Increased NFκ-B Activity in HCT116 Colorectal Cancer Cell Line Harboring TLR4 Asp299Gly Variant

Toll-Like Receptor 4 (TLR4), considered one of the most important TLR, recognizes lipopolysaccharide of gram-negative bacteria. Recognition of ligands by TLRs induces signaling pathways resulting in activation of transcriptional factors such as NF-κB which are involved in the expression of inflammatory cytokines and chemokines. To prevent an inappropriate immune response, a complex network of molecules negatively regulates TLRs and their associated signaling pathways. Two cosegregating single nucleotide polymorphisms of the human TLR4 gene, namely Asp299Gly and Thr399Ile, have been associated with hyporesponsiveness to inhaled LPS. The purpose of this study was to determine the impact of TLR4 gene variant on NF-κB activity in colorectal cancer cell line. HCT116 cells were transfected with wild-type and mutants Flag-CMV1-TLR4 expression vectors. Western blot analysis was performed to evaluate selected molecules involved in TLR4 signaling. NF-κB activity was assessed by dualluciferase reporter assay and cytokine profiles were evaluated by ELISA and Cytometric Bead Array method. Results showed that the activity of pNF-κB was higher in cells harboring TLR4 D299G compared to the other cells. However, the activity of pAKT, pERK1 and pIRAK was higher in wild-type. The results of cytokine measurements showed about four fold higher level of IL-8 in cells with wild-type TLR4. This study suggest that TLR4 Asp299Gly gene variant has an impact on TLR4 signaling and potentially on intestinal homeostasis due to impaired control signals at the epithelial cell level which may lead to chronic intestinal inflammation and interrupted intestinal homeostasis and may eventually lead to colorectal cancer. Copyright© 2012, Iranian Journal of Allergy, Asthma and Immunology. All rights reserved

5-Fluorouracil induce the expression of TLR4 on HCT116 colorectal cancer cell line expressing different variants of TLR4

Two common single nucleotide polymorphisms (SNPs) of the human TLR4 gene, namely Asp299Gly (D299G) and Thr399Ile (T399I), have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type (WT) and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC50-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells. © 2013 by School of Pharmacy

The nucleotide sequence of the bovine interleukin-5-encoding cDNA

The nucleotide (nt) sequence encoding the bovine homologue of interleukin-5 (boIL-5) was determined. Total cDNA generated from stimulated bovine peripheral blood lymphocytes was used to amplify the boIL-5 gene using primers based on the 5' and 3' untranslated regions of the ovine IL-5 cDNA sequence. The boIL-5 coding sequence is 405 by long, coding for a 15.2 kDa precursor protein of 134 amino acids (aa). Cleavage of a putative signal peptide of 19 aa yields a mature form of 13.1 kDa. Comparisons at the nt level revealed 96%, 81%, 74% and 73% identity with the ovine, human, mouse and rat IL-5 sequences, respectively, and 97%, 66%, 59% and 58% aa identity, respectively

The management of defective resin composite restorations:Current trends in dental school teaching in Japan

AIM: The aim of this article is to investigate the contemporary teaching of the management of defective direct resin composite restorations in dental schools in Japan. METHODS: A questionnaire relating to the teaching of the management of defective resin composite restorations was developed and e-mailed to 29 dental schools in Japan in 2010. RESULTS: Completed responses were received from 19 of the 29 invited schools (response rate = 66%). Eighteen schools (95%) report that they included the teaching of repair of direct defective resin composite restorations in their dental school programs. Thirteen schools reported that they included both clinical and didactic instruction on the repair of direct resin composite restorations. Fourteen schools did not teach any mechanical roughening of the exposed resin composite restoration surface before undertaking a repair. The most commonly reported treatment was acid etching with phosphoric acid (12 schools). The most commonly taught material for completing repairs was a flowable resin composite (16 schools). CONCLUSION: The teaching of repair of defective resin composite restorations is well established within many Japanese dental schools, to a greater extent than in some other regions of the world. The impact of this teaching on subsequent clinical practices in Japan should be investigated. Furthermore, it is concluded that there is a need for much stronger leadership in operative and conservative dentistry, ideally at the global level, to resolve differences in key aspects of operative procedures such as repairs

Research on cancer diagnosis in Malaysia: current status.

Cancer is a major morbidity and mortality concern in Malaysia. Based on National Cancer Registry data, the Malaysian population is estimated to bear a cancer burden of about 40,000 new cases per year, and a cumulative lifetime risk of about 1:4. Cancer research in Malaysia has to consider needs relevant to our population, and resources constraints. Hence, funding bodies prioritise cancers of high prevalence, unique to our community and posing specific clinical problems. Cancer diagnosis is crucial to cancer management. While cancer diagnosis research largely aims at improvements in diagnostic information towards more appropriate therapy, it also impacts upon policy development and other areas of cancer management. The scope of cancer diagnosis upon which this paper is based, and their possible impact on other R&D areas, has been broadly categorized into: (1) identification of aetiological agents and their linkages to the development of precancer and cancer (impact on policy development, cancer prevention and treatment), (2) cancer biology and pathogenesis (impact on cancer prevention, treatment strategies and product development), (3) improvements in accuracy, sensitivity and specificity in cancer detection, monitoring and classification (impact on technology development) and (4) prognostic and predictive parameters (impact on treatment strategies). This paper is based on data collected by the Working Group on Cancer Diagnosis Research for the First National Conference on Cancer Research Coordination in April 2004. Data was collated from the databases of Institutions/Universities where the authors are employed, the Ministry of Science, Technology and Innovation (MOSTI) and targeted survey feedback from key cancer researchers. Under the 7th Malaysia Plan, 76 cancer projects were funded through the Intensified Research in Priority Areas (IRPA) scheme of MOSTI, amounting to almost RM15 million of grant money. 47(61.8%) of these projects were substantially in cancer diagnosis, accounting for 65.6% (RM 9.7 million) of cancer project funds. The 8th Malaysia Plan saw a change in research strategy. The IRPA agency fielded several top-down projects which encouraged a multicentre and multidisciplinary approach. This resulted in larger funding per project i.e. RM32 million for 49 projects. There was also a surge of interest in drug development and natural products. Because of this shift in direction, cancer diagnosis projects constituted only 51% of IRPA-funded cancer projects. Nonetheless funding for cancer diagnosis research has exceeded that of the 7th Malaysia Plan, being RM12.5 million by March 2004. The majority of such research is carried out at the Universities, engaging a large number of young scientists and postgraduate students (51 MSc and 21 PhD). A lot of research findings presented at scientific meetings have not yet been published and there is a glaring shortage of patents and commercialization of research findings (such as creation of test kits). Because diagnosis is very much a part of clinical practice, many researchers felt satisfied and confident that their work will be translated into practice and will significantly improve diagnostic services in Malaysia. National guidelines and consensus development on at least three malignancies i.e. breast cancer, oral cancer and lymphoma, have substantial basis in local R&D work. Problems encountered in research included (1) insufficient funding to realize research objectives, (2) lack of local expertise (most research assistants are inexperienced BSc graduates with no or minimal research experience), (3) inadequate technical support from vendors during equipment failure, (4) inexperienced Institutional development units to assist in product development, (5) lack of venture capital for commercialization of findings, and (6) inadequate incentives to undertake research. Researchers pointed out that plans to promote research should include the establishment of (1) regional and national cancer tissue banks, (2) a National Cancer Research Institute, (3) a dedicated cancer research fund, (4) a registry of cancer researchers, (5) national research coordinators, (6) improved coverage by the National Cancer Registry, (7) more international collaboration, (8) a better career structure for researchers, (9) improved Institutional support for product realization, and (10) better recognition for cancer researchers