lymphopoietin–induced human asthmatic airway epithelial cell proliferation through an IL-13–dependent pathway

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays a pivotal role in the initiation of allergic airway inflammation. This cytokine is produced by several cell types, including human epithelial cells. OBJECTIVE We sought to determine the effect of TSLP on proliferation and repair of epithelial cells isolated from asthmatic patients and healthy subjects. METHODS Expression of TSLP receptor (TSLPR) and its response to inhaled corticosteroids was evaluated on bronchial biopsy specimens of healthy control subjects and asthmatic patients by means of immunohistochemistry. TSLPR, TSLP, and IL-13 mRNA expression was determined by means of quantitative PCR, and protein expression was measured by means of ELISA and Western blotting in epithelial cells isolated from asthmatic subjects compared with those isolated from healthy control subjects. The effect of TSLP on cell proliferation and wound healing was performed. RESULTS TSLPR is expressed by bronchial epithelial cells in bronchial biopsy specimens and in cultured cells, with no difference between asthmatic patients and healthy control subjects. Inhaled corticosteroids did not affect this expression. TSLP mRNA and protein levels were significantly higher in epithelial cells isolated from asthmatic patients compared with those from healthy control subjects. TSLP stimulated IL-13 production by bronchial epithelial cells. TSLP induced airway epithelial cell proliferation and enhanced epithelial injury repair. This effect was abrogated with IL-13 neutralization. CONCLUSIONS Our data indicate that epithelial cells express TSLPR and that TSLP induces bronchial epithelial cell proliferation and increases injury repair through IL-13 production. This suggests that TSLP and IL-13 loops play a homeostatic role on epithelial cell proliferation and repair.

Whole Cigarette Smoke Increased the Expression of TLRs, HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

The gingival epithelium is becoming known as a regulator of the oral innate immune responses to a variety of insults such as bacteria and chemicals, including those chemicals found in cigarette smoke. We investigated the effects of whole cigarette smoke on cell-surface-expressed Toll-like receptors (TLR)-2, −4 and −6, human β-defensin (HBD) and proinflammatory cytokine expression and production in primary human gingival epithelial cells. Whole cigarette smoke was shown to increase TLR2, TLR4 and TLR6 expression. Cigarette smoke led to ERK1/2, p38 and JNK phosphorylation in conjunction with nuclear factor-κB (NFκB) translocation into the nucleus. TLR expression following cigarette smoke exposure was down regulated by the use of ERK1/2, p38, JNK MAP kinases, and NFκB inhibitors, suggesting the involvement of these signaling pathways in the cellular response against cigarette smoke. Cigarette smoke also promoted HBD2, HBD3, IL-1β, and IL-6 expression through the ERK1/2 and NFκB pathways. Interestingly, the modulation of TLR, HBD, and cytokine expression was maintained long after the gingival epithelial cells were exposed to smoke. By promoting TLR, HBDs, and proinflammatory cytokine expression and production, cigarette smoke may contribute to innate immunity dysregulation, which may have a negative effect on human health.

Whole cigarette smoke promotes human gingival epithelial cell apoptosis and inhibits cell repair processes.

BACKGROUND AND OBJECTIVE Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. MATERIAL AND METHODS Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. RESULTS Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl-xL and caspase-3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. CONCLUSION Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl-xL proteins and caspase-3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.

Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa

We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeasts virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human β-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1β and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.

Effects of whole cigarette smoke on human gingival fibroblast adhesion, growth, and migration.

The aim of this study was to investigate the effects of a single exposure to whole cigarette smoke on human gingival fibroblast behavior. Normal oral mucosa fibroblasts were exposed once to whole cigarette smoke for 5, 15, or 30 min, and then were used to analyze cell adhesion, β1-integrin expression, cell growth and viability, cell capacity to contract collagen gel, and cell migration following wound infliction. Our findings showed that when gingival fibroblasts were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell adhesion, a decrease in the number of β1-integrin-positive cells, increased LDH activity in the target cells, and reduced growth. The smoke-exposed fibroblasts were also not able to contract collagen gel matrix and migrate following insult. Overall results demonstrate that a single exposure to whole cigarette smoke produced significant morphological and functional deregulation in gingival fibroblasts. This may explain the higher predisposition of tobacco users to oral infections and diseases such as cancer.

Cigarette smoke condensate increases C. albicans adhesion, growth, biofilm formation, and EAP1 , HWP1 and SAP2 gene expression

BackgroundSmokers are more prone to oral infections than are non-smokers. Cigarette smoke reaches the host cells but also microorganisms present in the oral cavity. The contact between cigarette smoke and oral bacteria promotes such oral diseases as periodontitis. Cigarette smoke can also modulate C. albicans activities that promote oral candidiasis. The goal of this study was to investigate the effect of cigarette smoke condensate on C. albicans adhesion, growth, and biofilm formation as well as the activation of EAP1, HWP1 and secreted aspartic protease 2.ResultsCigarette smoke condensate (CSC) increased C. albicans adhesion and growth, as well as biofilm formation. These features may be supported by the activation of certain important genes. Using quantitative RT-PCR, we demonstrated that CSC-exposed C. albicans expressed high levels of EAP1, HWP1 and SAP2 mRNA and that this gene expression increased with increasing concentrations of CSC.ConclusionCSC induction of C. albicans adhesion, growth, and biofilm formation may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced oral diseases.

Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects

To cite this article: Semlali A, Jacques E, Rouabhia M, Milot J, Laviolette M, Chakir J. Regulation of epithelial cell proliferation by bronchial fibroblasts obtained from mild asthmatic subjects. Allergy 2010; 65: 1438–1445.

Association between PARP-1 V762A Polymorphism and Breast Cancer Susceptibility in Saudi Population

Genetic aberrations of DNA repair enzymes are known to be common events and to be associated with different cancer entities. Aim of the following study was to analyze the genetic association of rs1136410 (Val762Ala) in PARP1 gene with the risk of breast cancer using genotypic assays and insilico structural predictions. Genotypic analysis of individual locus showed statistically significant association of Val762Ala with increased susceptibility to breast cancer. Protein structural analysis was performed with Val762Ala variant allele and compared with the predicted native protein structure. Protein prediction analysis showed that this nsSNP may cause changes in the protein structure and it is associated with the disease. In addition to the native and mutant 3D structures of PARP1 were also analyzed using solvent accessibility models for further protein stability confirmation. Taken together, this the first study that confirmed Val762Ala variant has functional effect and structural impact on the PARP1 and may play an important role in breast cancer progression in Saudi population.

Effect of prolonged hydroxytamoxifen treatment of MCF-7 cells on mitogen activated kinase cascade.

Resistance to the antiestrogen tamoxifen is the main stumbling block for the success of breast cancer therapy. We focused our study on cellular alterations induced by a prolonged treatment with the active tamoxifen metabolite hydroxytamoxifen (OHT). We show that a prolonged OHT treatment (for up to 7 days) led to a progressive increase in the level of phosphorylated p44/42 mitogen activated kinase (MAP kinase) induced by 10−7 M TPA stimulation, without any significant change in the protein level. This effect was also observed in MCF‐7 cells grown first in medium containing dextran‐coated charcoal‐treated FCS (DCC medium) for 20 days prior to OHT treatment, indicating a specific effect of the antiestrogen and not an effect of estrogen deprivation. It was prevented by cotreatment with estradiol and not observed in the estrogen receptor negative HeLa cell line, suggesting that it was mediated by the estrogen receptor. TPA induced phosphorylation of MEK1/2 was also raised by OHT treatment, without any change in their protein level or Raf‐1 and H‐Ras levels. When the MCF‐7R OHT resistant cell line was grown in antiestrogen containing medium, the level of phosphorylated p44/42 MAP kinase was also high but reversed when the antiestrogen was removed. The 2 other MAP kinase, JNK and P38 pathways were not affected in the same way by OHT treatment. In conclusion, our data reveal that a prolonged OHT treatment, by increasing p44/42 MAPK activity, affects a key step in the growth control of MCF‐7 cells, although not sufficiently to overcome the growth inhibitory effect of the drug.

Crosstalk between T cells and bronchial fibroblasts obtained from asthmatic subjects involves CD40L/α5β1 interaction

BACKGROUND Allergic asthma is characterized by infiltration of inflammatory cells into the airways. T cell-derived cytokines regulate both airway inflammation and remodelling. In the human airways, T cell-fibroblast interactions may have a role in regulating inflammation and remodelling. OBJECTIVES To evaluate the effect of bronchial fibroblast-T cell interaction on profibrogenic cytokine release and determine the nature of the molecules involved in this interaction. METHODS Human bronchial fibroblasts obtained from healthy and asthmatic donors were co-cultured with purified T cells derived from peripheral blood of the same subjects. IL-6 mRNA and protein levels were measured by real time PCR and ELISA. CD40, CD40L and alpha 5 beta 1 were evaluated by flow cytometry. Bronchial fibroblasts were stimulated with rsCD40L. Neutralisation was performed using neutralizing antibodies anti-CD40L and anti-alpha 5. RESULTS Contact of T cells with bronchial fibroblasts up-regulated IL-6 at both gene and protein levels. This effect was significantly higher in fibroblasts from asthmatics than those from controls. Blocking CD40L and alpha 5 beta 1 integrin showed a significant inhibition of IL-6 expression in asthmatics but not in healthy controls. Stimulation of fibroblasts with recombinant soluble CD40L up-regulated IL-6 production in asthmatics but not in controls. Adhesion to fibronectin, a alpha 5 beta 1 integrin ligand, is increased in fibroblasts from asthmatics compared to fibroblasts from controls. CONCLUSION These results showed that interaction of bronchial fibroblasts with T cells increases the production of profibrogenic cytokine IL-6. In asthmatic condition this interaction involves CD40L/alpha 5 beta 1. These results suggest that T cells and structural cells crosstalk in asthma may maintain local mucosal inflammation.