Fenofibrate impairs liver function and structure more pronounced in old than young rats.

INTRODUCTION: Since old animals are known to accumulate lipids in some organs, we compared effects of fenofibrate (FN) on systemic lipid metabolism, activity of liver marker enzymes and structure in young and old rats. MATERIAL AND METHODS: Young and old rats were fed chow supplemented with 0.1 % or 0.5 % FN. After 30 days, intraperitoneal glucose tolerance test (IPGTT) was performed, and blood and liver samples were collected. RESULTS: In young rats, 0.1 % FN, but not 0.5 % FN, decreased serum Chol by 74 %, and did not affect TG levels at either doses. In old rats, 0.5 % FN, but not 0.1 % FN, decreased Chol and TG level by 56 % and 49 %, respectively. In young rats, 0.1 % and 0.5 % FN increased serum activity of ALP by 227 % and 260 %, respectively, and did not affect AST and ALT activities. In old rats, only 0.5 % FN increased serum ALP activity by 150 %, respectively. In old rats, neither dose of FN affected serum AST activity, and only 0.5 % FN increased serum ALT activity by 200 %. The histological examination of liver structure revealed that both doses of FN impaired lobular architecture, expansion of bile canaliculi, and degeneration of parenchymal cells with the presence of cells containing fat droplets; administration of FN increased area occupied by collagen fibers. CONCLUSIONS: Although 0.5 % FN decreased serum Chol concentration, it increased serum ALP activity and impaired liver structure in both in both age groups of rats. Thus, FN treatment should be under the control of liver function, especially in older patients.

Expression of HTRA Genes and Its Association with Microsatellite Instability and Survival of Patients with Colorectal Cancer.

HtrA proteases regulate cellular homeostasis and cell death. Their dysfunctions have been correlated with oncogenesis and response to therapeutic treatment. We investigated the relation between HtrA1-3 expression and clinicopathological, and survival data, as well as the microsatellite status of tumors. Sixty-five colorectal cancer patients were included in the study. The expression of HTRA1-3 was estimated at the mRNA and protein levels by quantitative PCR and immunoblotting. Microsatellite status was determined by high-resolution-melting PCR. We found that the HTRA1 mRNA level was higher in colorectal cancer tissue as compared to the unchanged mucosa, specifically in primary lesions of metastasizing cancer. The levels of HtrA1 and HtrA2 proteins were reduced in tumor tissue when compared to unchanged mucosa, specifically in primary lesions of metastasizing disease. Moreover, a decrease in HTRA1 and HTRA2 transcripts' levels in cancers with a high level of microsatellite instability compared to microsatellite stable ones has been observed. A low level of HtrA1 or/and HtrA2 in cancer tissue correlated with poorer patient survival. The expression of HTRA1 and HTRA2 changes during colorectal carcinogenesis and microsatellite instability may be, at least partially, associated with these changes. The alterations in the HTRA1/2 genes' expression are connected with metastatic potential of colorectal cancer and may affect patient survival.

Distinct Expression Patterns of Two Tumor Necrosis Factor Superfamily Member 15 Gene Isoforms in Human Colon Cancer.

BACKGROUND: Tumor necrosis factor superfamily member 15 (TNFSF15) gene is involved in development of several cancers. It encodes two proteins: tumor necrosis factor ligand-related molecule 1A (TL1A) and vascular endothelial growth inhibitor 192 (VEGI-192). The main receptor for TL1A is death receptor 3 (DR3). AIMS: We investigated expression of TL1A, VEGI-192, and DR3 transcripts in different stages of colon cancer and compared them with survival of patients. We also aimed to reveal possible effects of microsatellite instability (MSI) and selected TNFSF15 single-nucleotide polymorphisms (SNPs) on expression of this gene. METHODS: Forty-five healthy individuals and 95 colon cancer patients were included in the study. Expression of VEGI-192, TL1A, and DR3 was measured by quantitative PCR. SNP and MSI analyses were performed on DNA isolated from normal or cancer tissue. RESULTS: Expression of VEGI-192 and TL1A was elevated in colon cancer, although the level of VEGI-192 decreased, while the level of TL1A increased with the progression of cancer. Patients with low expression of TL1A and/or high expression of VEGI-192 in tumor-transformed tissue showed longer survival. DR3 expression was decreased in the cancer, but it did not change with the tumor progression. Alleles T of rs6478108 and G of rs6478109 SNPs were associated with elevated expression of the TNFSF15 gene. There was no relation between the MSI status and TNFSF15 expression levels. CONCLUSIONS: Expression of the TNFSF15 gene isoforms was associated with the progression of colon cancer. Levels of TL1A and VEGI-192 transcripts can be considered as independent prognostic factors for colon cancer.

Optimal reference genes for qPCR in resting and activated human NK cells--Flow cytometric data correspond to qPCR gene expression analysis.

Natural killer cells (NK cells) are cytotoxic lymphocytes critical to the innate immune system engaged in rapid response against tumor or virus infected cells. After activation NK cells acquire enhanced cytotoxicity and are capable of producing cytokines to stimulate other immune cells. Quantitative PCR (qPCR) is a method of choice for gene expression analysis but the usage of reliable reference genes for the normalization process is critical. Commonly used reference genes may vary in expression level between different experimental conditions providing wrong quantitative results of the studied genes' expression levels. Fourteen potential endogenous control genes were analyzed by qPCR method in NK-92 cell line that shows characteristics of human natural killer cells and is often used in studies on biology of NK lymphocytes. NK-92 cells were stimulated with IL-2 or TNF for 2, 24 or 72 h. Results were analyzed with RefFinder, a program which enables evaluation and screening of reference genes and integrates the currently available major computational programs (Genorm, Normfinder, BestKeeper and Delta Ct). The most stable gene in activated and non-activated NK cells was B2M, followed by IPO-8 and GAPDH and the least stable were HPRT1, PPIA and RPL32. The normalization process was performed on SOD2 gene and the results of qPCR experiments were confirmed by flow cytometry. The flow cytometric data corresponded to the results of qPCR gene expression analysis performed for the reference genes qualified by RefFinder as the most stable.