Trend For Lower Expression Research Articles

Impact of Epithelial-Mesenchymal Immunophenotype on Local Aggressiveness in Papillary Thyroid Carcinoma Invading the Airway.

Primary thyroid tumours show different levels of aggressiveness, from indolent to rapidly growing infiltrating malignancies. The most effective therapeutic option is surgery when radical resection is feasible. Biomarkers of aggressiveness may help in scheduling extended resections such as airway infiltration, avoiding a non-radical approach. The aim of the study is to evaluate the prognostic role of E-cadherin, N-cadherin, Aryl hydrocarbon receptor (AhR), and CD147 in different biological behaviours. Fifty-five samples from three groups of thyroid carcinomas were stained: papillary thyroid carcinomas (PTCs) infiltrating the airway (PTC-A), papillary intra-thyroid carcinomas (PTC-B) and poorly differentiated or anaplastic thyroid carcinomas (PDTC/ATC). High expressions of N-cadherin and AhR were associated with higher locoregional tumour aggressiveness (p = 0.005 and p < 0.001 respectively); PDTC/ATC more frequently showed a high expression of CD147 (p = 0.011), and a trend of lower expression of E-cadherin was registered in more aggressive neoplasms. Moreover, high levels of AhR were found with recurrent/persistent diseases (p = 0.031), particularly when tumours showed a concomitant high N-cadherin expression (p = 0.043). The study suggests that knowing in advance onco-biological factors with a potential role to discriminate between different subsets of patients could help the decision-making process, providing a more solid therapeutic indication and an increased expectation for radical surgery.

Association of CCL7 Promoter Polymorphism with Responsiveness to Allergen in Cynomolgus Macaque Model of Asthma

BACKGROUND: C-C motif Ligand 7 (CCL7) has been reported to be associated with asthma severity in humans. Cynomolgus macaques (Macaca fascicularis; Mf) are often used as animal model of asthma but little is known about Mf genetic profile such as polymorphism. Our aim was to identify CCL7 polymorphism in Mf as a potential surrogate marker for identification of allergen responsiveness in the Mf model of asthma.METHODS: Real-time PCR was performed on archive of bronchoalveolar fluid samples previously collected from Mf that were exposed to allergen. Expression of CCL7 mRNA was evaluated, and sequencing technique was used to identify polymorphism in this gene.RESULTS: The results showed that CCL7 expression did not differ between Mf, despite a trend of lower expression in Mf that exhibited high response to allergen. By direct DNA sequencing of CCL7, 10 sequence variants were identified; three in promoter region (-460 G/A, -459 A/G, -456 -/A ), two in exon 1 (9 A/G, 65 G/C), four in intron 1 (135 T/C, 254 T/C, 420 T/C, 453 A/G), and one in intron 2 (1205 T/A).CONCLUSION: There was an association between Mf sensitivity to allergen with CCL7 promoter polymorphism at (-456 -/A). These results suggest that CCL7 may be a potential genetic marker to identify Mf sensitivity to allergen, which could be a useful tool to efficiently select for Mf model of asthma.KEYWORDS: asthma, CCL7, allergy, Ascaris suum, nonhuman primate

Immunohistochemical Assessment of the Expression of Biliary Transportation Proteins MRP2 and MRP3 in Hepatocellular Carcinoma and in Cholangiocarcinoma

Multidrug resistance-associated protein 2 (MRP2) is a multi-specific organic anion transporter predominantly expressed in the canalicular membrane of hepatocytes, epithelial cells from gallbladder and apical membranes of proximal tubular kidney epithelium whereas multidrug resistance-associated protein 3 (MRP3) is present in the basolateral membrane of hepatocytes and cholangiocytes. This study aims to detect the expression of these transporters in hepatocellular carcinoma (HCC) and in cholangiocarcinoma (CC), searching for evidences for future studies on differential diagnosis and on clinical essays. The immunohistochemical reactivity (IHC) of these transporters was assessed in tissue microarrays of 80 HCC and 56 CC cases using monoclonal antibodies and compared with anatomopathological (AP) variables. The positivity of MRP2 was observed in 92.3% of HCC and in 96.3% of CC. The detection of high MRP2 expression in HCC was not significantly different (p > 0.05) according to the size, number of nodules architectural pattern and growth pattern of HCC and CC. Regarding histological grades, 22/22 well moderately differentiated HCC versus 50/56 poorly differentiated HCC were positive for MRP2. A trend for lower expression in poor differentiation HCC was found. And 50/50 well/moderately differentiated CC versus 2/4 poorly/undifferentiated CC were positive for MRP2. This result showed a reduced expression (p = 0,0004) in poorly differentiated CC. MRP3 positivity was observed in 18.8% of HCC and was not significantly different according to AP parameters. MRP3 was expressed in 44.5% CC, with a trend for lower expression in less differentiated CC and significantly lower rates in the ductular histological subtype (p = 0.023). The high expression of MRP2 in HCC and in CC is conserved regardless most of the anatomopathological parameters, except for a trend of lower expression in less differentiated HCC and CC. The observation of lower MRP3 expression in less differentiated CC and, especially, in the histological subtype with expression of hepatic progenitor cell phenotypes leads to future opportunities to evaluate the expression of this marker in cholangiocarcinomas.

Changes in mitochondria fusion protein-2 hepatic expression in conditions of liver cirrhosis and acute on chronic liver failure

To determine the differential protein and mRNA expressions of mitochondria fusion protein-2 (Mfn2) in hepatic tissues in conditions of cirrhosis and acute on chronic liver failure using rat model systems,and to determine the correlative effects on production of adenosine triphosphate (ATP) and reactive oxygen species (ROS). A liver cirrhotic rat model (LC rats) was established by intraperitoneal injection of carbon tetrachloride (CCl4,in vegetable oil),and these mice were subsequently used (10 weeks later) to establish the acute on chronic liver failure rat model (LF rats) by injecting lipopolysaccharide and D-amino-galactose.Control groups (normal controls,NC rats) were established for each model by intraperitoneal injection of vegetable oil only.Protein expression of Mfn2 in liver was quantified by western blotting with fluorescence densitometry and immunofluorescence staining,and mRNA expression was measured by real-time fluorescence quantitative PCR.ROS levels in liver were measured by fluorescence spectrophotometry,and ATP content was measured by bioluminescence assay.Significance of inter-group differences was assessed by one-way ANOVA,and correlations were determined using bivariate statistical modeling. Mfn2 protein expression was significantly lower in the liver tissues from modeled rats than that from the control rats (LC:0.051+/-0.004 and LF:0.037+/-0.007 vs.NC:0.254+/-0.008;F=444.98,P less than 0.05).The mRNA expression followed the same trend of lower expression (LC:21.21+/-0.93 and LF:24.35+/-0.85 vs.NC:19.09+/-0.69; F=66.941,P less than 0.05).The ATP content in liver tissues was also significantly lower in the modeled rats (LC:2.07+/-0.05 mol/L and LF:1.81+/-0.11 mol/L vs.NC:3.24+/-0.08 mol/L; F =574.21,P less than 0.05).Lower Mfn2 expression was correlated with lower ATP content (r =0.982) and higher ROS content (r =0.803). Reduced Mfn2 expression in liver tissue may cause a decrease in ATP synthesis and increase in ROS generation,thereby disrupting metabolism and increasing oxidative stress in the liver under conditions of cirrhosis and liver failure.

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Introduction: Previous genome-wide expression studies demonstrated a subclass of children with septic shock characterized by repression of genes corresponding to the glucocorticoid receptor (GCR) signaling pathway. This subclass had higher illness severity and higher mortality, compared to other subclasses (Wong et al. BMC Med 2009 7:34) Hypothesis: We tested the hypothesis that decreased GCR expression correlates with the degree of illness severity in critically ill children. Methods: GCR expression was measured by flow cytometry in lymphocytes (Ly) and parallel cortisol level were collected. Mean fluorescence (MF) values minus background fluorescence (FMO) were analyzed for correlation with clinical data. Results: Cohort: 36 critically ill children, median age 86 months (IQR 24-127), 56% males, median PRISM III 6 (IQR 2-11), median # organ failures (OF) 1 (IQR 0-3), 13 subjects received cardiovascular support (shock group 36%) and 19 subjects received steroids (steroids group 53%). Specific findings: Shock group patients had lower GCR expression in CD8Ly (mean 812 vs 1076, p=0.05) and a trend for lower expression in CD4Ly (mean 731 vs 956, p=0.07), compared to non shock patients. Steroid administration did not correlate with GCR expression. Subjects with? 2 OF in the first 7 days of admission had lower GCR expression in CD4Ly (median 635 vs 897, p=0.045) and CD8Ly (mean 833 vs 1129, p=0.026) when compared with subjects with? 1 OF. Patients with a PRISM III value? 7 showed a trend for lower GCR expression in CD4Ly (mean 773 vs 977, p=0.08) and CD8Ly (mean 865 vs 1097, p=0.07) when compared with patients having PRISM III scores < 7. Cortisol? 7.5 mcg/dL were associated with a lower GCR expression when compared with cortisol levels < 7.5 (CD4Ly median 626 vs 859, p=0.045, CD8Ly median 754 vs 1004, p=0.024). Conclusions: This preliminary sudy suggests that patients with shock and increased illness severity have lower GCR expression in Ly, consistent with gene expression studies. If this association holds true across a larger sample size, it may have conceptual implications for critical illness-related corticosteroid insufficiency.Supported in part by the Nurturing Children’s Development Program, Procter & Gamble Co.

Mechanism of Resistance to Azacitidine in Myelodisplastic Syndromes.

AbstractAbstract 2810Azacitidine is a pyrimidine nucleoside analogue of cytidine and it is at present the standard treatment for myelodysplastic syndromes (MDS). A large number of studies performed to evaluate the mechanism of resistance/response to nucleoside chemotherapeutics like cytosine arabinoside or gemcitabine, have demonstrated the importance of the levels of expression of the enzymes involved in their metabolism. Despite the high clinical interest in azacitidine, little is known about its intracellular metabolism and scanty data are available in literature. Recently, differences in the expression of these enzymes has been reported in cell lines differentially sensitive to hypomethylating agents. Clinical responses to azacitidine are heterogeneous, mainly due to clinical characteristics of MDS patients, so there is a need to identify molecular markers that could predict and/or monitor the efficacy of this therapy. A better understanding of the biological mechanisms involved in the activation and DNA uptake of azacitidine is therefore necessary to possibly identify responsive MDS patients. Objectives:To measure the expression levels of the 5 enzymes involved in azacitidine metabolism and to correlate their gene expression with the clinical response to the drug. To test possible causes of differences in gene expression. Material and Methods:DNA and RNA was extracted from mononuclear cells of 39 IPSS high risk MDS patients treated with at least 6 cycles of azacitidine 75mg/m2/7 days every 28 days. Of them, 14 patients were responders and 25 non-responders to azacitidine according to IWG 2006 criteria. Gene expression was assessed with quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes of every selected gene will be acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Promoter methylation of UCK1 was evaluated by methylation specific PCR using specific primers. The UCK1 coding region was sequenced by Sanger method. Results:Gene expression of hENT1, UCK1, DCK, RNR1 and RNR2 was compared between the groups of responders and non-responders. Individually, none of the genes was differentially expressed in responders versus non-responders. Nevertheless, a trend for lower expression in non-responders was shown for the UCK1 gene (median responders: 12.4 versus median non responders: 10.7; P = 0.095). In order to verify whether the presence of aberrant methylation could impact in UCK1 gene expression, we measured the methylation status of a putative CpG island in the UCK1 gene promoter region. The CpG island was unmethylated in all the samples analyzed, including responders and non-responder cases, as well as healthy donors. Moreover, to verify whether the difference in gene expression could be due to nucleotide variants in the gene sequence, we sequenced the UCK1 sequence. We found one polymorphic locus (rs2296957) in exon 6 and additional five intronic SNPs (rs3904060 and rs7867616, Intron 1; rs2296956 and rs189473964, Intron 3; rs150900763, 5' UTR) at the intron/exon boundaries. The presence of an individual genotype at these loci was not associated with therapy response and UCK1 gene expression. Conclusions:Our results show that absence of clinical response to azacitidine could be partly correlated with lower expression of UCK1. This is most likely causing a scarce azacitidine monophosphorylation by UCK1 in non-responders patients. The methylation status of the UCK1 gene promoter is not affecting gene expression. In addition, the genetic polymorphisms found in the UCK1 sequence do not associate with therapy response nor with gene expression. Further studies on larger series of MDS cases are needed to confirm these results which could help driving therapeutic decisions. Disclosures:No relevant conflicts of interest to declare.

Effect of Sub-Atmospheric Oxygen on the Culture of Rabbit Limbal Epithelial Cells

Purpose: To investigate the effect of sub-atmospheric oxygen tensions on the maintenance and expansion of limbal epithelial cells in vitro.Materials and Methods: Limbal epithelial cells were isolated from rabbit corneas and cultured in 21%, 14%, 8%, and 2% oxygen in a co-culture system with Mitomycin C growth arrested 3T3 fibroblasts. The colony forming efficiency, proliferative ability, and cell cycle distribution of cells cultured in these different oxygen tensions were determined, and semi-quantitative RT-PCR was used to detect expression of putative limbal epithelial stem cell markers, such as ABCG2 and p63α.Results: Of the four different oxygen tensions studied, 14% and 2% supported the highest and lowest colony forming efficiency values, respectively. A greater proportion of cells were found in S and G2/M phases of the cell cycle in primary 14% and 8% oxygen cultures compared to atmospheric controls. Differentiation was promoted at oxygen tensions of 8% and below. Cells with a differentiated phenotype and limited proliferative capacity were observed in 2% oxygen. Semi-quantitative RT-PCR analysis showed that the putative limbal epithelial stem cell markers ABCG2 and p63α were expressed in 21%, 14%, and 8% oxygen, with a trend of lower expression in 8% oxygen being observed.Conclusions: Limbal epithelial cells are sensitive to in vitro changes in oxygen tension. A sub-atmospheric oxygen tension of 14% promoted the maintenance and expansion of cells with limbal epithelial stem cell characteristics in a feeder co-culture system and is, therefore, recommended for the culture of rabbit limbal epithelial cells. This may also have relevance for the culture of human limbal epithelial stem cells for therapeutic application.