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Exp Mol Pathol [journal]
- The Inflammasome in Alcoholic Hepatitis: Its Relationship with Mallory-Denk Body Formation. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Aug 19.
Recent studies indicate that the inflammasome activation plays important roles in the pathogenesis of alcoholic hepatitis (AH). Nod-like receptor protein 3 (NLRP3) is a key component of the macromolecular complex that is so called the inflammasome that triggers caspase 1-dependent maturation of the precursors of IL-1β and IL-18 cytokines. It is also known that the adaptor proteins including apoptosis-associated speck-like protein containing CARD (ASC) and the mitochondrial antiviral signaling protein (MAVS) are necessary for NLRP3-dependent inflammasome function. Steatohepatitis frequently includes Mallory-Denk body (MDB) formation. In the case of alcoholic steatohepatitis, MDB formation occurs in 80% of biopsies (French 1981; French 1981). While previous studies have focused on in vitro cell lines and mouse models, we are the first group to investigate inflammasome activation in AH liver biopsy specimen and correlate it with MDB formation. Expression of NOD1, NLRP3, ASC, NAIP, MAVS, caspase 1, IL-1β, IL-18, and other inflammatory components including IL-6, IL-10, TNF-α, IFN-γ, STAT3, and p65 was measured in three to eight formalin-fixed paraffin-embedded AH specimens and control normal liver specimens by immunofluorescence staining and quantified by immunofluorescence intensity. The specimens were double stained with ubiquitin to demonstrate the relationship between inflammasome activation and MDB formation. MAVS, caspase1, IL-18, and TNF-α showed increases in expression in AH compared to the controls (p<0.05), and NAIP expression markedly increased in AH compared to the controls (p<0.01). There was a trend that levels of NLRP3, ASC, caspase1, IL-18, IL-10, and p65 expression correlated with the number of MDBs found in the same field of measurement (correlation coefficients were between 0.62 and 0.93, p<0.05). Our results demonstrate the activation of the inflammasome in AH and suggest that MDB could be an indicator of the extent of inflammasome activation.
- Assessment of molecular testing in Fine-needle aspiration biopsy samples: an experience in Chinese population. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Aug 8.
Fine-needle aspiration biopsy remains the mainstay of the preoperative examination of thyroid nodules; however, it does not provide a definite diagnosis in up to 25% of nodules. Considerable studies have been done to find molecular markers to resolve this diagnostic dilemma. The aim of this study was to establish the distribution and frequency of common genetic alterations in a comprehensive set of benign and malignant thyroid nodules, also to illustrate the feasibility and role of testing for a panel of genetic alterations in improving the accuracy of cytology diagnosis in Chinese population. This study was conducted in 314 thyroid nodules comprising 104 papillary thyroid carcinoma, 13 suspicious nodules, 52 indeterminate nodules, and 145 benign nodules. Point mutations and RET/PTC rearrangements, were evaluated by pyrosequencing and TaqMan real-time PCR, respectively. After surgery, 115 nodules were confirmed to be conventional papillary thyroid carcinoma and 102 (88.70%) of these nodules harbored either BRAF(V600E) mutation (76.52%) or RET/PTC rearrangements (12.17%). RAS mutation was found in 1(33.33%) follicular thyroid carcinoma, 1(14.29%) follicular thyroid adenoma and 4 (10%) goiter. With the application of cytology and molecular testing, the diagnostic accuracy was further enhanced 98.82% in papillary thyroid carcinoma diagnosis, and was preoperatively increased to 76.92% and 84.00%, respectively, in nodules with suspicious and indeterminate cytology. In conclusion, molecular testing of a panel of genetic alterations in fine-needle aspiration biopsy can be effectively performed in clinical practice. It enhances the accuracy of cytology and is of particular value for indeterminate nodules in Chinese population.
- TNFα gene/protein in tumorigenesis of sporadic colon adenocarcinoma. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Aug 4; 97(2):285-291.
Inherited polymorphisms in immunomodulatory genes such as cytokines may contribute to variation in immunological response and genetic susceptibility for complex diseases, including cancer. TNFα can mediate tumor progression by inducing proliferation, invasion and metastasis of tumor cells. The aim of our study was to examine the allelic frequencies of TNFα promoter SNPs, -1031 T/C, -857 C/T, -308 G/A and -238 G/A, in patients with sporadic colon adenocarcinoma in order to investigate the possible role of these SNPs in susceptibility to sporadic colon cancer. Another aim of this study was to examine the influence of TNFα SNPs on TNFα mRNA and protein expression in colon tumors and their possible role in the development and progression of this type of tumor.The distribution of all four TNFα SNP genotypes in patients showed no significant difference compared to controls. No statistically significant difference in TNFα mRNA expression in tumors and corresponding normal mucous tissue was found (p=0.14). A statistically significant (p=0.028) difference was found in TNFα mRNA expression between histological grade 1 and histological grade 2 and 3 tumors. Additionally, a statistically significant correlation (p=0.03) was found between TNFα-857 C/T genotypes and TNFα mRNA expression in tumor tissue. TNFα mRNA expression was significantly higher in the tumor tissue of patients with -857 CT and -857 TT genotypes. Most of the tumors (78.26%) were positive for TNFα protein. No correlation was found between the TNFα protein expression and clinicopathological characteristics as well as TNFα genotypes. However, patients with TNFα protein negative tumors had longer survival but the result was not statistically significant (p=0.365).Our results suggest the role of TNFα as one of the immunomodulatory genes in the progression of sporadic colon cancer.
- Performance of HPV E6/E7 mRNA RT-qPCR for screening and diagnosis of cervical cancer with ThinPrep® Pap test samples. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Aug 4.
Recent research has shown that oncogenic human papillomavirus (HPV) DNA, which is currently used in the screening and diagnosis of cervical cancer, can be detected not only in high-grade cervical lesions, but also in low-grade cervical lesions and normal tissues. For this reason, HPV tests targeting the E6 and E7 mRNA of five oncogenic HPV strains (HPV genotypes 16, 18, 31, 33, and 45), which are known to be responsible for the oncogenesis of cervical cancer, have been commercialized using a real-time nucleic acid sequence based amplification (NASBA) assay. Previous data has shown that the real-time NASBA assay has higher clinical specificity than HPV DNA testing (97.1% vs. 53.7%). However, the sensitivity of the real-time NASBA assay was lower than that of HPV DNA testing (41.1% vs. 100%). Despite the fact that there are more than 16 oncogenic HPV genotypes known to cause cervical cancer (HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69), the commercialized real-time NASBA kit was designed to detect only five genotypes (16, 18, 31, 33, and 45). Therefore, in the present study, CervicGen HPV RT-qDX (Optipharm), a commercial diagnostic kit targeting a HPV E6/E7 mRNA based on RT-qPCR assay was evaluated with RNA extracted from ThinPrep® Pap samples, and the results were compared to real-time NASBA data. The sensitivity and specificity of the RT-qPCR assay were 92% and 98.6%, respectively, for the detection of cervical intraepithelial neoplasia CIN2(+) high-grade cervical lesions. Therefore, the CervicGen HPV RT-qDX assay showed a significantly higher sensitivity (91.1%) compared to the real-time NASBA assay (41.1%). In normal cytohistology cases, the specificity was 98.6% and 53.7% for HPV mRNA RT-qPCR and HPV DNA testing, respectively. These results demonstrate that HPV mRNA RT-qPCR better reflects clinical diagnosis. In conclusion, it is suggested that HPV mRNA RT-qPCR overcomes the shortcomings of lower specificity seen in the DNA assay and the lower sensitivity of the commercialized HPV mRNA real-time NASBA assay when testing from ThinPrep® Pap samples.
- Patient-tailored application for Duchene muscular dystrophy on mdx mice based induced mesenchymal stem cells. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Aug 4.
Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and regeneration of damaged tissues. However, isolating tissue specific-derived MSCs may cause pain and increased infection rates in patients, and repetitive isolations may be required. To overcome these difficulties, we have examined alternative methods for MSC production. Here, we show that induced pluripotent stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs) following exposure to SB431542. Purified iMSCs were administered to mdx mice to study skeletal muscle regeneration in a murine model of muscular dystrophy. Purified iMSCs displayed fibroblast-like morphology, formed three-dimensional spheroid structures, and expressed characteristic mesenchymal stem cell surface markers such as CD29, CD33, CD73, CD90, and CD105. Moreover, iMSCs were capable of differentiating into adipogenic, osteogenic, and chondrogenic lineages. Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels, and normal dystrophin expression levels were restored. This study demonstrates the therapeutic potential of purified iMSCs in skeletal muscle regeneration in mdx mice, and suggests that iPSCs are a viable alternate source for deriving MSCs as needed.
- Role of microRNA-34 family in cancer with particular reference to cancer angiogenesis. [REVIEW]
- Exp Mol Pathol 2014 Aug 4.
MicroRNA-34 is involved in pathogenesis in cancer by targeting different tumor-related genes. It could be a biomarker for predicting the prognosis of patients with cancer. In addition, miR-34 is involved in the tumor angiogenesis. Understanding the mechanism of the miR-34 in cancer and tumor angiogenesis will open horizons for development of anti-cancer and anti-angiogenesis drugs.
- Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Jul 30; 97(2):259-265.
Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs.We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Student's t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines.We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes.This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.
- Sineoculis homeobox homolog 1 protein is associated with breast cancer progression and survival outcome. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Jul 22; 97(2):247-252.
Sineoculis homeobox homolog 1 (SIX1) is one of the transcription factors that act as master regulators of development and is frequently dysregulated in cancer. This study explores the roles of SIX1 in tumor progression and as a prognostic determinant of breast cancer. Breast cancer specimens from 262 patients were selected for analysis of SIX1 protein by immunohistochemistry (IHC). The localization of SIX1 protein was detected in MDA-MB468 breast cancer cells using immunofluorescence (IF) staining. The survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazard models. SIX1 protein mainly showed cytoplasmic/perinuclear staining pattern in breast cancer using IHC in paraffin embedded breast cancer tissues and IF in MDA-MB468 cancer cells. The strongly positive rate of SIX1 protein was 61.8% (162/262) in breast cancer and 23.1% (12/52) in ductal carcinoma in situ (DCIS), which was significantly higher than adjacent normal breast tissues (6.7%, 3/45). SIX1 overexpression was positively correlated with clinical stage, lymph node metastasis, Her2 expression status, and disease-free survival (DFS) and 5-year overall survival (OS) rates of patients with breast cancer. Moreover, patients with late stage breast cancer and high SIX1 expression had poorer survival rates than those with low SIX1 expression. Further analysis using a Cox proportional hazard regression model revealed that high SIX1 expression emerged as a significant independent hazard factor for the DFS and OS rates of patients with breast cancers along with Her2 status and clinical stage. SIX1 may potentially be used as an independent biomarker for prognostic evaluation of breast cancer.
- Characteristic, polymorphism and expressing distribution of LCAT gene in Mongolian gerbil model for hyperlipidemia. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Jul 16.
This study aims to evaluate the genetic basis and activity of lecithin cholesterol acyltransferase (LCAT) in a novel Mongolian gerbil model for hyperlipidemia. Gerbils may be susceptible to high fat and cholesterol (HF/HC) diets, which can rapidly lead to the development of hyperlipidemia. Approximately 10-30% of gerbils that are over 8months old and fed controlled diets spontaneously develop hyperlipidemia. Using the HF/HC diet model, we detected triglycerides (TG), total cholesterol (TC), HDL (high density lipoprotein)-C, LDL (low density lipoprotein)-C and LCAT in both old (>8months) and young gerbils. The TC and HDL-C levels were two times higher in old gerbils compared with young gerbils (P<0.01). However, in the old group the LCAT activity fell slightly compared with the normal lipidemia group. It is reasonable to hypothesize that this may be associated with single nucleotide polymorphisms of the LCAT gene. We cloned this gene to investigate the sensitivity of the gerbil to the HF/HC diet and spontaneous hyperlipidemia. The entire LCAT gene was cloned by splicing sequences of RACE(rapid amplification of cDNA ends) and nest-PCR products (AN:KC533867). The results showed that the 3683 base pair gene consists of six exons and five introns. The LCAT protein consists of 444 amino acid (AA) residues, which are analogous to the human LCAT gene, and includes 24 signal peptide AA and 420 mature protein AA. Expression of LCAT was detected in the kidney, spleen and adrenal tissue, apart from the liver, by immunohistochemistry. The abundance of the protein was greater in the older group compared with the control group. Polymorphisms were analyzed by PCR-SSCP(PCR-single-strand conformation polymorphism) but none were found in 444 animals of the ZCLA closed population(a Chinese cultured laboratory gerbil population).
- The Impact of TP53 and RAS Mutations on Cerebellar Glioblastomas. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Jul 15.
Cerebellar glioblastoma (cGBM) is rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanism of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM.