Expression and Polymorphism of Toll-Like Receptor 4 and Effect on NF-κB Mediated Inflammation in Colon Cancer Patients

Our aim was to evaluate the association between the expression and the polymorphism of TLR4/NF-κB pathways and colon cancer. TLR4 (rs4986790, rs10759932, rs10759931 and rs2770150) were genotyped in blood samples from Colorectal patients and healthy controls. TLR4 and cytokines inflammatory expression were evaluated by real time PCR on 40 matching normal and colon tissues and the protein level by Immunohistochemistry. The high level of TLR4 expression in colon cancer tissues is mainly due to infections by bacteria in the human colon and leads to induction of an acute secretion of inflammatory cytokines mediated by NF-κB. Also, we report here a clear evidence for an association between TLR4 rs10759931 polymorphism (OR = 0.086, CI: 0.04–0.18, P = <0.00001). This polymorphism affects the entire population without being specific to either gender or to any age group. In contrast, the rs2770150 is associated with colon cancer in women aged over 50 years and is closely linked with the decreased levels of female sex hormones during the post-menopausal period (OR = 0.188, CI: 0.074–0.48, P = <0.00084). rs10759932 and rs4986790 appear to have any association with colon cancer. Our data suggest that TLR4 SNPs could possibly serve as biomarkers for decision making in colon cancer treatment.     

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 yeast's 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. Proinflammtory 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.     

Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase, CstNCED, isolated from Crocus sativus stigmas

Oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. ABA has been associated with dormancy and flower senescence, while also regulating plant adaptive responses to various environmental stresses. An NCED gene, CstNCED, was cloned from Crocus sativus stigmas. The deduced amino acid sequence of the CstNCED protein shared high identity with other monocot NCEDs, and was closely related to the liliopsida enzymes. At the N-terminus of CstNCED a chloroplast transit peptide sequence is located. However, its expression in chloroplast-free tissues suggested localization in other plastid types. The relationship between expression of CstNCED and the endogenous ABA level was investigated in the stigma and corms, where it was developmentally regulated. The senescence of the unpollinated stigma is preceded by an increase in ABA levels and CstNCED expression. In corms, a correlation was observed between CstNCED expression and dormancy. Furthermore, CstNCED expression was correlated with the presence of zeaxanthin in the dormant corms. When detached C. sativus leaves and stigmas were water and salt stressed, increases in CstNCED mRNA were observed. The results provided evidence of the involvement of CstNCED in the regulation of ABA-associated processes such as flower senescence and corm dormancy in monocotyledonous saffron.     

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.     

Proteomic analysis of major and minor allergens from isolated pollen cytoplasmic granules

Grass pollen is one of the most important vectors of aeroallergens. Under atmospheric conditions, pollen grains can release pollen cytoplasmic granules (PCGs). The allergens associated with these intrinsic subfractions induce, in laboratory animals as well as in asthmatic patients, allergic and inflammatory responses. The objectives of this study were to characterize the PCGs' intrinsic allergens and to compare them with those of pollen grains. The water-soluble proteins were extracted from pollen grains and their PCGs. IgE-binding proteins were analyzed and characterized through an allergomic strategy: 1- and 2-dimensional gel electrophoresis (1-DE and 2-DE), immunoblotting, using grass-pollen-sensitized patient sera, mass spectrometry (MS) analysis, and database searching. Several of the allergens listed in the IUIS nomenclature, Phl p 1, 4, 5, 6, and 12, were detected in pollen and PCG extracts, whereas Phl p 11 was found only in PCGs, and Phl p 2 as well as Phl p 13 were found only in pollen extract. Some other allergens not listed in the IUIS nomenclature were also characterized in both pollen and PCG extracts. Since the major grass pollen allergens were found in PCGs and because of their small size, these submicronic particles should be considered as very potent sensitizing and challenging respirable vectors of allergens.     

Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation

Background  

Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus.     

Immediate early gene X-1 (IEX-1), a hydroxytamoxifen regulated gene with increased stimulation in MCF-7 derived resistant breast cancer cells

The efficacy of tamoxifen in breast cancer treatment only lasts a few years and the tumor eventually recurs. We performed selective subtractive hybridization to isolate mRNAs that were differentially expressed in MCF-7 derived cells, in which resistance had been induced through long-term culture in the presence of hydroxytamoxifen (OHT). Among the 15 mRNAs found to be overexpressed, we focused on Immediate early gene X-1 (IEX-1) mRNA because of the recognized contribution of its expression to apoptosis or cell cycle progression, depending on the cell type and culture conditions. We observed that IEX-1 expression was stimulated by OHT, that the degree of increase was greater in resistant cells (four-fold versus 1.5-fold) and that this OHT regulation was estrogen receptor dependent. A detailed study of the IEX-1 promoter indicated that it involved NF-kappaB. Our cells were not cross-resistant to faslodex, a pure antiestrogen, which moreover was inefficient in regulating IEX-1 expression. Altogether, our data suggest that the greater IEX-1 expression in OHT resistant cells is related to their ability to grow in the presence of OHT. Knowledge on the capacity of OHT to stimulate gene expression and its NF-kappaB dependence should contribute to a better understanding of tamoxifen pharmacology and allow new drug strategies to be designed that would delay antiestrogen resistance acquisition.     

Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides

The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of beta-(1-->6)-galactofuranose fully substituted at O-2 by terminal residues of alpha-glucopyranose and alpha-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted beta-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.